You are on page 1of 121

Molecular Biology

Techniques Review Guide

II. SPECIMEN COLLECTION, HANDLING, PREPARATION AND

PROCESSING

A. Identify Methods for Collection, Preservation, and Transport

B. Assessment and Handling of Specimens

• Specimen Type

• Evaluate acceptability of specimens

• Evaluate appropriateness of test requested

• Corrective Actions

C. Record Pertinent Data

D. Maintaining Records and Laboratory Database

Table of Contents

13

A. Identify Methods for Collection, Preservation, and Transport

1. Blood, bone marrow, bodily fluids, tissues, cell swabs, and almost anything with nucleated cells are
appropriate specimen types for molecular testing.

• Red blood cells (RBCs) are not nucleated.

• Cerebral spinal fluid (CSF) has very few cells.

• Urea (from urine specimens) inhibits the polymerase chain reaction (PCR).

• It is very difficult to get nucleic acid from fecal material.

2. Anticoagulants

• EDTA (ethylene diaminetetracetic acid) (lavender top) is the preferred

anticoagulant for molecular tests.


• ACD (acid citrate dextrose) type A (yellow top) is also an acceptable

anticoagulant in the molecular laboratory. ACD is preferred for RNA isolation.

• Heparin is not used in the molecular laboratory as it inhibits DNA binding

enzymes and inhibits Taq polymerase. Heparin may interfere with restriction

enzyme (RE) digestion and therefore result in a pattern of partial digestion.

Some labs will accept specimens collected in heparin.

• Serum is collected in a red top vacutainer (no additive).

3. Storage conditions

• Specimens should be kept at cool temperatures.

• Specimens should be aliquoted to smaller tubes to avoid multiple freeze/thaws

• Tissue specimens for RNA work (gene expression) should be snap-frozen in

liquid nitrogen to prevent degradation of RNA or at least stored at -70C.

4. Shipping

• Samples should be protected from temperature extremes.

• Laboratories must comply with Bloodborne Pathogens shipping rules.

• A protective container with absorbent material will minimize chances of container

breaks and a sealed container/bag should be used to prevent leaks.

• All containers must be labeled as biohazard.

• Glass vacutainer tubes should be protected from breakage by wrapping in paper

towels or bubblewrap.

• Overnight mail service is best to ensure prompt delivery of specimens.

*The amount of specimen required for testing will vary based on specimen type, age of patient, clinical
presentation of patient and test required - most laboratories will not refuse a specimen due to

insufficient quantity, but rather will try anything.

Table of Contents

14

B. Assessment and Handling of Specimens

1. BLOOD

• 5 - 10 ml for Southern blot analysis

• .5ml - 3 ml for PCR

• 10 - 20 ml for RNA based tests

• The population of white blood cells can be enriched by isolating the buffy coat or

isolating mononuclear cells via ficoll hypaque gradient (use right one for desired

cell type)

o Can freeze mononuclear cells for storage for up to 2 months at -20C and

> 1 year at -70C (alone or in phosphate buffered saline)

• Blood may be stored 1-7 days at room temperature or at 2-8C

• -20C and -70C not recommended for storage of whole blood

2. BLOOD SERUM

• Serum should be separated from whole blood and frozen as soon as possible

(within 4 hours) so that any RNA viruses present will not degrade (this specimen

type usually used for infectious disease analysis)

• 1-2 ml is usually enough

• Serum is best shipped on dry ice overnight

• This specimen type also used for antibody detection (Western blotting)
3. BONE MARROW

• Similar requirements as whole blood

• Storage at -20C stable for 2 weeks

4. BUCCAL CELLS

• Buccal cells are used for PCR based tests only

• The cotton swab or brush used to collect the buccal cells should be stored in a

closed tube at room temperature or 4C

• There is no hemoglobin present in this sample type and thus it is stable longer

5. CULTURED CELLS

• 1-2 confluent T25 flasks of cultured cells required for testing

• T25s should be shipped full of media

• Cultured cells are appropriate for RNA testing only if the gene of interest is

expressed in that tissue

• Example cell types include muscle, amniocytes, tumor cells, fibroblasts

6. AMNIOCYTES

• Application is prenatal genetic diagnosis

o Amniocentesis usually performed at 16-18 weeks gestation

• DNA can be prepared from amniotic fluid directly or from cell cultures

• If preparing DNA directly from fluid – use ~10ml of unspun fluid or centrifuge to

obtain a cell pellet

o Small yields of DNA

Table of Contents
15

• Yield of DNA from 1 T25 flask of confluent cells ~25 ug (usually set up 2-3 flasks)

7. BLOOD SPOTS

• Screening cards (Guthrie)

• Dry well before shipping

• Protect from cross contamination

• Stable for 3 years at 4-25C

• Hemoglobin removal (time consuming) required{inhibitor of PCR}

• PCR based assays or infectious disease assays

8. SOLID TISSUE

• Ship tissues on ice and keep at -20 to -70 C for RNA based tests

• Tissue for DNA based tests is best shipped on ice - refrigerator temperatures ok

• Homogenize tissues to produce a suspension of single cells

• Snap freeze tissues in liquid nitrogen to preserve DNA

• Tissues can be stored at -20C for 2 weeks or at -70C for at least 2 years

• Thaw frozen tissues quickly

9. SPUTUM, PUS, URINE, CSF, ETC.

• Primarily used for infectious disease analysis

• Protect from cross contamination

• 4C or freeze if RNA

10. PARAFFIN SECTIONS


• Require special protocol to remove paraffin

11. SLIDES

• Cytogenetic preparations

• Tissue preparations

• Blood smears

• Used for PCR based tests

12. SAMPLES THAT DO NOT USUALLY WORK!

• Formalin fixed tissue

• Very old or lysed specimens

o Forensic testing or blood spot testing ok with these specimen types

16

C. Record Pertinent Data

1. Read label (name, date of birth, ID#) and cross check with all paperwork

2. Check storage and shipping conditions and amount of sample

• If blood sample is clotted, homogenize before extracting

• Look for leaks or damage

3. Generally, no specimen should be refused due to insufficient quantity

4. Be sure test requested is appropriate for sample type

5. Refuse all unlabeled specimens

6. Lysed samples} if PCR - try first, but if for Southern call for new sample

immediately

D. Maintaining Records and Laboratory Database

1. Record time/date collected, origin, specimen type, volume, specimen condition,


time/date received, initials, accession # from referral lab, accession # assigned,

test requested

2. Best to record everything in at least two places

3. Patient folder

4. Log book

5. Computer system

Table of Contents

17

III. PRINCIPLES AND TECHNIQUES

A. Nucleic Acid Preparation

1. Perform nucleic acid isolation, choosing the method by giving consideration to

specimen type and test requested

2. Re-suspend and Dilute/Determine Storage Conditions

3. Evaluate Quality and Quantity of Nucleic Acid

4. Employ and Document Corrective Actions

Table of Contents

18

1. Perform Nucleic Acid Isolation, Choosing the Method by Giving

Consideration to Specimen Type and Test Requested

RNA Extraction

A. RNA is extracted to evaluate EXPRESSION of a gene

B. Overview of steps required for RNA extraction:

1) disrupt cells or tissue

2) lyse cells in presence of protein denaturants

3) inactivate RNases
4) purify RNA away from DNA and protein

• guanidinium used to lyse cells and denature proteins thus inactivating

RNases

C. Guanidinium isothiocyanate (chaotropic agent) more effective than guanidinium

chloride in inhibiting ribonucleases

D. Guanidine thiocyanate and β mercaptoethanol are strong inhibitors of RNase

E. Procedure usually requires toxic/corrosive reagents

F. RNA is very easily susceptible to degradation by endogenous RNases

1. RNases are ubiquitous

2. RNases will not all be degraded by UV light or autoclaving

3. RNase high in pancreas and spleen, lower in liver and intestine

4. Whenever possible use sterile, disposable plasticware and always wear

gloves

5. Treat glassware with:

• diethylpyrocarbonate (DEPC)- inactivates ribonucleases by

covalent modification

• RNase Away

• NaOH

o if use NaOH, rinse well since NaOH destroys RNA

• bake at 200C overnight or 300C for 4 hours

• chloroform rinse

6. Treat liquids with DEPC except Tris buffers since Tris reacts with DEPC to

inactivate it

G. Does test require cellular RNA or viral RNA?


H. With cellular RNA be sure you have right tissue source since same RNA not in

every cell

I. Does test require total RNA (primarily ribosomal and transfer) or mRNA?

• mRNA isolation based on polyA tail

J. Mammalian cell contains ~ 15pg of RNA

K. ~80-85% of RNA contained within a cell is rRNA

DNA Extraction

A. Basic steps:

1) separate white blood cells (WBC) from red blood cells (RBC) or lyse RBC

2) lyse WBC

3) denature/digest proteins

4) pellet or separate proteins

Table of Contents

19

5) precipitate DNA with salt and alcohol

• hemoglobin strongly inhibits Taq polymerase and Proteinase K that is why

it is necessary to lyse the RBCs

• be gentle - don’t want to shear high molecular weight DNA

B. Typical yield: a normal diploid cell contains ~ 6-7 pg of genomic DNA

C. Organic isolation (rarely used because of toxic reagents)

• highly hydrophilic nucleic acids remain in aqueous phase and do not enter

organic phenol phase

• phenol(50):chloroform(49):isoamyl alcohol(1)

• phenol - denatures proteins, dissolves lipids, organic solvent


• chloroform - assists in dissolving remaining lipids, gives a cleaner

interphase between organic and aqueous phases, stabilizes boundary

between aqueous phase and pure phenol layer

• isoamyl alcohol - prevents foaming of mixture, aids in separation of

phases

• protein precipitates at interface of aqueous (top) and organic (bottom)

layers

• to ensure complete de-proteinization, extractions should be repeated until

no protein precipitate (white layer) is present at interphase of aqueous and

organic layers

• slow, labor intensive, need fume hood, vinyl gloves, use polypropylene

tubes

• precipitate with alcohol (ethanol or isopropanol: cold or room temp

depending on protocol)

D. Non-organic Isolation

• detergent (SDS) dissolves cell membranes and denatures proteins

o sodium dodecyl sulfate (SDS) – anionic detergent that denatures

proteins and makes them negatively charged by wrapping around

the polypeptides

• protein precipitate with salt solution {ammonium acetate, sodium chloride,

etc.}

• precipitate with room temperature alcohol so proteins will not precipitate

with the DNA, however for expected low yields, cold temperatures may be
used to precipitate DNA

• DNA can be “cleaned” prior to rehydration with rinses in 70% ethanol

E. Affinity Methods

1. Chelex (PCR grade DNA only)

• lyse cells by boiling

• impure, toasts proteins but may not get rid of all inhibitors

• Chelex has a high affinity for polyvalent metal ions, prevents DNA

degradation during boiling

2. Solid support (silica) or metal beads – (kits!)

20

2. Re-suspend & Dilute/Determine Storage Conditions

Note: Don’t want a lot of freeze/thaws

DNA

• resuspend DNA in water, Tris buffer or TE buffer(10mM Tris, 1mM EDTA) - be

sure to record amount of buffer

• TE buffer preferred since EDTA binds magnesium which is necessary for DNase

and RNase activity

• genomic DNA stable at 4C for weeks in TE

• can also store genomic DNA at -20/-80C in salt/ethanol

• PCR grade DNA is not stable for storage

RNA

• -80C or -20C in H2O, 0.1%DEPC H20 or TE (RNA dissolves easier in H2O than

in a salt solution)
• for long term storage keep RNA pellet in 70% ethanol at -70C

3. Evaluate Quality and Quantity of Nucleic Acid

A. Gel electrophoresis for evaluation of size and quantity (integrity test gel)

• compare to known standards

• good DNA - above marker for 20-60 Kb

• good RNA - 28S:18S rRNA =2:1

o eukaryotic 28S rRNA band is 5.0 kb in size

o eukaryotic 18S rRNA band is 1.87 kb in size

o if 18s band is more intense than 28S, or if there is a smear in the low

molecular weight range, then RNA is degraded

o DNA contamination is evident as very high molecular weight bands

• good quality DNA should be high molecular weight DNA - not a smear!

B. Spectrophotometry

• light of a certain wavelength is shined through a cuvette containing the sample of

interest and the amount of light that makes it through to the other side of the

cuvette is measured. Measuring how much light went in (incident light) and how

much light goes through (transmitted light) to calculate the amount of light

absorbed by the sample in the cuvette

• maximum absorption by nucleic acids (DNA and RNA!) is at 260nm

• maximum absorption by proteins is at 280 nm

• absorption at 230 nm may indicate presence of organic compounds,

thiocyanates, or phenol

• absorption at 270 nm may indicate presence of phenol


• absorption at 320/325 nm may indicate particulates in the solution or dirty

cuvettes

• spectrophotometry measurements are quantitative for relatively pure nucleic acid

preps in ug amounts

Table of Contents

21

• concentration of nucleic acids calculated from the optical density (OD) (OD=

absorbance when the path length of the light is 1 cm) at 260 nm

• 1 OD of ds DNA = 50ug/ml

• 1 OD of ss DNA = 33 ug/ml

• 1 OD of ss RNA = 40 ug/ml

• [DNA] = A260 x dilution factor x 50 ug/ml

• equation assumes a 1cm pathlength spectrophotometer cuvette and a neutral pH

• calculation based on Lambert-Beer law, A=ECl (A= absorbance, C=

concentration of DNA, l =pathlength of cuvette and E = extinction coefficient

• [RNA] = A260 x dilution factor x 40 ug/ml

C. Fluorimetry

• Quantitate ds DNA with concentrations of 2-500 ng/mL greater than 1kb in length

• Hoescht (pronounced “herkst”) dye binds with high specificity to AT rich regions

of DNA (toxic dye)

• Fluorimeter laser detects amount of Hoescht dye (bound Hoechst excitation

maximum is 365 nm, emission maximum of 458 nm whereas unbound stain =356
nm and 492 nm respectively for excitation and emission maximum)

• This method detects RNA less than 1% of the time

• Advantage: requires less sample and more sensitive than spectrophotometry

• must calibrate the fluorimeter with known concentrations of high molecular weight

dsDNA

D. Purity

• the ratio between A260nm and A280nm (A260/A280) used to estimate sample

purity

• pure DNA = 1.8 and pure RNA = 2.0

• acceptable ratio = 1.7-2.0

• 260 = nucleic acids, 280 = proteins, 230 = phenol, urea, salts , 325 = background

scatter,dirty cuvettes

• < 1.7 = too much protein or phenol

• >2.0=potential alcohol contamination

• low A260/A230 for RNA = contamination with guanidine thiocyanate or Bmercaptoethanol

22

4. Employ and Document Corrective Action

A. Low yield

1. repeat extraction if more sample is available

• adjust reagent amounts for low cell numbers

2. double check quantitation (pipetting or spectrophotometer error)

3. low yield may be due to insufficient time for resuspension

B. Poor quality and/or purity


1. repeat extraction if sample available

2. were reagents or specimen contaminated?

3. try to clean up with Proteinase K, phenol/chloroform, alcohol precipitation

C. Request new sample if required or proceed with testing depending on laboratory

protocol

D. Document all corrective actions

Table of Contents

23

B. Nucleic Acid Manipulations

1. Perform Restriction Enzyme (RE) Digests

2. Clone and Label Nucleic Acid

3. Prepare Nucleic Acid Probes

4. Probe Labeling Methods

5. Determine Labeling Efficiency and Separation of dNTPs from Labeled Probe

6. Cloning Procedures

7. Prepare cDNA

Table of Contents

24

1. Perform Restriction Enzyme (RE) Digests

• also known as restriction endonucleases

• naturally occurring in bacteria

• recognize and cleave specific sequences within double stranded DNA

• Type II (contain separate restriction and methylation systems) are the main type

of RE used in labs
• RE have an absolute requirement for magnesium

• most recognize palindromic sequences (in other words, the restriction enzyme

cut sequence reads the same in both directions)

• most RE cut within recognition sequence so leave usually 5’-phosphate, 3’-

hydroxyl overhangs, or blunt ends

• isochizomers - different RE that recognize same sequence (cleavage site may

differ) and may have different methylation sensitivities

• 1 unit of RE Activity = amount of enzyme required to completely digest 1 ug of

DNA in 1 hour at appropriate temperature, with a specific buffer in a 50 ul

reaction volume

• ideally, a gene of interest should be digested with an enzyme that will cut the

gene into a few fragments (1-3) that range in size from 1.0 – 10.0 kb

DNA methylation

• addition of methyl (CH3) groups to DNA bases (usually to cytosine)

• methyl sensitive enzymes will not cleave methylated recognition sequence

DNA substrates

• bacterial plasmid DNA (requires more RE because it is supercoiled, closed

form, usually 3-10kb in size), genomic DNA (high molecular weight linear) and

DNA from bacteriophage lambda (48 kb and linear)

Contaminants

• dose dependent

• phenol, chloroform, protein, alcohol, EDTA, RNA, detergents, excess salts

Uses
• Southern Blots

• Restriction Fragment Length Polymorphisms (RFLPs)

• predigest before PCR

• digest plasmids for probe preps

Table of Contents

25

Reaction components

A. DNA

B. Enzyme

• ADD ENZYME LAST

• KEEP ENZYME ON ICE

• be sure to mix all reaction components (gently)

• spin or vortex gently so as not to inactivate the enzyme

• 5-10 units of RE/ ug DNA to be digested

• volume not >1/10 total reaction volume because usually stored in glycerol

o glycerol lowers freezing point of enzyme

o excess glycerol in a reaction may cause diminished enzyme

activity, processivity or star activity

Buffer

• specific for each enzyme, but comes from manufacturer

• provides optimal pH and ion requirements for enzyme

• 10X buffer supplied should be 1X final concentration


H2O

• bring final volume of reaction up

• practical reaction volume is not less than 10 ul (more likely to have

pipetting errors below this volume)

Additives

• BSA (bovine serum albumin) - enzyme stabilizer/carrier protein

• DTT (dithiothreitol) - enzyme stabilizer

• Triton X-100 - stabilizes some enzymes

• spermidine can be added to improve digestion of <1.8 genomic DNA

o spermidine is polycationic and will bind negatively charged

contaminants

• most digests at 37C

• the method used to stop a RE digest depends on next application and enzyme

used

o add gel loading dye

􀂃 gel loading dye contains glycerol, a dye (bromophenol blue

or xylene cyanol), EDTA and sodium dodecyl sulfate (SDS)

o heat inactivation

o addition of 10mM EDTA (since EDTA binds Magnesium)

o phenol extraction and ethanol precipitation

26

• to check for RE digest efficiency, a small aliquot of the digest reaction is run out

on a test gel
o run .5-1.0 ug on a EtBr (ethidium bromide) gel for complete digestion

o a smear is evidence of complete digestion

o a single high molecular weight band indicates non-digestion

o may see evidence of bands - repeat sequences

o check that fluorescent intensity the same across all wells that were loaded

with same amount of DNA and cut with same enzyme

o partial digestion – may see higher MW bands

o star activity - enzyme cuts more than it should (overdigestion)

􀂃 star activity can be due to too high glycerol concentration or

incorrect salt concentrations

Restriction Enzyme Digestion Set-Up Calculations

Example: Set up a RE digest of 10 ug of DNA using 10 units of RE per ug of DNA.

DNA concentration is .5 ug/ul.

RE concentration is 50 units/ul. RE buffer = 10X

Total RE digest volume = 30 ul

1. Volume of DNA sample that equals 10 ug of DNA :

.5 ug/1 ul = 10 ug/ x ul x = 20 ul DNA

2. Volume of RE needed:

10 ug DNA x 10 units/ug RE x 1 ul/50 units = 2 ul RE

3. Volume of RE buffer:

x (10) = 1(30) x = 3 ul

4. Volume of distilled H2O to add:

20 ul of DNA + 2 ul RE + 3 ul buffer = 25 ul

so need 5 ul of H2O to bring volume up to 30 ul

2. Clone and Label Nucleic Acid


Enzymes Used for Cloning

Definitions and notes

Processivity: number of dNTPs that a polymerase can incorporate continuously

without dissociating from primer template

Fidelity: error rate of polymerase

• enzymes have specific pH, ionic, and temperature requirements

• do not expose enzymes to increased temperatures or many freeze/thaws

• excess glycerol will impair enzyme activity

o not to exceed 10% of enzyme volume

Star Activity: additional cleavages at imperfect recognition sequences

Table of Contents

27

Polymerases: 1 unit is amount of enzyme required to incorporate 1 nmol NTP or

dNTP into acid soluble product in 1 hour at recommended temperature

DNA polymerases -

• DNA Polymerases are enzymes that add deoxyribonucleotides to 3’ hydroxyl

end of a primed dsDNA – synthesis is in a 5’-3’ direction with respect to the

synthesized strand – each nucleotide that is incorporated is complementary to

the template strand.

• reaction components: template, primer with free 3’OH group attached to

template, dNTPs, Mg (magnesium)

• Many DNA polymerases have an exonuclease activity (3’ to 5’)

• takes out 1 nucleotide at a time


• proofreading function

• some DNA polymerases also have 5’ to 3’ exonuclease activity

• takes out 1 to several nucleotides at a time

• most are mesophilic (active at 37-42C) or thermostable

• examples:

DNA polymerase I (holoenzyme)

• 3’ and 5’ exonuclease activity, low processivity

DNA polymerase I (Klenow)

• either or both exonuclease activity removed (remove with protease

treatment or by deleting gene), low processivity

T4 DNA polymerase

• ss and ds 3’ to 5’ exonuclease activity

• low processivity

• lacks 5’ to 3’ exonuclease activity

• used for 3’ end labeling

T7 DNA polymerase

• ss and ds 3’ to 5’ exonuclease activity

• high processivity therefore enzyme of choice for DNA synthesis from

primers on long template

• also used for 3’ end labeling

• can be modified to remove 3’ to 5’ exonuclease activity which

increases its processivity and rate of DNA synthesis


Taq Polymerase (Thermus acquaticus)

• thermostable

• 5’ exonuclease function

• requires Mg and primer with free 3’ hydroxyl

• inhibited by EDTA

• used for amplification techniques

• stable at 95C (1/2 life =45 minute)

• polymerizes DNA at rate of 100 nucleotides per second per molecule

at 70C

28

Tth DNA polymerase (Thermus thermophilus)

• thermostable

• 5’ exonuclease activity

• reverse transcriptase activity

• requires Mg, primer with free 3’ hydroxyl for polymerase activity

• requires Mn for reverse transcriptase activity

• used for amplification techniques

RNA Polymerases -

• require DNA template, NTPs and appropriate promoter

• will transcribe DNA (ds or ss) into RNA using NTPs

• only DNA downstream of the specific promoter will be transcribed


• examples: T7 RNA Polymerase, E.coli RNA polymerase, T3 RNA polymerase

and SP6 RNA polymerase

• reaction components: TrisCl, MgCl2, DTT or B-mercaptoethanol, DNA

template with promoter, {primer not required}, NTPs, BSA, RNA polymerase

{spermidine required for SP6}

• stop reaction with EDTA or heat

• applications: RNA probes

Reverse Transcriptases

• use RNA as template to make cDNA

• deoxyoligos used as primers on RNA (usually mRNA)

• use DNA as template to make DNA (DNA dependent DNA polymerase)

o dNTP incorporation is very slow

o this DNA polymerase lacks 3’-5’ exonuclease function

• RNase H activity:

o ribonuclease (RNase) H is an endonuclease specific for the RNA of a

RNA:DNA hybrid

o expressed in every cell

o 3’ and/or 5’ exoribonuclease activity

• reaction components: TrisCl, MgCl2, DTT, KCl, mRNA, oligonucleotide,

dNTPs, BSA, reverse transcriptase

• stop reaction with EDTA, heat

• can get rid of RNA with NaOH incubation overnight


• intermediate processivity

• other uses: labeling 3’ end of DNA fragments with 5’ protruding ends

29

Nucleases: 1 unit is amount of enzyme required to produce 1 nmol acid soluble

nucleotides from nucleic acid at specified conditions

Nucleases

• most common group are the restriction enzymes

• degrade phosphodiester bonds in nucleic acids

Endonucleases: recognize and cut within specific sequence within nucleic acid

o DNase I - endonuclease that attacks ss or ds DNA at pyrimidines, used for

nick translation

o RNase I - endoribonuclease cleaves 3’ of A,C, G and U

o RNase A - endoribonuclease cleaves 3’ at pyrimidines , used to remove

RNA during procedures for the isolation of plasmid or genomic DNA

Exonucleases: degrade DNA from free 3’ hydroxyl or 5’ phosphate ends

DNA Ligases

• repair ss nicks in ds DNA

• join 5’ phosphate and 3’ hydroxyl of ds DNA RE fragments having blunt ends

or homologous cohesive ends

E. Coli ligase

• uses NAD as source of energy

• will not ligate blunt ends

T4 DNA ligase
• will ligate blunt ends

• uses ATP as energy source

• inhibited by >150mM NaCl concentrations

• more commonly used ligase

Ligation reaction components: TrisCl, MgCl2, DTT, DNA, ATP or NAD, BSA,

DNA ligase

• reaction temperature: 12C - 15C

o higher temperatures = difficult annealing ends

o lower temperatures = lower ligase activity

o blunt ends = room temperature

• reaction stop with EDTA or heat

30

RNA Ligases

T4 RNA ligase

• joins ss 5’ phosphate end of DNA or RNA to ss 3’ hydroxyl end of DNA or

RNA

• reaction components: HEPES, MgCl2, DTT, ssDNA or ss RNA, ATP, BSA,

T4 RNA ligase

• used for labeling 3’ end of RNA, circularizing oligonucleotides, ligating

oligomers

Alkaline phosphatases

• catalyze hydrolysis of 5’ phosphate residues from DNA, RNA, rNTPs and

dNTPs
• the dephosphorylated products possess 5’hydroxyl termini which can then

be radioactively labeled (used for Maxam and Gilbert sequencing)

• prevents vector religation to reduce background in cloning

• require zinc for activity

• example: bacterial alkaline phosphatase and calf intestine phosphatase

3. Prepare Nucleic Acid Probes

• a probe is a labeled piece of nucleic acid used in hybridization to detect sequences

of closely related sequence

• RNA probes produce stronger signals than DNA probes

• labeled with reporter molecule which allows visualization

o 32P, biotin, digoxigenin - see Probe Labels

4. Probe Labeling Methods

A. Nick Translation

• low levels of DNase introduce ss nicks into ds DNA (ss DNA not appropriate

for this method)

• DNA Polymerase I will add nucleotides to the 3’-hydroxyl end that is created

when the ds DNA is nicked. DNA Polymerase I also has a 5’ to 3’

exonuclease activity, therefore can remove nucleotides from the 5’ end of the

strand. The simultaneous removal of nucleotides from the 5’ end and the

addition of nucleotides to the 3’ end results in the translation (movement) of

the nick.

o 3’ end of nicked molecule serves as primer for new DNA synthesis by

DNA Polymerase (5’-3’ synthesis)


o 5’-3’ exonuclease activity of DNA polymerase removes next nucleotide as

each nucleotide is added

• synthesis incorporates dNTPs, 1 is labeled (usually CTP)

• no net DNA synthesis, but labeled dNTPs are incorporated into probe of

interest

• reaction components: TrisHCl buffer, MgCl2, TE buffer, labeled dNTP,

unlabeled dNTPs, DNA Pol I, DNase I, (keep all ice cold while using), DNA

• reaction at 15C (1-3 hours)

Table of Contents

31

o “snapback” DNA occurs at higher temperatures

• stop reaction with EDTA

• store labeled probe at -20C in presence of EDTA (high activity probes should

not be stored longer than 3 days)

• large amount of template required (0.5 ug/reaction)

• protocol not suitable for small size fragments of DNA (<200bp)

• troubleshooting:

o insufficient labeling: reaction time too short or DNase I too low

o degraded DNA: reaction time too long, DNase I too high, or prolonged

storage

B. Random Primed Labeling

• Oligonucleotides serve as primers for initiation of DNA synthesis on singlestranded

DNA or RNA. Synthesis is carried out using Klenow since this


enzyme does not have a 5’-3’ exonuclease activity. Labeled nucleotides are

incorporated during synthesis.

• overview of procedure

o RE digest DNA

o denature DNA (must remove RE buffer from DNA because Mg will inhibit

denaturation)

o anneal probe primers (random 6-9mers, all possible hexanucleotides)

o add Klenow (no exonuclease function) and dNTPs ( 1 is labeled)

• reaction components: RE digested ss DNA, dNTPs, labeled dNTP, Klenow,

TrisHCl, MgCl2, TE buffer, random hexanucleotides

• reaction temperature: room temperature or 37C

• stop reaction with EDTA

• store labeled probe at -20C in presence of EDTA (do not store high activity

probes for longer than 3 days)

• small quantities of template DNA required (25-50 ng)

• reaction appropriate for small fragments (200 bp)

• higher specificity than nick translation

• usually produces shorter probes than nick translation generated probes

C. End Labeling

3’ end labeling

Terminal (deoxynucleotidyl) transferase (TdT)- (also known as terminal

transferase)

• this enzyme does not require a template


• radioactive or nonradioactive tags

• requires divalent cations (incorporation depends on which cation used)

• preferable primer = ss DNA

• reaction components: sodium cacodylate {poisonous arsenic compound},

divalent cation, DTT, DNA, dNTPS, BSA, terminal deoxynucleotidyl

transferase

32

Klenow Fragment

• reaction components: RE digested DNA, labeled dNTP, unlabeled dNTP,

(dNTP used depends on RE used), Klenow

• stop reaction with EDTA or heat

T4 DNA Polymerase

• reaction components: TrisCl, MgCl2, DTT, RE digested DNA (can do RE in

T4 buffer or if done in incompatible buffer, be sure to repurify with phenol

extraction, ethanol precipitation and resuspend in TE), dNTPs, BSA, T4

• reaction temperature: 11C

• stop reaction with EDTA or heat

• costs more than Klenow

T7 DNA Polymerase

• same as T4

5’ end labeling

T4 polynucleotide kinase
• transfers gamma phosphate of ATP to 5’ hydroxyl end of DNA and RNA

• enzyme inhibited by phosphate buffer or ammonium salts

• enzyme doesn’t work as well with DNA purified from agarose

• other reaction components:TrisCl, MgCl2,ATP,DTT, BSA

• PEG improves rate and overall efficiency of reaction

• reactions stopped by EDTA or heating at high temperature

• labeled separated from unlabeled by filtration on Sephadex G-100 or

through spin column with Sephadex G-50

D. Amplification Labeling

• probes can be prepared by PCR

o primers hybridize to a target sequence of DNA, Taq Polymerase elongates

the 2 primers and through repetitive cycles of temperature changes in a

thermal cycler, multiple copies of the target sequence are made

o a labeled nucleotide can be incorporated during PCR

o length of the probe defined by the 5’ ends of the primers

o labeled probe can be precipitated with ethanol

• PCR generated probes have greater specificity than nick translation or

random primed labeled generated probes

o add DNA sequences non-complementary to template to 5’end of

oligoprimers

􀂃 these sequences will become incorporated into ds PCR product

o small mismatches tolerated

o need promoter sequence so amplicon can be transcribed


E. Psoralen-biotin Labeling

• Psoralen has a high affinity for DNA

33

• This is a non-radioactive DNA labeling method

• Biotin will covalently link to DNA when denatured DNA is incubated with

psoralen-bioten in the presence of UV light.

F. Probe Labels:

Radioactive

half life = time for 50% of sample to decay, shorter 1/2 life = greater max specific

activity

32P

• most common for radioactive labeling

• highest specific activity

• 1/2 life = 14.3 days

• β emission } requires use of protective acrylic shield

• radioactive probes can be stored and re-used

• either the alpha or gamma phosphate is transferred depending on the

labeling procedure

33P

• 1/2 life = 25.4 days

• requires use of protective acrylic shield

35S
• 1/2 life = 87.4 days

• used for more stable, but lower specific activity probes

• β emission

• lower energy than 32P - sharper image on autorads

• less dangerous than 32P

3H

• lowest specific activity

• 1/2 life = 12.43 years

• weak β emission (not used for most autorads)

• used for in situ hybridization

Others: 14C ( 1/2 life = 5370 years, β emission) and 125I (1/2 life = 60 days, γ and

X-ray emission)

34

Non-radioactive

Direct

• reporter molecule is bound directly to nucleic acid probe so that probetarget

hybrids can be detected immediately after hybridization

• reporter molecule must be able to survive post-hybridization washes

and must not interfere with hybridization

Indirect

• reporter molecule is introduced enzymatically or chemically which is

later detected by affinity cytochemistry


• reporter molecule must be able to survive post-hybridization washes

and must not interfere with hybridization

• Haptens: biotin (small, water-soluble vitamin), digoxigenin

o Haptens are alone incapable of causing production of antibodies,

but are capable when combined with larger antigenic molecules

called carriers

o Labeling efficiency determined by blotting serial dilutions on a

membrane and detecting with antibodies

• Antibodies used for hapten labeled probes:

o avidin - 4 binding sites and high affinity for biotin

o streptavidin - similar to avidin, advantage over avidin is near

neutral isoelectric point which leads to reduction in non-specific

binding and therefore reduced background signals

• Example enzymes used for hapten probes are alkaline phosphatase,

acid phosphatase, horseradish peroxidase, and luciferase

Fluorescent reporter molecules

• direct or indirect labeling can be used

• fluorescein, rhodamine, etc

• ABI PRISM™ fluorescent labeling reagents allow simultaneous

detection of three amplicons using three (3) 5’ fluorescein reporters

(HEX, TET, and FAM) and a rhodamine 3’ quencher {multiplex PCR}

Colorimetric reactions

• Conjugated enzymatic activity when in the presence of a specific


chemical substrate will produce a colored precipitate to allow direct

visualization of hybridized probe on a membrane

• Example of colorimetric reaction: alkaline phosphatase +BCIP (5-

Bromo-4-chloro-3-indolylphosphate) + NBT (nitroblue tetrazolium) =

blue

• Colorimetric probes can not be re-used

35

Chemiluminescent

• detect nucleic acids on membranes

o detection is based on the association of an enzyme conjugated

antibody with digoxigenin, biotin or fluorescein labeled probes.

Light production is catalyzed by horseradish peroxidase (HRP) or

alkaline phosphatase (AP).

• A chemiliumiscent substrate (example: luminol, CSPD, and AMPPD)

) is dephosphorylated by HRP or AP and thus generates an unstable

anion that emits light as it decomposes. Blots are exposed to X-ray

film to visualize signals.

o CSPD when dephosphorylated will decompose and emit light at

475 nm

o AMPPD when dephosphorylated will decompose and emit light at

477 nm

Example protocol

o Incubate membrane in blocking solution

o Incubate in alkaline phosphatase conjugated anti-fluorescein


antibody

o Wash membrane

o Incubate in chemiluminescent substrate

o Drain excess substrate

o Expose to X-ray film

• strong signals, low background

• labeled probes stable for several months

• membrane cross-linked before probing

• direct labeling with HRP or indirectly with a hapten in which case HRP

added as an anti-hapten HRP conjugate

5. Determine Labeling Efficiency and Separation of dNTPs from Labeled Probe

• measure radioactivity: nucleotides >60 bp are quantitatively precipitated in strong

acids - dNTPs and NTPs stay in solution : separate by filtration on discs of glass

microfiber paper - measure counts with scintillation counter

• gel filtration: separate DNA and dNTPS based on molecular weight (most efficient

method)

• Sephadex G50 columns

o size exclusion chromatography (column chromatography)

o column plugged with glass wool, add Sephadex, apply sample, wash column

(TE buffer), collect fractions in microcentrifuge tubes, determine radioactivity in

fractions

o DNA > 100 nucleotides

o can also use spin columns (packing and running the column achieved by

centrifugation instead of gravity)


• Ethanol precipitation

o ammonium acetate or lithium chloride and ethanol

o DNA recovered in pellet, unincorporated dNTPs in supernatant

Table of Contents

36

Calculations

• %incorporation = incorporated cpm/total cpm x 100

• specific activity = total incorporated cpm/total ug DNA

• specific activity = amount of radioactivity for a known amount of

DNA, reported as counts per minute (cpm) per ug of nucleic acid

6. Cloning Procedures

• Cloning procedures involve the isolation of gene and then making multiple

copies of the gene.

• A gene and vector are cut with restriction enzymes to produce sticky ends.

The gene and vector are then mixed together with ligase to insert the gene

into the vector. The vector is grown in a bacteria to make lots of copies. The

vector is later purified from the bacteria and finally the gene of interest

removed from the vector for further use as a probe or other genetic

experiments.

• Vectors are DNA molecules capable of accepting a DNA insert and replicating

in a host cell.

• Vector types:

1. plasmid
• A plasmid is a self-replicating, circular DNA.

• This vector is usually 4-8 kb in size.

• All plasmids contain an origin of replication, a selectable marker, and a

cloning site.

• Plasmids allow for the insertion of up to 10kb of sequence.

2. cosmid

• Cosmids are synthetic cloning vectors, plasmids that use ability of

bacteriophage lambda infectious particles to take up large pieces of

DNA and introduce that DNA into bacterial cells.

• An insert of ~ 50 kb of sequence can be incorporated into a cosmid.

3. lambda phage

• Lambda phage is a bacterial virus.

• Sequences <20kb can be inserted into lambda phage.

4. yeast artificial chromosome (YAC)

• Sequences up to 1000 kb can be incorporated into YACs.

Cloning Steps and Notes

Cloning overview:

• Vector DNA and DNA of interest are digested with one or two restriction

enzymes.

• Digested DNAs are purified and then ligated.

• Bacterial cells are transformed with the ligated vector:DNA of interest

mixture.
• DNA of interest is isolated and purified away from bacterial cells and

vector.

Table of Contents

37

Restriction enzyme notes:

• A circular DNA molecule with n restriction sites will produce n fragments

when digested.

• A linear DNA molecule with n restriction sites will yield n+1 fragments

when digested.

• A commonly used DNA ligase is T4 DNA ligase (see Cloning and labeling

enzymes: ligases)

Transformation

• Transformation is the incorporation of DNA into bacterial cells that leads to a

change in phenotype.

• Transformation is the process by which bacterial cells take up foreign DNA.

o If the foreign DNA has an origin of replication that is recognized by the

host cell DNA polymerases, the bacteria will replicate the foreign DNA

along with their own DNA.

• Bacteria which are able to uptake DNA are called competent. Competent

cells are produced by treatment with calcium chloride in the early log phase of

growth. The bacterial cell membrane is permeable to chloride ions, but is nonpermeable

to calcium ions. As the chloride ions enter the cell, water

molecules accompany the charged particle. This influx of water causes the

cells to swell and is necessary for the uptake of DNA. The exact mechanism
of this uptake is unknown.

• Transformation, the uptake of DNA into the bacterial cells, is accomplished by

heat shock or electroporation.

o Heat shocking the bacterial cells allows uptake of the DNA and expression

of the bacterial genes known as heat shock genes. DNA uptake is

reduced at higher temperatures, and extremely high temperatures results

in bacterial death.

• Transformation is usually combined with a selection technique in order to

select for the bacteria which have incorporated the foreign DNA.

o The selectable phenotype is most often antibiotic resistance.

o Selection of cells containing a plasmid DNA and the DNA sequence of

interest usually involves growing the bacterial cells on media containing

an antibiotic (antibiotic selection).

􀂃 Plasmids used for cloning are engineered to contain the genes for

antibiotic resistance. For example, if the bacterial transformation is

plated onto media containing ampicillin, only bacteria which contain

the plasmid DNA will metabolize ampicillin and form colonies.

o Another selection method involves taking advantage of the E.coli lacZ

gene.

􀂃 The object of transformation is to clone foreign DNA of interest into

a known vector and to isolate those cells which have the

recombinant molecules, not the cells which contain a nonrecombinant

vector.

􀂃 The E. coli lacZ operon is incorporated into many cloning vectors.

The polylinker regions of these vectors are engineered inside the


38

lacZ gene coding region, but it does not interrupt the reading frame

or the functionality of the lacZ gene product: B galactosidase.

􀂃 In recombinant vectors which have an insert DNA molecule cloned

into one of the restriction enzyme sites in the polylinker, this insert

DNA results in an altered lacZ gene and a non-functional

galactosidase.

• B galactosidase protein production is determined by using IPTG

and X-Gal.

o IPTG is the lacZ gene inducer and is necessary for the

production of the galactosidase. The usual substrate for the

lacZ gene protein product is galactose, which is metabolized

into lactose and glucose.

o X-Gal is a colorless, modified galactose sugar. When this

molecule is metabolized by the galactosidase, the resultant

products are a bright blue color.

o When IPTG and X-Gal are included in a transformation

experiment, blue colonies represent bacteria containing nonrecombinant

vector DNA since the lacZ gene region is intact.

Presence of IPTG induces production of galactosidase which

cleaves X-Gal and results in the blue color. White colonies

contain recombinant DNA since a nonfunctional

galactosidase is induced by IPTG which is unable to cleave

the X-Gal.

Plasmid DNA Isolation


• Once DNA has been transformed into bacteria, the DNA can be isolated

using CsCl density gradients or alkaline lysis methods.

• CsCl density gradients are time consuming.

• Many kits (spin columns) available from a number of vendors. Procedures

based on alkaline lysis method and adsorption of DNA onto columns in

presence of high salt.

• Example isolation protocol by the alkaline lysis method:

o Liquid bacterial cultures are centrifuged to concentrate the bacteria.

o Bacteria cell pellet is then resuspended in a high osmotic strength medium

containing a high concentration of glucose or sucrose and EDTA.

􀂃 EDTA binds metal ions which are necessary to maintain the

integrity of the bacterial cell membrane.

􀂃 High sugar concentration dehydrates the bacterial cells, making

them more susceptible to lysis (next step).

o Bacterial cells are resuspended in a solution of TE, NaOH and SDS.

􀂃 EDTA destroys the bacterial cell wall and inhibits nucleases.

􀂃 NaOH denatures bacterial chromosomal DNA. Plasmid DNA is

also denatured, but the concentration of NaOH is kept low so that

the plasmid DNA is not completely denatured.

􀂃 SDS lyses bacterial cell well and membrane and denatures

bacterial proteins.

39

o A neutralizing solution is then added. The solution contains some type of

salt (ammonium or potassium acetate) to precipitate proteins and acetic


acid to neutralize (potassium acetate). High salt causes denatured

proteins, chromosomal DNA, cellular debris and SDS to precipitate while

the shorter plasmid DNA renatures correctly and stays in solution.

o Centrifugation at this step will pellet the bacterial DNA and protein while

the plasmid DNA will remain in the supernatant.

o The next steps involve purification of the supernatant to concentrate the

plasmid DNA and removal of contaminating RNA and proteins.

􀂃 Kit-based purification involves binding matrices. The matrix has a

high binding affinity for DNA and a low binding affinity for protein

and RNA. The plasmid solution is added to the binding matrix and

then the matrix is washed. Plasmid DNA bound to the matrix is

eluted (after the wash steps) with a low salt solution.

􀂃 Non-kit purification involves treatment of the solution with RNase

and then an alcohol precipitation.

o Purified plasmid DNA is quantified at A260 and qualify is assessed with

restriction enzyme digestion and gel electrophoresis.

7. Prepare cDNA

• cDNA is a single stranded piece of DNA that is complementary to the mRNA of

the coding strand of DNA

• cDNA probes are produced by primer extension of ssDNA or by reverse

transcribing RNA

• cDNA is prepared from mRNA

o need pure mRNA

􀂃 mRNA ~1-5% total cellular RNA

􀂃 distinguish mRNA from other cellular RNAs by the presence of the


polyA tail

• hybridize total RNA with oligo dTs to use as primers for synthesis of ss DNA by

reverse transcriptase

• 1st strand DNA made serves as template for mRNA (cDNA)

• restriction digest cDNA out

• ligate cDNA into vector and propagate in bacteria

• mRNA copies 3’ to 5’ so cDNA synthesis is 5’-3’ - necessary to get full length of

transcript

• cDNA can also be prepared from a total RNA sample using random hexamers as

primers and a reverse transcriptase

• cDNA stored -20C

Table of Contents

40

C. In Vitro Nucleic Acid Amplification

1. PCR – Polymerase Chain Reaction

2. Preventing PCR Contamination

3. PCR Quality Assurance and Quality Control

4. PCR Troubleshooting

5. Reverse Transcriptase PCR (RTPCR)

6. Ligase Chain Reaction (LCR)

7. Transcription-mediated Amplification (TMA)

8. Strand Displacement Amplification (SDA)

9. Nucleic Acid Sequence Base Amplification

10. Real-time PCR


11. Detection/Analysis of PCR Amplifications

Table of Contents

41

1. PCR - Polymerase Chain Reaction

• technique by which a template is amplified many thousand or million fold

• typical reaction components: target sample, thermostable polymerase, two

oligonucleotide primers, dNTPs, reaction buffer, magnesium

• primers are short, single stranded DNAs (oligonucleotides) that are used to

initiate DNA synthesis.

• Primers are oriented with the 3’ ends facing each other since DNA synthesis

occurs in the 5’ to 3’ direction.

• sense strand DNA codes for mRNA

• 5’ = upstream

• 3’ = downstream

Three Steps to PCR

1. Denature

• ds DNA is made ss

• 90-95C

• initial denature step 3-5 minutes

• DNA must be denatured completely, if only partial then it will “snap back”

very quickly

2. Anneal
• primers attach to ss DNA

• 40-60C

• specific temperature is 5C below primer Tm

• affects specificity of amplification

• too high temperature = no annealing of primer to target DNA

• too low temperature = non-specific annealing

3. Extend

• primers extended by incorporation of dNTPS by Taq polymerase enzyme

• 70-75C

• 1 minute per kb of target length

Reaction Components

1. Oligo primers

• 18-28 nucleotides in length

• 50-60% GC

• Tm of primer pair should match closely

• Tm = 4(G+C) + 2(A+T)

• avoid stretches of polypurines, polypyrimidines

• avoid internal secondary structure

• avoid 3’ end complementarity

• optimal final primer concentration varies depending on protocol (0.1-

0.5uM)
Table of Contents

42

o To determine primer concentration, first calculate molar extinction

coefficient of the primer at A260, using the formula (8400 x T) +

(15200 x A) + (12010 x G) + (7050 x C), where T, A, G, and C are

the number of times that each base is present in the primer. Molar

extinction coefficient is the A260 of a 1M solution of a primer.

Second, measure the A260 of the primer solution. Divide this A260

value by the molar extinction coefficient to get the molar

concentration of the primer.

2. dNTPs

• provide energy and nucleosides for synthesis of DNA

• too many dNTPs will result in misincorporation by Taq

• concentration should be around 200uM for each dNTP

3. Taq

• thermostable polymerase

• half life less than 5 minutes at 100C, but a half life up to 40 minutes at 95C

• use 0.5 to 2.5 units per 100 ul of reaction (increased concentration of Taq

will result in nonspecific product and reduced yield)

4. Enzyme Buffer

5. MgCl2

• Magnesium is cofactor of Taq

• concentration .5mM to 5mM (usually 1.5mM)


6. Template

7. Thermal Cycler

• reliable

• must be able to maintain at least 3 temperatures

• must change temperatures (ramp) in a definable time

• type of reaction tubes affect rate at which heat transfers from thermal

cycler to reaction mix

Cycle Number

• depends on template concentration

• usually 25-35 cycles

• copy number limited by diminishing polymerase activity after 25-20 cycles

Enhancing Agents

• dimethyl sulfoxide (DMSO) and betaine commonly used enhancing reagents

for PCR; facilitate strand separation

• glycerol, BSA,

• spermidine -increases specificity and reproducibility of Taq-mediated PCR

o neutralizes and stabilizes DNA phosphate backbone

Inhibitors of PCR

• ionic detergents (SDS), phenol, heparin, xylene cyanol, bromophenol blue,

hemoglobin

43

2. Preventing PCR Contamination

A. Separate areas for set-up, master mix preparation, amplification and postamplification
B. Use positive displacement pipettes

C. Use pipettes dedicated to work area and specimen type

D. Incorporate dU into PCR products followed by UNG (uracil DNA glycosylase)

digestion

• prevents amplicons from serving as templates

• DNA amplicons are distinguished from target DNA because target DNA

contains deoxythymidine while amplicons have deoxyuridine

• UNG is inactivated at 95C for 10 minute after mixing with PCR mixture

E. Aliquot reagents

F. Change gloves frequently

G. Separate tubes in rack

H. Use gauze to open tubes (prevent amplicon aerosols)

I. UV irradiation of PCR mixtures prior to amplification induces pyrimidine dimers

in DNA so contaminating amplicons can’t serve as effective templates (not

effective for short amplicons)

J. Depurinate work area with HCl (2M)

K. Bleach (10%) more effective than HCl for degrading DNA

3. PCR Quality Assurance and Quality Control

• positive control

• negative control (mandatory to rule out false +)

• dispense negative controls last for an indication of quality of reagents added

to all tubes

• reagent control
• irrelevant or internal positive (will help rule out nonspecific priming and

monitor contamination)

• label and document tubes and positions on worksheet

• thermal cycler maintenance

o keep cover closed when not in use

o check program before use

o avoid power failures

o perform and document system checks

o perform temperature checks

• use pure water

• avoid freeze/thaw cycles for primers and enzymes

• keep a consistent and organized work pattern

Table of Contents

44

4. PCR Troubleshooting

A. No Amplification

• check reaction setup

• check pipettor

• check detection method

• check for inhibitors (heme, agarose, phenol, EtBr, heparin, bromophenol

blue)

• increase Mg+2 concentration


• increase template concentration

• increase denaturation temperature

• check buffer pH

• increase concentration of Taq, primers, and/or dNTPs

• increase number of cycles

• decrease annealing temperature

• add enhancers

• use nested PCR

B. Nonspecific Amplification

• check basis for expected size

• check primers for repeat sequences

• check for homology with mitochondrial DNA (want nuclear)

• increase primer specificity

• increase annealing temperature

• increase number of cycles

• decrease concentration of Mg+2, Taq, primers and/or dNTPs

C. Primer Dimers

• amplification artifact

• fragment length = sum of two primers

• seen when there is too little template

D. To Increase Reaction Specificity


• decrease Mg+2

• decrease dNTPs

• decrease Taq Polymerase

• decrease primers

• decrease number of cycles

• decrease cycle times

• increase annealing temperature

• add enhancers

• increase ramp speed

• use hot-start PCR, nested PCR or touchdown PCR

Table of Contents

45

5. Reverse Transcriptase PCR (RTPCR)

• RNA is template for amplification

• must first reverse transcribe RNA to cDNA to provide DNA template for

thermostable polymerase

• RNA template must be pure and high quality

• Use a reagent control (no RNA)

• Use a no RT enzyme control (detects contamination of patient RNA sample)

• can amplify total RNA or mRNA

• detect gene expression or detect RNA viral infections


• can’t use dUTP/UNG system for carryover prevention

• 4 steps: 1) RNA isolation 2) reverse transcription 3) PCR amplification 3)

product analysis

• 2 separate reactions

A. reverse transcribe RNA to cDNA

o primer options: polyT oligo (polyA tail),random hexamers,

sequence specific

o enzyme options: reverse transcriptase or Tth DNA polymerase (Tth

has both reverse transcriptase and DNA polymerase activity)

B. cDNA amplification

o Taq polymerase, dNTPs, 3’ and 5’ primers, etc. (same as standard

PCR)

6. Ligase Chain Reaction (LCR)

• repeated cycles of probe (primer) hybridization and ligation to make multiple

copies of target

• target sequence is not duplicated, only provides a template for probe

hybridization

• there are 2 complementary pairs of probes

• after probes hybridize to target, ligase joins the probes - thermal denaturation

then makes original template and ligated probes available for ligation in next

round

• use high concentrations of probes and ligase compared to target

• thermostable DNA ligase and cycling protocol


• 2 sets of long primers that are full length of target DNA except for 1 base are

annealed to template

• ligation of primers occurs only if complementary (no mutation)

• each cycle results in doubling of target

• 1 primer labeled (probe)

• used to detect single bp mutation or make multiple copies of a target

• nonspecific binding

o use blocking salmon sperm DNA

o single base 3’ overhangs on primers

o 5’ phosphorylation on ligating primers

o non-complementary tails on primers

Table of Contents

46

7. Transcription-mediated Amplification (TMA)

• transcription-based target amplification

• primary product is RNA

• target is rRNA

• procedure uses 2 enzymes and 2 primers

A. Two Primers

o first primer has promoter sequence for RNA polymerase and a

region complementary to target sequence

o second primer is complementary to cDNA made by first primer

B. Two Enzymes : reverse transcriptase and RNA polymerase


o reverse transcriptase extends first primer to make a cDNA so now

have RNA:cDNA complex

o RNase H activity of reverse transcriptase degrades RNA to make

the cDNA available for annealing by the second primer

o after annealing of second primer, a ds DNA is made

o RNA polymerase makes RNA copies of the DNA molecule so that

the primary amplification is RNA and the product is primarily RNA

• 3 basic steps: sample preparation, amplification and detection

• amplification step is isothermal (usually 42C)

• detection of RNA amplicons uses the hybridization protection assay (HPA)

o probes that are specific to the amplicons are chemiluminescently

labeled with acridinium ester

o hybridization of probe to amplicons is done at 60C for 15 minutes,

then an alkaline selection reagent is added

􀂃 acridinium ester that is not associated with hybridized probe

will hydrolyze in presence of selection reagent

􀂃 acridinium ester associated with hybridized probe is

protected from hydrolysis and will generate light when

exposed to detection reagents in a luminometer

8. Strand Displacement Amplification (SDA)

• targeting infectious diseases

• utilizes DNA polymerase, engineered restriction sites and dual primer sets for

amplification

• not an isothermal reaction


• Little et al, Clin Chem, 1999; 45:777-784

Table of Contents

47

9. Nucleic Acid Sequence Based Amplification

• Isothermal

• 90 mins

• target = RNA

• sequence specific primer with T7 RNA polymerase promoter

• extension of T7 primer to form cDNA by reverse transcriptase

• Degrade template RNA by RNase H

• Second primer hybridizes to cDNA

• Extenstion by reverse transcriptae to give double stranded DNA

• RNA molecules synthesized from DNA with T7 by RNA polymerase

10. Real-time PCR

• Real-Time PCR is also known as quantitative real time polymerase chain

reaction (QRT-PCR)

• kinetic polymerase chain reaction

• Usually, real-time PCR is combined with reverse transcription polymerase

chain reaction to quantify low abundance messenger RNA (mRNA)

• RT-PCR follows the general steps as PCR, but the DNA in RT-PCR is

quantified after each round of amplification

• This is what makes it real time: as the PCR product of interest is produced,
the thermal cycler camera detects the amount of fluorescent dye (product

accumulation) and quantifies the number of substrates present in the initial

PCR mixture as relative fluorescent units.

• There are two methods used for quantification in RT-PCR

o The first method consists of using fluorescent dyes which intercalate with

double-stranded DNA Example: SYBR Green

􀂃 The fluorescence of the dyes increases as the product accumulates

with each amplification

􀂃 Record the amount of fluorescence emission at each cycle. Draw a

graph between the log of the template starting amount and the

increase the fluorescence during the PCR reaction.

􀂃 A linear relationship should be observed.

􀂃 SYBR Green is the most widely used double-stranded DNA-specific

dye for real time PCR (fluoresces much more brightly than ethidium

bromide and less toxic). SYBR Green binds to the minor groove of

the DNA double helix. In solution the unbound dye fluoresces very

little. SYBR® Green remains stable under PCR conditions and

does not quench under the optical filter of the thermalcycler.

􀂃 Advantages of using non-specific detection (fluorescent dyes):

Simple, relatively inexpensive, safe, however Drawback is that both

specific and nonspecific products generate signals

o The second method utilizes modified DNA oligonucleotide probes which

fluoresce when they hybridize with a complementary DNA Example:

Molecular Beacons or TaqMan Probes, FRET Hybridization Probe,

Scorpion® Primers
Table of Contents

48

􀂃 These probes are labeled with both a reporter fluorescent dye and

a quencher dye.

For example: a TaqMan probe consists of two types of

fluorophores, which are the fluorescent parts of reporter proteins:

While the probe is hybridized or unattached and before the

polymerase enzyme starts working, the quencher fluorophore

(usually red at 3’ end) reduces the fluorescence from the reporter

(R) fluorophore (usually green at 5’end). This is accomplished by

Fluorescence Resonance Energy Transfer (FRET), which is the

inhibition of one dye caused by another.

􀂃 Once the TaqMan® probe has bound to the template DNA, after

denaturation and after primers anneal to the DNA, Taq adds

dNTPs and removes the Taqman® probe from the template. This

separates the quencher from the reporter, so that the reporter gives

off energy. This energy is quantified using a computer. The more

times the denaturing and annealing takes place, i.e. the more target

and product, the more signal produced.

11. Detection/Analysis of PCR Amplifications

Gel Electrophoresis

• Polyacrylamide or agarose

• Visualize product with ethidium bromide or silver staining

Hybridization methods

• Southern blot or dot-blot


• Radioactive or non-radioactive probes

Amplification labeling

• PCR products labeled during PCR reaction

o Radiolabeled, fluorescent or biotinylated primers

• Detection

o Gel electrophoresis and auotradiography

o Phosphorimaging

o Fluorescent detection

Real-time detection

• Intercalating dyes added to PCR master mix (Ethidium bromide)

o Fluorescence of Ethidium bromide increases in double stranded DNA and

is detectable during amplification

Table of Contents

49

D. Electrophoretic and Other Nucleic Acid Separation Techniques

1. Electrophoresis

2. Polyacrylamide Gel Electrophoresis (PAGE)

3. Agarose Gel Electrophoresis

4. Pulsed-Field Gel Electrophoresis (PFGE)

5. RNA Gels

6. Denaturing Gradient Gel Electrophoresis (DGGE)

7. Mutation Detection Enhancement (MDE)

8. Single Stranded Conformational Polymorphism (SSCP)

9. Heteroduplex Analysis (HEX or HA)


10. Gel Set-up

11. Factors Affecting Rate of Migration

12. Electrophoresis Buffers

13. Gel Staining

14. Electrophoresis Troubleshooting

Table of Contents

50

1. Electrophoresis

Electrophoresis is the separation of proteins or nucleic acids on the basis of size.

o The charge to mass ratio is the same for DNA molecules of different lengths so it

is size of DNA that determines migration rate.

• Separation is accomplished via a sieving material (agarose or polyacrylamide) in an

electric field (electrophoresis chamber).

• Water is electrolyzed during electrophoresis so that protons (H+)are generated at the

anode (positive) and hydroxyl (OH-) ions are generated at the cathode (negative).

• DNA has net negative charge (-2) at neutral pH (phosphate molecules in backbone

of DNA molecules) and thus will migrate toward the positive end (anode) in an

electrophoresis chamber. The buffers used during electrophoresis are basic, thus

the net negative charge on DNA is retained and DNA will migrate toward the anode.

• Linear double-stranded molecules migrate at rate inversely proportional to log10 of

their number of base pairs

• Linear DNA moves through a gel at a rate proportional to the voltage applied.

• Voltage gradient is dependent on the size of the electrophoresis chamber, the size

of the gel, and the volume and strength of the electrophoresis buffer.
2. Polyacrylamide Gel Electrophoresis (PAGE)

• gives high resolution of fragments

• best resolution for 5-500 bp fragments

• can distinguish 1 bp differences under denaturing conditions

• run with TBE buffer

• vertical format

• difficult to handle

• neurotoxic

• polyacrylamide gels formed by polymerization of acrylamide monomers into long

chains which are further covalently attached by a crosslinking agent (N,N’ methylene

bisacrylamide), polymerization initiated by free radicals provided by ammonium

persulfate and stabilized by TEMED

• most important parameter for successful separation is polymerization reaction

• denaturing gel

o used for the separation of single-stranded DNA

o polymerized in presence of urea which suppresses base pairing in nucleic acids

o use SDS in gel: SDS is an anionic detergent which denatures proteins and

makes them negatively charged

• non-denaturing - separation of ds DNA fragments

o run at low voltage to prevent denaturation of small fragments by heat generated

by current

• 12-15% best for short oligonucleotides


• 5-8% for sequencing

• pore size of polyacrylamide gel determined by total % of acrylamide (sum of weights

of acrylamide + cross linker) - expressed as %T

Table of Contents

51

o as %T↑, pore size ↓

o acrylamide and bisacrylamide break down in solution to acrylic acid, which

affects mobility of fragments through a gel

o protect acrylamide solutions from light

% polyacrylamide recommended for resolving dsDNA on nondenaturing gel

3.5 100-1000 bp 5.0 75-500 bp

8.0 50-400 bp 12.0 35-250 bp

15.0 20-150 bp 20.0 5-100 bp

3. Agarose Gel Electrophoresis

• Agarose is typically purchased in powder form. Agarose is weighed out and mixed

with a buffer according to the amounts required to make a certain percentage

agarose gel. The agarose and buffer are heated by microwave or boiling to form a

liquid which is then poured into a gel casting tray. When gelled, agarose forms a

matrix that is used to separate DNA molecules.

• Agarose gels have a lower resolving power than polyacrylamide, but provide for

greater range of separation.

• Agarose gels are more porous than polyacrylamide so they can be used to separate

larger fragments.

• Agarose gels work best for separating DNA fragments 200bp - 50kb in size.
• Agarose gels are usually run in a horizontal format with TAE or TBE buffer.

• The typical voltage for an agarose gel is 4 - 10 v/cm.

• 6% agarose is best for separating small bp differences.

• 0.5% agarose is best for separation of larger DNA fragments.

% Agarose recommended for electrophoresing linear DNA

0.3 5000-60,000bp 1.25 300- 5000 bp

0.4 2000 -30,000 bp 1.50 200 - 4000 bp

0.7 8000 – 10,000 bp 2.00 100- 2500 bp

1.0 500 - 10,000 bp

4. Pulsed-Field Gel Electrophoresis (PFGE)

• PFGE is used to separate larger DNA up to 10,000 kb.

• PFGE works by repeated changes of electrical current direction.

o This current direction change forces molecules to reorient before continuing to

move “snakelike” through the gel.

• PFGE is a slow process and the gels used are very fragile.

Table of Contents

52

5. RNA Gels

• before loading RNA - denature ssRNA to abolish secondary structure

a. heat RNA sample to 65C in presence of formaldehyde and formamide

b. maintain denatured RNA in gel electrophoresis by adding formaldehyde to gel

c. glyoxal can be used in place of formaldehyde to denature RNA

• ethidium bromide stained RNA gels - impedes transfer


• RNA electrophoresis buffer = MOPS {3-(N-morpholino) propanesulfonic acid

• stain RNA gels with EtBr (transfer may be impeded if EtBr is present), acridine

orange or Stains All (cationic carbocyanine dye)

• transfer RNA gels immediately after separation

o must remove formaldehyde prior to transfer (soak gel in DEPC treated water or

transfer buffer)

6. Denaturing Gradient Gel Electrophoresis (DGGE)

• A mutation in DNA will change the migration rate compared to “normal” DNA.

• DGGE performed on polyacrylamide gels which are poured with a denaturant

gradient from high percentage of denaturant at the anode (positive) end of the gel to

a low percentage of denaturant at the cathode (negative) end.

• Denaturant used is usually either urea or formamide.

• There will be a point in the gel where DNA begins to dissociate (melt) and therefore

the mobility of the DNA decreases. The point of denaturation depends on: length of

DNA and GC content.

• GC clamps can be applied to the 5’ends of DNA fragments to stabilize the fragment

and allow fragments of DNA to travel farther in the gels.

7. Mutation Detection Enhancement (MDE)

• MDE gels are high-resolution gels formulated to separate DNA based upon

conformational differences. MDE gels used for heteroduplex or single-strand

conformational polymorphism analysis.

8. Single Strand Conformational Polymorphism (SSCP)

• SSCP is based on detection of conformational intrastrand differences between


DNAs which have different sequences.

• ss DNA fragments fold into 3D shape that depends on the sequence within the

fragment.

• The rate of migration through a non-denaturing polyacrylamide gel is dependent on

size of fragment and on the 3D shape (conformation) of the fragment so that

separation is a function of shape of DNA

• SSCP is a method for mutation detection in short (<300 bp) fragments

• The overall procedure is: PCR and label target area, denature, snap cool, separate

by electrophoresis in non-denaturing gel (without SDS) run slowly at RT or 4C.

• Result will be 4 single stranded bands: the sense and antisense strand of the

normal and the variant.

Table of Contents

53

• Re-annealing of ss DNA is prevented by keeping the concentration of DNA in the

buffer very low thus, detection requires radioactive labeling or ethidium bromide or

silver staining.

• This method is also appropriate for RNA.

9. Heteroduplex Analysis (HEX or HA)

• This method also referred to as DSCP (double stranded conformational

polymorphism).

• HEX separates DNA in a polyacrylamide gel based on conformational differences

between double-stranded heteroduplexes and double-stranded homoduplexes of

DNA.
• Single bp mismatches alter mobility of DNA fragment duplexes in non-denaturing

acrylamide gels.

• Overall procedure

o Amplification (PCR) of target DNA and wild type DNA is done using the same

primers.

o The two PCR products (wild type and mutant samples for example) are

combined in a nondenaturing buffer. This mixture is then denatured and slowly

cooled to room temperature.

􀂃 As the mixture cools, the complementary DNA strands will anneal to form

homoduplexes. The non-complementary strands will anneal to form

heteroduplexes. The 3D shape of homoduplexes is different than the 3D

shape of heteroduplexes. Heteroduplexes usually have less mobility in a

gel than homoduplexes.

• Results : If target DNA is wild type then all duplexes will be homoduplexes and will

comigrate to same place in gel. If a mutation is present, heteroduplexes will form

and thus migrate differently than the homoduplexes.

• HEX will detect 1bp mismatch.

homo duplexes _________ and ___x____

_________ ___x____

heteroduplexes _________ and ___x____

____x____ ________

can use SSCP and Heteroduplex Analysis to get 100% detection of mutations

Table of Contents

54
10. Gel Set-up

• lower % = bigger pieces of DNA

• higher % = better resolution

• use appropriate molecular weight markers/ladders to determine size of test

fragments

• document sample loading order

• document electrophoresis conditions and results (photography)

• can store previously made gels in SaranWrap (in drawer in dark if include EtBr)

• gels should be cooled to 55C in H2O bath before pouring to prevent warping

• Horizontal gels should typically be poured between 3 to 4 mm thick to achieve the

best resolution

• monitor electrophoresis progress via dyes in loading buffer and watch temperature

o common tracking dyes are xylene cyanol and bromophenol blue

o gel loading dyes/buffers serve 3 purposes

1. increase density of sample so sinks in well

2. add color to sample for visualization

3. contain dyes that in electric field move toward anode at predictable rates

o gel loading dyes/buffers are comprised of the dye (bromophenol blue or xylene

cyanol) and glyerol, EDTA, and SDS

o bromophenol blue moves faster than xylene cyanol

o bromophenol blue typically migrates with DNA around 0.5 kb

o xylene cyanol typically comigrates with DNA around 5 kb in length

o use bromocresol green for alkaline gels because displays more vivid color than
bromophenol blue

11. Factors Affecting Rate of Migration

A. % of sieving material

• In general, the higher the concentration of agarose/polyacrylamide, the higher

the resolving power for small fragments, but the lower resolving power of the gel

for larger fragments.

• higher concentration of agarose = quicker gel hardens

• decreased gel thickness = increased rate of migration of fragments

• lower % of agarose will separate higher molecular weight fragments of DNA

• higher % of agarose will give better resolution of lower molecular weight

fragments

B. DNA conformation

• linear (form III), superhelical circular (form I), nicked circular (form II)

• closed circular supercoiled DNA moves faster than linear - supercoiling winds

DNA up giving them smaller size

Table of Contents

55

C. Electromagnetic Forces (voltage and direction)

• at low voltage, migration of linear DNA proportional to voltage applied, as voltage

increases the mobility of high MW fragments increases differentially

• effective range of separation in agarose decreases as voltage increases

• can increase velocity by a) decreasing distance between electrodes b)

decreasing gel thickness or c) decreasing buffer volume


• In general, higher voltage (5V/cm) can be applied to separate and resolve

smaller size DNA fragments. Larger size DNA fragments should be separated at

lower voltage to achieve better resolution (1-2V/cm).

D. Temperature/base composition

• agarose gels not significantly affected by either (no mobility change from 4-30C,

usually run at RT)

• will get better resolution with cooler gels

E. Intercalating dyes

• EtBr if in gel : reduces electrophoretic mobility of linear DNA by ~15%

• dye intercalates between stacked base pairs, extending length of linear and

nicked circular DNA and making them more rigid

• EtBr migrates toward cathode (opposite of DNA)

• extended electrophoresis can deplete gel of EtBr

F. Buffer Composition, Ionic Strength, and Volume

• too few ions (i.e. buffer is not added) will result in reduced or no movement of

DNA

• too high ionic strength (i.e. 10X concentration used instead of 1X concentration)

will result in excess heat where gel may melt or DNA may denature

• decreased buffer volume = increased speed of migration

G. Salt Concentration

• DNA resuspended in a high salt buffer will exhibit retarded migration and broader

bands in comparison to DNA resuspended in a lower salt buffer.

12. Electrophoresis Buffers


A. TAE Buffer (Tris-Acetate with EDTA)

• alkaline pH

• use when DNA is to be recovered

• TAE is used for resolving large DNA fragments (>10,000 bp).

• may require re-circulation (more easily exhausted)

• ds linear DNA 10% faster in TAE vs. TBE

• resolution of supercoiled DNAs better in TAE

• low buffering capacity

Table of Contents

56

B. TBE Buffer (Tris-Borate EDTA)

• alkaline pH

• TBE is used to resolve smaller DNA fragments (50 – 10,000 bp)

• higher buffering capacity than TAE (slower rate of buffer depletion)

• avoid if going to purify DNA from gel

• better resolution of smaller DNA

• high ionic strength

• precipitate formed if stored for long time

C. TPE (Tris-phosphate EDTA)

• similar properties as TBE

D. Alkaline electrophoresis buffer


• used to electrophorese denatured single-stranded DNA

• example: 50mN NaOH, 1mM EDTA

13. Gel Staining

A. Ethidium bromide

• fluorescent

• stains nucleic acids by intercalating between bases

• minimum amount of DNA detected by EtBr stained gels is ~2ng in 0.5 cm wide

band (usual width)

• can stain gel during electrophoresis, but including EtBr in gel while running will

retard migration by ~15%

• can stain gel after electrophoresis with gel immersed in buffer or H2O containing

0.5 ug/ml EtBr for 30-45 minutes at RT {25 ul of 10mg/ml in 500 ml H2O}

o destaining not necessary, but will reduce background-soak stained gel in

H2O or 1mM MgSO4 for 20 minutes at RT (needed if detecting small

amounts of DNA)

• fluorescence of EtBr:DNA > EtBr unbound

• low affinity of EtBr for ss DNA

• observe stained gel on 302 nm transilluminator

• when excited at 302nm, EtBr emits light at 590nm

• need appropriate filter for photography

B. Silver Staining

• sensitivity similar to EtBr


• silver stain powder is toxic

• best with polyacrylamide gels

• high background with agarose

Table of Contents

57

C. SYBR Green I and II

• SYBR Green I is a fluorescent stain used to detect nucleic acids in agarose or

polyacrylamide gels. SYBR Green I is less mutagenic than ethidium bromide.

• SYBR Green I will stain double stranded DNA whereas SYBR Green II will stain

single stranded DNA and RNA.

• A yellow filter is required for photography of SYBR Green stained gels.

D. Methylene Blue

• this stain has low sensitivity and thus often requires overloading of gel for

detection

• not as hazardous as EtBr

• view on white light

E. Acridine Orange

• DNA and RNA

• View with UV transilluminator

14. Electrophoresis Troubleshooting

• decrease width of well = increased band intensity

• wider well = better resolution


• poor resolution = wrong concentration of agarose

• fuzzy bands = diffusion of DNA through gel

• distortion of DNA bands = result of dried agarose on gel comb teeth

• smearing = overloading, high voltage, or sample well torn

• melted gel = buffer omitted or exhausted

• >500 ng DNA = overload = trailing and smearing

• incomplete digested DNA will not move out of well

• “smiling” - lanes in center of overheated gel run faster than lanes at side

o caused by uneven dissipation of heat by gel, sides are cooler than center,

samples run faster at higher temperatures

• if DNA degraded, no large fragments, only small ones that migrate as a smear

Table of Contents

58

E. Transfer of Nucleic Acid to Selected Solid Matrix

1. Membranes

2. Pre-Transfer

3. Transfer Methods

4. General Notes

5. Fixation

Table of Contents

59

1. Membranes

A. Nitrocellulose
• fragile, brittle

• not used for re-probing

• binds ss DNA efficiently in high ionic strength solutions (20X SSC, 20X SSPE

used for transfer) {SSC= sodium chloride sodium citrate, SSPE = salt-sodium

ctirate phosphate EDTA}

• color may be more intense on nitrocellulose vs. nylon membrane

• baking is preferred over UV for immobilizing nucleic acid to membrane

B. Nylon

• strong, durable

• can strip old probe by treatment with alkaline solution and then re-probe

• can purchase neutral or positively charged membranes

o positively charged membranes bind DNA covalently at high pH

• disadvantage = higher background than nitrocellulose

• transfer ss or ds DNA

• DNA will bind to nylon in low ionic strength solutions

• Higher binding capacity versus nitrocellulose

• Nylon membranes not recommended for Western blotting since it is difficult to

block non-specific binding sites due to high protein binding capacity (i.e. you’ll get

background!)

DO NOT TOUCH MEMBRANES WITH FINGERS (interferes with blotting process)

Southern Blotting - transfer DNA from gel to membrane

Northern Blotting - transfer RNA from gel to membrane


Western Blotting - transfer protein from gel to membrane

Protein/protein hybridization technique used for detection of antigens in a mixture of

proteins

1. First separate protein mixture according to size using SDS PAGE (sodium

dodecyl sulfate polyacrylamide gel electrophoresis)

2. Next transfer proteins to membrane and immobilize

3. Hybridize with specific antibody

4. Indirect detection with an enzyme linked to a second antibody and substrate

5. Can directly detect using fluorescence-labeled antibody

Table of Contents

60

2. Pre-Transfer

A. Depurinate DNA

• with hydrochloric acid (low concentration e.g. 0.2M)

• reduces size of fragments to transfer

• hydrolyzes some of purines ( A and G) and randomly breaks large fragments into

smaller ones

• carry out depurination quickly with no agitation to minimize diffusion of pieces out

of the gel

B. Denature DNA

• with 0.5N NaOH/1.5M NaCl

• this step not necessary with RNA since RNA is already single stranded

C. Neutralize

• with 0.5M Tris-HCl pH7.2 /1.5M NaCl


• requirement for nitrocellulose membranes

• a lot of protocols use an alkaline solution as transfer buffer for nylon membranes

􀂃 no matter which method of transfer used, recommend equilibrating both the gel

and the membrane in transfer buffer prior to transfer

3. Transfer Methods

A. Capillary Transfer

• DNA diffuses from gel to membrane by capillary action

• rate of transfer depends on size of DNA fragments and concentration of agarose

• transfer buffer drawn up from a reservoir and passes through gel into stack of

paper towels that is weighted, DNA moves from gel with the movement of buffer

• can also make a sandwich that includes from bottom to top saran wrap, gel,

membrane, 2 pieces of Whatman Chromatography paper, stack of paper towels,

weight

• does not work with polyacrylamide gels } pore sizes are too small for effective

transfer

• recommended for nitrocellulose membranes

B. Vacuum

• application of negative pressure to draw the nucleic acid down through the gel

onto a membrane

• standard for dot blots and slot blots

• uses little transfer buffer

• process takes short time (~30- 45 minutes)


• more efficient than capillary transfer

Table of Contents

61

C. Electroblotting

• requires use of special equipment (need cooling mechanism and an apparatus

that can hold large volumes of buffer), so not usually used unless capillary or

vacuum transfer are not sufficient

• electric current (20-40 volts) passes through gel so DNA moves from gel to

membrane

• preferred method for PAGE

• requires special power supply and lots of transfer buffer

• process takes several hours

• not practical for nitrocellulose membranes because of high ionic strengths of

buffers required to bind nucleic acid to these types of filters

D. Dot blots

• bind DNA or RNA to membrane without first separating components by

electrophoresis

• many nucleic acids applied to membrane and then hybridized with one probe

• pipet nucleic acid to membrane as a small spot

• dot blot manifold - apparatus consisting of top block with sample wells,

membrane, middle block, lower block (which can be connected to vacuum pump)

• disadvantage compared to Southern = less discrimination between correct


hybridization and cross hybridization

• DNA denatured with heat prior to applying to filter

• don’t press too hard when applying sample because it causes background

• keep dots small

• after DNA is applied to filter, the filter is treated with denaturing solution which is

then neutralized to maintain ss DNA

• if use positively charged membrane, DNA will bind covalently at high pH,

therefore samples for dot or slot blotting can be applied in alkaline buffer which

will promote both denaturation and binding to the membrane

E. Reverse Dot blot

• probes are applied to membrane

• many probes on membrane and then hybridized with one nucleic acid

62

4. General notes:

• air bubbles between layers inhibit transfer

• membrane should be wet without white spots before performing transfer

• note which side of membrane is nucleic acid binding side

• membranes should be labeled with waterproof pen

• don’t use alkaline solution for RNA transfer because RNA hydrolyzes after

prolonged exposure

• paper towels, chromatography paper and membrane should be cut to exact size

of gel
• gel is compressed after transfer

• when using an alkaline transfer buffer and a positively charged membrane, DNA

becomes covalently linked to the membrane {fixation not necessary}

• saran wrap with designs are not appropriate for capillary transfer (the design

transfers as well!)

5. Fixation

• ensures DNA remains adhered to membrane

• UV crosslinking

o ~ 1 minute

o DNA side up

o 254nm

􀂃 overirradiation = decreased hybridization signal

􀂃 preferred over baking for RNA

• Baking

o 80C 2 hours

Table of Contents

63

F. Pre-hybridization and Hybridization Materials and Conditions

1. Pre-hybridization

2. Hybridization

3. Post-hybridization Washes

4. Stringency

5. Southern blot Analysis

6. Troubleshooting Southern blot


Table of Contents

64

Note: Document all conditions (reagents, amounts, temperatures, time, procedures,

etc.) on appropriate worksheets throughout pre-hybridization, hybridization and posthybridization.

1. Pre-hybridization

equilibrates membrane and minimizes nonspecific binding of probes

• use pre-hybridization solution at same temperature as hybridization and wet

membrane in solution that will be used for hybridization

• complete wetting of membrane necessary prior to hybridization to reduce

possibility of high background (no white spots should be visible)

• do not let membrane dry out during hybridization procedure otherwise get

high background

• pre-hybridization solution may contain blocking agent for sites on membrane

that may bind nonspecifically

o blocking DNA examples: salmon sperm, herring sperm, human Alu

repeats, LINE-1, Cot1,

o other blocking solutions: Denhardts (Ficoll, polyanion polymer, and

BSA)casein (milk protein)

2. Hybridization

• depends on ability of ss nucleic acid to anneal to complementary ss nucleic acid

just below the melting temperature of the nucleic acids

• formation of complementary base pairs between labeled probe and unlabeled

target DNA

• Tm = temperature at which one half of the DNA is present in ss form


o calculated by A260

o higher GC content = higher Tm

Hybridization solution

• SSC– provides ionic conditions and can be manipulated to affect stringency (see

below) (SSCP or SSPE can also be used)

• dextran sulfate - increases rate of hybridization

• SDS - decreases background of hybridization

• Denhardts solution – blocking agent to reduce background

• phosphate buffer or sodium pyrophosphate - maintains neutral pH

3. Post-Hybridization Washes

• remove excess/unbound probe

• salt (SSC or SSPE) and detergent solutions (SDS)at high temperatures

Table of Contents

65

4. Stringency

• conditions that favor perfect heteroduplexes

• hybridize under reduced stringency to favor annealing

• higher stringency for washes to remove unbound probe

Factors that affect stringency, hybridization stability and hybridization rates:

A. Temperature: ↑temperature =↑stringency

***important***

• hybridization = small optimum temperature range


o maximum rate of hybridization occurs 20-25C below Tm for DNA:DNA and

at 10-15C below Tm for DNA:RNA hybrids

• higher temperatures without formamide

• lower temperature = poor hybridization

• too high temperature = stripping of probe

B. Destabilizing Agents

• formamide concentration ↑ formamide concentration = ↑ stringency

o solution polarity is lowered(positive charges and negative charges can’t

stay together so only well matched probe and target will bind and stay

together)

o organic solvent lowers thermal stability of DNA

o 0.72C for each 1% formamide

o use in denaturation to lower Tm of DNA so hybridization can be performed

at lower temperatures

o 50% formamide has no effect on hybridization rate, but higher or lower

concentrations will decrease the hybridization rate

• other destabilizing agents

o urea

o tetramethylammonium chloride (TMAC)

􀂃 when using TMAC, the Tm is a function of length of target/probe rather

than a function of base composition so in effect can reduce the Tm of

high GC oligonucleotides.

C. Salt Concentration: ↑ salt concentration = ↓ stringency

• small changes with noticeable affects


• overcomes electrostatic repulsion between 2 ssDNA

• balances action of formamide

D. Ionic strength

• Tm increases ~17C for each 10-fold increase in monovalent cations

between 0.01 and 0.40M NaCl (hybrid stability)

• Optimal hybridization rate occurs at 1.5M Na+

Table of Contents

66

E. Time: ↑time = ↓ stringency

• too little wash time = cross hybridization

􀂃 too much wash time = reduced signal

F. Probe Length

• hybrid stability not influenced with probes >500 bp

• hybridization rate is directly proportional to probe/target length

• shorter probes require higher stringency

• longer probes = more hybridization time, more stable, easier to visualize

G. Base Composition

• (%GC) - need to know to decide appropriate temperatures

• A & T pairs are held by 2 Hydrogen bonds so are less stable than G & C

pairs which are held together by 3 Hydrogen bonds (hybrid stability)

• Base composition has little effect on hybridization rate, except repetitive

sequences will increase hybridization rate


H. pH

• little effect on hybridization rate between pH 5-9, however all solutions should

be pH’d prior to use

I. Dextran Sulfate

• used in hybridization solutions because it favors binding of probe to

membrane

• in aqueous solutions does not allow macromolecules access to hydrating

water, therefore there is an apparent increase in probe concentration and

consequently higher hybridization rates {solution crowder}

• increased solution viscosity increases hybridization rate

J. Mismatched Pairs

• Tm reduced ~1°C for each 1% mismatch (hybrid stability)

• Hybridization rate reduced by a factor of 2 for each 10% of mismatching

• Tm for DNA:DNA hybrids

o 81.5+16.6(log10[NA+]) +0.41(%GC)-0.72(%form)-(500/length)

• Tm for oligonucleotides = add 2° for each AT pair, and 4°C for each GC pair

Probe considerations

• nucleic acid probes must be ss for hybridization - denature

• RNA probes, although they are single-stranded are denatured prior to

hybridization to reduce 2° structure of ssRNA

o denature RNA with glyoxal and DMSO or formaldehyde and formamide

• determine activity if radioactively labeled


• advantages RNA probes vs. DNA probes

o lack of competition of probe:probe hybrids

o high specific activity

67

o readily defined length

o higher thermal activity of RNA:RNA vs. DNA:DNA = increased sensitivity

• thermal stability - RNA:RNA> DNA:RNA > DNA-DNA

5. Southern blot Analysis

• Clear, clean autoradiogram with no background

• Determine size of bands of interest using a size/molecular weight marker

• Popular size standard is lambda (λ) phage DNA which has been cut with HindIII,

1Kb marker or 100 bp molecular weight ladder

• This gives a pattern of fragments from 125 bp – 23.1 kb

• Compare band pattern of patient to band pattern of normal control

• Correlate results with clinical information

• Generate preliminary report

• Repeat Southern blot when:

o Too much background

o Bands masked by background

o Size of bands do not match with positive and/or negative controls

o Bands did not adequately separate

o Sizes do not agree with standard size marker

6. Troubleshooting Southern blot


A. False negatives

• Co-migration of normal and rearranged bands

• Rearranged bands too big or too small

B. False positives

• Incomplete restriction enzyme digestion

• Polymorphisms

• Extra bands may be due to insufficient wash stringency

• Extra bands may be due to probe containing nonspecific sequences (probe

design)

• Plasmid DNA sequences in patient DNA sample due to in vivo or in vitro bacterial

growth

• Plasmid DNA sequences usually around 3-5 kb in size, strip probe and rehybridize

with vector probe to confirm

C. Poor signal

• Probe specific activity too low

• Inadequate depurination

• Inadequate transfer buffer

• Not enough target DNA

• Inadequate fixation of membrane

• Transfer time too short

Table of Contents

68
D. Insufficient transfer

• Probe concentration too low

• Incomplete denaturation of probe and/or target

• Blocking agents interfere with hybridization

• Wash too stringent

• Hybridization temperature too low

• Hybridization temperature too short

• Inappropriate membrane used

E. Spotty background

• Unincorporated nucleotides not removed from labeled probe

• Precipitated hybridization buffer / particles in hybridization buffer

• Agarose dried on the membrane

F. High background

• Insufficient pre-hybridization blocking

• Membrane allowed to dry out during hybridization or washing

• Membranes stuck together during hybridization

• Air bubbles during hybridization

• Volume of post-hybridization wash solution insufficient

• Hybridization temperature too low

• Probe length too short

• Probe concentration too high


• Inadequate pre-hybridization time

• Probe not denatured

• Inappropriate membrane type

• Dextran sulfate sometimes causes background

• Not enough SDS in wash solutions

69

G. Detection Systems

1. Gel Staining (See Section D. 13)

2. Detection

Table of Contents

70

Note: Document all reagents, amounts, temperatures and procedures throughout

detection on appropriate worksheet

1. Gel Staining (See Section D. 13)

2. Detection

A. Autoradiography

• exposure of membrane to x-ray film (coated with silver halide crystal emulsion)

• intensifying screens and film cassettes (light proof) used

o intensifying screens are used to capture excess energy of emitters and

return that energy to the film as multiple photons of light

• x-ray film between membrane and intensifying screen

• high energy β particles pass through film and hit screen, are converted to light

and intensify image


• β particles from sample convert silver ions on film to silver atoms and produce an

image of the sample

• developing of film produces blackening in the film’s emulsion where the sample

emitted β particles

• perform this procedure in the darkroom under a safelight

• exposure time and temperature vary

o film exposure should be performed at low temperatures (-70C)

o there is reduced light scattering at lower temperatures

• hybridization side of membrane, filter etc. should be in contact with the film

• bring film to room temperature before developing to avoid condensation

• troubleshooting:

o poor developer = reduced intensity image

o poor fixer = cloudy film

o poor band resolution = sample and film not in contact or characteristic of

label used

o partial or complete fogging of film = light leak, old cassette, expired

chemicals

Film cassette

Intensifying Screen

Film

Sample

Film Cassette

Table of Contents
71

B. Chemiluminescence (Luminography) (also see non-radioactive probe label, Section

II-B)

• chemical reaction produces light

• non-radioactive detection

• certain substrates of horseradish peroxidase and alkaline phosphatase emit

photons during enzyme reaction

• emitted light exposes x-ray film

• choose appropriate substrate

C. Photography

• Polaroid or computer image to document electrophoresis

• Polaroid or computer image to document colorimetric reactions

• 35 mm or computer image for fluorescent labeled probe hybridization

• special filters required for different stains and fluorochromes

D. Phosphorimagers

• instrument used to assess amount of energy released by radioactively labeled

probes

• can directly analyze membranes that have been hybridized with radioactive

probes

• measures amount of radioactive emission corresponding to each band on the

membrane

• generates a value expressed in counts per minute


72

H. DNA Sequencing

1. Sequencing

2. Maxam & Gilbert

3. Sanger Dideoxy Termination Method (Enzymatic Method)

4. Automated DNA Sequencing

5. Example Automated Systems

6. Sources of Error When Sequencing

7. General Notes for Sequencing

Table of Contents

73

1. Sequencing

• gold standard for molecular testing

• high resolution denaturing polyacrylamide gel electrophoresis to resolve ss

nucleotides up to 1 bp (thinner gels = higher resolving power) (35S higher

resolution vs. 32P labeled fragments)

• denaturing conditions maintained by making gels with urea and running gels

at 65C

2. Maxam and Gilbert

• method rarely used

• based on base-specific cleavage of DNA

o DNA is cleaved by piperidine at modified bases

o chemical degradation with piperidine

• dependent on end-labeling ss or ds DNA


• target DNA to be sequenced is analyzed for each of the 4 bases (A,T,C, and G)

in 4 separate reaction mixtures

o labeled DNA fragments are divided into 4 aliquots and chemical reactions

(hydrazine and dimethyl sulfate or formic acid) result in a modification in

one of the 4 bases

o run each of the 4 aliquots on a denaturing polyacrylamide gel

• procedure:

a. label DNA of interest at 5’ end

b. denature the DNA

c. cleave DNA with a base specific chemical modifier. The cleaved base

detaches and thus a break in the DNA is produced at this site

􀂃 cleave guanine with dimethyl sulphate

􀂃 cleave guanine and adenine with formic acid

􀂃 cleave cytosine and thymine with hydrazine

􀂃 cleave cytosine by hydrazine with 5M NaCl

d. load the four reactions onto four separate lanes of a polyacrylamide gel and

electrophorese

e. read sequencing gel from bottom to top (smallest to largest fragments)

• hazardous chemicals

3. Sanger Dideoxy Termination Method (Enzymatic Method)

• Principle is interrupting the synthesis of the 5’ to 3’ strand at specific bases.

Different size DNA fragments are thus formed and the size of the fragments

represent the position of the bases.

• does not required single stranded DNA


• requires annealing of primer to template strand that will be sequenced

o primer must have no significant secondary structure and bind to single site

in template

o must know sequence adjacent to anneal primer

Table of Contents

74

• divide primer: template hybrid into 4 aliquots that have 4 dNTPs (1 is labeled)

and 1 ddNTP

o dNTP = deoxynucleotide triphosphate

o ddNTP = dideoxynucleotide triphosphates

􀂃 ddNTPs have an H atom instead of the usual OH group in the C3

position of the sugar component of the nucleotide

• elongate primer withKlenow, Taq, or T7 DNA polymerase

o Klenow sometimes creates false terminations, inability to travel long

distances, ineffective if encounters secondary structure of dNTPs

• random incorporation of ddNTPs into growing chain

o ddNTPs have no 3’OH group so synthesis stops after incorporation

o when a ddNTP is incorporated into the DNA chain, no bond between the 3’

position of the ddNTP and the next nucleotide is possible so the synthesis

is interrupted

• 4 separate chain termination reactions, each containing a different labeled

ddNTP

• run 4 reactions side by side on denaturing PAGE until bands resolved by one

nucleotide
• detection by autoradiography

4. Automated DNA Sequencing

• fluorescently labeled primers or ddNTPs

• chain termination principles with 4 reactions

• 4 different fluorochrome-labeled bases

• laser scans fragments and produces characteristic emission for each fluorophore

• each base is represented by a colored peak on the computer screen specific for

the base (example: A=green, T=red, C= blue and G=black)

• computer records data

5. Example Automated System

• Visible Genetics System

• Perkin Elmer Applied Biosystems – ABI PRISM® 3700 DNA Analyzer

• The ABI PRISM® uses capillary electrophoresis to separate DNA fragments.

• DNA molecules are fluorescently labeled with ABI PRISM BigDye™ or

Rhodamine terminators.

• Fluorescently labeled molecules are detected in solution at the ends of the

capillaries where there is laser excitation of the fluorophores.

• Labeled fragments are separated based on the fluorescence spectrum by a

spectrograph and are then imaged with a CCD (charge-coupled device) camera.

The CCD image is translated into a computer which generates an

electropherogram.

• The ABI PRISM® includes DNA Sequencing Analysis software.


Table of Contents

75

6. Sources of Error When Sequencing

• poor quality template

• poor template preparation

• primer design

• background signal

• computer analysis

• dirty equipment

• low quality reagents

• technologist error

7. General Notes for Sequencing

• DNA fragments/products are separated using a polyacrylamide gel or with

specialized, optimized polymers provided with automated systems.

• Non automated systems: Sequencing gels are silver stained (silver nitrate is

highly toxic in powder form).

• Gel plates must be thoroughly cleaned for gel electrophoresis

• Wrinkles should be not be present in the gel when drying prior to

autoradiography.

• A technical problem often encountered when sequencing is compression

o This is more of a problem with manual sequencing versus automated

sequencing.
o Compression occurs when bases of ss DNA interact to form secondary

structures.

o Compression occurs more often in palindrome areas or in areas of high

GC content.

o Compression is evidenced as co-migration of bands in different lanes and

anomalous wide spacing of bands above the region of compression.

o It is important to sequence both strands.

o It may be possible to resolve compression by running gels at the highest

temperature possible. Caution should be exercised however, as very high

temperatures may result in cracked gel plates and fuzzy bands.

o Formamide may be used in gel preparation to reduce secondary

structures and reduce compression. Note that these gels require more

TEMED, slower electrophoresis and higher voltage.

• Another technical problem is bands across four lanes

Table of Contents

76

IV. SUMMARIZE RESULTS AND REPORT TO

DESIGNATED AUTHORITY

A. Interpret Results

1. Evaluate Interpretability of Results and Necessity of Repeating Analysis

2. Correlate Test Results with Other Lab Results and/or Clinical Information

3. Interpretation of Result Requirements

Table of Contents

77

1. Evaluate Interpretability of Results and Necessity of Repeating Analysis


Each component of the test should be reviewed before results are reported including:

• nucleic acid extraction worksheet (quantitation, purity, quality)

• restriction enzyme digestions worksheet and photograph

• gel electrophoresis worksheet and photograph

• transfer and hybridization information

• hybridization photograph/autoradiograph

2. Correlate Test Results with Other Lab Results and/or Clinical Information

Recommend reviewing the following molecular tests including:

• indications for testing

• specimen requirements

• test performed (PCR vs. Southern, etc.)

• controls

• test interpretation

• criteria for repeating the test

• Example of Assays:

Fragile X

Duchenne and Becker Muscular Dystrophy

B/T Gene Rearrangement

Cystic Fibrosis

bcl2

bcr abl

3. Interpretation of Result Requirements


• Knowledge of the disease being tested for

• Type of defect being tested: deletion, point mutation, trinucleotide repeat,

methylation status

• Specific mutations associated with specific ethnic groups or gender

• Method of choice to test for the specific defect

• Incidence of the disease, mutation

Table of Contents

78

IV. SUMMARIZE RESULTS AND REPORT TO DESIGNATED

AUTHORITY

B. Record Results

1. Items to Remember to Review

2. Tabulate Results

Table of Contents

79

1. Items to Remember to Review :

• cross check all patient identifiers

• correlate results with flow cytometry, cytogenetics, hematology, virology etc. as

appropriate

• write interpretation of results

• provide all required information to supervisor and/or director

2. Tabulate Results

• Periodically tabulate normal and abnormal results


• % normal vs. abnormal data must be available for all tests in accordance with a

specific question on CAP molecular pathology inspection checklist

• Cross check patient identifiers and be sure all information is included in file folder

before filing

• Genetic testing information must be kept forever

• State requirements for maintaining patient data for other molecular tests vary

• Do not file results in an area of high access - limit access to patient information

• Never report results directly to a patient

80

V. LABORATORY PRACTICE

A. Prepare Reagents

B. Select, Operate and Maintain Laboratory Equipment and Instruments

C. Select Sterilization/Decontamination Procedures

1. Biological contamination

2. Nucleic acid contamination

D. Design and Implement a System to Maintain Adequate Stocks of Laboratory

Supplies and Chemicals, Including Monitoring of Expiration Dates

E. Use Correct Procedures to Store, Handle and Dispose of Biological, Chemical,

Radioactive, Sharps and Glass Materials/Waste

F. Implement Established Procedures for General Laboratory Safety

1. Universal precautions for biological hazards

a. primary barriers including gloves, goggles, masks and lab coat

b. secondary barriers including biological safety cabinet

2. Material Safety Data Sheets


3. Emergency procedures including chemicals or radioactive spills, fire, etc.

G. Employ Appropriate Cleaning Procedures for Laboratory Glassware,

Instruments, and Equipment

H. Ensure Laboratory Quality Control and Continuous Quality Improvement in All

Areas, and Comply with Any Federal, State and/or Local Regulations

1. Verify reproducibility and accuracy of results, including correlation with indication

for study

2. Maintain patient confidentiality and security of patient records

3. Verify equipment maintenance and repair

4. Participate in proficiency testing

5. Attend continuing educational opportunities

6. Maintain acceptable turnaround time

7. Provide evidence of monthly review of quality indicators to ensure problems were

identified and corrected

8. Participate in site laboratory inspections by an accrediting agency

9. Follow protocols to ensure proper identification of patient material through the

complete process, from accession to final report

Table of Contents

81

I. Ethical and Professional Standards

1. Demonstrate an attitude of responsibility and respect toward patients

2. Demonstrate a respectful and cooperative attitude toward professional

colleagues

3. Demonstrate an honest and forthright manner in carrying out professional tasks

4. Explain the purpose of the requested test to other healthcare personnel


J. Participate in training

82

1. Storage and labeling of reagents

• all reagents should be labeled with name of reagent, pH if necessary, date

prepared, expiration date, preparer’s initials, storage conditions, safety

precautions

• storage coding

National Fire Protection Rating System

Health Hazards (blue)

0= No hazard

1 = Can cause irritation if left untreated

2= Can cause injury. Requires prompt treatment

3= Can cause serious injury despite medical treatment

4= Can cause death or major injury despite medical treatment

Flammability (red):

0=Will not burn

1=Ignites after considerable preheating

2=Ignites if moderately heated

3=Can be ignited at all normal temperatures

4=Very flammable gases or very volatile flammable liquid

Reactivity (yellow)

0= Normally stable. Not very reactive with water.

1 = Normally stable. Unstable at high temperature and pressure. Reacts with

water.

2 = Normally unstable but will not detonate.


3 = Can detonate or explode, but requires strong initiating force or heating.

4= Readily detonates or explodes.

Fire -

RED

Reactivity

-YELLOW

Other –

WHITE

Health

– BLUE

Table of Contents

83

In the diamond designated other (white), the following descriptions may appear

OX = oxidizer

ACID = acid

ALK = alkali

COR = corrosive

-W- = Use no water

Hazard Definitions

COMBUSTIBLE. Flash point above 100F. Will not ignite when exposed to ignition

source below 100F.

FLAMMABLE. Flash point below 100F. Vapors will ignite when exposed to ignition

source below 100F.

EXPLOSIVE. Capable of causing an explosion when struck or shaken or when in

contact with another chemical (example, picric acid).


OXIDIZER. A chemical that can initiate or promote combustion.

IRRITANT. A chemical that causes a reversible inflammatory effect on living tissue

at the site of contact.

SENSITIZER. A chemical that causes an allergic reaction in normal tissue after

repeated exposure.

CORROSIVE. Causes visible destruction to living tissue at the site of contact (i.e.,

mineral acids).

TARGET ORGAN EFFECTS. Chemically induced changes to liver, kidneys,

reproductive organs, nervous systems, respiratory system, blood, or fetus.

2. Math/Chemistry

{c= concentration, v= volume}

• math calculations in which there is no change in concentration = ratio or

proportion

• 50ml/42 cm2 = x ml/100 cm2

• math calculations in which there is a change in concentration - c1v1=c2v2

• molar: describes concentration in moles per liter of a solution

• molarity = # moles/Liter (moles of solute per liter of solvent)

• mole = number of grams of a substance equal to its atomic or molecular weight

• formula weight – also known as the molar mass, refers to the mass of atoms in a

compound

• molality = more precise than molarity, less convenient = #moles/kg of solvent

• normality = # equivalent weights/Liter (expresses concentration in terms of

weight per unit volume)


• equivalent weight = amount of an element or compound that will combine with or

replace one mole of hydrogen in a chemical reaction

• as a general rule, equivalent weight of an element is equal to the molecular

weight divided by the valence

• milliequivalent = equivalent weight expressed in milligrams, 1eq = 1000mEq

Table of Contents

84

• weight per volume = grams/100 ml {10% NaCl = 10g of NaCl per 100 ml of

solution}

• (v1 x c1) + (v2 x c2) + (v3 x c3) = vf x cf {f=final}

• molecular weight x molarity = grams/Liter

• dilution factor = volume of sample / total volume of solution

• 1 cm3 = 1 ml

• multiply 2 numbers with same base - write the base with the exponents added

{am x an = a m+n}

• raise an exponential number to higher power - multiply 2 exponents {(am)n = amxn

• divide one number by another number with same base - write the base with the

exponent of the divisor subtracted from the exponent of the dividend - { am/an =

am-n}

• dilution - # parts of material being diluted in the total # of parts of the final product

Math, Formulas, Conversions Etc.

Tera (T) = 1012 Giga (G) = 109


Mega (M) = 106 Kilo (k) = 103

milli (m) = 10-3 micro (u) = 10-6

nano (n) = 10-9 pico (p) = 10-12

femto (f) = 10-15 atto (a) = 10-18

average MW of a nucleotide = 330pg/pmol

average MW of a base pair = 660pg/pmol

To convert picomoles to ug for dsDNA = pmol x # base pairs x 660 pg/1pmol x 1ug/106

pg = ug

To convert picomoles to ug for ssDNA = pmol x # base pairs x 330pg/1pmol x

1ug/106pg = ug

Decay – atom loses particles to become another element

Anion = negatively charged ion

Cation = positively charged ion

isotope = different form of a single element

radioactivity = emission of energy due to decay of an atomic nucleus (alpha particles,

beta particles or gamma rays)

85

3. Select, Operate and Maintain Laboratory Equipment and Instruments

READ MANUAL FOR EACH PIECE OF EQUIPMENT!

spectrophotometer

• measure DNA/RNA concentrations

• usually require a warm-up period

• must be blanked with same concentration of solution that nucleic acid is

resuspended in
• quartz cuvettes must be free from dust and fingerprints

• be sure volume of solution to be analyzed is at appropriate level in cuvette so

that light passes through

• avoid specimen mix-up as cuvettes are often unmarked

• take absorbance readings within linear range of the instrument

centrifuge

• must always be balanced

• always place microfuge tubes in same orientation so know where to expect

small pellets

• centrifuge bucket covers should always be used to minimize formation of

aerosols

• perform regular speed check

balance

• balanced on level countertop

• tared before each use

• clean

• put in area of low traffic and minimal air flow

• calibrate twice per year with NIST (National Institute of Standards and

Technology) Class “S” standard weights

automated pipettes

• know upper and lower limits of pipette

• know which tips are appropriate


• know which is “stop” and which is “release”

• regularly cleaned with .25N hydrochloric acid/.5N sodium hydroxide

pH meter

• use reagents same temperature as solution to be pH’d

• make sure electrode is completely immersed

• make sure electrode is saturated with potassium chloride solution

• standardize with known pH buffers within range of the unknown solution

• <6 = acidic >8 basic

Table of Contents

86

incubators and waterbaths

• always check temperatures

• keep clean!

Geiger counter

• measures radioactivity

• regularly scheduled calibration

thermal cycler

• perform temperature checks on regular basis

beta particle counter

• quantitate radioactive probes

• replace batteries on regular basis

• calibrate with known standards


electrophoretic equipment

• clean as needed (don’t use alcohols with acrylic electrophoresis equipment)

• regular voltage check

• do not connect or disconnect leads when the power is on

• always turn off power before opening gel apparatus

• double check leads are appropriately connected (black to black and red to

red)

thermometers

• thermometers should be certified by NIST

• maintain daily log displayed on the instrument - log should also include

acceptable range of temperatures

4. Select Sterilization/Decontamination Procedures

a. Decontamination and sterilization

• decontaminate with heat, liquid disinfectants, vapors and gases, and radiation

• most effective method of sterilization is steam under pressure at 121-132C in

the autoclave

• alcohols are good surface disinfectants, but they don’t kill spores

• bleach is active against all microorganisms including bacterial spores, but

must be used as freshly diluted solution (10%) {within 24 hours} {strong

oxidizing agent and is corrosive to metal}

• other liquid decontaminants are glutaraldehyde, formaldehyde and phenolic

compounds {toxic chemicals}


Table of Contents

87

b. Nucleic acid contamination

• incorporate dU into PCR products followed by UNG (uracil DNA glycosylase)

digestion

• UV irradiation of PCR mixtures prior to amplification induces pyrimidine

dimers in DNA so contaminating amplicons can’t serve as effective templates

• depurinate work areas with 2M hydrochloric acid

• wipe work areas with freshly made 10% bleach (more effective than HCl for

degrading DNA)

• regularly clean pipets with .25N hydrochloric acid/.5 N sodium hydroxide

• commercial products (AMP erase)

• treat glassware and solutions (except Tris buffers) that will be used for RNA

testing with DEPC

• wear gloves throughout procedures to minimize contamination with

endogenous nucleases

5. Waste

storage, handling and disposal of biological, chemical, radioactive, sharps and glass

materials and waste

6. READ MSDS SHEETS!

• provide information about chemicals including toxicity, flammability, storage,

emergency and first aid procedures

• ethidium bromide is inactivated with activated charcoal slurry (some labs treat
ethidium bromide solutions with sodium hypochlorite {bleach})

• place all syringes/needles, scalpel blades and other sharp items in puncture

resistant containers

• refrigerators and freezers used to store ethanol should be explosion proof

• contact Radiation Safety Officer for proper storage, handling and disposal of

radioactive materials

• Chemical fume hoods

• do not store chemicals in the hood, front shield should be at height specified by

certification sticker on hood, but always below eye level

• keep acid and base spill kits available for small spills

• store acids in separate cabinet

• store flammables in dedicated cabinet

• clean all small spills immediately

• biological waste should be disposed of in marked containers according to

laboratory/hospital policy

Table of Contents

88

7. Universal Precautions

Health care professionals and students who, in the course of their duty or study,

come in contact with blood or body fluids must take protective measures to

prevent accidental infection with blood borne diseases such as hepatitis B and

acquired immunodeficiency syndrome (AIDS). Since the majority of patients

who are infected are unidentified, these recommendations apply to ALL blood
an body fluid specimens, regardless of whether they are known or suspected to

harbor infectious agents. That is to say, you should consider ALL blood

and body fluids to be potentially infected.

GUIDELINES FOR UNIVERSAL PRECAUTIONS

1. HANDWASHING: Wash hands before and after handling any blood or body

fluid specimen. Wash hands immediately when contaminated with any

blood or body substance.

2. GLOVES: Gloves MUST be worn when performing venipuncture.

Gloves MUST be worn when touching mucous membranes or

non-intact skin, and for handling items or surfaces soiled with

blood or body fluids. Hands should be washed before gloving

and after gloves are removed.

Gloves MUST be worn when contact with blood or body

substances is anticipated.

Gloves MUST be changed after contact with each patient or after

handling items or surfaces soiled with blood or body fluids.

3. GOWNS: Gowns should be worn during procedures that are likely to

generate splashes of blood or body fluids, or when clothing is likely to b e

soiled by blood or body fluids.

4. GOGGLES: Protective eyewear such as goggles or plastic splash glasses

should be worn when generation of droplets or splashes of blood or other

body substances is anticipated.

5. MASKS: Masks should be worn for all invasive procedures.

6. NEEDLES AND SHARPS: Precautions must be taken to prevent injuries

caused by needles, scalpels and other sharp instruments.


A. Needles should not be recapped, purposely bent or broken by hands,

removed from disposable syringes, or otherwise manipulated by

hand.

B. After use, disposable syringes and needles, scalpel blades, and

other sharp items must be placed in puncture-resistant containers for

disposal. Disposal containers must be located as close as practical

to the area of use. (DO NOT OVERFILL DISPOSAL

CONTAINERS!)

Table of Contents

89

7. CLEANSING SPILLS OF BLOOD OR BODY FLUIDS: Blood and body

fluids spilled onto environmental surfaces should be cleansed with a

disinfecting procedure such as the following:

A. The person cleansing the spill should don gloves. Pour disinfectant

on the spill before blotting.

B. The majority of spills should be wiped up with absorbent paper

towels. These towels should be disposed of in a sturdy plastic trash

liner that should then be sealed.

C. Residual material on the environmental surface should be

disinfected by pouring a small amount of appropriate high-level

disinfectant solution onto the area. The solution should remain in

contact with the area for ten (10) minutes. The area may then be

routinely mopped up. This treatment kills all known viruses.

D. In most circumstances, appropriate high-level disinfectant means

bleach diluted 1:10 (10% bleach solution).


8. General Safety Notes

• biological safety cabinets

o Class I - protects worker, does not prevent contamination of sample

o Class II - protects both worker and specimen

o Class III - gas-tight, provides highest level of protection

o high efficiency particulate air (HEPA) filter - removes particulates

• no eating, drinking or smoking in the laboratory

• do not keep food and drink in the laboratory

• never pipet by mouth

• do not sniff chemicals

• know location of fire extinguisher, fire blanket, eye wash, shower station and

exits

• get vaccinated for Hepatitis B (most frequently reported laboratory-acquired

infection)

9. Emergencies

• Procedures for emergencies will vary by laboratory and hospital - review these

procedures on a regular basis

10. Chemical Hazards

• acrylamide – neurotoxin as powder or liquid

• ethidium bromide – mutagen and teratogen

• phenol – highly corrosive and toxic

• use volatile chemicals such as phenol, formaldehyde, chloroform and ether in a


fume hood

Table of Contents

90

11. Radioactivity

• take appropriate precautions to minimize exposure

• lab coats, safety glasses, plastic gloves

• spread absorbent paper under work area so spills can be more easily cleaned up

• used Plexiglass shields when using 32P and 125I

• shield not required for 3H and 35S since particle emissions not sufficient intensity

to penetrate skin

12. Cleaning and Glassware

• use NIST Class A glassware

• RNA testing requires special cleaning

• be sure glassware is free of soap residue by checking with pH paper (if detergent

is basic) or use an indicator like bromo-sulfone-phthalein

• always rinse glassware with pure water

13. CAP MOLECULAR PATHOLOGY CHECKLIST

Review most recent College of American Pathologists Molecular Pathology Checklist

(www.cap.org) for detailed information about regulations/requirements for the following

topics:

PROFICIENCY TESTING

QUALITY CONTROL AND QUALITY IMPROVEMENT

REQUISITIONS
SPECIMEN HANDLING

SPECIMEN PROCESSING

REPORTING OF RESULTS

RECORDS

REAGENTS

CONTROLS

INSTRUMENTS AND EQUIPMENT

PERSONNEL

PHYSICAL FACILITIES

LABORATORY SAFETY

RADIATION SAFETY

Table of Contents

91

VI. TEST YOURSELF ANSWERS

1. D

2. A

3. A

4. B

5. C

6. A

7. A

8. B

9. B

10. C

11. C
12. A

13. B

14. D

15. D

16. A

17. B

18. A

19. A

20. C

21. C

22. C

23. D

24. A

25. D

26. D

27. C

28. D

29. A

30. C

Table of Contents

92

VII. REFERENCES

Adams, Dany Spencer (2003). Lab Math: A handbook of measurements, calculations,

and other quantitative skills for use at the bench. Cold Spring Harbor, New York:

Cold Spring Harbor Laboratory Press.


Alberts, B. et al (Eds). Molecular biology of the cell. Third Edition. New York: Garland

Publishing, Inc.

Ausubel, F.M. et al (Eds). (1994). Short Protocols in molecular biology. Third Edition.

Wiley and Sons, Inc.

Ausubel, F. M. et al (Eds). (1994). Current protocols in molecular biology. John Wiley

and Sons, Inc.

Barch, M.J., Knutsen, T. and Spurbeck, J.L. (1997). The AGT Cytogenetics Laboratory

Manual, Third Edition. New York: Lippincott-Raven.

Brown, T.A. (1991). Essential Molecular Biology. Oxford University Press.

Campbell, J.M and Campbell, J.B. (1990). Laboratory mathematics: Medical and

biological applications. Philadelphia: The C.V. Mosby Company.

Coleman, W.B. and Tsongalis, G.J. (Eds). (1997). Molecular diagnostics. Totowa, New

Jersey: Humana Press.

Davies, K.E. (Ed.) Human genetic disease analysis: A practical approach. 2nd Edition.

New York: IRL Press.

Elles, Rob (Ed.) (1996). Molecular diagnosis of genetic diseases. Totowa, New

Jersey: Humana Press, Inc.

Farkas, D.H. (1993). Molecular biology and pathology: A guidebook for quality control.

New York: Academic Press, Inc.

Farkas, D.H. (1997, March). Clinics in laboratory medicine: DNA technology in the

clinical laboratory. Volume 17, Number 1. Philadelphia: W.B. Saunders Company.

FMC BioProducts Catalog. (1998). 800-341-1574. Rockland, ME.

Fuscaldo, A. A., Erlick, B.J. and Hindman, B. (Eds). (1980). Laboratory safety: Theory

and practice. New York: Academic Press, Inc.

Guilfoile, P. (2000). A photographic atlas for the molecular biology laboratory.


Englewood, CO: Morton Publishing Company.

Harwood, A. (1996). Basic DNA and RNA Protocols. Totowa, New Jersey: Humana

Press.

Herrington, C.S. and McGee, J.O’D. (1992). Diagnostic molecular pathology: A

practical approach. Volume II: Cell and tissue genotyping. New YOrk:IRL Press.

Knutson, G. et al. (1996?) Non-radioactive, chemiluminescent detection of

immunoglobulin gene rearrangements by Southern blotting. Paper presented at the

1996 U.S. and Canadian Academy of Pathology, Washington D.C. DAKO

Corporation, Carpinteria, CA.

Lee. H., Morse, S. and O. Olsvik. (Eds). (1997). Nucleic acid amplification

technologies: Application to disease diagnosis. Boston: BioTechniques Books, Div.

Eaton Publishing

Lunn, G. and Sansone, E.B. (1987). Ethidium bromide destruction and decontamination

of solutions. Analytical Biochemistry, 162: 453-458.

Matteson, K.J. et al. (1997). Molecular study guide. Biloxi, MS: Foundation for Genetic

Technology.

Table of Contents

93

McPherson, M.J., Quirke, P. and Taylor, G.R. (Eds). (1991). PCR: A practical

approach. New York: IRL Press.

NCCLS. (1997). Molecular diagnostic methods for genetic diseases; proposed

guideline. NCCLS document MM1-P (ISBN 1-56238-340-X). NCCLS, 940 West

Valley Road, Suite 1400, PA 19087.

Newton, C. R. and Graham, A. (Eds.) PCR. Second Edition. New York: Springer-

Verlag.
Nonradioactive In Situ hybridization application manual. (1996). Second edition.

Boehringer Mannheim.

Nucleic acid isolation and purification. (1998). Boehringer Mannheim.

Passarge, Eberhard (1995). Color atlas of genetics. New York: Thieme Medical

Publishers.

PCR Applications Manual. Boerhringer Mannheim

Promega Protocols and Applications Guide, Third Edition. (1996). Promega

Corporation.

Bruce A. Roe, Department of Chemistry and Biochemistry, The University of Oklahoma,

Norman, Oklahoma 73019 broe@ou.edu

http://www.genome.ou.edu/protocol_book/protocol_adxF.html

Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989). Molecular cloning: A laboratory

manual. Cold Spring Harbor Laboratory Press.

Thompson, M.W., McInnes, R.R. and Willard, H.F. (1991). Thompson and Thompson

Genetics in medicine. Philadelphia: W.B. Saunders Company.

Zyskind, J.W. and Bernstein, S.I. (1992). Recombinant DNA Laboratory Manual. New

York: Academic Press, Inc.

A lot of companies have excellent protocol descriptions and illustrations on their web

sites!!

VIII. ABBREVIATIONS

AMPPD disodium 3-(4-methoxyspiro[1.2-dioxetane.3,2’-tricyclo[3.3.1.1]-

decan]4-ylphenyl phosphate

bDNA branched DNA

bp base pair

BSA bovine serum albumin


cDNA complementary DNA

cpm counts per minute

CSPD 3-(4-methoxyspiro[1,2-dioxetane.3,2’-(5’-

chloro)tricyclo[3.3.1.1].decan]4-yl)phenyl phosphate

DNA deoxyribonucleic acid

dNTP deoxynucleotide triphosphate

ds double stranded

DEPC diethylpyrocarbonate

DSCP double stranded conformational polymorphism

DTT dithithreitol

EDTA ethylene diaminetetracetic acid

EtBr or EB ethidium bromide

H2O water

HCl hydrochloric acid

HEX heteroduplex analysis

HRP horseradish peroxidase

kb kilobases

LCR ligase chain reaction

Mg, Mg++, Mg+2 magnesium

Mn manganese

MW molecular weight

NaOH sodium hydroxide

NASBA nucleic acid sequence based amplification

NTP nucleotide triphosphate

OD optical density (A260)


PAGE polyacrylamide gel electrophoresis

PCR polymerase chain reaction

PFGE pulsed field gel electrophoresis

RBC red blood cell

RE restriction enzyme

RFLP restriction fragment length polymorphism

RNA ribonucleic acid

RT room temperature

RT PCR reverse trancriptase polymerase chain reaction

SDS sodium dodecyl sulfate

Southern(s) Southern Blot

ss single stranded

SSC sodium chloride sodium citrate or salt sodium citrate

Table of Contents

95

SSCP single stranded conformational polymorphism

SSPE sodium chloride sodium phosphate EDTA or salt-sodium

phosphate EDTA

TAE tris-acetate-EDTA

Taq Taq Polymerase

TBE tris-borate-EDTA

TE Tris EDTA

Tm thermal melting point

TMA transcription mediated amplification

TMAC tetramethylammonium chloride


UNG uracil-N-glycosylase

WBC white blood cell