Tools

used in
Molecular Biology
 MOLECULAR BIOLOGY – Molecular biology techniques

1 ISOLATION OF DNA
 Sources of DNA include

• Blood
• Buccal cells
• Cultured cells (plant and animal)
• Bacteria
• Biopsies
• Forensic samples i.e. body fluids, hair follicles,
bone & teeth roots.
 DNA isolation is a routine procedure to collect DNA for
subsequent molecular analysis. There are three basic steps
in a DNA extraction:
• Cell disruption:- This is commonly
achieved by grinding or sonicating the
sample. Removing membrane lipids by
adding a detergent.
• Isolation of DNA:- Removing proteins
by adding a protease (optional but
almost always done).
• Precipitating the DNA :-usually ice-cold
ethanol or isopropanol is used. Since
DNA is insoluble in these alcohols, it will
aggregate together, giving a pellet upon
centrifugation. This step also removes
alcohol soluble salt.
 Basic rules
• Blood – first lyse (explode) the red blood cells with a gentle detergent
such as Triton-X-100.

• Wash cells – haemoglobin (and other pigments) inhibits restriction

enzymes and TAQ polymerase.

• Work on ice to slow down enzymatic processes.

• Wear gloves to protect your samples from you!!

• Autoclave all solutions and store in fridge (except SDS and organic
solvents!)

• Keep all pellets & supernatants until you have the DNA you want.
 Getting to the DNA
• Cells – lyse all cells in presence of :

• NaCl so that DNA is stabilised and remains as a double helix,

• EDTA which chelates Mg++ and is a co-factor of DNAse which
chews up DNA rapidly.
• anionic detergent SDS which disrupts the lipid layers, helps to
dissolve membranes & binds positive charges of chromosomal
proteins (histones) to release the DNA into the solution.
• Include a protease (proteinase K) to digest the proteins
• incubate the solution at an elevated temperature (56oC to
inhibit degradation by DNAses) for 4-24 hrs.
 Getting rid of the protein
• Organic solvent extraction using
equal volume phenol:chloroform
(24:1)

• Protein at the interface after centrifugation

(10000 rpm at 10o c for 10 min.)
 Precipitating the DNA
• add 2.5 - 3 volumes ice-cold 95% ethanol to
the DNA & leave at -20oC overnight.
• Centrifuge sample at 10000 rpm ,10 min., 4 0C.
• Wash DNA pellet to remove excess salt in 70%
EtOH and air-dry.
• Resuspend in sterile distilled water(pH7.4)
• Store at 4oC or frozen at -20oC long term.
 Quantifying the DNA

• The amount of DNA can be quantified using the formula:

DNA concentration (g/ml) = OD260 x 100 (dilution factor) x 50 g/ml

1000
• Nucleic acids have a peak absorbance in the ultraviolet range at about 260
nm

• 1 A260 O.D. unit for dsDNA = 50 µg/ml

• 1 A260 O.D. unit for ssDNA = 33 µg/ml

• 1 A260 O.D. unit for RNA = 40 µg/ml

 DNA purity
• The purity of the DNA is reflected in the OD260:OD 280
ratio and must be between 1.6 and 2.00.

< 1.6 – protein contaminated

> 2.0 – chloroform / phenol contaminated

• Repurify sample.
 Summary
• Sample for DNA extraction
• Lysis of cells at elevated temperature +
detergent + enzyme in salt buffer
• Removal of cellular proteins
• Precipitation of nucleic acids with ethanol
• Quantitation and purity measurement of
DNA
 High MW Genomic DNA Isolation
Typical Procedure Phenol Extraction
1 Cell Lysis • mix sample with equal volume
– 0.5% SDS + proteinase of sat. phenol soln
K (55oC several hours) • retain aqueous phase
2 Phenol Extraction • optional chloroform/isoamyl
– gentle rocking several alcohol extraction(s)
hours

 aqueous phase
(nucleic acids)
 phenolic phase
(proteins)

ORGANIC PHASE SEPARATION

 High MW Genomic DNA Isolation
Typical Procedure EtOH Precipitation
1 Cell Lysis • 2-2.5 volumes EtOH, -20oC
– 0.5% SDS + proteinase K • high salt, pH 5-5.5
(55oC several hours) • centrifuge or ‘spool’ out
2 Phenol Extraction
– gentle rocking several
hours
3 Ethanol/ salt Precipitation
4 RNAse followed by
proteinase K

5 Repeat Phenol Extraction

and EtOH ppt
 PLASMID DNA
Natural Bacterial Transformation/ conjugation

Also possible to experimentally

S. Pneumoniae ‘transforming’ DNA is a ‘transform’ plasmid vectors into bacteria -
plasmid see later
 PLASMID DNA ISOLATION
Alkaline lysis denaturation/ renaturation protocol

Bacteria lysed in Protein denaturation (SDS)

Single stranded plasmid DNA
SDS + strong NaOH Single stranded genomic DNA
buffer
DENATURATION
Potassium pH
acetate NEUTRALISATION

Aqueous (double
stranded plasmid
DNA) Small multi-copy plasmid DNA
Centrifugation quickly re-anneals in solution
Large single copy genomic
SEDIMENTATION DNA fails to re-anneal and
forms precipitate with proteins

Pellet (proteins
and genomic
DNA)
 PLASMID DNA ISOLATION
Aqueous (double
stranded plasmid
DNA) 1. Phenol/ CHCl3 extraction & Ethanol/ Salt precipitation

or 2. Solid phase/ silica extraction ‘miniprep’

Pellet (proteins
and genomic
DNA) In presence of alkaline
chaotropic salts, denatured
plasmid DNA binds to silica
beads in the column

Wash buffers used to remove impurities

&
DNA eluted (and re-natured in H2O)

Quick relatively pure double stranded

Centrifugation steps plasmid DNA
 PLASMID DNA ISOLATION
Aqueous (double
stranded plasmid
DNA)

or 3. Anion exchange column-based chromatography

Pellet (proteins
and genomic
DNA)

Altering the pH and ionic conditions removes impurities

leading to high [salt] elution and EtOH or isopropanol
precipitation

Extremley pure double stranded plasmid

DNA
 Isolation of RNA
Special Considerations
Guanidinium thiocyanate

• RNAse inhibitors!
• extraction in guanidine salts
• phenol extractions at pH 5-6
• (pH 8 for DNA)
• selective precipitation of high MW
forms (rRNA, mRNA) with LiCl
• oligo-dT column for mRNA’s
• treatment with RNase-free DNase
 Using UV spectroscopy to analyze DNA/ RNA
• Nucleic acids absorbs UV light with a major peak at 260nm (max)
Absorbance

Beer-Lambert equation
A = cl
260

Wave Length ()

• Absorbance extinction coefficients () vary depending on the nucleic acid structure

A260

Isolated ss RNA/ DNA ds DNA

nucleotides  = 25  = 20

• A260 / A280 ratio indicates sample purity

• Detection
Pure RNA = 2.0 • Quantitation
Pure DNA = 1.8 • Assessment of purity
So now we have isolated DNA ... but it is still too long to work with:

2 how to fragment it?

- mechanical shearing
(no control)

or ...
MOLECULAR SCISSORS - TYPE II RESTRICTION ENDONUCLEASES
Hamilton Othanel Smith 1968

cohesive ends

SPECIFIC CUT SPECIFIC JOINING (LIGATION)

Ability to join two foreign pieces of DNA together
 END

COHESIVE ENDS
BLUNT
 3 RECOMBINANT DNA TECHNOLGY
The plasmid as DNA ‘vector’ (vehicle)

Possible to insert ‘interesting’

DNA’s into a plasmid using
restriction endonucleases
 RECOMBINANT DNA TECHNOLGY
The plasmid as DNA ‘vector’ (vehicle)

The recombinant plasmid containing the

‘interesting’ DNA sequence can now be
propagated/ amplified by experimentally
transforming the recombinant plasmid
into bacteria and allowing these bacteria
to multiply and produce more
recombinant plasmid

Specialized strains of bacteria can be permeablised

by electroporation or heat shock

Therefore specific DNA fragments can

be selectively propagated i.e. cloned
RECOMBINANT DNA TECHNOLGY
Specialized plasmid cloning vectors

Muliple Cloning Site (MCS) contains many

restriction sites to maximize target DNA
cloning potential

Plasmids contain genes that confer antibiotic

resistance so that only successfully
transformed bacteria are propagated
3 Why not clone whole genomes?

i.e. together they represent a ‘Genomic

DNA Library’

Each bacterial colony represents an amplified

clone containing a recombinant plasmid
harbouring a distinct region of the genome

Also possible to do this using cDNA copies of transcribed mRNAs resulting a

‘cDNA Gene Expression Library’
Bacteriophage Lambda
vectors

 phage linear DNA genome

cos cos

Non-essential region that can be substituted by

DNA to be cloned (approx 20Kb)

Cosmids, phosmids, BACs and YACs to clone larger DNA fragments

 DNA ISOLATION AMPLIFICATION

4 How to visualize DNA?

GEL ELECTROPHORESIS NUCLEIC ACID HYBRIDIZATION

size distinction sequence distinction
 Gel electrophoresis
• The basic principle is that DNA, RNA, and
proteins can all be separated by means of
an electric field.
• In agarose gel electrophoresis, DNA and
RNA can be separated on the basis of size
by running the DNA through an agarose
gel.
• Proteins can be separated on the basis of
size by using an SDS-PAGE gel, or on the
basis of size and their electric charge by
using what is known as a 2D gel
electrophoresis.
 fragmented DNA GEL ELECTROPHORESIS

- -
-
+

agarose

D-galactose 3,6-anhydro
L-galactose n
Ethidium Bromide SYBR® Safe
on blue light
Genomic DNA on gel:

23 kb
9,5 kb
Plasmid DNA on gel:
 NUCLEIC ACID HYBRIDIZATION

labeled probe

Fluorescence In Situ Hybridization

(FISH) Metaphase spread chromosomes
on a slide
Probe labeling by incorporation of modified (d)NTPs

radioactive labeling

AUTORADIOGRAPHY
32
P

Fluorophores

Biotin-11-dUTP Streptavidin conjugation

Y anti-DIG
antibody
Enzymes
alkaline phosphatase
horseradish peroxidase

(DIG) chemiluminiscence
 DIG
DIG
DIG
DIG

DIG

HRP
Y light
substrate HRP
Y light
substrate HRP
Y light
DIG substrate HRP
YY substrate
HRP
light
DIG
substrate
light
DIG
DIG

s
D

u
IG

bs
tra
DI
G

te
Y

HR
lig
ht

P
 DNA denaturation

Melting (denaturation) temperature

depends on these major factors:
- GC content (and therefore AT content)
- sequence length
- gaps in the annealed strands
- salt concentration GC rich
Temp Temp
- pH AT rich
- organic solvents (DMSO, formamide...)
GC rich
 CHROMOSOME PAINTING – MULTI COLOR FISH

Translocated chromsome segment

Particularly useful when diagnosing chromsomal abnormalities in certain forms of

cancer (region specific barcoding on left and whole chromsosome paints on right)
How to analyze specific form of genes in genomic DNA?

e.g. successful intergration of a transgene into the genome of a

transgeneic animal (mouse)
 Southern blotting

• Southern blot is a method for probing for the presence of a

specific DNA sequence within a DNA sample.
• DNA samples are separated by gel electrophoresis and then
transferred to a membrane by blotting via capillary action.
• The membrane is then exposed to a labeled DNA probe that
has a complement base sequence to the sequence on the DNA
of interest.
• less commonly used due to the capacity of other techniques,
such as PCR.
• Southern blotting are still used for some applications such as
measuring transgene copy number in transgenic mice, or in the
engineering of gene knockout embryonic stem cell lines.
 Southern blot – transfer of DNA to membrane
e.g. fragmentation by restriction
endonucleases

fragmented
DNA

Figure 8-38 (part 1 of 4) Molecular Biology of the Cell (© Garland Science 2008)
Figure 8-38 (part 2 of 4) Molecular Biology of the Cell (© Garland Science 2008)
 Labeled probe has sequence
homology to DNA of interest e.g.
the hopefully integrated
transgene

Figure 8-38 (part 3 of 4) Molecular Biology of the Cell (© Garland Science 2008)
 e.g. bands reveal
integrated transgenes
and size shows whether
integration was correct

Figure 8-38 (part 4 of 4) Molecular Biology of the Cell (© Garland Science 2008)
 SOUTHERN BLOT
combines DNA fragmentation,
gel electrophoresis and hybridization
to analyze specific DNA sequences
Same procedure blotting RNA used to
confirm gene mRNA expression called
NORTHERN BLOTTING
Similar principle used to blot proteins that are then
detected by specific antibodies - WESTERN
BLOTTING

Figure 8-38 Molecular Biology of the Cell (© Garland Science 2008)

GENOMIC or cDNA EXPRESSION LIBRARY

Hybridization
y
ntar to filter
leme
omp
c
isolate
DNA
cloning
 genomic library

hybridisation
.

experimentation

sub-clone

restriction
digestion
 How to see genes ... How to see specific How to clone
forms of genes? and amplify
... where in ... where on genes?
tissues? chromosomes?

in situ hybridization southern blot recombinant DNA

genomic libraries
RFLP
 Northern blotting
• The northern blot is used to study the expression patterns of a
specific type of RNA molecule as relative comparison among a set
of different samples of RNA.
• RNA is separated based on size and is then transferred to a
membrane then probed with a labeled complement of a sequence of
interest.
• The results may be visualized through a variety of ways depending
on the label used. Most result in the revelation of bands
representing the sizes of the RNA detected in sample.
• The intensity of these bands is related to the amount of the target
RNA in the samples analyzed.
• It is used to study when and how much gene expression is occurring
by measuring how much of that RNA is present in different samples.
• one of the most basic tools for determining at what time, and under
what conditions, certain genes are expressed in living tissues.
 Western blotting
• In western blotting, proteins are first separated by size, in a thin gel
sandwiched between two glass plates in a technique known as SDS-PAGE
sodium dodecyl sulphate polyacrylamide gel electrophoresis.

• The proteins in the gel are then transferred to a nitrocellulose, nylon or other
support membrane.

• This membrane probed with solutions of antibodies. Antibodies specifically

bind to the protein of interest & visualized by a variety of techniques,
including colored products, chemiluminescence, or autoradiography.

• Antibodies are labeled with enzymes. When a chemiluminescent substrate

is exposed to the enzyme it allows detection.

• Using western blotting techniques allows not only detection but also
quantitative analysis.
 Polymerase chain reaction (PCR)

• The polymerase chain reaction is an extremely

versatile technique for copying DNA.
• PCR allows a single DNA sequence to be
copied (millions of times), or altered in
predetermined ways.
• PCR has many variations, like reverse
transcription PCR (RT-PCR) for amplification of
RNA, and real-time PCR (QPCR) which allow for
quantitative measurement of DNA or RNA
molecules.
 PCR Analysis

The process follows the principle of DNA replication

53
 PRIMER
• A primer is a strand of nucleic acid that serves as a starting
point for DNA synthesis.
• These primers are usually short, chemically synthesized
oligonucleotides, with a length of about twenty bases. They
are hybredized to a target DNA, which is then copied by the
polymerase.
• minimum primer length used in most applications is 18
nucleotides.
• Replication starts at the 3'-end of the primer, and copies the
opposite strand.
• In most cases of natural DNA replication, the primer for DNA
synthesis and replication is a short strand of RNA .
 Applications of PCR
•A common application of PCR is the study of patterns of gene expression.

•The task of DNA sequencing can also be assisted by PCR.

•PCR has numerous applications to the more traditional process of DNA

cloning.

•An exciting application of PCR is the phylogenic analysis of DNA from

ancient sources

•A common application of PCR is the study of patterns of genetic mapping

•PCR can also used in Parental testing, where an individual is matched

with their close relatives.
 Molecular markers
• Molecular marker are based on naturally occurring polymorphism in
DNA sequence(i.e. base pair deletion, substitution ,addition or
patterns).
• Genetic markers are sequences of DNA which have been traced to
specific locations on the chromosomes and associated with particular
traits.
• It can be described as a variation that can be observed.
• A genetic marker may be a short DNA sequence, such as a
sequence surrounding a single base-pair change (single nucleotide
polymorphism, SNP), or a long one, like mini satellites.
 Some commonly used types of genetic
markers are
• RFLP (or Restriction fragment length polymorphism)
• AFLP (or Amplified fragment length polymorphism)
• RAPD (or Random amplification of polymorphic DNA)
• VNTR (or Variable number tandem repeat)
• Micro satellite polymorphism, SSR (or Simple sequence
repeat)
• SNP (or Single nucleotide polymorphism)
• STR (or Short tandem repeat)
• SFP (or Single feature polymorphism)
• DArT (or Diversity Arrays Technology)
• RAD markers (or Restriction site associated DNA markers)
There are 5 conditions that characterize a
suitable molecular marker
• Must be polymorphic
• Co-dominant inheritance
• Randomly and frequently distributed
throughout the genome
• Easy and cheap to detect
• Reproducible
 Molecular markers can be used for
several different applications including
• Germplasm characterization,
• Genetic diagnostics,
• Characterization of transformants,
• Study of genome
• Organization and phylogenic analysis.
• Paternity testing and the investigation of crimes.
• Measure the genomic response to selection in
livestock
RFLP (Restriction fragment length polymorphism)

RFLPs involves fragmenting a sample of DNA by a restriction enzyme,

which can recognize and cut DNA wherever a specific short
sequence occurs. A RFLP occurs when the length of a detected
fragment varies between individuals and can be used in genetic
analysis.

Advantages:
• Variant are co dominant
• Measure variation at the level of DNA sequence, not protein
sequence.
Disadvantage:
• Requires relatively large amount of DNA
Restriction Fragment Length Polymorphism (RFLP)

EcoRI
3’AGCTAGCGTGCTGTGATGTAGCTGATGCTGAATTCTGCGATGTT’5
5’TCGATCGCACGACACTACATCGACTACGACTTAAGACGCTACAA’3

3’AGCTAGCGTGCTGTGATGTAGCTGATGCTGAATGCTGCGATGTT’5
5’TCGATCGCACGACACTACATCGACTACGACTTACGACGCTACAA’3

SNP
Restriction digestion by EcoRI

3’AGCTAGCGTGCTGTGATGTAGCTGATGCTG AATTCTGCGATGTT’5
5’TCGATCGCACGACACTACATCGACTACGACTTAA GACGCTACAA’3

3’AGCTAGCGTGCTGTGATGTAGCTGATGCTGAATGCTGCGATGTT’5
5’TCGATCGCACGACACTACATCGACTACGACTTACGACGCTACAA’3
RFLP
Diagnosis of Genetic Diseases by RFLP
AFLP ( Amplified fragment length polymorphism)
In this analysis we can amplify restricted fragments and reduces
the complexity of material to be analyzed (approx 1000 folds).it
can be used for comparison b/w closely related species only.
Advantages:
• Fast
• Relatively inexpensive
• Highly variable
Disadvantage:
• Markers are dominant
• Presence of a band could mean the individual is either
homozygous or heterozygous for the Sequence - can’t tell
which?
 RAPD ( Random amplification of polymorphic DNA)

Random Amplification of Polymorphic DNA. It is a type of PCR

reaction, but the segments of DNA that are amplified are random.
Advantages:
• Fast
• Relatively inexpensive
• Highly variable
Disadvantage:
• Markers are dominant
• Presence of a band could mean the individual is either
homozygous or heterozygous for the Sequence - can’t tell which?
• Data analysis more complicated
Micro satellite polymorphism, SSR or Simple
sequence repeat
Microsatellites, Simple Sequence Repeats (SSRs), or
Short Tandem Repeats (STRs), are repeating
sequences of 1-6 base pairs of DNA.
Advantages:
• Highly variable
• Fast evolving
• Co dominant
Disadvantage:
• Relatively expensive and time consuming to develop
 SNP
• A single-nucleotide
polymorphism (SNP,
pronounced snip) is a DNA
sequence variation occurring
when a single nucleotide — A,
T,C, or G — in the genome (or
other shared sequence) differs
between members of a species
or paired chromosomes in an
individual.
• Used in biomedical
research ,crop and livestock
breeding programs.
 STR
• A short tandem repeat (STR) in DNA occurs when
a pattern of two or more nucleotides are repeated
and the repeated sequences are directly adjacent to
each other.
• The pattern can range in length from 2 to 16 base
pairs (bp) (for example (CATG)n in a genomic region)
and is typically in the non-coding intron region
• Used in forensic cases.
• used for the genetic fingerprinting of individuals