DNA and RNA isolation and purification (course readings

10 and 11)

I. Genomic DNA preparation overview

II. Plasmid DNA preparation

III. DNA purification

• Phenol extraction
• Ethanol precipitation

IV. RNA work

What do we need DNA for?

•Detect, enumerate, clone genes

•Detect, enumerate species
•Detect/sequence specific DNA regions
•Create new DNA “constructs” (recombinant DNA)

What about RNA?

•Which genes are being transcribed?
•When/where are genes being transcribed?
•What is the level of transcription?
 DNA purification: overview

cell harvest and

cell growth lysis

DNA concentration DNA purification

Bacterial genomic DNA prep: cell extract
Lysis:

• Detergents
• Organic solvent
• Proteases
(lysozyme)
• Heat

“cell extract”
 Genomic DNA prep: removing proteins and RNA

chloroform

Need to mix gently! (to avoid shearing breakage of the

genomic DNA)
Add the enzyme RNase to degrade RNA in the aqueous layer
 2 ways to concentrate the genomic DNA

70% final conc.

“spooling” Ethanol precipitation

Genomic DNA prep in plants --
how get rid of carbohydrates?
CTAB:

Cationic detergent

CH3
CH3

(low ionic N+ Br-

conditions)
CH3
C16H33
(MC 6.61-6.62)
Plasmids: vehicles of recombinant DNA

Bacterial cell

genomic DNA plasmids

Non-chromosomal DNA
Replication: independent of the chromosome
Many copies per cell
Easy to isolate
Easy to manipulate
Plasmid purification: alkaline lysis

Alkaline
conditions
denature DNA

Neutralize:
genomic DNA
can’t renature
(plasmids
CAN because
they never
fully separate)
 DNA purification: silica binding

Binding occurs in presence of high salt

concentration, and is disrupted by
elution with water
DNA purification: phenol/chloroform extraction

1:1 phenol : chloroform

or
25:24:1 phenol : chloroform : isoamyl alcohol

Phenol: denatures proteins, precipitates form at interface

between aqueous and organic layer

Chloroform: increases density of organic layer

Isoamyl alcohol: prevents foaming

 Phenol extraction

1. Aqueous volume (at least 200 microliters)

2. Add 2 volumes of phenol:chloroform, mix well
3. Spin in centrifuge, move aqueous phase to a new tube
4. Repeat steps 2 and 3 until there is no precipitate at phase
interface
5. (extract aqueous layer with 2 volumes of chloroform)
 Ethanol precipitation (DNA concentration)
Ethanol depletes the hydration shell surrounding DNA…
• Allowing cations to interact with the DNA phosphates
• Reducing repulsive forces between DNA strands
• Causing aggregation and precipitation of DNA

• Aqueous volume (example: 200 microliters)

-- add 22 microliters sodium acetate 3M pH 5.2
-- add 1 microliter of glycogen (gives a visible pellet)
-- add 2 volumes (446 microliters) 100% ethanol
-- mix well, centrifuge at high speed, decant liquid
-- wash pellet (70% ethanol), dry pellet, dissolve in appropriate
volume (then determine DNA concentration)
 DNA purification: overview

cell harvest and

cell growth lysis

DNA concentration DNA purification

 DNA --------------> mRNA --------------> protein

Lots of information in mRNA:

When is gene expressed?

What is timing of gene expression?
What is the level of gene expression?

(but what does an mRNA measurement really say about

expression of the protein?)

Isolation of RNA -- Course reading 11

RNA in a typical eukaryotic cell:
10-5 micrograms RNA

80-85% is ribosomal RNA

15-20% is small RNA (tRNA, small nuclear RNAs)

About 1-5% is mRNA

-- variable in size
-- but usually containing 3’ polyadenylation
The problem(s) with RNA:
RNA is chemically unstable -- spontaneous cleavage of
phosphodiester backbone via intramolecular transesterification

RNA is susceptible to nearly ubiquitous RNA-degrading enzymes

(RNases)

RNases are released upon cell lysis

RNases are present on the skin
RNases are very difficult to inactivate
-- disulfide bridges conferring stability
-- no requirement for divalent cations for activity
 Common sources of RNase and how to avoid them

Contaminated solutions/buffers

USE GOOD STERILE TECHNIQUE

TREAT SOLUTIONS WITH DEPC (when possible)
MAKE SMALL BATCHES OF SOLUTIONS

Contaminated equipment

USE “RNA-ONLY” PIPETS, GLASSWARE, GEL RIGS

BAKE GLASSWARE, 300°C, 4 hours
USE “RNase-free” PIPET TIPS
TREAT EQUIPMENT WITH DEPC
Top 10 sources of RNAse contamination
(Ambion Scientific website)

1) Ungloved hands
2) Tips and tubes
3) Water and buffers
4) Lab surfaces
5) Endogenous cellular RNAses
6) RNA samples
7) Plasmid preps
8) RNA storage (slow action of small amounts of RNAse
9) Chemical nucleases (Mg++, Ca++ at 80°C for 5’ +)
10) Enzyme preparations
Inhibitors of Rnase
DEPC: diethylpyrocarbonate

alkylating agent, modifying proteins and nucleic acids

fill glassware with 0.1% DEPC, let stand overnight at room

temp

solutions may be treated with DEPC -- add DEPC to 0.1%,

then autoclave (DEPC breaks down to CO2 and ethanol)
Inhibitors of Rnase
Vanadyl ribonucleoside complexes
competitive inhibitors of RNAses, but need to be removed
from the final preparation of RNA

Protein inhibitors of RNAse

horseshoe-shaped, leucine rich protein, found in cytoplasm of
most mammalian tissues
must be replenished following phenol extraction steps
Making and using mRNA (1)
Making and using mRNA (2)
Purifying RNA: the key is speed

Break the cells/solubilize components/inactivate RNAses by the

addition of guanidinium thiocyanate (very powerful denaturant)

Extract RNA using phenol/chloroform (at low pH)

Precipitate the RNA using ethanol/LiCl

Store RNA:
in DEPC-treated H20 (-80°C)
in formamide (deionized) at -20°C
Selective capture of mRNA: oligo dT-cellulose

Oligo dT is linked to cellulose matrix

RNA is washed through matrix at high salt concentration

Non-polyadenylated RNAs are washed through

polyA RNA is removed under low-salt conditions

(not all of the non-polyadenylated RNA gets removed

Other methods to capture mRNA

Poly(U) sepharose chromatography

Poly(U)-coated paper filters

Streptavidin beads:

•A biotinylated oligo dT is added to guanidinium-treated

cells, and it anneals to the polyA tail of mRNAs

•Biotin/streptavidin interactions permit isolation of the

mRNA/oligo dT complexes
How good is the RNA prep?

The rRNA should form 2 sharp bands in ethidium bromide-

stained gels (but mRNA will not be visible

Use radiolabelled poly dT in a pilot Northern hybridization--

should get a smear from 0.6 to 5 kb on the blot

Use a known, “standard” gene probe (e.g. GAPDH in

mammalian cells) in Northern hybridization--there should be
a sharp band with no degradation products
 In vitro amplification of DNA by PCR
I. Theory of PCR
II. Components of the PCR reaction
III. A few advanced applications of PCR

a) Reverse transcription PCR (for RNA measurements)

b) Quantitative real-time PCR
c) PCR of long DNA fragments
d) Inverse PCR
e) MOPAC (mixed oligonucleotide priming)

Molecular Cloning, p. 8.1-8.24

 What is PCR?

• Polymerase Chain Reaction--first described in 1971 by

Kleppe and Khorana, re-described and first successful
use in 1985
• Allows massive amplification of specific sequences that
have defined endpoints
• Fast, powerful, adaptable, and simple*

• Many many many applications

* usually
 Why amplify specific sequences?
• To obtain material for cloning and sequencing, or for
in vitro studies
• To verify the identity of engineered DNA constructs
• To monitor gene expression
• To diagnose a genetic disease
• To reveal the presence of a micro-organism
• To identify an individual
• Etcetera, etcetera
 What you need for PCR:

1. Template DNA that contains the “target sequence”

2. Primers: short oligonucleotides that define the ends

of the target sequence

3. Thermostable DNA polymerase

4. Buffer, dNTPs

5. A thermal cycler
 A typical PCR program:

Denaturation: denature template strands (94°C for

2-5 minutes), can also add your DNA polymerase at
this temp. for a “hot start” (adding DNA pol to a hot
tube can prevent false priming in the initial round of
DNA replication)

Annealing: The default is usually 55°C. This

temperature variable is the most critical one for
getting a successful PCR reaction. This is the best
variable to start with when trying to optimize a PCR
reaction for a specific set of primers. Annealing
temperatures can be dropped as low as 40-45°C,
but non-specific annealing can be a problem
 A typical PCR program:

Extension: generally 72°C, this is the operating

temperature for many thermostable DNA
polymerases.

Number of cycles: Depends on the number of

copies of template DNA and the desired amount
of PCR product. Generally 20-30 cycles is
sufficient.
 How it works:
a simple PCR reaction, first cycle
(Can also be
Single-stranded)
94°C
50°C
Cycles of
denaturation,
primer annealing,
72°C and primer
extension by DNA
polymerase
 a simple PCR reaction, second cycle

new like
reactions first
cycle
a simple PCR reaction, third cycle

PCR animation:
http://www.dnai.org/b/index.html
http://www.dnalc.org/ddnalc/resources/shockwave/
pcranwhole.html
 Choosing primers:
• Should be 18-25 (17-30?) nucleotides in length (giving specificity)
• Calculated melting temperature varies depending on the method
used (55-65°C using the Wallace Rule, eg. see MC), but should be
nearly identical for both primers
• Avoid inverted repeat sequences and self-complementary
sequences in the primers, avoid complementarity between primers
(‘primer dimers’)
• Have a G or C at the 3’ end (a G/C “clamp”)
• Many computer programs exist for helping meet these criteria (ex:
Biology Workbench, workbench.sdsc.edu)
 Thermostable DNA polymerases
(See Molecular Cloning table 8-1)

• Isolated from thermophilic bacteria and archaea (T. aquaticus is

a bacterium, not an archaeon)
• Bacterial enzymes (e.g. Taq) good for routine reactions and
small PCR products, fidelity of replication is somewhat low
• Archaeal enzymes (e.g. Pfu) also good for routine reactions and
best for cloning: 3’--5’ exonuclease activity provides very high
fidelity, and enzymes are very stable to heat
 Thermal cyclers
Standard: heat block, “ramp” times fairly long (10 -20 seconds to
change temperature), 30 cycle PCR lasts 2-3 hours.
Advantage: easily automated, heat blocks can PCR up to
384 samples at a time
Disadvantage: relatively slow
New: reactions are being sped up significantly
--capillary tubes heated and cooled by blasts of air--30
cycle-PCR done in >30 minutes (harder to scale up)
--fluid flow cells: channels force liquid through
temperature gradients, very fast (but still not widely available)
 Sources of problems in PCR
• Inhibitors of the reaction from the the template DNA
preparation (protease, phenol, EDTA, etc)
• Cross-contamination by DNA from sources other
than the template added
– if this becomes a problem:
• Work in a laminar flow hood (decontaminate using UV
light 254 nm)
• Use PCR dedicated pipettors (with barrier tips), PCR
dedicated reagents
• Centrifuge tubes before opening them to prevent
spattering, pipet contamination
 Controls to include in difficult PCRs:
Bystander template Target Specific Bystander DNA:
DNA DNA DNA primers not recognized by
Positive primers
controls
Target DNA:
1 + - + + known to contain
primer
2 - - + + recognition
Negative sequences
controls

3 - - - +
4 + - - +
 Hot Start of PCR reactions

• Witholding some component of the reaction until the

denaturing temperature is reached (94°C)
• This helps prevent non-specific priming, which may
occur at the low temperatures (room temp.) -- the non-
specific priming could give artifactual amplification as
PCR block temperature rises

A) Wait until 94°C to add enzyme --or--

B) Use enzyme bound to an inactivating enzyme antibody
that releases at high temperature --or--
C) Use wax beads containing Mg++ that can only be released
at high temp.
 Touchdown PCR
• Useful if your primers are not 100% complementary to your
template DNA (e.g. degenerate oligos), or when there are multiple
members of the gene family you are amplifying
• Allows you to selectively amplify only the best sequences (with
the least mismatches) while minimizing non-specific PCR products

• Start with 2 cycles at an annealing temperature about 3°C higher

than the calculated primer melting temperatures.
• Progressively reduce the annealing temperature by 1°C at 2 cycle
intervals
 Trouble-shooting:

-- Very little product

-- No PCR product
-- Multiple bands
Etc.

(see Molecular cloning, tables

8-4 and 8-5)
III. Special applications for PCR

A. Reverse transcription PCR (for RNA

measurements)

B. Quantitative (real-time) PCR

C. PCR of long DNA fragments

D. Whole genome amplification

E. Inverse PCR

E. MOPAC (mixed oligonucleotide priming)

Amplification of RNA (monitor gene expression): reverse
transcription PCR (RT-PCR)
Step 1: generate a 1st strand cDNA using reverse transcriptase (catalyzes
synthesis of DNA from an RNA template)

A)

B)

C)

Step 2: normal PCR (from cDNA) using gene-specific primers

 Quantitative
(real time)
PCR
The more target DNA there is,
the more probe anneals, the
more it is cleaved (by Taq’s 5’-
3’ exonuclease activity)

Fluorescence measurements are

done simultaneously with PCR
cycles, yields an instantaneous
measurement of product levels
 Quantitative
Real Time
(QRT) PCR
Position of the
steep part of the
curve varies
depending on the
amount of
template DNA or
RNA, can measure
more
variation over 5 or template less
6 orders of template

magnitude
 Another quantitative measure of double stranded DNA in a PCR
reaction: binding of SYBR Green Dye

Non-fluorescing SYBR
green dye

Fluorescing SYBR
green dye

From the Molecular Probes website

(www.probes.com)
 Use of a quenching dye to reduce measurement of “primer
dimer” artifacts in QRT-PCR

QSY quencher dye: it

absorbs fluorescence from
sybr green dyes in the
vicinity--prevents
accumulation of signal from
primer dimers
Always do your controls!
QRT-PCR using Sybr green dye fluorescence

Standard curve: what is “threshold” for specific number of DNA

molecules?

(From the Invitrogen website)

 PCR of long sequences (>2 kb)

Long DNAs are difficult to amplify

– Breakage of the DNA
– Non-processive behavior of DNA polymerase
– Misincorporation by error prone DNA
polymerases
 PCR of long sequences (>2 kb)

Changes to protocol that assist in long PCR

– Make sure DNA is exceedingly clean
– Use DNA polymerase “cocktail”: Taq for it’s
high activity, and Pfu for its proofreading
activity (it can actually correct Taq’s mistakes)
– Increase time of extension reaction (5-20
minutes, compared to the standard 1 minute for
short PCRs)
•Amplified product longer than 3 kilobases with high fidelity
•10 times fewer mutations than with conventional PCR

•Taq DNA pol (no proofreading) plus an archaeal DNA pol (does
proofreading)

•Betaine (amino acid analogue with several small

tetraalkyammonium ions)--reduces non-specific amplification
products--reduces non-complementary primer-template interactions?
(unknown how it works)
Whole genome amplification: multiple displacement
amplification (MDA)

Applications: forensics, embryonic disease diagnosis,

microbial diversity surveys, etc.

How it works:
Strand-displacement amplification used by rolling-circle
replication systems.

Phi29 DNA polymerase (very low error rate) and random

hexamer primers, low temperature! (30°C)
Whole genome amplification : multiple displacement amplification
(MDA)

20-30 micrograms human DNA can be recovered from 1-10 copies

of the human genome

Distribution of products appears to be random sampling of the

available template (and this is good!)
 Inverse PCR:
sequencing “out”
from known
sequence
 “Vectorette” PCR

First primer: known sequence

Vectorette primer: only in vectorette-ligated sequence--it cannot
anneal until there is a single round of primer extension from the
specific primer

http://www.bio.psu.edu/People/Faculty/Akashi/vectPCR.html
 MOPAC: Mixed oligonucleotide primed
amplification of cDNA
If you only have a protein sequence, and you want to clone the
gene for the protein:

1. Design oligonucleotides based on deduced mRNA (and DNA)

sequence (but since multiple codons can encode the same
amino acid, this gets complicated quickly)
2. program your oligo synthesizer to make primer sets that are
randomized for the degenerate positions of each codon
3. use universal nucleotides like inosine, which base pairs with C,
T, and A (limits degeneracy)
4. Do your PCR and hope for the best