POLYMERASE CHAIN REACTION - PCR

POLYMERASE CHAIN REACTION - PCR
A `licence` to do molecular biology
A key central technique that has revolutionised molecular and consequently cell biology
POLYMERASE CHAIN REACTION
This lecture:
Principles of PCR and applications
PCR - the basics
How to optimise PCR and troubleshoot problems
A simple rapid, sensitive and versatile in vitro method for selectively amplifying defined sequences/regions of DNA/RNA from an initial complex source of nucleic acid - generates sufficient for subsequent analysis and/or manipulation
Human diploid cell contains 6 X 10-9 base pairs 
`average` gene size ~ 10,000bp = 1/300,000 
600bp fragment = 1/1,000,000
Amplification in a normal PCR will be perhaps a million fold
WHAT IS PCR?
Cloning of genes or gene fragments
same species or homologous genes from different species (DOP-PCR)
Genetic diagnosis - Mutation detection
basis for many techniques to detect gene mutations (sequencing) - 1/6 X 10-9 bp
Paternity testing
Mutagenesis to investigate protein function
Quantitate differences in gene expression
Reverse transcription (RT)-PCR
Identify changes in expression of unknown genes
Differential display (DD)-PCR 
Forensic analysis at scene of crime
Industrial quality control
APPLICATIONS OF PCR
Requires the binding of short sequences of DNA (oligonucleotides/amplimers/primers) to complementary sequences flanking the desired target region
the action of an enzyme (DNA polymerase) to synthesise new exact copies of the target DNA.
HOW DOES PCR WORK?
5`
3`
5`
3`
5` 3`
3` 5`
ANNEALING
37°C - 65°C
EXTENSION
72°C
25-35 CYCLES
DENATURATION
93°C - 95°C
DENATURATION
93°C - 95°C
Denaturation; 93°C - 95°C
30 secs – 1min

Annealing; 37°C - 65°C
30 secs – 1min
depends on the melting temperature of duplex

Extension/Polymerisation; 72°C
1min (+ 30secs per 500bp DNA)
EACH PCR CYCLE HAS THREE STEPS
TYPICAL REACTION MIXTURE
25 or 50ls in a micro Eppendorf (0.5ml) tube
CYCLING PARAMETERS
Denaturation; 93°C - 95°C
30 secs – 1min
Annealing; 37°C - 65°C
30 secs – 1min depends on the duplex
Extension; 72°C
1min
(+ 30secs per 500bp DNA)
25-35 cycles
Final extension 2-10mins
PCR
Agarose gel electrophoresis
The final product
UV visualisation
3-4 hours
ALWAYS REMEMBER!
PCR is a highly sensitive technique – contamination with unwanted DNA can be a problem

Always run NEGATIVE controls

Include a positive control if appropriate
Use dedicated filtered tips and positive displacement pipettes
Dedicated areas?
Can use UV cabinets
The specific method should be ROBUST
Re-optimise for each set of primers
OPTIMISE PCR CONDITIONS AT THE OUTSET
X

Starting nucleic acid - DNA/RNA
Tissue, cells, blood, hair root, saliva, semen
Thermo-stable DNA polymerase
e.g. Taq polymerase
Oligonucleotides
Design them well!
Buffer
Tris-HCl (pH 7.6-8.0)
Mg2+
dNTPs (dATP, dCTP, dGTP, dTTP)
OPTIMISING PCR – THE REACTION COMPONENTS
Starting nucleic acid - DNA/RNA
Tissue, cells, blood, hair root, saliva, semen
Thermo-stable DNA polymerase
e.g. Taq polymerase
Oligonucleotides
Design them well!
Buffer
Tris-HCl (pH 7.6-8.0)
Mg2+
dNTPs (dATP, dCTP, dGTP, dTTP)
OPTIMISING PCR – THE REACTION COMPONENTS
Tissue, cells, blood, hair root, saliva, semen
Obtain the best starting material you can.
Some can contain inhibitors of PCR, so they must be removed e.g. Haem in blood
Good quality genomic DNA if possible
Blood – consider commercially available reagents Qiagen– expense?
Empirically determine the amount to add
THE RAW MATERIAL
Starting nucleic acid - DNA/RNA
Tissue, cells, blood, hair root, saliva, semen
Thermo-stable DNA polymerase
e.g. Taq polymerase
Oligonucleotides
Design them well!
Buffer
Tris-HCl (pH 7.6-8.0)
Mg2+
dNTPs (dATP, dCTP, dGTP, dTTP)
OPTIMISING PCR – THE REACTION COMPONENTS
Number of options available
Taq polymerase
Pfu polymerase
Tth polymerase
How big is the product?
100bp 40-50kb
What is end purpose of PCR?
Sequencing - mutation detection
Need high fidelity polymerase
integral 3’ 5` proofreading exonuclease activity
Cloning (TA cloning?)
CHOOSE YOUR POLYMERASE WITH CARE
TA CLONING OF PCR PRODUCTS REQUIRES As
Starting nucleic acid - DNA/RNA
Tissue, cells, blood, hair root, saliva, semen
Thermo-stable DNA polymerase
e.g. Taq polymerase

Oligonucleotides
Design them well!
Buffer
Tris-HCl (pH 7.6-8.0)
Mg2+
dNTPs (dATP, dCTP, dGTP, dTTP)
OPTIMISING PCR – THE REACTION COMPONENTS
Length ~ 18-30nt (21nt)
Base composition; 50 - 60% GC rich
pairs should have equivalent Tms

Tm = [(number of A+T residues) x 2 °C] + [(number of G+C residues) x 4 °C]
Initial use Tm–5°C
Avoid internal hairpin structures
no secondary structure
Avoid a T at the 3’ end
Avoid overlapping 3’ ends – will form primer dimers
Can modify 5’ ends to add restriction sites etc
PRIMER DESIGN IS VITAL
PRIMER DESIGN
Use specific programs
OLIGO
Medprobe

PRIMER
DESIGNER
Sci Ed software
Also available on the internet
http://www.hgmp.mrc.ac.uk/GenomeWeb/nuc-primer.html
Starting nucleic acid - DNA/RNA
Tissue, cells, blood, hair root, saliva, semen
Thermo-stable DNA polymerase
e.g.Taq polymerase
Oligonucleotides
Design them well!
Buffer
Tris-HCl (pH 7.6-8.0)
Mg2+
dNTPs (dATP, dCTP, dGTP, dTTP)
OPTIMISING PCR – THE REACTION COMPONENTS
TITRATE YOUR Mg2+ CONCENTRATION!
Normally, 1.5mM MgCl2 is optimal
Best supplied as separate tube
Always vortex thawed MgCl2
Mg2+ concentration will be affected by the amount of DNA, primers and nucleotides
USE MASTERMIXES WHERE POSSIBLE
Taken from -http://info.med.yale.edu/genetics/ward/tavi/PCR.html
“ALL BLOCKS AND TUBES ARE EQUAL BUT SOME ARE MORE EQUAL THAN OTHERS!”
G. Orwell (not!)
Taken from -http://info.med.yale.edu/genetics/ward/tavi/PCR.html
THE PERFECT RESULT
If not………………………troubleshoot
Qiagen PCR methods
ADDITIVES?
Depends on the PCR
Can be used where products are diffuse or absent
DMSO
Formamide
Glycerol

QIAGEN – Q
Stratagene - Perfect Match
http://taxonomy.zoology.gla.ac.uk/~rcruicks/additives.html
Many useful sites:
PCR Jump Station
http://www.highveld.com/pcr.html

http://www.protocol-online.net/molbio/
http://info.med.yale.edu/genetics/ward/tavi/PCR.html

Additives
http://taxonomy.zoology.gla.ac.uk/~rcruicks/additives.html
PCR ON THE NET