Pcr

POLYMERASE CHAIN REACTION

The Invention of PCR • A 'licence' to do molecular biology • A key central technique that has revolutionised molecular and consequently cell biology • Invented by Kary Mullis in 1983. • First published account appeared in 1985. • Awarded Nobel Prize for Chemistry in 1993.

Did He Really Invent PCR? • The basic principle of replicating a piece of DNA using two primers had already been described by Gobind Khorana in 1971: – Kleppe et al. (1971) J. Mol. Biol. 56, 341-346.

• Progress was limited by primer synthesis and polymerase purification issues. • Mullis properly exploited amplification.

What is the Polymerase Chain Reaction? • 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 • The defined sequence may represent a small part of a large and complex mixture of DNAs: e.g. a specific exon of a human gene. • It can be thought of as a molecular photocopier.

A Molecular Photocopier • A photocopier capable of duplicating a part of a sentence: • “The next day was quite a different day. Instead of being hot and sunny, it was cool and misty. Pooh didn’t mind for himself, but when he thought of all the honey the bees wouldn’t be making, making a cold misty day always made him feel sorry for them.” A.A. Milne, 1928.

• The words in blue must be unique for the copier to locate the correct piece of text.

How Powerful is PCR? • PCR can amplify a usable amount of DNA (visible by gel electrophoresis) in ~2 hours. • The template DNA need not be highly purified — a boiled bacterial colony. • The PCR product can be digested with restriction enzymes, sequenced or cloned. • PCR can amplify a single DNA molecule, e.g. from a single sperm.

How Powerful is PCR? • PCR can amplify a usable amount of DNA (visible by gel electrophoresis) in ~2 hours. • The template DNA need not be highly purified — a boiled bacterial colony. • The PCR product can be digested with restriction enzymes, sequenced or cloned. • PCR can amplify a single DNA molecule, e.g. from a single sperm.

The Basics of PCR Cycling • 30–35 cycles each comprising: – denaturation (95°C), 30 sec. – annealing (55–60°C), 30 sec. – extension (72°C), time depends on product size.

DENATURATION 93°C - 95°C

ANNEALING 37°C - 65°C

25-35 CYCLES DENATURATION 93°C - 95°C

EXTENSION 72°C

EACH PCR CYCLE HAS THREE STEPS • Denaturation; 30 secs – 1min

93°C - 95°C

• Annealing; 37°C - 65°C 30 secs – 1min depends on the melting temperature of duplex • Extension/Polymerisation; 72°C 1min (+ 30secs per 500bp DNA)

TYPICAL REACTION MIXTURE 25 or 50µls in a micro Eppendorf (0.5ml) tube COMPONENT

VOLUME

Final Concentration

10 X PCR Buffer

5µl

1X

10 X dNTPs (2mM)

5µl

200µM

Forward primer

(10pmols/µl)

5µl

1µM (50pmols/50µl)

Reverse primer

(10pmols/µl)

5µl

1µM (50pmols/50µl)

Genomic DNA template

2µl

1µg

Thermostable polymerase (2U/µl)

0.5µl

1 unit

H2O (to 50µl Final volume)

27.5µl

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

3-4 hours

The final product

UV visualisation

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

How many copies? • No target products are made until the third cycle. • The accumulation is not strictly a doubling at each cycle in the early phase. • At 30 cycles there are 1,073,741,764 target copies (~1× 109). • The final number of copies of the target sequence is expressed by the formula (2n-2n)x n- no of cycles. 2n – first product obtained after cycle 1 and second products obtained after cylce 2 with undefined length. x- no. of copies of the original template. • There are also 60 other DNA copies.

How many cycles? • Increasing the cycle number above ~35 has little positive effect. • The plateau occurs when: – The reagents are depleted – The products re-anneal – The polymerase is damaged

• Unwanted products accumulate.

• http://www.dnalc.org/ddnalc/resources/pcr. html

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 • 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)

THE RAW MATERIAL 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

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)

CHOOSE YOUR POLYMERASE WITH CARE 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?)

TA CLONING OF PCR PRODUCTS REQUIRES As A

A PCR product

Taq - yes T

T

pGEM-T pCR 2.1-TOPO

Pfu -

no

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)

PRIMER DESIGN IS VITAL •Length ~ 18-30nt (21nt) •Base composition; 50 - 60% GC rich pairs should have equivalent Tms Tm = [(number of A+T residues) x 2 °C] + residues) x 4 °C]

[(number of G+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 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

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)

TITRATE YOUR Mg2+ CONCENTRATION! 1

1.5

2

2.5

3

3.5

4 mM

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

Qiagen PCR methods If not………………………troubleshoot

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

PCR ON THE NET 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