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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 EXTENSION
93°C - 95°C
72°C
EACH PCR CYCLE HAS THREE STEPS

• 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)
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 0.5µl 1 unit


(2U/µl)

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 Taq - yes
product
T T Pfu - no

pGEM-T
pCR 2.1-TOPO
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] + [(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

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