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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
37°C - 65°C
25-35 CYCLES
DENATURATION EXTENSION
93°C - 95°C
72°C
EACH PCR CYCLE HAS THREE STEPS
• 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
3-4 hours
A A
PCR Taq - yes
product
T T Pfu - no
pGEM-T
pCR 2.1-TOPO
OPTIMISING PCR – THE REACTION COMPONENTS
• 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
Use specific
programs
OLIGO
Medprobe
PRIMER
DESIGNER
Sci Ed software
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
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