Learning Outcomes

:
• At the end of this lecture, students will be able to: • define PCR • Describe components required for a PCR reaction and their function • Elaborate basics of PCR cycling • Tell how to check on PCR product • Explain factors affecting PCR reaction • Explain uses of PCR and their application in life sciences

What is polymerase chain reaction (PCR)?
 It is a techniques used selectively to amplify a particular segment of DNA  The segment may represent a small part of a large and complex mixtures of DNAs eg. Region A of the DNA molecule shown below

 Acts like a molecular photocopier

The Invention of PCR
• • • • Invented by Kary Mullis in 1983 First published in 1985 Awarded Nobel Prize for Chemistry in 1993 Basic principle of replication a piece of DNA using 2 primers was first described by Gobind Khorana(1971) J. Mol. Biol. 56, 341-346 • Progress was limited by primer synthesis and polymerase purification issues • Mullis properly exploited amplification

PCR Thermal cycler
-Available commercially
-First introduced in 1986 - Reactions can be made in individual tubes or 96 well plates

What is required for PCR?
(a) DNA polymerase -heat tolerant -length fragment -Error rate -Ends of fragments-blunt or 3’-A overhang - first thermostable enzyme, isolated from the bacterium Thermus aquaticus, was called Taq polymerase - Taq polymerase lacks 3'-5' exonuclease activity (proofreading) and so introduces errors into the newly made strands - Its half-life at 95°C is 35-40 minutes - Pfu, Vent, Deep Vent etc. which posess 3' - 5' exonuclease activity (proofreading) have been isolated and are now commercially available

(b) -

Template Usually DNA, can be RNA (RT-PCR) Contains sequence to be amplified can come from a variety of sources (genomic DNA, other cloned DNA fragments or libraries of DNA fragments) (c) Primers Must flank the region to be amplified Must anneal to opposite strands of DNA primers can be constructed in the lab, or purchased from commercial suppliers • The length (and sequence composition) of the oligonucleotide primer also determines the temperature at which we anneal the denatured template and primers to form specific hybrids which can be extended by the DNA polymerase. • Tm = 2oC ( A + T) + 4 oC ( G + C) Usually primer length is 18-30 bp, single stranded most critical component of the PCR reaction -determine the specificity Higher GC content is better (>50% GC) Primers should be GC rich at 3’ end Not self complementary No primer dimers

What is required for PCR?

What is required for PCR?
(d) Buffer - this generally contains the buffering agents necessary to regulate the pH and salt concentration of the reaction mixture - Supplied with the DNA polymerase - Depends on the specific enzyme used (e)MgCl2 - DNA polymerases require magnesium - Also contributes to the specificity of primer binding - The optimum MgCl2 must be determined (1-5 mM)

What is required for PCR?
(f) dNTPs (deoxynucleotides) - dATP,dTTP, dGTP and dCTP - Required for DNA synthesis - Usually 0.2 mM (higher concentration can increase the error rate of Taq polymerase)

PCR cycle
The basic reaction consists of three steps:  Template denaturation - double-stranded DNA template is denatured by heating to 95oC/94oC  Annealing - The single stranded template is allowed to hybridize to short single stranded oligonucleotide primers - Annealing temp. affects specificity of binding, at too high a temperature primers will remain dissociated, at too low a temp. there will be mismatch hybrid. - Determination of Tm ( melting temperature) for template/primer hybrid can be calculated using the formula; Tm = (4x [G + C]) + (2 x [A + T]) oC  Extension - These oligonucleotides are then recognized by DNA polymerase and the strands are replicated. Extension : ~ 72oC – 74oC

Amplifications are carried out in cycles. Denature, anneal and extend-repeat the cycle 30-35 times

Initial denaturation Temperature

Time

PCR cycle
• At the end of the cycle, we have doubled the number of DNA strands corresponding to the target sequences downstream of the specific primers • The doubling of the number of DNA strands corresponding to the target sequences allows us to estimate the amplification associated with each cycle using the formula Amplification = 2n where n=number of cycles

Has it worked?
• Check PCR product by agarose gel electrophoresis • Is the product the size you expected? • Is there more than one band  if yes – requires optimisation

PCR and product analysis

Preparing PCR reaction mix

PCR

Loading of PCR products

Verification of PCR product on agarose gel

Agarose gel electrophoresis

How to optimise PCR reaction?
• • • • Annealing temperature of the primers The concentration of Mg2+ in the reaction The extension time, denaturation time Amount of template

How big a target DNA to be amplified?
• Usually in the size range of 100-1500 bp • Longer targets are amplifiable > 25 kb • Requires modified reaction buffer, cocktails of polymerases and longer extension times • Limited by the intergrity of the starting target

How powerful is PCR?
• PCR can amplify an usable amt of DNA in ~ 2 hours • The template need not be highly purified-a boiled bacterial colony • The PCR product can be digested with restriction enzymes, cloned or sequenced • PCR can amplify a single DNA molecule

Studying PCR products
• agarose gel electrophoresis • direct sequencing analysis • cloning PCR products – 3’OH ends of PCR products have extra adenosine overhang, to overcome – remove with exonuclease, clone into vector with T overhang, design primers that contain restriction sites

Applications of PCR
• To study minute quantities of DNA
– forensic analysis – genetic fingerprinting – archaeology and palaeontology (preserved and fossilised material)
• Clinical diagnosis
• • type mutations rapidly – RFLP diagnosis of pathogenic diseases

Applications of PCR
• Quantification of mRNA
– RT-PCR – Measure relative amounts of mRNA in tissues

Compare different genomes
– RAPD – random amplified polymorphic DNA analysis) – Phylogenetics

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