The Polymerase Chain Reaction (PCR)

Topics　　• What is the Polymerase Chain Reaction?　　• History and (pre-history) of PCR　　• How PCR works　　• Optimising PCR　　• Fidelity, errors and cloning　　• PCR primer design　　• Applications of PCR

What is the Polymerase

Chain Reaction?　　• It’s a means of selectively amplifying a particular segment of DNA.　　• The segment 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, 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.

Gene Analysis Prior to PCR?　　• Southern blotting (1975) permitted rudimentary mapping of genes in unrelated individuals (RFLPs, insertions & deletions).　　• DNA sequencing (1978) required genes to first be cloned into plasmid or λ vectors.　　• Gene library construction and screening could take many months and libraries had to be made for each individual analysed.

The Invention of PCR　　• 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.