Polymerase Chain Reaction (PCR)

What is PCR?:

Use of DNA polymerase to selectively amplify a segment of DNA from a much larger sample.

Xeroxing DNA, start with one page and get many.

Examples of what PCR is used for:

Forensics, DNA typing from very small samples

Clinical diagnostics e.g. detection of HIV, detection of some microbial infections, detection of whether an individual carries a mutation predisposing them to some sort of cancer or genetic disease.

Research, mapping and sequencing of genomes, cloning, basic research

Who invented PCR?• PCR was invented in 1985 by Kary

Mullis. Advances offered include• The use of thermostable DNA

polymerases. The first thermostable DNA polymerase to be used was isolated from the bacterium Thermus aquaticus.

• Thermal cyclers or PCR machines. • These two developments led to the

automation of PCR. Thermus aquaticus

How does the PCR technique work?• PCR is an in vitro technique for the amplification of a region

of DNA which lies between two regions of known sequence. • PCR amplification is achieved by using oligonucleotide

primers. – These are typically short, single stranded oligonucleotides which are

complementary to the outer regions of known sequence.

• The oligonucleotides serve as primers for DNA polymerase and the denatured strands of the large DNA fragment serves as the template. – This results in the synthesis of new DNA strands which are

complementary to the parent template strands. – These new strands have defined 5' ends (the 5' ends of the

oligonucleotide primers)

• The oligonucleotide directed synthesis of daughter DNA strands can be repeated if the new duplex is denatured (by heating) and additional primers are allowed to anneal (by cooling to an appropriate temperature).

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

The steps of the PCR reaction: • Template denaturation • Primer annealing • Primer extension • These steps comprise a single "cycle" in

the PCR amplification methodology. After each cycle the newly synthesized DNA strands can serve as templates in the next cycle.

Template denaturation: Making the DNA single stranded

• The initial denaturation of template is accomplished at 95-100 °C. – Supercoiled plasmids are tougher to melt and may

require boiling for several minutes, or may be initially denatured by using base (NaOH, followed by pH neutralization).

• Denaturation during the PCR experiment (i.e. second cycle onward) is usually accomplished at temperatures of 92-95 °C (usually empirically determined).

Primer annealing temperature: Getting the primer to stick to the template DNA

• Primer annealing temperature is an important parameter in the success of the PCR experiment.

• The annealing temperature is characteristic for each oligonucleotide: – it is a function of the length and base composition of

the primer as well as the ionic strength of the reaction buffer.

– Estimates of the annealing temperature can be calculated in several different ways.

– These calculated annealing temperatures are a starting point for the PCR experiment, but ideal annealing temperatures are determined empirically.

Primer extension: Building the new strand of DNA

• Primer extension is usually performed at 72 °C, or the optimum temperature of the DNA polymerase.

• The length of time of the primer extension steps can be increased if the region of DNA to be amplified is long, however, for the majority of PCR experiments an extension time of 2 minutes is sufficient to get complete extension.

Number of cycles• The number of cycles is usually between

25 and 35. – More cycles mean a greater yield of product. – However, with increasing number of cycles

the greater the probability of generating various artifacts (e.g. mispriming products).

– It is unusual to find procedures which have more than 40 cycles because rxn is no longer exponential