PCR

Basics

PCR amplifies DNA fragments specified by a set of primers

These primers are designed to match either end of a piece of DNA, typically in a gene or region of interest. Taq polymerase is the enzyme which replicates the DNA starting at the location where the primers anneal, adding bases to match the template strand. The reaction is performed in a buffer containing Mg and other chemicals which aid the Taq enzyme

Temperature cycling
Once the reaction is made up, it is put in a thermal cycler. The reaction is heated to denature the two DNA strands. It is cooled to a temperature where the primers anneal to their matching locations on the DNA. This annealing temp determines the specificity of the reaction. The reaction is then warmed to a temperature where the Taq efficiently extends the DNA completing the two strands. The extension time is determined by the DNA lengths : longer DNA requires more extension time.

Multiple cycles are run (25-35) to amplify the DNA. Ideally each cycle doubles the amount of DNA present.

Components of a PCR reaction

Buffer containing Mg (some people add the Mg separately)

dNTP = dATP, dCTP, dGTP and dTTP which are incorporated into the DNA as it is replicated

forward and reverse primers

DNA

Water to bring things to volume

Taq polymerase

Polymerases

There are a variety of polymerases available. These differ in the fidelity of nucleotide incorporation (how many errors the the polymerase introduces) during PCR. We use a number of polymerases. For routine genotyping we use either Promega Taq or homemade lab taq. For higher accuracy amplification, we use Dynazyme EXT which is a mixture of fast (low fidelity) and proofreading (high fidelity) polymerases. For much of the C.elegans work and for real time PCR, Amplitaq gold is used. This polymerase is coupled to an enzyme which keeps the polymerase inactive until a 10 min soak at 90 C activates the polymerase. This hot start is thought to decrease the amount of background amplification of primers which misanneal during the initial heat up.

Master mixes

Make up a master mix for the number of reactions which are using the same primers containing everything but the DNA. Usually you need 10% extra to make up for pipetting losses. Aliquot the master mix to each tube and then add the DNA's.

Include one reaction which contains no DNA (negative control to prove there is no contamination in the reagents which might amplify).

Include one reaction which contains DNA known to amplify (positive control).

PCR machine

Everyone keeps their own programs on the PCR machine. Use a program which first denatures DNA, then runs 30-35 cycles of PCR and then cools to 4 C. This is a method which links other programs. The denature step is 95 C for 1 min or so. Check the PCR program to change annealing temperature or times. Typical PCR conditions are: 95 C 20 s / 53 C 30 s / 72 C 1 min repeated for 35 cycles. Run the method program which calls the PCR program
Once the method is running, push the MORE button to find out how long the reaction will take.