Gradient PCR Machines

Master Cycler, Eppendorf, New York, USA

Veriti , Applied Biosystem, USA

Polymerase chain reaction (PCR) is a method that allows exponential amplification of short DNA sequences. It involves the use of 2 primers, each about 20 nucleotides in length that are complementary to a defined sequence on each of the two strands of the DNA to be amplified. First, 1 double stranded is denatured to 2 single strand DNAs. The primers bind to the single strand DNAs and the DNA polymerase makes a complementary strand to convert the single strand DNA to double strand DNA. Now 1 double strand DNA has becomes 2. This completes one cycle. In the next cycle, the 2 double strand DNAs are denatured to 4 single strand DNAs and all the 4 of them are converted to double strand DNAs. Likewise, in 30 cycles that takes about 2 hours, 1073741824(=2³⁰) double strand DNA molecules can be from single DNA molecules.

For doing PCR, the test tube containing the reaction components are kept at 95C for 1 min for denaturation of the template DNA, at variable temperatures for 1 min for annealing of the primers, and at 72C for 2 min for new strand synthesis. The annealing temperature is variable depending on the primers used for the particular PCR and each PCR would require empirical standardization of this temperature. In normal PCR machine, only one annealing temperature can be used at a time. So if 8 temperatures are to be tried the PCR should be done 8 times!. This will not only take time but can also introduce human errors between the runs. Using gradient PCR machine, different reactions with different annealing temperatures can be performed at one time and the conditions can be optimized quickly and reliably.