Real-time PCR Machine

  LC480, F. Hoffmann-La Roche Ltd, Basel, Switzerland.

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. So, if PCR is started with one double stranded DNA molecule (= one copy of a gene), there will be 1024 double strand DNA molecules at the end of 10 cycles. In other words, if we know how many (or how much) DNA molecules are present after 10 cycles, we can tell how many DNA molecules were present at the beginning of the PCR. That is, we can quantify the gene number. This requires Real time PCR machine in which progression of PCR can be monitored in real time using florescent dyes and CCD camera. Real time PCR has tremendous applications in gene expression studies and molecular diagnosis of genetic diseases and pathogens.