Amplifying and analyzing the genome
PCR is performed in a thermal cycler machine and involves three phases:
- Melting: denaturing of the DNA duplex at a high temperature, which yields single stranded DNA.
- Annealing: Primers anneal to their single stranded target DNA sequences.
- Extension: DNA polymerase extends the primers by polymerizing dNTPs into the phosphate backbone, to build a complementary DNA strand.
These steps repeat until the DNA concentration is optimally high.
Quantitative real time PCR (qPCR) is a variant of PCR method where the amount of PCR product formed is detected after each cycle by utilizing either fluorescent dyes that intercalate between double stranded DNA or fluorescently-tagged oligonucleotide probes that change signal intensity as a result of amplification1. qPCR has advantages over traditional PCR, with respect to DNA quantification, because results can be obtained faster and with less variability due to sensitive fluorescent chemistry and elimination of post-PCR detection procedures.
The basic principles of PCR/qPCR have been adapted for experiments with varying start materials. Direct PCR amplifies DNA directly from sample tissue (plant, animal, human, blood) without prior DNA purification. Reverse Transcription PCR (RT-PCR) first transcribes complementary DNA (cDNA) from RNA starting material and subsequently amplifies it.
PCR can be used to detect the presence or absence of specific DNA sequences, amplify large amounts of DNA for downstream use, or mutagenize coding DNA, for further genetic manipulation, mutational analysis, sequencing, genotyping, or cloning.