Variants of PCR - Pretreatments and Extensions

Pretreatments and Extensions

The basic PCR process can sometimes precede or follow another technique:

• RT-PCR (or Reverse Transcription PCR) is used to reverse-transcribe and amplify RNA to cDNA. PCR is preceded by a reaction using reverse transcriptase, an enzyme that converts RNA into cDNA. The two reactions may be combined in a tube, with the initial heating step of PCR being used to inactivate the transcriptase. The Tth polymerase (described below) has RT activity, and can carry out the entire reaction. RT-PCR is widely used in expression profiling, which detects the expression of a gene. It can also be used to obtain sequence of an RNA transcript, which may aid the determination of the transcription start and termination sites (by RACE-PCR) and facilitate mapping of the location of exons and introns in a gene sequence.

• Ligation-mediated PCR uses small DNA oligonucleotide 'linkers' (or adaptors) that are first ligated to fragments of the target DNA. PCR primers that anneal to the linker sequences are then used to amplify the target fragments. This method is deployed for DNA sequencing, genome walking, and DNA footprinting. A related technique is Amplified fragment length polymorphism, which generates diagnostic fragments of a genome.

• Methylation-specific PCR (MSP) is used to identify patterns of DNA methylation at cytosine-guanine (CpG) islands in genomic DNA. Target DNA is first treated with sodium bisulfite, which converts unmethylated cytosine bases to uracil, which is complementary to adenosine in PCR primers. Two amplifications are then carried out on the bisulfite-treated DNA: One primer set anneals to DNA with cytosines (corresponding to methylated cytosine), and the other set anneals to DNA with uracil (corresponding to unmethylated cytosine). MSP used in Q-PCR provides quantitative information about the methylation state of a given CpG island.