Wild-Type Blocking PCR to Detect Low-Frequency Somatic Mutations
筆記録
Wild-type blocking polymerase chain reaction, or WTB-PCR detects point mutations by selectively amplifying low-abundance mutant alleles while inhibiting the amplification of high-abundance wild-type alleles in DNA samples.
WTB-PCR utilizes locked nucleic acids, or LNA-containing single-stranded, modified oligonucleotide complementary to the sample's wild-type DNA strand that binds specifically to the complementary strand. LNA binding blocks the activity of DNA polymerase and inhibits the elongation of the wild-type template DNA.
To perform WTB-PCR, take a master mix containing dNTPs, LNAs, and target-specific forward and reverse primers in nuclease-free distilled water.
Add a thermostable DNA polymerase-containing solution to the tube, and pipette the mix into the well of a PCR plate. Add the genomic DNA sample containing wild-type and mutant DNA into the well, and begin PCR.
During the PCR, high temperature-mediated denaturation separates the double-stranded DNAs. The separated strands act as templates for the primers to anneal, while the LNA binds to its complementary wild-type DNA strand and forms the blocker-wild-type DNA hybrid. At a higher temperature, DNA polymerase adds dNTPs and extends the primer-DNA templates.
A higher melting temperature of the LNA-DNA hybrid than the DNA-DNA complex keeps it intact. The modification of LNA eventually blocks the DNA polymerase from extending the LNA-DNA hybrid, which inhibits its elongation.
Over several PCR cycles, LNA-mediated blocking increases the number of mutant-type amplicons relative to its wild-type variant in the sample, helping their selective detection.
