COLD-PCR for DNA variation detection (CAT#: STEM-MB-0201-WXH)

The principle of COLD-PCR is based on the obs ervation that there is a critical denaturation temperature ( Tc ) for each DNA sequence that is lower than its melting temperature ( Tm ). PCR amplifi cation efficiency for a DNA sequence drops abruptly if the denaturation temperature is set below its Tc. Studies have shown that PCR reaction yields no detectable amplification product when PCR denaturation temperature is set to below the Tc.
The COLD-PCR method takes advantage of this characteris-tic of PCR amplifi cation to selectively enrich the minority alleles differing by one or more nucleotides at any position of a given sequence. In COLD-PCR, an intermediate annealing temperature is used during PCR cycling to allow cross-hybridization of mutant and wild-type alleles; heteroduplexes, which melt at lower temperatures than homo-duplexes, are then selectively denatured and amplifi ed at Tc, while homo-duplexes remain double stranded and do not amplify efficiently. By fixing the denaturation temperature at Tc, mutations at any position along the sequence are enriched during COLD-PCR amplifi cation.

• DNA variation detection.
• Detection of mutations in oncology specimens, especially in heterogeneous tumours as well as bodily fluids.
• Assessment of residual disease after surgery or chemotherapy.
• Disease staging.
• Molecular profiling for prognosis or tailoring therapy to individual patients.
• Prenatal diagnosis of hereditary diseases.

1.Denaturation stage. DNA is denatured at a high temperature – usually 94 °C (201 °F).
2.Intermediate annealing stage. Set an intermediate annealing temperature that allows hybridization of mutant and wildtype allele DNA to one another.
3.Melting stage. These heteroduplexes will more readily melt at lower temperatures. Hence they are selectively denatured at the Tc.
4.Primer annealing stage. The homo-duplex DNA will preferentially remain double stranded and not be available for primer annealing.
5.Extension stage. Since the heteroduplex DNA is used as template, a larger proportion of minor variant DNA will be amplified and be available for subsequent rounds of PCR.