The #fastCOLDPCR is a faster adaptation of the CO amplification at Lower Denaturation temperature PCR (#COLDPCR) as the name implies. As it is fast, it has got its own demerits as well! For the basics of COLD PCR please see full COLD PCR.
Principle
Preferential amplification of mutant alleles with COLD-PCR can be achieved for most of the point mutations even without performing the intermediate #CrossHybridization step at 70C. Thus rapid PCR amplification performed at the #Tc instead of 94C/ 98C discriminates strongly towards the lower-Tm allele. If two alleles differing by a single base-pair, A:T instead of G:C, are present, the A-allele is enriched preferentially during cycling, presumably because the A:T amplicon has a slightly lower Tm than the G:C amplicon.
For mutation enrichment to occur, the full COLD PCR protocol requires the build-up of substantial PCR product to achieve efficient cross-hybridization, which restricts the enrichment to the late stages of PCR and lengthens the cycling time. In #fastCOLDPCR the cycle is the same as any 3-step PCR cycle with the Tc set as the denaturation temperature, so it results in higher enrichments than #fullCOLDPCR.
Protocol
FIGURE: #fastCOLDPCR general cycling conditions!
a. Mutant allele denaturation: The temperature is raised to the Tc (~80-90 C), to denature the mutant alleles preferentially over the wild-type sequences. Because critical denaturation is performed at every PCR cycle, the differential enrichment of mutation-containing alleles is compounded exponentially, and results in a large difference in overall amplification efficiency between mutant and wild-type alleles, at the end of the cycling.
d. Primer annealing: The temperature is reduced to appropriate Ta or generally 55 C to allow primers to bind and replicate the preferentially denatured sequences.
e. Extension: The temperature is raised to 72 C for the Taq to extend the 3' end of the bound primers and the cycle continues until your 'molecule of interest' enriches!
Do look for signature melt curve peaks!
Merits
1. It is a lot faster than #fullCOLDPCR and #iceCOLDPCR owing to omission of the hybridization step.
2. Fast COLD PCR has the highest enrichment potential for Tm-reducing mutations (~20 fold).
Demerits
1.Fast COLD PCR is unable to enrich both Tm-equivalent and Tm-increasing mutations.
2. In order to enrich for all possible mutations, including deletions/insertions, the fast COLD PCR program cannot be used.
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