Discuss about COLD-PCR - (Oct/30/2011 )
i was searching online for some PCR techniques and came across a technique known as COLD-PCR.
i was just wondering if any of you guys had done it and how was your results?
Since my project has something to do with genotyping, i feel it might be a good technique to use.
i would really like to know how was it for you guys who had done it before starting it.
please feel free to share and discuss!
I've seen a presentation of a person who did it and it looked impressive.
But the main advantage as I understand is in the preferential amplification of minority variants. Thus great for multiplying low-proportion mutants in tumor samples or so. But If I'm not mistaken, downstream aplication must be used to analyse the result, restriction or sequencing, (or real-time with two probes) it's just enrichment.
For a simple genotyping wt 100%, mut 100%, heterozygote (wt 50%, mut 50%) I wouldn't use it. We genotype our mice with HRM.
What are you going to genotype?
Hi! thanks for the reply!
i'm also using HRM to genotype BRAF V600E mutation using patient samples.
Just asking, does ur genotyping using HRM gives a good and distinct results? Mine doesn't and i can't figure out what went wrong.
Hence, me and my mentor decided to try out COLD-PCR to see whether it works better or not.
So you have a somatic mutation, that's present in a small portion of cells. That is a good reason to use COLD-PCR as the minor allele would be enriched. But you need to detect it somehow.
HRM on variable portion (mainly small) of mutated cells wouldn't be robust enough, I think, but this may change if you do HRM after COLD-PCR, as there would be more of the mutant. But still as in any other HRM you wouldn't be sure if the difference you see is really your mutation, or some other sequence variation. Can you do a restriction?