I was considering about using a FAM-labelled Forward Primer, a GeneScan™
500 ROX™ Size Standard or GeneScan™ 400HD ROX™ Size Standard, and a PCR fragment size of about 200-300 bp? It really makes any difference the PCR fragment size picked? There is any criteria to be followed when choosing the PCR fragment size in order to dectect LOH? Which are the differences between a 6-FAM and 5-FAM dye?
In order to provide all the information, I am going to use the peak scanner software.
Any suggestion is welcome
Hi,
We use 500 LiZ size standard in a 3730XL DNA analyser. Fragment size-200-250 is good enough and we use 5-FAM dye. I think ROX is also fine.
Thank You so much for Your answer Kalpita ,
I would like to ask You something else. One of my DNA samples or patients is homozygous for all the analyzed SNPs within my gene of interest and I was wondering if it’s just homozygosity (2 identical copies or forms , one inherited from each parent) or LOH (loss of the entire gene or part of it). For this reason I want to perform this technique. My question is whether I could distinguish between homozygosity and LOH? Because, if the case of homozygosity the peaks or alleles would be overlapping each other (theoretically they are supposed to be the same size) and if I have the case of LOH I would just see one allele of the same size as the previous ones. As in both cases they should have the same allele size,Could I distinguish between homozygosity and LOH?
Perhaps the intensity and the height of the peaks it’s much higher in the case of having homozygosity than in the case of having LOH? I am using the Peak Scanner Software in order to analyze the data.
Any suggestion?
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