Hello, I am new in molecular, I have a doubt with the PCR that I am doing, to amplify the fragment of a gene in patients with mutation, why do you have heterozygotes if you are amplifying the coding strand and not both strands, ie, only oligos are used for the coding strand.
In some only one band is seen, but I would expect that for the case of patients with many trinucleotide repeats (the mutation is due to expansion of a trinucleotide) only one very large band would be seen instead of two, and for the case of patients without the mutation only one band but with much fewer base pairs.
In PCR you are amplifying both strands of DNA, not just the coding strand. One primer will be from each strand. So with genomic DNA from diploid organisms you will observe the heterozygosity if it is present.
In analysis of long triplet expansions the length and secondary structure problems make pcr very difficult in detecting rtriplet repeat expansions. The pcr product often needs blotting and probing or very specific conditions to amplify so often in pcr only the normal allele can show. Do not forget also that one band can be generated from 2 normal alleles as well as 1 normal small allele and one large invisible allele.
Hi Jude
in diploid organisms, running PCR for an heterozygous target just means that you have amplification of part of both chromosomes, not due to the double helix state of the genomic template.
fred
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