I am trying to sequence mitochondrial DNA using DNA extracted via blood DNA extraction kit. I thought of using mitochondrial specific primers to amplify the area of interest. But certain mitochondrial regions are highly homologous to chromosome 5.
The primers which i designed are specific for mitochondrial regions but they are also specific to chromosome 5 (only 2 or 3 mismatches in one of the primer ) and they give similar PCR product size too.
I am wondering if these 2 or 3 mismatches will be sufficient to amplify only the mitochondrial DNA or will it amplify both chromosome 5 and mitochondrial DNA.
Hi Praveenan
since mtDNA and DNA are extracted at once, I'm not sure that your primers will ensure distinct amplification, maybe you'll have a mix of both amplifications even with a smaller amount with the mismatch one.
have you the possibility to test by sequencing the results?
maybe you could increase the specificity by increasing stringency by use of DMSO at 5%, increasing Tm by 2-3°c and decreasing amount of the mismatched primer in the mix?
all the best
fred
Hi Dr. Frederic,
Thank you so much for your answer, much appreciated.
I am planning to sequence the samples. Since my target is highly homologous with chromosome 5, i pretty sure i won't be able distinguish them both.
Maybe i will do something like Nested PCR and initially get the mitochondrial specific region then i will use these primers to sequence my area of interest. Any thoughts/suggestions on this?
Best regards,
Praveenan.
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