2 replies to this topic

[Ameya P]

Hi all,

I am interested in determining a SNP responsible for a disease in humans, So, I decided to amplify the exon and then sequence it using my PCR primers. The good news is that all that worked, my sequencing results gave me the expected results for my positive sample.

What I would like to know is whether sequencing can help, if I want to determine carrier status in my sample. If a person were

a carrier, then the PCR product (ideally) should contain both the polymorphism and the wild type nucleotide in equal amounts. So, would a sequencing read tell me that both the bases are present in the person?

Thanks

[elpollodiablo]

gt_ameya, on 06 December 2010 - 10:53 PM, said:

Hi all,

I am interested in determining a SNP responsible for a disease in humans, So, I decided to amplify the exon and then sequence it using my PCR primers. The good news is that all that worked, my sequencing results gave me the expected results for my positive sample.

What I would like to know is whether sequencing can help, if I want to determine carrier status in my sample. If a person were

a carrier, then the PCR product (ideally) should contain both the polymorphism and the wild type nucleotide in equal amounts. So, would a sequencing read tell me that both the bases are present in the person?

Thanks

Yes, you'd see two peaks on top of each other if that person was heterozygous.

[Ameya P]

elpollodiablo, on 06 December 2010 - 11:59 PM, said:

Yes, you'd see two peaks on top of each other if that person was heterozygous.

Thanks for your reply.
If sequencing is so sensitive (the gold standard to detect nucleotide changes), why is it still not used for diagnostic purposes???? Why is it still used for confirmatory purposes alone?