Sequencing SNPs basics - Can anyone explain? (Sep/21/2006 )
I'm thinking of sequencing some known SNPs. How do I go about finding primers and all this new technology for sequencing? Can I do a PCR with a 60 bp product and sequence that? Or just use those primers for sequencing? I'm looking for as cheap and high through put as possible as I have 47 SNPs I want to examine in 100 samples. Thanks
I would say sequencing is the most costly method for genotyping of 47 SNPs and 100 samples. A suitable high throughput method is the taqman genotyping analysis. You can check Applied Biosystems website for further info. They provide a software for you to design the probe and primers.
Other methods include RFLP which requires convenient restriction site at the SNP sites and may be costly, and allele specific PCR which is not very accurate.
Your PCR primers should work fine as sequencing primers. I wouldn't recommend having such small PCR fragments (~60 bp) because sequencing tends to be junk for the first 20-30 bases so would lose a lot of the sequence quality. I sequence fragments of approx 500bp using an ABI 3730. Im not sure if you have heard of Sequenom Inc. Worth a look if you have a large bundle of SNPs, very high throughput and highly accurate at genotyping.
you can use pyrosequencing.
I am not quite sure if it is possible but if you have DHPLC in your lab you could use it to see if your samples have the polimorfism or not and then you sequence the positives... but I agree with one of the post in here: 60 bp is to short and the first 20-30 bp are crap in the sequencing... maybe you shoul alongate your product..