MadSci Network: Genetics |
Hello, How much you need to sequence depends of what you want to know. If you assume you have a gene with defined variants, then you need to screen for the positions in the gene that are most likely associated with one or another variant. In other words, you define genotypes and find within the sequence of the gene the SNPs or indels that are the signature of each genotype (the most genotype-specific portion). You need to obtain a different pattern for each genotype. What I would suggest is to have a more systematic approach. Check what human SNPs are known for your gene. If there are many, you can assume you would not find more in your cohort and stop with sequencing. If you think you could discover more, then sequence the whole gene for a part of your cohort. Once you have your set of SNPs, screen them, but not by sequencing, by fragment analysis : you interrogate one position at a time in the genome. This is much faster and cheaper... as most of the sequence of your gene is probably conserved, you do not need this information. Check at Applied Biosystems for their "SNaPshot" kit. You can also use double-dye oligos (so-called TaqMan assay) in a real-time PCR format... About DNA extraction, many kits exist for saliva samples. The companies I have here are mostly european ones, but check Qiagen for their "QiaAmp DNA mini/micro" kits to extract DNA from buccal swabs. I would not recommend to make your own method like phenol extraction or alcohol precipitation. About the price indications, you can assume that the price per sample is : - DNA extraction : 3 - 4 $ - PCR : 1 - 2 $ - sequencing 1kb : +- 5 $ - 10 SNP screening : +- 5 $ - TaqMan assay (1 SNP) : 2 - 3 $ Hope that helps...
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