MadSci Network: Genetics

Re: How do you determine how much of a gene to sequence when genotyping?

Date: Fri Sep 14 01:52:09 2007
Posted By: Sebastien De Landtsheer, Undergraduate, Immunology, Laboratoire National de Santé
Area of science: Genetics
ID: 1184000447.Ge


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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