genotyping - how to determine a individual homozygous or heterozygous? (Apr/17/2008 )
Hi all,
Recently I 'd like o genotyping a gene of my interest, first now I have gotten several samples at hand, but I don't know how to determine the allele feature, homozygous or heterozygous they are 
. Coud some one give me a method or information to deal with this question?
Thanks in advance.
Andy
Your question is a bit confusing... What is it that you want to genotype? Is it a known SNP? Is it a known mutation? If you give us a little bit more information we might be able to help you.
A fast and easy way is to PCR your gene, the entire gene if it is small, or in fragments if it is large, and then sequence the PCR products. You will need good, clean PCR products for this. Heterozygous nucleotide sites will show two roughly equal peaks in the chromatogram, while homozygous sites will have a single peak. It is best to have a known homozygous control to distinguish heterozygosity from spurious signals generated during the sequencing reactions. If even a single site has two peaks, then you have two alleles (with a strict definition of an allele, disregarding whatever the protein sequence may be).
Recently I 'd like o genotyping a gene of my interest, first now I have gotten several samples at hand, but I don't know how to determine the allele feature, homozygous or heterozygous they are
. Coud some one give me a method or information to deal with this question?Thanks in advance.
Andy
you can make DETECTION OF DNA IN POLYACRYLAMIDE GELS. it's so useful method in our lab
It depends on the alleles you are looking at, SNPs are easily detected by sequencing, but can also sometimes be done by dHPLC. Small mutations dHPLC is good, or RFLP if you have time and the right RE's to design it. Larger mutations can be done by designing primers so that a mutant homozygous will give you a product of one size and the wild-type homozygous another and a hemizygous will have both (use 3 primers in the reaction, one in common to both reactions, and the others unique to the mutation or wild-type respectively)
Anyway, the first step is to sequence the mutation and see what you have. Once you have this information, we will probably be able to help a bit more.