Hi everyone,
I have a question regarding sequencing SNPs. I have currently creating two SNPs on one gene and these SNPs are about 8kb apart from each other on two different exons. I would like to have these SNPs on separate Alleles. As I am isolating heterozygous for each SNP I will not be able to find out if these SNPs are in the separate allele just by sequencing beside the fact that they are also 8kb apart. As the gene is not expressed in Stem Cells and the gene editing is being done in Stem Cells I would not be able to isolate the RNA, make cDNA and do the TOPO cloning for the `1kb cDNA. It would be great if anybody has any thoughts on this.
Thank you,
Mina
If the SNP has been documented in a haplotype database like HapMap, you may be able to determine if they are part of the same allele or occur separately.
If that's not available, I wonder if you could use a FISH or NanoString based strategy to probe whether these SNPs are different chromosomes or not
Dear Mina,
Please read for the PCR technique of allele-specific amplifications in our articles:
Shirak A., Golik M., Lee B-Y, Howe A.E., Kocher T.D., Hulata G., Ron M. and Seroussi E. (2008). Copy number variation of lipocalin family genes for male-specific proteins in tilapia and its association with gender. Heredity 101: 405-415.
Curzon A. Y., Shirak A., Dor L., Zak T., Perelberg A., Seroussi E. & M. Ron. A duplication of the Anti-Müllerian hormone gene is associated with genetic sex determination of different Oreochromis niloticus strains. Heredity (2020)
Best Regards
Andrey
if you're sure to get both SNP in the same sample and at heterozygous state, just make a long PCR with the amplicon containing the SNP and sequence each strand beginning, you'll see if the signal is clear.
fred
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