4 replies to this topic

[tonyqcc]

Hi guys.

I am having a small problem here...

My project is to extract and analyse some SNP (single nucleotide polymorphism) of two genes, namely BDNF (brain-derived neurotrophic factor) and ApoE (apolipoprotein E) of human subjects. I am kinda lost on the feasible (yet not too expensive) ways to do so. For BDNF, the whole gene is 247-AA long & 26kDa, located at 11p13; while it is 317-AA & 36kDa, located at 19q13.2 for ApoE. My samples are human whole blood samples.

Is it possible if i would like to selectively cut and sequence only the 2 specific genes via PCR, and send the whole gene for sequencing (since BDNF is only app. 750bp, and ApoE is only app. 950bp long)? What types of PCR should i use?

Thanks

Regards,
Tony.

[pcrman]

If the coding region is short as in the case of your two genes, yes you can amplify the whole gene and sequence the product. But if you have many sample to genotype, that will be costly. Since there are not many SNPs on a gene, the cheapest way is to either do allele specific PCR or do regular PCR followed by restriction digestion.

[tonyqcc]

dear pcrman.

thanks for your explanations. however, for BDNF alone, i checked and found out that there are more than 700 SNPs reported for this gene (though only around 20 on the active gene site if im not mistaken). So instead of doing 6 times of genotyping for the particular 6 SNPs i want to look into, can i just proceed and do one genotyping for the whole gene sequencing for that particular gene? As per mentioned earlier the active gene site for BDNF is 247AA-long.

thanks.

[phage434]

How many samples do you have? Next gen sequencing is currently bar coding samples up to at least 384 samples/lane, and might be a cost effective way to sequence your PCR products.

[DrLeo]

I used to screen for SNPs in some genes. And share some experience, you have 2 choices:
1.PCR to get whole gene and send with various primers pairs which will be used for sequencing each target SNP. (1 pair of primer for 1 or 2 SNPs which are located near to each other)
2.Design different primers and use PCR to get separate sequences which contain one or more SNPs for each sequence (if they are located near to each other).

You should consider that, the sequencing process cannot be performed well if you have a long sequence and especially if that sequence contains "poly T"
Therefore, you should divide the whole gene into short fragments which contains the target SNPs (by PCR directly or by various primer pairs in sequencing)
Hope this can help you.