Hi. I am planning a screening study for apt1a3 gene in a family with clinical manifestations. The protein is about 1013 amino acids long, so its coding part is 3042 nucleotides. Numerous mutations have been reported in its coding sequences. It seems that designing ~5 -6 pairs of primers for Sanger sequencing of cDNA might be a cost effective alternate to WES. I am not a pathologist, so need an expert advice whether or not different mutations affect mRNA synthesis and its level. Can the mutant forms be expected to express sufficiently high for capturing mutations using mRNA and cDNA, or we will be compromising on the quality of our findings by avoiding WES? Thanks.
Hey,
I hope I got your question right.
Sure you can use some sort of sequencing like Sanger to detect your mutations, but if you would like to study the effect of the mutations you should probably go for a RNA sequencing. Also this would give you at least a hint on the expression of the gene itself when mutated. Always keep in mind, that there is a strong hint of protein expression when the mRNA amount is going up, but its never safe to assume the protein amount!
WES would, in my opinion, only make sense if you are searching for other mutations in other genes to see correlations, that have previously not been shown.
Hope this helps a bit, feel free to ask any further questions.
Regards,
André
You typically don't do exome sequencing for a single gene. You can use targeted amplicon sequencing with custom DNA panels rather than using exome sequencing. If you do go end up going with exome sequencing, remember that there will be a bioinformatics burden whereas with sanger this is non-existant.
As you are saying, you want to detect mutations in a single gene of around 3042 bp long. In that case, you can amplify your gene part by part using specific primers. Then go for the Sanger sequencing. If your gene contain any SNP, you can digest the amplicon with specific Restriction enzyme followed by Sanger sequencing. Hope this will be more cost effective.
If you can get the cDNA sequence you can get your functional sequence and as like as your mRNA sequence. No need to do the exom sequence also. But if you want to do the SNP then exom is fine because you have to get the proper position.
Exom sequencing, is a genomic technique for sequencing all of the protein-coding regions of genes in a genome.
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