I am looking to do DNA sequencing for mutated genes BRCA1 , BRCA2 in lung cancer so i need best methods,to determine Mutations in these genes .by use PCR .Technique.can some one told me about that ??
Dear Mohammed Mhawish,
You can amplify the exons of these genes along while ensuring that the intron exon boundaries are covered and then get these PCR products sequenced to find mutations.
Are you looking for some mutations in particular, for a specific population? or will you be targeting the whole gene itself?
Hi,
The answer of Ameya is good in theory but for BRCA1 and 2 might be difficult to perform...
Too many isoforms, too many exons and no real hotspots...
So depending on your budget and what appliances are available you have different solutions...
if you only need to screen for mutation at a specific domain just go with the explanation Ameya gave you.
If you need to screen for all mutations the best way would certainly be using Next Gen sequencing with a custom enrichment of all BRCA1 and 2 exons... however it requires money, personal and infrastructure.
An easier method would be to design amplicons for all your exons, check all amplicons with a screening method (DGGE, HRM...) to find specific samples and amplicons which presented a change. And then sequence the amplicons you found altered to understand which alteration is present.
Hope my explanations were clear enough,
Best of luck,
As the above researchers told that you have to amplify the product you can go for SSCP. in sccp there is no need of sequencing. if you find any mutation then go for sequencing of that particular allele.
You can find systematic genetic evaluation steps for hereditary breast cancer here:
http://www.ncbi.nlm.nih.gov/books/NBK1247/
However Sir Ludovic Barault artfully provides information you need.
Hi ,
BRCA1 and BRCA2 all exons mutations had been studied and sequenced in breast cancer by my colleague in University of Baghdad , genetic engineering institute. so you can use this study to start yours.
thanks
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