Hi,
My plan is to design a taqman qPCR assay to detect multiple point mutations/ SNPs in a single reaction. I have designed serotype/variety specific qPCR assays before, but not to detect SNPs.
I was searching through literature, but couldn't found what I am exactly looking for. So, to explain things better, I am giving a bit more detail about what I am thinking -
Consider I have the following sequence:
ATG GTA CGA TGC
The TA and GC can have different type of point mutations. Like-
TA → AA/CA/TG/TC
GC → AC/GT/GA
At first, I was thinking about allele-specific probe. But I guess, it will not work here, due to the multiple combinations of SNPs. I found 8-9 different SNP combinations in literature already, which cannot be covered in a single qPCR reaction (as far I know, you can run 4/5 different dyes in a single qPCR, not more than that).
So, I am kind of stuck here and confused about how to proceed on.
Would really appreciate your input here.
Thanks.
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I have also tried few other thoughts, like -
1. Degenerate primer/probe: will not work, as qPCR cannot detect the SNP itself then and I have to use sequencing, which I am trying avoid.
2. qPCR-Melting curve analysis: possibly will not work, as it will be tough to multiplex and not really suitable if I want to run the reaction with clinical specimen.
Hello
Add to Milli suggest , look at the links here
Good Luck
http://www.idtdna.com/pages/decoded/decoded-articles/core-concepts/decoded/2016/08/24/multiplex-qpcr-how-to-get-started
http://dyes.gene-quantification.info/
Thank you Milli, for your answer. :)
I have actually considered designing probe with degenerate nucleotides before. However, I am afraid, degenerate primer/probe will not work in this case, as it cannot detect the SNP from the qPCR/PCR. I have to do the sequencing of PCR product to know about the SNP.
I also considered melting-curve analysis with qPCR. But, that will not work either, as it would be tough to multplex.
I have already designed a RFLP assay to detect the SNP. But, I need to make the process faster, like in a single qPCR/PCR.
Thank you Saleh, for your input.
I know the quite details about the first link (IDT).
But, I have some lacking about the very basic of the dyes. Second link will be more useful to me.
Thanks :)
I am not sure, whether the following article (in the link) can help you or not. We are using TAC card for the detection of drug resistance mutation and is working well.
Article Integrated Microfluidic Card with TaqMan Probes and High-Res...
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