Sometimes two genes contain high Homology[95~98%],it is difficult for Real Time PCR (SYBR Green method)to design primers,and it seems not possibile to distinguish the Major gene .Can somebody give me some advice to deal with it?
You are correct, it is very difficult to distinguish genes of similar homology by SYBR Green methodology. There are a couple of solutions to this problem though, as many people use real-time RT-PCR to distinguish SNP's, which are only 1 base pair change.
1) Your best option is to purchase a TaqMan primer/probe set (used exclusively for SNP analysis). These add an extra level of specificity to your product by introducing a small probe between the primers that is identical to the isoform you are interested in. You can also make your own primer probe sets as well, if you want to chose the exact region of interest that is different to exploit for your PCR amplification. ABI makes the best primer/probes in my opinion, and they are very helpful in guiding you through the process of choosing the correct sets for your needs (http://www.invitrogen.com/site/us/en/home/Products-and-Services/Product-Types/Primers-Oligos-Nucleotides/applied-biosystems-custom-primers-probes.html)
2) The other option, though trickier, is to create SYBR green primers that bind in areas of your genes that are different. This requires some quality control though to make sure you are not amplifying both targets. This type of control could include dissociation curves, agarose gels for product size validation, and sequencing of your products. I have done this before, but it takes a little work in the start-up to be confident you are amplifying what you think you are.
Good luck with your experiments!
~Adam
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