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how to distinguish one base mismatch by real time PCR (SYBR green)

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#1 veer2



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Posted 07 June 2009 - 11:54 PM

Hello, I am working on miRNA real time PCR detection by SYBR green methods. I meet some problem on real time PCR:
1. how to distinugish the miRNA family which has only one base mismatch, for example let-7 family. I have designed two individual primer for let-7a and let-7d, but both primer could detect let-7a and d. Should I increase the anealling temerature or any other methods? how to improve the specificy up to base pair mismatch by real time PCR

2. I heard the two steps real time pcr (anealing and exension at 60C together) and three steps real time PCR (60C anealing and extension at 72C), which one is better specificity? or what is the difference between above two program?

Thanks in advance

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