Hi, I am just wandering if it is possible to do nested PCR with the same primer used primarily? would appreciate suggestion on this matter.
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Generally speaking, it is absolutely not recommended to do nested PCR with the same primers. However, sometimes one doesn't have a choice... In the end it will depend on what your purpose is, what limitations you're facing and what alternatives you may have. But with the limited information you give, it is difficult to give you a better answer. Finally, my personal opinion is that nested PCR should be banned as much as possible. If you are planning on using nested PCR for detection purposes, I'd recommend using real time PCR instead (i.e. qPCR, not to confuse with RT-PCR which should only stand for reverse transcription PCR). |
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Hi, sorry, I'm a bit confused: Nested PCR actually implies a different set (within the first amplicon) of primers. Your question is a bit contradictory. Please explain! Wo |
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Hi, Thanks for your reply. I am very sorry that I confused you all. Actually, I am looking for splice variants and I am following a paper and their protocol. They did nested PCR after the first PCR but they haven't mentioned about the nested primers so I was trying to know if it was possible with the same primers. I hope I have made your confusion clear... So, in this case, how do I design nested primers (inner primers)? Thanks |
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Hi Rosh, a possible solution depends a bit on the properties of your splice variants. If they have different size, you might be able to see them directly after the PCR with 2 primers. If agarose gels don't have enough resolution, a capillary electrophoresis device (eg sequencer) might help. If you know the sequence of your products, you also might be able to distinguish them with a restriction enzyme. For a nested PCR, you might design one or two primers in a way that you only get a product when one of the proposed species is present. I think, you'll have to confirm all variants positively and by (exemplary) sequencing. Primer design is easy. as you only have a few different cDNAs in your sample from the first amplification round, don't worry about Tm and GC, just focus on the sequence of your target(s). Placing the mismatches at the end of the primers and using a non-proofreading polymerase might do the trick. BTW, a second PCR with the same primers I would not consider to be "nested" and rather name "reamplification". But you'll amplify all the garbage from the first round that has incorporated your 2 primers, that's why nested PCR with another set of primers within the first amplicon has been invented. HTH Wo |
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