Designing primers for qPCR

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7.7 years ago

casley_queiroz ▴ 20

When designing primers for qPCR, which sequence is better to consider, CDS or full gene?

gene • 4.2k views

ADD COMMENT • link updated 7.6 years ago by Biostar 20 • written 7.7 years ago by casley_queiroz ▴ 20

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Do you with full gene mean also intronic sequences, or CDS + UTR?

ADD REPLY • link 7.7 years ago by WouterDeCoster 47k

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Just intron + exon, wihout UTR.

ADD REPLY • link 7.7 years ago by casley_queiroz ▴ 20

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qPCR for cDNA I assume? Do you expect introns in your mature transcript/cDNA? Or are you talking about qPCR on genomic DNA?

ADD REPLY • link 7.7 years ago by WouterDeCoster 47k

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Thank you for your support. I got an answer. Ideally designing between an exon + intron + exon. Thus eliminates the risk of losing a amplification if primers were designed in intro, because this would not be the cDNA.

ADD REPLY • link 7.7 years ago by casley_queiroz ▴ 20

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Definitely not in an intron, best having intron spanning amplicon (one primer in exon n and the other in exon n+1) or a primer spanning the exon-exon junction. Are you sure you understand what you are doing?

ADD REPLY • link 7.7 years ago by WouterDeCoster 47k

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I think I really understood, I drew one of the primers in the junction of two exon and the other within outher exon (exon 2 or 3 for exemplo).

ADD REPLY • link 7.7 years ago by casley_queiroz ▴ 20

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Okay, I just came across this posts so this might be of interest for you: http://eu.idtdna.com/pages/decoded/decoded-articles/pcr-qpcr/decoded/2016/06/28/use-splice-junctions-to-your-advantage-in-qpcr

ADD REPLY • link 7.7 years ago by WouterDeCoster 47k

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