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I am trying to design a primer for TMPRSS2 PCR reaction. However, this gene is in reverse position like in the picture I'll show.enter image description here

From the nucleotide sequence I got from NCBI, should I reverse the sequence and start designing the primer, or do you have any other solution? Thank you!

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  • $\begingroup$ Welcome to the site. Please take a tour and visit the help center for more information about the site and what we do here, as well as about good questions and answers. Can you please specify if you aim to amplify genomic DNA or coding sequence only (e.g. off RNA/cDNA). $\endgroup$
    – bob1
    Commented Jan 11, 2023 at 19:13

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If your goal is simply PCR amplification than it makes no difference whether you flip the sequence or not: By convention the 'forward' primer refers to the primer that is designed against the anti-sense strand and your 'reverse' primer is designed so it is complementary to the sense strand. In practice however the PCR reaction obviously doesn't care what we call the primers, it will amplify the region that is encompassed by them, producing double-stranded DNA fragments that are indistinguishable regardless if the sequence/design was flipped or not.

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  • $\begingroup$ Thanks for your answer. I am considering to use the original sequence (unflipped) retrieved from NCBI RefSeq to cover the whole region I'd like to amplify. $\endgroup$
    – Hieronimus Adiyoga
    Commented Jan 12, 2023 at 6:31

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