Degenerate Primers and dI - (Jun/18/2007 )

We regularly use some primers with degeneracy as high as 36,864-fold; they actually work surprisingly well considering the level of degeneracy.

But substituting deoxyinosine for 'N' in these primers would reduce the degeneracy to 144- and 546-fold for the forward and reverse, respectively. My question is, is it worth paying the $8/dI cost for substituting dI for 'N' positions in these primers?

Any personal experiences with this much appreciated.

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topic bump

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does it work well or surprisingly well but not good?

if it works well then no need to incur greater expense.

we have worked with dI and it was worth the extra cost to get reduced degeneracy in our case. you don't need to replace all Ns with Is, just select some strategic positions for them.

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some pairs work surprisingly well, others only acceptably well.



thanks for the advice; how do you decide what those strategic positions are?

also, how do you go about determining the T_m for primers containing dI?

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we didn't replace positions where it was only 2 choices. we also didn't want to have too few bases between degenerate positions.

melting temperature was determined by the company that prepared the primers.

(my colleague was the one who actually devised and used them, i'll be able to ask him (if i remember) when i come back from vacation).

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i spoke with my coworker. he said that he replaced all Ns (only) with I. he followed advice from IDT and had them no closer than 3 bases apart.

he determined Tm with the designer at the IDT website.

he was able to significantly reduce degeneracy this way.

after isolating the pcr products from these primers he subcloned then sequenced to find the actual bases for those positions.

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