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Site-directed Mutagenesis using Dut-Ung system - Troubleshooting a mutagenesis (Mar/14/2002 )


I have been trying over the last few months to introduce a single point mutation in a looped portion of an RNA, using the dut-ung method of mutagenesis.  So far, I have had little success.  I have a feeling that this problem is due to the fact that intramolecular secondary structure is being formed by the mutagenic DNA oligo (length = ~25 bases) and/or the complementary single-stranded DNA, thus preventing the annealing of the two molecules to occur.

Is this a logical hypothesis?  And if so, what can be done to prevent such intramolecular interactions from occuring, so that the mutagenesis can proceed?

Any help on this topic will be greatly appreciated.


depending on ur mutagenesis oligo, u can raise the annealing/ extension temp.

if ur primers have 2ndary structures then it shoudl come off once u raise the temp.

but like i said it depends on ur oligo