silincing box, no amplification
Posted 16 September 2011 - 03:12 AM
I have to amplify a T-DNA of 11700 bp that was inserted in a genomic DNA plant. This T-DNA has a silencing box that is formed by two sequences of 1214 bp completely complementary (sense and antisense) and these latter are spaced by a specific sequence (732 bp) that form a hairpin.
When I do a PCR I can't amplify the entire box because of sequence complementarity between the two arms. Obviously during the denaturation at 95°C the double strand is open but when the temperature go down to 62°C (to permit the annealling of primers) it happens that the specificity and the length of the two sequence is grater than my primers.
I mean, if a used a forward primer designed on the sense arm and the reverse on the antisense arm I can't amplify.
But strangely if I use as forward primer a primer designed on the hairpin the PCR work well. So it seams that a primer on the hairpin stabilize and linearizes the strand.
Primers are not the problem because a tested a lot of different primers.
How can I solve my problem? What kind of technique can I use?
Thanks in advance
Posted 16 September 2011 - 04:37 AM
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Posted 19 September 2011 - 05:00 AM