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silincing box, no amplification


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#1 cece23

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Posted 16 September 2011 - 03:12 AM

Hi everyone

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.

hairpin
sense ------------------antisense



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.

Any suggestion?

How can I solve my problem? What kind of technique can I use?

Thanks in advance

Emanuele

#2 phage434

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Posted 16 September 2011 - 04:37 AM

Do you need to detect this sequence, or do you need to copy the sequence and use it in another construct? Detecting it could be done with one primer outside the hairpin and another in the loop. Amplifying will probably require doing the amplification in two pieces, then using ligation to put the pieces together.

#3 cece23

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Posted 18 September 2011 - 11:20 PM

I need to detect and sequencing the entire sequence, from left to right border. The problem is that I think (I am pretty sure) I have more than one T-DNA copy, probably inserted in tandem within the plant genome. For this reason I can't amplify the entire sequence in more than two pieces, because then I don't know how assembling the different pieces. I mean which piece come before? And if there are two identical sequences I will amplify it just like one.

#4 phage434

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Posted 19 September 2011 - 05:00 AM

You should be able to determine whether there are tandem repeats by doing a PCR with outward directed primers on the left and the right of the hairpin region, which will amplify only if there is a repeat. If the repeat is longer than about 1000 bp, you won't be able to easily use sequencing to determine the sequence in any case.




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