primer annealing question

[jcosborn06]

I'm using pcr to screen TOPO TA transformants, and have results that may suggest my insert transformed in with the wrong orientation. I'm using the M13 Reverse primer included with the kit and a Forward primer that hybridizes to my insert to check the orientation.

My question is this: if my insert is backward, will pcr primers that hybridize to the insert still anneal to it? I'm essentially asking if primers can anneal to a primer's reverse sequence (e.g. can a hypothetical primer that anneals to AAATTTGGG also anneal to GGGTTTAAA?). I'm not aware that primers have any directionality that causes them to anneal only in a 5' to 3' orientation, so i suspect that this is possible.

Any input is much appreciated.

[kmunson779]

Primers are DNA oligos -- they have the same directionality that any DNA molecule has (i.e. 5' and 3' ends) and will only anneal to complimentary nucleic acid.

[arammik]

Dear jcosborn06

Your kit has provided the TOPO TA vector which has the M13 priming sites flanking the region that would accept the insert. The idea is that if your insert has been accepted into the backbone of the vector, the positive reaction indicated by successful amplification would be the result of the priming of your insert-specific primer onto the sense strand (for example) and the M13 reverse to the anti-sense strand, thereby amplifying the region between the M13 priming region and the gene-specific priming region, if ofcourse the insert was accepted in the right orientation. If not, the gene specific priming region would now be in the antisense strand very close to the M13 region. Since both primers now bind the antisense strand, you will receive no amplification and hence a negative reaction. Hence you will receive a positive reaction only in samples with the right orientation.

CHeers!!