PCR: Can you get amplification if only one primer anneals? - Same size pcr product using 2 different primer sets (Jul/25/2007 )

Hi everyone,
Would love to hear your thoughts on the following situation:

I have designed two different primer sets to amplify the same gene. Both sense primers are in exon 1 but anneal to different parts of the exon. They are next to each other but don't overlap. One antisense primer is in the 5' end of exon 6 and the other is in exon 7. The product for exon 1-6 primer set is 671bp and for exon 1-7 is 3.4kb (exon 6 is quite large). When I do the pcr using each of the primers sets and the same cDNA I get an amplicon that is the same size for both of the primer sets. Is it possible that only my sense primer is annealing and amplification is only happening in the forward direction and I have any alternative product? Any thoughts on this would be appreciated.
Thanks

Not sure I understand your question exactly but if only one primer is annealing (the forward or reverse) you will get no amplification. The maximum amount of product would be single stranded DNA at the same concentration as the template.
Sat

I am wondering if I am getting only a ssDNA product. I know there are techniques like asymmetric pcr that result in only one of the strands being amplified so I thought that may be happening to my reaction inadvertently. If I do not have a full length cDNA then my reverse primer can't anneal so I amplify only with the forward primer. I am really not familiar enough with the technique to know if this is possible. I am just trying to figure out an explanation for getting the same size product with 2 different primer sets.

Hi,

it is true, you can amplify with only one primer, but this is going to be linear reaction and the product size depends on the polymerase speed. I doubt that you would see bands at all, as you don't get a exponential amplification and, even if you see a band, it wouldn't be clear cut.

I would go for unspecific binding. Did you Blastcheck your primers?

They are specific for my gene and I have also had one of the products sequenced and it is the correct product. It is quite a conundrum. I have never been able to amplify the full length cDNA and because it is baboon I don't know the sequence. Parts of it are very homologous to human bet there are enough differences in alternative splicing that I need the know the speicifc sequence for baboon.

Could you sequence the products for both primer sets?
Maybe they have the same length by chance, but represent different transcripts due to alternative splicing (e. g. skipping of internal exons).
You also could test your hypothesis by setting up a pcr with either only your sense or one of your antisense primers. In the first case you should get a pcr product of similar size and amounts as before. Setting up the reaction with only your antisense primers should lead to no product if you are right.

Maybe one of your primers binds in both directions?
Have you blasted your primer sequences and checked wether they are specific for your gene of interest?

Try sequencing your product fully. If it's cloned, you should be able to see what's happening at the ends where the primers are.

BLAST your primers but don't always trust it. Sometimes BLAST shows no matching but somehow the primers anneal to nonspecific regions.

You can gel-purify your fragment and (if there are any restriction sites) try to cut it with a rare-cutting enzyme before sequencing it. Or just sequence it if it's easier.