Pair of primers (SYBR) with more than one amplicon product(?) - (Mar/27/2013 )

Hi everyone,

I have a question.

I am trying to design a pair of primers in a repetitive sequence, but i am facing the issue below:

The forward primer is ok and align in only one site in the sequence.

But the reverse primer align in four sites downstream the sequence amplifying 4 amplicons: 213bp, 425bp, 637bp and 849bp.

My question is: at the end of PCR, how many amplicons will I have?

Theoretically I know that I will have 4 amplicons, but I was thinking if the first aligment of the reserve primer (213bp) will allow the other bigger amplicons to be produced.

What do you think about it?

Thanks,

Thinker

does the second primer aneal to the sequence that is 100% complementary (in all 4 cases) ?

bigudukaz on Wed Mar 27 12:15:41 2013 said:

Yes bigudukaz
It is a repetitive sequence, so the primer align perfectly in all 4 downstream sequence.

You will amplify all of the 4 products and the melting curve will look funny. Probably the only option would be to use probe for the shortest fragment - you will get around the problems because of 4 amplicons forming and you still should be able to quantify gene expression.

There will be the most of shorter fragments and least the longest ones.

I haven't tried such experiment yet, but time will come.

You may be able to select the shorter sequence by using a shorter extension time. It will be difficult to selecct the longer sequence. The PCR reaction will favor the short sequence, and you may find it difficult to produce much of the longer sequences, but will likely get some of the longer fragment.