How do you estimate PCR product size ?

[aniruddho]

Hello everyone

I am in a bit of a dilemma.

I am isolating soil genomic DNA and am using fungal specific primers to isolate certain families of fungi that I plan to clone and sequence. The soil genomic DNA mix that I extract is a mixture of all sorts of DNA and using fungal specific primers I hope to locate various fungi specific to those primers. In other words, there is not fixed product size that I am working with. The way these primers have been designed is through multiple sequence alignments of these fungi. These primers have been used before in other papers.

My reverse primer is (5' to 3') is say X and my forward primers for 4 classes of fungal specific primers are (5' to 3') A,B,C,D.

For each soil genomic DNA samples, I set-up 4 PCR reactions, A and X, B and X, C and X, D and X.

I had some very basic questions:

- When I BLAST my forward primer, I obtain results for various fungal species specific to those primers. They give me lengths and the starting point for that primer.

- When I BLAST the reverse primer, do I reverse complement it or reverse it or just keep it the way it is to obtain results ? This is a very basic question but I am confused !

After that, do I, for example, pick one of the BLAST matches common to BOTH the primers, search for my primer matches in THAT particular sequence and THEN estimate product size ? Even so, that's just one estimate. how do I get ballpark figures for all sorts of species for that particular family specific primer ?

Thanks a lot !

[nullall]

for the reverse primer, just keep it the way it is

[SebaQ]

Agreed, for the reverse just keep it as it is, 5' to 3'. The BLAST algorithm will automatically "reverse" it and match it where it's supposed to match.

To estimate the product size, I think you can try this:

Blast your primers as you did before, and save the sequence for every fungical match you get with your desired e-value. Since you're using primers got from a multiple sequence alignment, they're likely to have some mismatches.

[It'd be recommended you save the sequence and a few kilobases upstream and downstream your matched gene, just in case].

Anyway, now that you've got a number of results for the primer X, A, B, C and D, check if both primers you're going to make the PCR with (X-A, X-B, X-C and X-D) are able to amplify something with the sequences got from the BLAST results. And don't forget to check the primers with themselves (A-A, B-B, etc) in case they match something within your sequences and amplify undesired products. From these bioinformatical results you can have an idea of what size your products are. For this analysis i'd recommend a program other than BLAST, like Vector NT, Amplifix, etc, since their interfaces are more quick and friendly

Well, that's somethig I came up with now. I guess it might work ^^. Hope it will.

Best Regards

[aniruddho]

Hello nullall and SebaQ

Thank you very much for your suggestions... SebaQ... your suggestion of combining the primers was pretty nifty ! thanks for that info! I got what I wanted !!