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Asymmetric PCR for generation of single stranded DNA - (Jan/24/2005 )

Hi All,

Does any one have any experience doing assymetric PCR? I am hoping to amplify a single stranded DNA from my double stranded DNA (a PCR product).

Currently, I am trying doing it via the protocols suggested by the current protocols molecular biology(book). Did a primary PCR, then gel purify the DNA from contaminating primers, then did a secondary PCR using only one of the primers.

Is there a way to tell apart the single stranded DNA apart from a double stranded DNA.

-TanC-

Hello! Can you not make it in one step, just use a reduced amount of one of the primers so it is exhausted after a while. In the gel there must be two bands then?! Thus you can extract the right (smaler one). I think you need more cycles so. Perhaps you should reduce the denaturation temperature in the second "pcr-step" to make shure the amplicon is amplified and not the template.

-nabla-

QUOTE (nabla @ Jan 25 2005, 12:26 AM)
Hello! Can you not make it in one step, just use a reduced amount of one of the primers so it is exhausted after a while. In the gel there must be two bands then?! Thus you can extract the right (smaler one). I think you need more cycles so. Perhaps you should reduce the denaturation temperature in the second "pcr-step"  to make shure the amplicon is amplified and not the template.

Dear Nabla,

Yeah I have done the PCR step with reduced primers, i.e. 1:100 ratio like you suggested.

Got a weak band of the correct size..can't tell if it's a ssDNA or dsDNA...unfortunately, no double bands.

by denaturation in second step, you mean lower the temperature to like say 80oC from 95oC. I assume you mean, denaturation is not so important at this stage, coz most DNA should be ssDNA and hence no need to break the H-bonds of dsDNA?

Ok...will give it a try..

thanks for the input. smile.gif

-TanC-

Hello! What do you mean with correct size? If the band is dsNDA mustn't it have the double size than the ssDNA? 80 C is perhaps too low, better would something about 88-90 C, I suppose. I must confess I have not tried it before, just read about it somerwhere. You should consider that you have increase the cycle number !! You only dublicate your amplicon at the step the second primer is gone!!

QUOTE
I assume you mean, denaturation is not so important at this stage, coz most DNA should be ssDNA and hence no need to break the H-bonds of dsDNA?


That is not exactly what I meant. You amplicon (ds) is just smaler than the gDNA (e.g.) thus less tmperature is needed to make it ss again.
I hope I could help!?

-nabla-

Thanks for the tips..tried it, didn't see any bands.....

Assuming I successfully amplify the ssDNA, I suspect I may not even detect it on the agarose gel.
I reckon that ethidium bromide does not intercalate with single stranded DNA, but only dsDNA. Coz the etbr needs to get into (nick) one of the bases of the DNA, for ssDNA it will cut the DNA randomly.

Anyway, I am trying out a ssDNA detection kit from invitrogen.

wish me luck

cool.gif

-TanC-

biggrin.gif You could perform a PCR with a biotinylated forward primer and a normal reverse primer so one strand of your amplicon is biotinylated and then bind the product to streptavidin coated magnetic beads and denature one of the strands with NaOH solution while leaving the biotinylated strand bound to the beads.

Just another way that works, how were you going to purify the ssDNA? I don't think the conventional binding columns can handle ssDNA.

Good luck!

Nick biggrin.gif

-methylnick-