Asymmetric PCR for generation of single stranded DNA
Posted 24 January 2005 - 02:57 PM
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.
Posted 24 January 2005 - 11:26 PM
Posted 25 January 2005 - 11:59 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.
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.
Posted 25 January 2005 - 10:48 PM
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!?
Posted 04 February 2005 - 10:40 AM
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
Posted 04 February 2005 - 04:47 PM
Just another way that works, how were you going to purify the ssDNA? I don't think the conventional binding columns can handle ssDNA.