genomic DNA PCR - RE digestion of gDNA before amplification (Sep/14/2005 )

Hello,
I want to PCR amplify a no-coding region from genomic DNA (1.5 kb). After several failed attemps to amplify the mentioned region I decided to perform PCR from gDNA digested with a restriction enzyme that doesn't cut in the sequence of interest. I think that digestion could somehow make gDNA a better template. Maybe cut DNA could be more accesible to Taq or could have less secondary stucture or could be denaturated easier... I don't know.

To make sure that I'm actually amplifying the sequence of my interest I want to perform digestion of template gDNA with a restriction enzyme that cut within the amplicon and then I would PCR it. I expect that if my primers are specific they won't amplify that template.

Please tell me what do you think about it or if you've got another ideas to amplify difficult templates.

Is the area you want to amplify high GC? This could be causing problems with the template melting and ultimately the PCR. You could try adding betaine or DMSO to help break up secondary structures. I also find that a 10 minute hot start works (adding the taq after the 10 minutes are up). Once you get some amplification of your 1.5 kb in the first round, the normal melting time (30 seconds - 1 minute) should be enough since the template is small.

quote=Marvilla,Sep 15 2005, 02:17 PM]
Hello,
I want to PCR amplify a no-coding region from genomic DNA (1.5 kb). After several failed attemps to amplify the mentioned region I decided to perform PCR from gDNA digested with a restriction enzyme that doesn't cut in the sequence of interest. I think that digestion could somehow make gDNA a better template. Maybe cut DNA could be more accesible to Taq or could have less secondary stucture or could be denaturated easier... I don't know.

To make sure that I'm actually amplifying the sequence of my interest I want to perform digestion of template gDNA with a restriction enzyme that cut within the amplicon and then I would PCR it. I expect that if my primers are specific they won't amplify that template.

Please tell me what do you think about it or if you've got another ideas to amplify difficult templates.

At what concentration do you use DMSO in your PCRs?,

Besides DMSO are there other agents used in PCR to overcome problems with secondary structure? Please tell me how do you use it (concentration)?

I generally use 5-10% DMSO for PCRs from genomic DNA. You should also consider that amplification might be very inefficient in the first few cycles, so either try 35 PCR cycles or re-amplify your product (do 20 cycles, then take some of that as template in another PCR).

Digestion should help with the PCR as well.

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