SSCP questions

Lana Perko perkolk at genesisakl.genesis.co.nz
Sun Sep 24 20:01:05 EST 1995


In article <199509211619.JAA01670 at net.bio.net>,
fgarbrec at HENDRIX.JCI.TJU.EDU ("Frederick Garbrecht") wrote:
> 
> We have been performing some SSCP experiments using PCR fragments in 
> a "mutation search" that we are performing on one of our patients, 
> and  there have been some problems that I have been at a loss to 
> solve. [We have basically been doing the non-radioactive PhastGel 
> method using PCR fragments which have been denatured by heating
> in the presence of formamide and then snap frozen,  with either 
> silver detection or detection on a fluorimager (primers are 
> FITC labeled)].
> 
> First, since we run a non-denatured control to identify dsDNA on the 
> gel, I can always see a strong dsDNA band in all lanes (usually more intense than 
> the denatured band(s)  present in the same lane.  Shouldn't the 
> heating and formamide effectively convert the vast majority (all???) 
> of the ds to ssDNA?
> 
> We also consistently see an additional band that runs lower (faster) on the gel than the 
> single dsDNA band in the lanes containing samples that have been 
> formamide treated, but not in the non-treated, dsDNA control lane.  
> I'm not sure what it is, any ideas?  
> 
> Thanks for any help,
> 
> Frederick Garbrecht
> Thomas Jefferson University
> Neoplastic Diseases
> fgarbrec at lac.jci.tju.edu

I have performed SSCP genotyping of TAP2 haplotypes using gamma 32P
labelled probes. Initially results consisted of multiple bands running
faster or slower than the non-treated ds DNA control. 

My suggestion to you is to remove your *primers* away from your PCR product
before running out on a gel. This can make a dramatic difference, see Cai
and Touitou. Nucleic Acid Res 21; 3909-3910 (1993) .

I removed the primers by  Magic DNA Clean-Up columns. Autorad results
showed two bands for homozygotes and four bands for heterozygotes, the
bands  generally ran faster (lower) than unpurified PCR products. 

Lana Perko
perkolk at genesisakl.genesis.co.nz



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