Need tips for sequencing through poly-c tail


Tue Feb 4 13:29:05 EST 1997



On Sun, 2 Feb 1997 ku at helix.nih.gov wrote:

> Scott Hodgson wrote:
> > 
> > In order determine the sequence at the 5' end of a mRNA I carried out 5'
> > RACE (BRL kit) and cloned the product.
> > 
> > The 5'RACE procedure involves tailing of the first strand cDNA with dCTP
> > and terminal transferase.  Subsequently, an internal gene specific primer
> > and an anchor primer that sits on the tail are then used in a PCR.
> > 
> > I need to sequence through the tail to get to the region of interest but
> > have been unable to do so.   My reactions look fine until they reach the
> > far side of the tail where I am seeing what looks like two or three
> > superimposed and slightly offshifted sequences. I have tried two cycle
> > sequencing kits (USB Thermosequenase and Perkin Elmer Amplicycle) with
> > similar results.  Sequencing in from the opposite side is not a convenient
> > alternative.
> 
> It sounds to me like you are seeing the "stuttering" classic with cycle
> sequencing of microsatellites that I think results from prematurely
> terminated chains synthesized during one cycle of the PCR serving as
> primers in subsequent rounds of PCR. These "primers" dont necessarily
> anneal in perfect register on the template since they are able to form
> stable hybrids further 3' on the template. This essentially results an
> in vitro expansion of the polyC tract. One possibility is to raise the
> annealing temperature in the hope that this will reduce the priming by
> these "primers", but I think your best option is to sequence the old
> fashioned way with a single round of denaturation and extension eg with
> Sequenase.
> 
> 
> Karen
> 
> 
> Karen Usdin
> Section on Genomic Structure and Function
> Laboratory of Molecular and Cellular Biology
> NIDDK
> National Institutes of Health
> 
> 


What about trying some DMSO - 10% worked well for 70% GC for me

Ian




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