Need tips for sequencing through poly-c tail

ku at helix.nih.gov ku at helix.nih.gov
Sun Feb 2 10:37:09 EST 1997


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



More information about the Methods mailing list