PCR of a GC Rich, Tandem Repeat Region: HELP!

Paul N Hengen pnh at kaylor.ncifcrf.gov
Tue Oct 17 14:20:42 EST 1995


Petros wrote:

: I am trying to amplify by PCR a highly GC rich region with a high number of 
: 15bp tandem repeats.  The template is genomic DNA.  The expected size 
: of the fragment is approximately 1.2Kb. The polymerase I am using is 
: Taq DNA Polymerase.	
:
: I have tried two different sets of primers, (both of which are from 
: published papers and hence have been used before successfully), under 
: various conditions such as:
: - 2 cycle PCR i.e. only denaturation and extension cycles
: - a variety of different annealing temperatures, magnesium concentrations
:   and primer concentrations
: - Nested PCR using the two sets of primers (one set flanks the other set 
: thus the PCR is carried out first with the flanking primers and that PCR 
: product is then used as the template for another PCR reaction with the 
: other, inner, set of primers).
:
: Note: The two sets of primers flank the tandem repeat region.
:
: I have tried many, many conditions and I have had only two kinds of 
: results: 1) a blank gel or 2) a gel with many smaller, non-specific junk 
: bands.
:
: Does anyone have any suggestions for getting such a PCR to work?
: Any suggestions would be highly appreciated.

I don't know if you've actually tried this, but the use of a KCl-free
buffer might help you get results. Some people at NIH tried for some
time to sequence through the repeats involved with fragile X syndrome
and only got it to work by removing potassium, which stabilized a REALLY
funky folded over secondary loop structure.

@article{Woodford1995,
author = "K. Woodford
     and M. N. Weitzmann
     and K. Usdin",
title = "The use of {K}+--free buffers eliminates a common
cause of premature chain termination in {PCR} and {PCR} sequencing",
journal = "Nucleic Acids Res.",
volume = "23",
number = "3",
pages = "539",
comment = "potassium free buffer for PCR",
year = "1995"}

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