problems sequencing T rich template

drodenhi at JULIAN.UWO.CA drodenhi at JULIAN.UWO.CA
Fri Jun 23 10:14:47 EST 1995


We are sequencing DNA templates which are T-rich, as a result of chemical
conversion of unmethylated cytosines to uracil, which are then amplified as
a thymidine by PCR. The PCR products are cloned and then sequenced using
Pharmacia's T7 sequencing kit.

Our problem is that when we have runs of T's in the template we are seeing
dual termination sites in the sequence when the sequence "leaves" the T run
and continues to the site of a purine A or G. So, for example, at the site
where we would expect to see a G, a T is also present at the same point in
the sequencing gel. Interestingly, the intensities of the "G" and "T" at
that point in the gel are roughly equivalent.  This phenomemon appears to
be reaction-specific, it is not due to the presence of polymorphism or
mutation, nor does it appear to due to a shortage T's in the mix. All other
aspects of the sequencing experiments are giving us very acceptable
results.

If anyone has any suggestions and or explanations these would be
appreciated. One suggestion already raised is that the DNA template has
been rendered less flexible due to the runs of Ts which are present.
Comments?

Thanks in advance

David I. Rodenhiser
University of Western Ontario, Canada,
drodenhi at julian.uwo.ca

..........................................................................
Sincerely,
Dr. David Rodenhiser
Assistant Professor, Dept. of Paediatrics,
Molecular Medical Genetics Program,
Child Health Research Institute,
Children's Hospital of Western Ontario
800 Commissioner's Road East,
London, Ontario, Canada, N6C 2V5
..........................................................................
drodenhi at julian.uwo.ca
tel: 519-685-8430
fax: 519-685-8186





More information about the Methods mailing list