=91Gid Day, Eh!
We recently invested a rather healthy chunk of money for a series of
custom dye-primers to sequence a gene with a number of known =
polymorphic
sites. The template is a PCR product, fairly G+C rich and could be
considered as a =93difficult template=94. Up until now we=92ve been =
using the
dye-terminator PRISM-FS kit and the data has been quite good except =
for
the fact that we can=92t always identify the heterozygotes even after =
close
scrutiny of the data at the polymorphic sites (a minor inconvenience, =
I
know, but it was really starting to bug me). For a number of reasons
dye-primers are suppose to be better at heterozygote detection so I
thought we should give it a try. So far our experience has proven to =
be
a dismal failure. The problem is that more often than not the data =
is
junk. On the gel image the sequences are really blue (I mean so blue
that you don=92t see any other color). I only have experience with
dye-terminator sequencing and I don=92t know if this is normal for
dye-primers. Is it possible that the =93C=94 reaction is overloaded =
(I think
that=92s the blue one). The primers were resuspended at the correct
concentrations and we are following the instructions for the =
appropriate
dye-primer kit. The funny thing is that for some template/primer
combinations the data isn=92t half bad (but not great). Even though =
the
gel image is saturated with blue bands the software still seems to =
pick
up the other signals. I don=92t know if our problems are related to =
the
chemistry or if it has something to do with the template.
I would appreciate some advice from anyone using dye-primers for
heterozygote detection, especially those using custom primers (I=92m =
afraid
that if I don=92t get this working soon they=92re going to take the =
cost of
the primers out of my pay cheque).
Shaun Tyler
DNA Core Facility
Laboratory Centre for Disease Control
Health Canada
Ph#: (613) 941-6441
FAX#: (613) 957-1358
E-mail: styler at hpb.hwc.ca
