Dye-primers and heterozygotes

Shaun Shaun
Mon Jan 20 13:33:19 EST 1997

=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 =
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 =
the fact that we can=92t always identify the heterozygotes even after =
scrutiny of the data at the polymorphic sites (a minor inconvenience, =
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 =
a dismal failure.  The problem is that more often than not the data =
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 =
dye-primer kit.  The funny thing is that for some template/primer 
combinations the data isn=92t half bad (but not great).  Even though =
gel image is saturated with blue bands the software still seems to =
up the other signals.  I don=92t know if our problems are related to =
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 =
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

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