automated sequencing of genomic dna

Bob Bob
Fri May 30 08:34:54 EST 1997


We have directly sequenced bacterial genomic DNA as well as plant DNA
for the detection of mycoplasma-like organisms.  The first method uses
two primers (one labeled) in a single step procedure.  This method was
called SAS (Simultaneous Amplification and Sequencing) and is published
in Journal of Microbiological Methods 17(1993):103-113.
  The second more useful procedure uses only one labeled primer in a
single step linear amplification reaction (Direct Genomic Sequencing,
DGS).  This method allows you to sequence into unknown stretches of your
genomic DNA.  We used this method extensively in our lab to directly
sequence 16S rRNA genes and their flanking regions (Letters in Applied
Microbiology 23:218-222).   We thought this was a great technique but
had difficulty in getting it published. The reviewers didn't think it
was a novel technique.  We had positive feedback when we presented the
method at the ASM meetings in 1993 and finally published it in the
Proceedings of the Japan Academy, Vol. 69, Ser. B., No.8 (1993):
208-211.
  The drawback with each of these techniques is that you have to use a
lot of DNA (from 0.01 - 1.0 ug). The DGS technique was the standard
sequencing method for my lab until we purchased a LiCor sequencer.  One
day when I have time we will try to adapt the technique to the LiCor
machine. 
  If anyone is interested in the techniques send me an email with your
address and I will forward the reprints describing the method.   


Dr. Bob Forster
Centre for Food and Animal Research
Agriculture and Agri-Food Canada
Ottawa, ON  K1A 0C6

forster at em.agr.ca
_________________________________________
"and remember, if the women don't find you handsome,
they should at least find you handy." Red Green, Possum Lodge



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