You write:
=> Anybody have any experience trying to get sequence directly
=> from purified chromosomal DNA? We have been trying to do so with a
=> 9.2 Mb linear chromosome without success. Our central core
=> sequencing facility says they just need more DNA and then they
=> will get sequence. Making as much as they want will be a bother,
=> and if sequencing directly off of a chromosome of this size is not
=> possible, I'd rather spend my time on some other strategy.
=>
=> I should probably mention that we suspect that the
=> region we are trying to sequence contains a transposon.
=> Inverse PCR attempts to clone the region of interest gave
=> anomolous results, among other clues.
=>
=> Thanks.
=>
=> Laura Kasman
=> University of Georgia - Athens
=> Dept. of Entomology
=>
Dear Laura,
Absolutely positively no way can this be done without
some intermediate step such as PCR. You cannot make sufficient
DNA and get it at a high enough concentration for it to work
in cycle sequencing with one of the Taq polymerases. The largest
size templates we and others have been able to sequence off of
are purified BACs (approx. 150Kbp). NO WAY can this be done
off the 3 Billion base paired human genome or even off the ~4Mbp
E. coli genome.
Sorry........but I'd suggest you look at making PCR primers
and getting the PCR product pure enough for sequencing. Hope this
helps but you central core sequencing facility is quite wrong in
suggesting that they can sequence directly off genomic DNA.
Cheers and good luck,
B. Roe
***************************************************************************
Bruce A. Roe, Ph.D Department of Chemistry and Biochemistry
University of Oklahoma, Norman, OK 73019-0370, U.S.A.
Phone: (405) 325-4912 or 7610; FAX: (405) 325-7762; e-mail: broe at uoknor.edu
********************** http://dna1.chem.uoknor.edu/ ***********************
