GC-rich Sequencing

Zhonglin Chai zchai at COBRA.PATH.MONASH.EDU.AU
Sat Jul 26 21:24:29 EST 1997

You may have something to do with the reaction. You may just increase the
temperature for both annealing and extension. I successfully sequenced GC
rich region using Perkin Elmer's PRISM dye terminator kit (AmpliTaq FS),
simply by annealing at 58degreeC, and extending at 72degreeC (no sequencing
obtained if using 50degreeC annealing and 60degreeC for extension).

Hope this of help

>I am working with excessively GC-rich templates (to 86%) and am in GREAT
>need of sequencing through these regions. I am using plasmid DNA with
>the insert of interest (very clean) but my sequence (on ABI 377) fades
>when it enters the higher GC-rich region (conveniently located after a
>76% GC-rich flanking region next to CTG repeats). Can anyone suggest any
>approaches which might be useful? I'm using 7-deaza-guanosine in the
>sequencing reaction and hot-starts.

Dr. ZhongLin Chai
Department of Pathology and Immunology
Monash University Medical School
Alfred Hospital
Commercial Rd, Prahran, VIC 3181, AUSTRALIA
Telephone: (61 3) 9276 2698 (lab)
           (61 3) 9276 2696 (office)
           (61 3) 9384 1305 (home)
           0419 87 1940 (mobile)
           + 61 419 87 1940 (international mobile)
Fax:       (61 3) 9529 6484
email:     zchai at cobra.path.monash.edu.au

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