Large DNA fragment sizing using native acrylamide gels on an automated DNA sequencer and GENESCAN software.
We have investigated the potential of the PE Applied Biosystems Model 373 Automated DNA Sequencer and GENESCAN software to size minisatellite alleles ranging in size from 230 bp to 2.5 kbp. We report on the use of a native (non-denaturing) acrylamide gel system and fluorescent dUTP labeling of PCR products. The observed variability in size calling ranged from +/- 0.4-bp standard deviation (SD) at the lower end of the size range to +/- 37.5-bp SD for the largest allele. Both within-gel and between-gel variability in sizing increased with larger alleles, in particular when sizes exceeded 2 kbp. Size-calling differences were observed dependent on the method used to fluorescently label the PCR products and with the fluorescent dye type and concentration used in incorporation. The benefits and limitations of the current GENESCAN software in sizing large DNA fragments are also discussed.
Flinders University of South Australia.
C R
CR
McEvoy
R
R
Seshadri
F A
FA
Firgaira
D000483:Alleles; D004247:DNA / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes / Q000737:chemistry; D011905:Genes, ras / Q000235:genetics; D006801:Humans; D018895:Microsatellite Repeats / Q000235:genetics; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000295:instrumentation* / Q000379:methods; D012984:Software
D017931:DNA Primers; D005456:Fluorescent Dyes; D004247:DNA