High-speed, multicolor fluorescent two-dimensional gene scanning.
Two-dimensional gene scanning (TDGS) is a method for analyzing multiple DNA fragments in parallel for all possible sequence variations, using extensive multiplex PCR and two-dimensional electrophoretic separation on the basis of size and melting temperature. High throughput application of TDGS is limited by the prolonged time periods necessary to complete the second-dimension electrophoretic separation step--denaturing gradient gel electrophoresis--and the current need for gel staining. To address these problems, we constructed a high-voltage, automatic, two-dimensional electrophoresis system and used this in combination with thinner gels to reduce two-dimensional electrophoresis time about 80%. Instead of gel staining, we used three different fluorophores to simultaneously analyze three samples in the same gel. These improvements greatly increase TDGS speed and throughput and make the method highly suitable for large-scale single-nucleotide polymorphism discovery and genetic testing.
McGrath
S B
SB
Institute for Drug Development, Cancer Therapy and Research Center, 8122 Datapoint Drive, Suite 700, San Antonio, Texas 78229, USA.
Bounpheng
M
M
Torres
L
L
Calavetta
M
M
Scott
C B
CB
Suh
Y
Y
Rines
D
D
van Orsouw
N
N
Vijg
J
J
D048868:Adaptor Proteins, Signal Transducing; D019313:BRCA1 Protein / Q000235:genetics; D002352:Carrier Proteins; D004247:DNA / Q000032:analysis* / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006801:Humans; D000070957:MutL Protein Homolog 1; D009363:Neoplasm Proteins / Q000235:genetics; D009687:Nuclear Proteins; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
D048868:Adaptor Proteins, Signal Transducing; D019313:BRCA1 Protein; D002352:Carrier Proteins; C501486:MLH1 protein, human; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D016159:Tumor Suppressor Protein p53; D004247:DNA; D000070957:MutL Protein Homolog 1