TY - JOUR
T1 - High-speed, multicolor fluorescent two-dimensional gene scanning
AU - McGrath, Sean B.
AU - Bounpheng, Mangkey
AU - Torres, Loyda
AU - Calavetta, Marco
AU - Scott, Charles B.
AU - Suh, Yousin
AU - Rines, David
AU - Van Orsouw, Nathalie
AU - Vijg, Jan
N1 - Funding Information:
This work was supported by NIH grants AG13319 and CA78564 (through J.V.), a grant from the San Antonio Cancer Institute (to J.V.), and research funds from Accelerated Genomics Co. (San Antonio), CBS Scientific Co. (Del Mar), Metagentech Co.,Ltd. (Seoul, South Korea), and Hitachi Genetic Systems (San Francisco).
PY - 2001/9/1
Y1 - 2001/9/1
N2 - 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.
AB - 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.
KW - Genetic testing
KW - High-voltage denaturing gradient gel electrophoresis
KW - Multi-color fluorescence
KW - Single-nucleotide polymorphism discovery
KW - Two-dimensional DNA electrophoresis
KW - Two-dimensional gene scanning
UR - http://www.scopus.com/inward/record.url?scp=0034760837&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0034760837&partnerID=8YFLogxK
U2 - 10.1006/geno.2001.6649
DO - 10.1006/geno.2001.6649
M3 - Article
C2 - 11707076
AN - SCOPUS:0034760837
SN - 0888-7543
VL - 78
SP - 83
EP - 90
JO - Genomics
JF - Genomics
IS - 1-2
ER -