Fast high-throughput screening of angiotensin-converting enzyme insertion/deletion polymorphism by variable programmed electric field strength-based microchip electrophoresis.
An insertion (I)/deletion (D) polymorphism in angiotensin-converting enzyme (ACE) has been associated with susceptibility to various diseases in numerous studies. Traditionally, slab gel electrophoresis (SGE) after polymerase chain reaction (PCR) has been used to genotype this ACE I/D polymorphism. In this study, single- and multi-channel microchip electrophoresis (ME) methods based on variable programmed electric field strength (PEFS) (i.e., low constant, high constant, (+)/(-) staircase, and random electric field strengths) were developed for fast high-throughput screening of this specific polymorphism. The optimum PEFS conditions were set as 470V/cm for 0-9s, 129V/cm for 9-13s, 470V/cm for 13-13.9s, 294V/cm for 13.9-16s, and 470V/cm for 16-20s for single-channel ME, and 615V/cm for 0-22.5s, 231V/cm for 22.5-28.5s, and 615V/cm for 28.5-40s for multi-channel ME, respectively. In the multi-channel PEFS-ME, target ACE I/D polymorphism DNA fragments (D=190bp and I=490bp) were identified within 25s without loss of resolving power, which was ∼300 times faster than conventional SGE. In addition, PCR products of the ACE gene from human blood samples were detected after only 10 cycles by multi-channel PEFS-ME, but not by SGE. This parallel detection multichannel-based PEFS-ME method offers a powerful tool for fast high-throughput ACE I/D polymorphism screening with high sensitivity.
Department of Chemistry, Graduate School, Kyung Hee University, Yongin-si, Gyeonggi-do 17104, Republic of Korea.
Yucheng
Y
Sun
Kohwang Medical Research Institute, School of Medicine, Kyung Hee University, Seoul 130701, Republic of Korea.
Su-Kang
SK
Kim
Department of Chemistry, Graduate School, Kyung Hee University, Yongin-si, Gyeonggi-do 17104, Republic of Korea.
Peng
P
Zhang
Department of Chemistry, Graduate School, Kyung Hee University, Yongin-si, Gyeonggi-do 17104, Republic of Korea.
Nain
N
Woo
Department of Chemistry, Graduate School, Kyung Hee University, Yongin-si, Gyeonggi-do 17104, Republic of Korea; Department of Applied Chemistry and Institute of Natural Sciences, Kyung Hee University, Yongin-si, Gyeonggi-do 17104, Republic of Korea. Electronic address: shkang@khu.ac.kr.
Seong Ho
SH
Kang
D001483:Base Sequence; D046209:Electrophoresis, Microchip / Q000191:economics / Q000295:instrumentation / Q000379:methods*; D004867:Equipment Design; D057166:High-Throughput Screening Assays / Q000191:economics / Q000295:instrumentation / Q000379:methods; D006801:Humans; D054643:INDEL Mutation; D016254:Mutagenesis, Insertional; D007703:Peptidyl-Dipeptidase A / Q000235:genetics*; D011110:Polymorphism, Genetic; D017384:Sequence Deletion
D007703:Peptidyl-Dipeptidase A
Angiotensin-converting enzyme (ACE); DNA; Microchip electrophoresis; Polymorphism; Programmed electric field strength (PEFS)