High-speed screening of polymerase chain reaction products by capillary electrophoresis.
In an effort to develop capillary electrophoresis (CE) for high-throughput polymerase chain reaction (PCR) molecular diagnostics, a method was developed to rapidly screen small PCR products of similar molecular weights. The assay of interest required the separation of two PCR products (375 and 400 bp) in an assay of TGF-beta 1 knockout mice to determine the genotype of neonates. Using a commercially available CE instrument, the two PCR products were separated in 12 min with a replaceable gel buffer, a 20-cm effective length DB-17 capillary, and 185 V/cm field strength. With the coinjection of a 20-bp ladder, the sizes of the PCR products were determined from the electropherogram without using a calibration plot and curve-fitting program. Faster separation was obtained using the combination of a short effective length capillary and high field strength. The two PCR products were separated in 82 s with a 7-cm effective length capillary and 556 V/cm. A 60% buffer further reduced the separation time in about a minute. This high-speed separation, with minimum postrun data processing, is highly desirable for the high-throughput screening of PCR products using a single-capillary CE system.
Chan
K C
KC
Chemical Synthesis and Analysis Laboratory, SAIC Frederick, National Cancer Institute-Frederick Cancer Research and Development Center, Maryland 21702, USA, chan@ncifcrf.gov
Muschik
G M
GM
Issaq
H J
HJ
Garvey
K J
KJ
Generlette
P L
PL
D000818:Animals; D004247:DNA / Q000737:chemistry; D004252:DNA Mutational Analysis / Q000379:methods; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods*; D005838:Genotype; D051379:Mice; D016133:Polymerase Chain Reaction; D016212:Transforming Growth Factor beta / Q000235:genetics
D016212:Transforming Growth Factor beta; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; C024403:GGCC-specific type II deoxyribonucleases