DNA mutation detection with chip-based temperature gradient capillary electrophoresis using a slantwise radiative heating system.
A simple and robust chip-based temperature gradient capillary electrophoresis (TGCE) system was developed for DNA mutation/single-nucleotide polymorphism (SNP) analysis using a radiative heating system. Reproducible, stable and uniform temperature gradients were established along a 3 cm length of the electrophoretic separation channel using a single thermostated aluminium heater plate. The heater was slightly slanted relative to the plane of the glass chip at 0.2-1.3 degrees by inserting thin spacers between the plate and chip at one end to produce differences in radiative heating that created the temperature gradient. On-chip TGCE analyses of 4 mutant DNA model samples amplified from plasmid templates, each containing a single base substitution, with a wide range of melting temperatures, showed that mutations were successfully detected under a wide temperature gradient of 10 degrees C and within a short gradient region of about 3 cm (3.3 degrees C cm(-1) gradient). The radiative heating system was able to establish stable spatial temperature gradients along short microfluidic separation channels using simple peripheral equipment and manipulation while ensuring good resolution for detecting a wide range of mutations. Effectiveness of the system was demonstrated by the successful detection of K-ras gene mutations in 6 colon cancer cell lines.
Zhang
Hui-Dan
HD
Department of Cell Biology, Key Lab of Cell Biology of Ministry of Public Health, China Medical University, Shenyang, 110001, China.
Zhou
Jing
J
Xu
Zhang-Run
ZR
Song
Jin
J
Dai
Jing
J
Fang
Jin
J
Fang
Zhao-Lun
ZL
D001483:Base Sequence; D004247:DNA / Q000032:analysis* / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000295:instrumentation* / Q000379:methods; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006361:Heating / Q000295:instrumentation* / Q000379:methods; D008969:Molecular Sequence Data; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D004247:DNA