Microchip electrophoresis: a method for high-speed SNP detection.
As a trial practical application, we have applied optimized microfabricated electrophoresis devices, combined with enzymatic mutation detection methods, to the determination of single nucleotide polymorphism (SNP) sites in the p53 suppressor gene. Using clinical samples, we have achieved robust assays with quality factors as good as conventional electrophoresis in approximately 100 s. This is 10 and 50 times faster than capillary and slab gel electro-phoresis, respectively. The method was highly accurate with an average error of mutation site measurement of only +/-5 bp. No clean-up of the digestion mixtures was needed prior to injection. This greatly simplifies sample handling relative to capillary instruments, which is important for high-throughput screening applications. Following identification, absolute mutation determination of the screened samples was achieved in a second microdevice optimized for four-color DNA sequencing. Total run time was 25 min in this second device and sequencing data were in full agreement with ABI Prism 377 sequencing runs which required 3.5 h. The tandem application of microdevices for location then full characterization of SNPs appears to confirm many of the improvements claimed for future application of microdevices in practical scaled screening for mutational analysis.
Schmalzing
D
D
Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02139, USA.
Belenky
A
A
Novotny
M A
MA
Koutny
L
L
Salas-Solano
O
O
El-Difrawy
S
S
Adourian
A
A
Matsudaira
P
P
Ehrlich
D
D
D004252:DNA Mutational Analysis / Q000379:methods*; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004706:Endodeoxyribonucleases / Q000378:metabolism; D016158:Genes, p53; D006801:Humans; D066298:In Vitro Techniques; D008904:Miniaturization; D020411:Oligonucleotide Array Sequence Analysis; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods
D018076:DNA, Complementary; D004706:Endodeoxyribonucleases; C035571:endodeoxyribonuclease VII