Abstract:
: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.
journal_name
Nucleic Acids Resjournal_title
Nucleic acids researchauthors
Schmalzing D,Belenky A,Novotny MA,Koutny L,Salas-Solano O,El-Difrawy S,Adourian A,Matsudaira P,Ehrlich Ddoi
10.1093/nar/28.9.e43keywords:
subject
Has Abstractpub_date
2000-05-01 00:00:00pages
E43issue
9eissn
0305-1048issn
1362-4962pii
gnd044journal_volume
28pub_type
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