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An integrated method for mutation detection using on‐chip sample preparation, single‐stranded conformation polymorphism, and heteroduplex analysis
Author(s) -
Vahedi Golnaz,
Kaler Karan,
Backhouse Christopher J.
Publication year - 2004
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200405957
Subject(s) - heteroduplex , single strand conformation polymorphism , dna , microbiology and biotechnology , genotyping , polymerase chain reaction , mutation , biology , genetics , gene , genotype
This work integrates rapid techniques for mutation detection by producing single‐stranded DNA and (renatured) double‐stranded DNA on‐chip, labeling these with fluorescent DNA stains and then performing two complementary methods of mutation detection‐single stranded conformation polymorphism (SSCP) analysis and heteroduplex analysis (HA). This involves the denaturation of double‐stranded polymerase chain reaction (PCR) product into single‐stranded DNA, the mutation analysis of the single‐stranded DNA by SSCP and the rehybridized double‐stranded DNA by HA. These steps were performed entirely on‐chip within several minutes of operation. The combination of these two mutation detection methods on‐chip provides a highly sensitive method of mutation detection for either genotyping or screening. Many mutation analysis methods rely upon fluorescently labeled samples from a PCR with fluorescently labeled primers. By labeling on‐chip we not only attain improved signal strength, but the method is considerably more versatile. Although we used PCR products in this work, this method could be used to analyze DNA from any source. We believe that this combination of several procedures on a single chip represents a significant step in the development of higher levels of integration upon microfluidic devices.