Genotyping of mutation in the beta-globin gene using DNA microarrays.
Genotyping using DNA microarrays is a cost-efficient method compared with real-time PCR and DNA sequencing. Here, a DNA microarray-based method using allele-specific oligo hybridization is demonstrated. This method relies on immobilization of probes that are specific for wild-type sequences or the mutated sequences, respectively. The method makes use of agarose film-coated glass slides, unmodified DNA probes, target preparation using T7 in vitro transcription, labeling of target using biotin labels, and detection using alkaline phosphatase precipitation reaction. Visualization is performed using a desktop computer scanner. Because the biotin/strepavidin chemistry is utilized, the method described here is compatible with many different detection methods. The demonstrated colorimetric detection of mutations has an expected error frequency of about 5 x 10(-7) per mutation or better. Given this low error frequency, an array diagnosing 100 different mutations would misclassify about 1 patient in 100,000.
Dufva
Martin
M
Fluidic Arrays Systems And Technology (FAST), DTU Nanotech, Department of Micro and Nanotechnology, Technical University of Denmark, Kongens Lyngby, Denmark.
Poulsen
Lena
L
D001483:Base Sequence; D001609:Beta-Globulins / Q000235:genetics*; D004247:DNA / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000295:instrumentation* / Q000379:methods; D004867:Equipment Design; D019544:Equipment Failure Analysis; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods* / Q000639:trends; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*
D001609:Beta-Globulins; D004247:DNA