Single-nucleotide polymorphism genotyping on optical thin-film biosensor chips

^†Department of Genetics, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510; ^‡ThermoBioStar, Inc., Louisville, CO 80027; ^¶Department of Pathology, Oklahoma City Veterans Affairs Medical Center and University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104; and ^∥Denver Veterans Affairs Medical Center, Denver, CO 80220

Contributed by David C. Ward, July 29, 2003

Abstract

Single-nucleotide polymorphisms (SNPs) constitute the bulk of human genetic variation and provide excellent markers to identify genetic factors contributing to complex disease susceptibility. A rapid, sensitive, and inexpensive assay is important for large-scale SNP scoring. Here we report the development of a multiplex SNP detection system using silicon chips coated to create a thin-film optical biosensor. Allele-discriminating, aldehyde-labeled oligonucleotides are arrayed and covalently attached to a hydrazinederivatized chip surface. Target sequences (e.g., PCR amplicons) then are hybridized in the presence of a mixture of biotinylated detector probes, one for each SNP, and a thermostable DNA ligase. After a stringent wash (0.01 M NaOH), ligation of biotinylated detector probes to perfectly matched capture oligomers is visualized as a color change on the chip surface (gold to blue/purple) after brief incubations with an anti-biotin IgG-horseradish peroxidase conjugate and a precipitable horseradish peroxidase substrate. Testing of PCR fragments is completed in 30–40 min. Up to several hundred SNPs can be assayed on a 36-mm² chip, and SNP scoring can be done by eye or with a simple digital-camera system. This assay is extremely robust, exhibits high sensitivity and specificity, and is format-flexible and economical. In studies of mutations associated with risk for venous thrombosis and genotyping/haplotyping of African-American samples, we document high-fidelity analysis with 0 misassignments in 500 assays performed in duplicate.

Footnotes

↵ †† To whom correspondence should be addressed. E-mail: david.ward{at}yale.edu.
↵ § R.J. has financial interest in and is an employee of ThermoBiostar.
Abbreviations: SNP, single-nucleotide polymorphism; HRP, horseradish peroxidase; VTE, venous thromboembolism; RFLP, restriction fragment length polymorphism; MTHFR, methylene tetrahydrofolate.