title: Single-nucleotide polymorphism genotyping on optical thin-film biosensor chips.
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.
authors.affiliation: Department of Genetics, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510, USA.
authors.forename: Xiao-Bo
authors.initials: XB
authors.lastname: Zhong
authors.forename: Robert
authors.initials: R
authors.lastname: Reynolds
authors.forename: Judith R
authors.initials: JR
authors.lastname: Kidd
authors.forename: Kenneth K
authors.initials: KK
authors.lastname: Kidd
authors.forename: Robert
authors.initials: R
authors.lastname: Jenison
authors.forename: Richard A
authors.initials: RA
authors.lastname: Marlar
authors.forename: David C
authors.initials: DC
authors.lastname: Ward
mesh_terms: D015374:Biosensing Techniques; D044383:Black People / Q000235:genetics; D002886:Chromosomes, Human, Pair 17; D020022:Genetic Predisposition to Disease; D005838:Genotype; D006801:Humans; D055095:Optics and Photonics; D020641:Polymorphism, Single Nucleotide; D012680:Sensitivity and Specificity; D013923:Thromboembolism / Q000235:genetics; D001741:Black or African American