A Microsphere-Based Assay for Multiplexed Single Nucleotide Polymorphism Analysis Using Single Base Chain Extension

Abstract

A rapid, high throughput readout for single-nucleotide polymorphism (SNP) analysis was developed employing single base chain extension and cytometric analysis of an array of fluorescent microspheres. An array of fluorescent microspheres was coupled with uniquely identifying sequences, termed complementary ZipCodes (cZipCodes), which allowed for multiplexing possibilities. For a given assay, querying a polymorphic base involved extending an oligonucleotide containing both a ZipCode and a SNP-specific sequence with a DNA polymerase and a pair of fluoresceinated dideoxynucleotides. To capture the reaction products for analysis, the ZipCode portion of the oligonucleotide was hybridized with its cZipCodes on the microsphere. Flow cytometry was used for microsphere decoding and SNP typing by detecting the fluorescein label captured on the microspheres. In addition to multiplexing capability, the ZipCode system allows multiple sets of SNPs to be analyzed by a limited set of cZipCode-attached microspheres. A standard set of non-cross reactive ZipCodes was established experimentally and the accuracy of the system was validated by comparison with genotypes determined by other technologies. From a total of 58 SNPs, 55 SNPs were successfully analyzed in the first pass using this assay format and all 181 genotypes across the 55 SNPs were correct. These data demonstrate that the microsphere-based single base chain extension (SBCE) method is a sensitive and reliable assay. It can be readily adapted to an automated, high-throughput genotyping system.

BARCSoySNP23: a panel of 23 selected SNPs for soybean cultivar identification

“…The SBE protocol used here was essentially that described by Chen et al (2000) which involves (a) an initial PCR amplification of the SNP-containing genomic fragment, before (b) using a SNP-specific SBE oligonucleotide capture probe designed to anneal next to the SNP, (c) using a biotinlabeled dideoxy terminator in the extension step, and finally (d) using microsphere-based flow cytometry for SNP allele identification. Details of each step follow:…”

“…The hybridization of the capture probe to the microsphere was conducted as described by Chen et al (2000). Hybridization procedures for binding the ten extended SBE capture probes (with their ZipCode) to their ten specific microspheres (with the corresponding anti-ZipCode) were carried out in a 50 ll total reaction volume, including 49.4 ll 1· TMAC [3 M tetramethylammonium chloride, 50 mM Tris-HCl (pH 8.0), 4 mM EDTA (pH 8.0), 0.1% Sarkosyl] and 0.06 ll microsphere with 3,000 microspheres of each type in the reaction mix.…”

The emerging importance of DNA mapping and other comprehensive screening techniques, as tools to identify new drug targets and as a means of (cancer) therapy personalisation

“…Analysis by a flow cytometer of the bead-associated fluorescence determines the nucleotide at the SNP site. Sequence tags or 'ZipCodes' have also been used for SNP analysis by OLA [58] and single base extension [59] assays. A previous method involves a fluoresceinated oligonucleotide reporter sequence added to a 'capture' probe by OLA.…”

“…Flow cytometric analysis of the microspheres simultaneously identifies both the microsphere type and the fluorescent green signal associated with the SNPs genotype [58]. In the single base extension assay, ZipCode at the 5′ end of the capture fluorescent oligonucleotide probe allows the DNA polymerase reaction product to be captured by its complementary sequence (cZipCode) which has been coupled to a specific fluorescent microsphere [59]. Both assays can be used with standard flow cytometers (e.g., B-D FACSCalibur) with individual loading, or with the much less expensive bead-designed Luminex system.…”

Rapid characterization of DNA oligomers and genotyping of single nucleotide polymorphism using nucleotide-specific mass tags

“…This new research era requires the urgent development of accurate, high throughput technologies for determining many genetic variations or polymorphic sites in the human genome (2)(3)(4). About 80% of these polymorphic sites are single nucleotide polymorphisms (SNPs) with an abundance of one in every 100-300 bp (5)(6)(7)(8). Because of their associations with many genetic diseases, the identification of SNPs has high priority in functional genomic studies.…”