Flow Cytometric Platform for High-Throughput Single Nucleotide Polymorphism Analysis

Abstract

We have developed a rapid, cost-effective, high-throughput readout for single nucleotide polymorphism (SNP) genotyping using flow cytometric analysis performed on a Luminex 100 flow cytometer. This robust technique employs a PCR-derived target DNA containing the SNP, a synthetic SNP-complementary ZipCode-bearing capture probe, a fluorescent reporter molecule, and a thermophilic DNA polymerase. An array of fluorescent microspheres, covalently coupled with complementary ZipCode sequences (cZipCodes), was hybridized to the reaction products and sequestered them for flow cytometric analysis. The single base chain extension (SBCE) reaction was used to assay 20 multiplexed SNPs for 633 patients in 96-well format. Comparison of the microsphere-based SBCE assay results to gel-based oligonucleotide ligation assay (OLA) results showed 99.3% agreement in genotype assignments. Substitution of direct-labeled R6G dideoxynucleotide with indirect-labeled phycoerythrin dideoxynucleotide enhanced signal five- to tenfold while maintaining low noise levels. A new assay based on allele-specific primer extension (ASPE) was validated on a set of 15 multiplexed SNPs for 96 patients. ASPE offers both the advantage of streamlining the SNP analysis protocol and the ability to perform multiplex SNP analysis on any mixture of allelic variants.

Pazopanib-induced hyperbilirubinemia is associated with Gilbert's syndrome UGT1A1 polymorphism

“…In the rare instance when a patient had a TA-repeat number that was not 6 or 7 (o1%), the genotype call for that patient was treated as missing data. The remaining polymorphisms were genotyped using Illumina (San Diego, CA, USA) GoldenGate platform (Fan et al, 2003), a single base chain extension assay modified by GlaxoSmithKline (Research Triangle Park, NC, USA) (Taylor et al, 2001), TaqMan SNP Genotyping assays (Applied Biosystems, Foster City, CA, USA; Livak et al, 1995), or sequencing.…”

Variants in the gene encoding C3 are associated with asthma and related phenotypes among African Caribbean families

“…These additional SNPs were genotyped by flow cytometric analysis performed on a Luminext 100 flow cytometer as described elsewhere. 30 After all SNPs were genotyped and analyzed, a nonsynonymous SNP (rs11569472) that fell within the most significant (five-SNP) haplotype (see Results and Discussion) was genotyped by the TaqMan probe-based 5 0 nuclease assay using Assays by Designt (Applied Biosystems, Foster City, CA, USA). However, this marker was not polymorphic in 64 founders and was therefore dropped from further studies.…”

Single nucleotide polymorphism genotyping: biochemistry, protocol, cost and throughput

“…Except for clinical applications, accuracy and sensitivity seldom are an issue, as the accuracy and sensitivity of all commercially available techniques have been tested and verified by many independent groups. For example, there are many reports for SBE with MS detection, 12,16,17,20,76,95 SBE with microarray 23 and microbeads [96][97][98][99] detection, the FP-TDI, [100][101][102] the TaqMan assay, 64,88,103 pyrosequencing, [104][105][106] the Invader assay 46,107-109 and ligation-based methods. 53,84,110,111 The issue becomes more salient when users intend to try out newly developed but not widely used methods.…”