Large-scale human genotyping requires technologies with a minimal number of steps, high accuracy, and the ability to automate at a reasonable cost. In this regard, we have developed a rapid, cost-effective readout method for single nucleotide polymorphism (SNP) genotyping that combines an easily automatable single-tube allele-specific primer extension (ASPE) with an efficient high throughput flow cytometric analysis performed on a Luminex 100 flow cytometer. This robust technique employs an ASPE reaction using PCR-derived target DNA containing the SNP and a pair of synthetic complementary capture probes that differ at their 3' end-nucleotide defining the alleles. Each capture probe has been synthesized to contain a unique 25-nucleotide identifying sequence (ZipCode) at its 5' end. An array of fluorescent microspheres, covalently coupled with complementary ZipCode sequences (cZipCodes), was hybridized to biotin-labeled ASPE reaction products, sequestering them for flow cytometric analysis. ASPE offers both an advantage of streamlining the SNP analysis protocol and an ability to perform multiplex SNP analysis on any mixture of allelic variants. All steps of the assay are simple additions of the solutions, incubations, and washes. This technique was used to assay 15 multiplexed SNPs on human chromosome 12 from 96 patients. Comparison of the microsphere-based ASPE assay results to gel-based oligonucleotide ligation assay (OLA) results showed 99.2% agreement in genotype assignments. In addition, the microsphere-based multiplex SNPs assay system was adapted for the identification of bacterial samples by both ASPE and single base chain extension (SBCE) assays. A series of probes designed for different variable sites of bacterial 16S rDNA permitted multiplex analysis and generated species- or genus-specific patterns. Seventeen bacterial species representing a broad range of gram-negative and gram-positive bacteria were analyzed within 16 variable sites of 16S rDNA sequence. The results were consistent with the published sequences and confirmed by direct DNA sequencing.

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.

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. [Primer sequences used in this study are available as online supplementary materials at www.genome.org.]

BACKGROUND:We have developed a rapid, high throughput method for single nucleotide polymorphism (SNP) genotyping that employs an oligonucleotide ligation assay (OLA) and flow cytometric analysis of fluorescent microspheres.METHODS:A fluoresceinated oligonucleotide reporter sequence is added to a "capture" probe by OLA. Capture probes are designed to hybridize both to genomic "targets" amplified by polymerase chain reaction and to a separate complementary DNA sequence that has been coupled to a microsphere. These sequences on the capture probes are called "ZipCodes". The OLA-modified capture probes are hybridized to ZipCode complement-coupled microspheres. The use of microspheres with different ratios of red and orange fluorescence makes a multiplexed format possible where many SNPs may be analyzed in a single tube. Flow cytometric analysis of the microspheres simultaneously identifies both the microsphere type and the fluorescent green signal associated with the SNP genotype.RESULTS:Application of this methodology is demonstrated by the multiplexed genotyping of seven CEPH DNA samples for nine SNP markers located near the ApoE locus on chromosome 19. The microsphere-based SNP analysis agreed with genotyping by sequencing in all cases.CONCLUSIONS:Multiplexed SNP genotyping by OLA with flow cytometric analysis of fluorescent microspheres is an accurate and rapid method for the analysis of SNPs.