| | The GoldenGate genotyping assay is based on a highly multiplexed, allele-discriminating, extension reaction. |
| | Two allele-specific oligos (ASOs) and a locus-specific oligo (LSO) are used to query each SNP. |
| | The pooled assay oligos are hybridized to genomic DNA in a carefully controlled process. |
| | ASOs whose 3' end base pairs with the SNP position are efficiently extended by a DNA polymerase. |
| | ASOs whose 3' end mismatches the SNP position are not efficiently extended. |
| | Thus, extension by DNA polymerase imparts allele selectivity to the assay. |
| | The successfully extended ASOs terminate at the 5' phosphate of their cognate LSOs. |
| | Upon addition of DNA ligase, the successfully extended ASOs are ligated to the LSOs. |
| | The ligation process combines information carried by the allele-specific extension product (as determined by the DNA polymerase) with information for hybridization to a bead type in the Sentrix array matrix (through the unique address sequence in each LSO |
| | After thermocycling, the PCR product is captured on a solid support, and single stranded fluorescently labeled material is eluted and hybridized to the Sentrix array matrix. |
| | The unique address sequences in the LSOs will hybridize to complementary sequences on the beads in the Sentrix array matrix. |
| | This last process de-multiplexes the information about the genotype calls generated in solution by separation on a Sentrix array matrix. |
| | Illumina's Sentrix array matrix consists of 96 array bundles configured in a microplate-compatible format. |
| | Each array bundle contains nearly 50,000 individual light-conducting fiber strands.. |
| | The fiber strands are chemically etched to create a microscopic well at the end of each strand. |
| | Most of the wells are filled, each with a single 3-micron bead. Covalently attached to each bead are several hundred thousand copies of an oligonucleotide probe. |
| | Consequently, each SNP genotype determination is the result of data averaged from multiple beads, greatly reducing the possibility of error. |
