| | The Illumina® Infinium® I Assay enables unlimited multiplexing from 10,000 to hundreds of thousands of SNPs per sample, dependent only on the number of features (bead types) on the array. This is accomplished by combining whole-genome amplification (WGA) |
| | sample preparation with direct, array-based capture and enzymatic scoring of the SNP loci. Locus discrimination is provided by a combination of sequence-specific hybridization capture and array-based primer extension. Allelic discrimination is provided b |
| | y the single base specificity of the primer extension reaction (ASPE) using two different allele-specific (probe sequence differs only at 3' base) bead types per locus. Signal amplification of the incorporated label further improves the overall signal-to- |
| | noise ratio of the assay. |
| | The assay begins with the amplification of the DNA sample by several thousand fold. This whole-genome amplification process uniformly increases the amount of the DNA sample without introducing large amounts of amplification bias. The amplified DNA sample |
| | is then processed for fragmentation using an endpoint procedure, to avoid overfragmentation of the sample. The DNA sample is then precipitated and resuspended in hybridization buffer. After heat denaturation of the resuspended sample, the sample is applie |
| | d to the Sentrix BeadChip. Genomic loci of interest are selectively captured on the Sentrix BeadChip during hybridization. After hybridization, mis- and nonhybridized DNA is washed away from the BeadChip. Allele-specific extension of the oligos on the Be |
| | adChip, using the captured DNA as a template, incorporates detectable labels on the BeadChip and determines the genotype call for the sample. After staining of the detectable labels, the BeadChip is washed and imaged on the Illumina BeadArray Reader. |
| | The Sentrix Human-1 Genotyping BeadChip is a siliconbased array device composed of 12 individual arrays arranged in parallel on a single slide, enabling a single sample to simultaneously interrogate all the arrays at once. Each individual array in the mat |
| | rix may hold over 24,000 different oligonucleotide probe sequences that are in turn attached to 3-micron beads assembled into the micro-wells of the BeadChip substrate. Because the micro-wells outnumber the probe sequences, multiple copies (average approx |
| | imately 15-30) of each bead type are present in the array. This built-in redundancy improves robustness and measurement precision. The Sentrix BeadChip manufacturing process includes hybridization-based quality control of each array feature, allowing cons |
| | istent production of high-quality, reproducible arrays. |
| | The Infinium I Assay uses Tecan's GenePaint automated slide processor to process BeadChips. The GenePaint system employs a capillary gap flow-through chamber to enable reagent entrapment and exchange over the BeadChip?s active surface. Washing, blocking, |
| | extension, and signal amplification are all performed by simple reagent additions to the flow cell. Addition of a new reagent displaces the entrapped reagent from the flow cell. For maximum flexibility, these additions can be performed either manually or |
| | via the Tecan Genesis robot. The optional automated robotic processing and single-use reagent tube barcoding assure maximum consistency from slide to slide. |
| | BeadChips are imaged using the Illumina BeadArray Reader, a twochannel, 0.8 ?m resolution confocal laser scanner. The BeadArray Reader can automatically (via menu-driven software) scan a BeadChip and create an image file. |
