Genotyping

The GeneChip Mapping Assay genotypes up to 1 million human single nucleotide polymorphisms (SNPs) on a single array, using a single polymerase chain reaction (PCR) primer. The GeneChip Mapping Assay is a highly parallel genotyping platform that takes advantage of GeneChip brand microarrays by coupling a highly reproducible generic sample preparation method with allele-specific hybridization. About 250 ng of genomic DNA is digested with one of two restriction enzymes and ligated to adaptors recognizing the cohesive four base overhangs. All fragments resulting from restriction enzyme digestion, regardless of size, are substrates for adaptor ligation. A generic primer, which recognizes the adaptor sequence, is used to amplify ligated DNA fragments and PCR conditions are optimized to preferentially amplify fragments in the 250-1000 bp size range. The amplified DNA is labeled and hybridized to GeneChip arrays. The arrays are washed and stained on a GeneChip fluidics station and scanned on a GeneChip Scanner 3000.

The biochemical approach relies on the position of restriction endonuclease sites in the genome and the position of resulting 20 to 1000 bp size fragments; therefore, the genome-wide distribution of SNPs in the GeneChip Mapping 20K 2.0 Array is random, but not completely uniform. Gaps are most frequently associated with telomeres and centromeres due to the paucity of SNPs discovered by The SNP Consortium (TSC) in these regions.

Principles of Allele-Specific Hybridization on GeneChip Probe Arrays

Allele-specific hybridization (ASH) is a method of allele discrimination in genotyping. By synthesizing probes on the array corresponding to both of the two possible alleles at each SNP and hybridizing the target to the array, we can determine whether a SNP is AA, AB, or BB by analyzing the resulting signal from the allele-specific probes. Affymetrix synthesize 25-mer probes corresponding to a perfect match of the A allele sequence (PMA) and to the perfect match of the B allele sequence (PMB). In addition to this, to determine specificity in binding, a mismatch probe is synthesized for each allele (MMA and MMB). The mismatch probe differs from perfect match probe by a single base substitution in the center. In fact, for each SNP we tile 40 different 25 bp oligonucleotides, each with a slight variation in perfect matches, mismatches, and flanking sequence around the SNP.