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AFFYMETRIX GENOTYPING
The GeneChip Mapping Assay genotypes up to 50,000 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.
The biochemical fractionation method devised for the GeneChip Mapping Assay
is shown
in Figure 1. About 250 ng of genomic DNA is digested with one of two
restriction enzymes( XbaI or HindIII) 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.
Figure 1. GeneChip
Mapping Assay
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. An example of hybridized array image is shown in
Figure 2.
Figure 2. Example of
Allele-Specific Hybridization with 40 probes/SNP
This information was adapted from the
Affymetrix Mapping Assay Manual.
For
more information on the SNPs chosen for the Mapping Arrays, please see:
GeneChip Human Mapping 10K Array Xba 142 2.0
Data Sheet
GeneChip Human Mapping 100K Array
Data Sheet
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