Perlegen Assay Design Protocol
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Perlegen Assay Design Protocol
Description: Long-range PCR assays were designed using Oligo primer design software
(Molecular Biology Insights). Primers were selected to have similar
stringency and to map uniquely to NCBI Build 33. From a collection of
all suitable candidate primers, we used custom software to select a
minimum spanning set having maximum coverage with minimal overlap
between adjacent amplicons.
Genotyping arrays of 25-bp oligonucleotides were designed as four sets
of 20 features (80 features per SNP), corresponding to forward and
reverse strand tilings of sequences complementary to each of two SNP
alleles. A set of 20 features consisted of five sets of 4 features
where the location of the SNP within the oligonucleotide varies from
position 11 to position 15. A set of four features consisted of
sequences where A, C, T, or G is substituted at position 13. Thus,
each set of four features provided one perfect match to the sequence
of the corresponding SNP allele and three features with a single-base
mismatch for that allele. Mismatch probes were used to measure
background, and by comparison with the signal for the perfect match
probes, to detect the presence or absence of a specific PCR product.
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