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Robust and Accurate Single Nucleotide Polymorphism Genotyping by Dynamic Allele Specific Hybridization (DASH)
Dalarna University, School of Health and Social Studies, Medical Science.
2001 (English)In: SNP 2000 : Third International Meeting on Single Nucleotide polymorphism and Complex Genome Analysis, Taos, New Mexico, USA, 2001Conference paper, Published paper (Refereed) Published
Abstract [en]

Dynamic allele specific hybridization (DASH) [1] is a method for genotyping single nucleotide polymorphisms (SNPs), insertion/deletions (indels), and other subtle sequence variations. Allele discrimination is based upon the detection of stability differences in duplex DNAs (oligonucleotide probes hybridized to PCR product targets) involving fully matched or allelic-mismatched structures. The procedure involves PCR amplification of a short sequence spanning a variant position. One PCR primer is biotinylated allowing subsequent facile affinity (streptavidin) capture of one strand of the PCR product onto a solid support. An unlabelled oligonucleotide probe, complementary to one of the alleles, is then annealed to this target DNA at low temperature in the presence of a double-strand specific intercalating dye. Samples are then steadily heated through a temperature range while continually monitoring fluorescence, i.e., the amount of duplexed probe-target material. A melting temperature profile is thus derived, indicating the presence of perfectly matched or subtly mismatch probe-target duplexes, or a heterozygous mixture of the two. An extensive testing and validation study was performed in order to maximize the utility of DASH. This study involved design and application of DASH assays for 89 randomly selected SNPs. Nonoptimized assay designs (worst-case scenario) resulted in 79 functional assays from which genotypes could be clearly determined. Statistical analysis of many variables in these assays revealed that the presence or absence of secondary structures in the target sequence was a critical factor for DASH performance. By identifying key bases involved in secondary structure formation and then by altering these in the PCR primers, the formation of secondary structure could be minimized. Six of the failed DASH assays were redesigned following this strategy, and full recovery of all six assays was achieved (bestcase scenario). Subsequent replication and method comparison studies demonstrated that DASH achieves a genotyping accuracy better than 99.9%, and a reproducibility of 100%. No post-PCR assay failures have yet been encountered. These findings, along with intrinsic low cost (less than 25c/genotype) and high flexibility, validate DASH as an effective procedure for SNP genotyping.

Place, publisher, year, edition, pages
Taos, New Mexico, USA, 2001.
Series
Human mutation, ISSN 1098-1004 ; 4
Identifiers
URN: urn:nbn:se:du-2302OAI: oai:dalea.du.se:2302DiVA, id: diva2:521659
Conference
SNP 2000 : Third International Meeting on Single Nucleotide polymorphism and Complex Genome Analysis , Taos, New Mexico, USA, 2001
Available from: 2006-09-19 Created: 2006-09-19 Last updated: 2012-04-24Bibliographically approved

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CiteExportLink to record
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Citation style
  • apa
  • ieee
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