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Single-molecule dilution and multiple displacement amplification for molecular haplotyping.

Philip Paul·Josh Apgar
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Abstract

Separate haploid analysis is frequently required for heterozygous genotyping to resolve phase ambiguity or confirm allelic sequence. We demonstrate a technique of single-molecule dilution followed by multiple strand displacement amplification to haplotype polymorphic alleles. Dilution of DNA to haploid equivalency, or a single molecule, is a simple method for separating di-allelic DNA. Strand displacement amplification is a robust method for non-specific DNA expansion that employs random hexamers and phage polymerase Phi29 for double-stranded DNA displacement and primer extension, resulting in high processivity and exceptional product length. Single-molecule dilution was followed by strand displacement amplification to expand separated alleles to microgram quantities of DNA for more efficient haplotype analysis of heterozygous genes.

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AllelesAntigensBacteriophagesDNADNA-Directed DNA PolymeraseElectrophoresis, Polyacrylamide GelGene AmplificationHaplotypesHeterozygoteHot TemperatureHumansIndicator Dilution TechniquesLeukocytesNucleic Acid Amplification TechniquesNucleic Acid HybridizationOligonucleotide Array Sequence AnalysisPolymerase Chain ReactionPolymorphism, Single NucleotideReference StandardsSequence Analysis, DNATemplates, GeneticTime FactorsJournal ArticleTechnical Report

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Philip Paul, Josh Apgar. 2005. Single-molecule dilution and multiple displacement amplification for molecular haplotyping.. https://doi.org/10.2144/05384st01

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