Single-molecule dilution and multiple displacement amplification for molecular haplotyping

Biotechniques. 2005 Apr;38(4):553-4, 556, 558-9. doi: 10.2144/05384ST01.

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.

Publication types

  • Technical Report

MeSH terms

  • Alleles
  • Antigens / genetics
  • Bacteriophages / genetics
  • DNA / blood
  • DNA / genetics*
  • DNA / metabolism
  • DNA-Directed DNA Polymerase
  • Electrophoresis, Polyacrylamide Gel
  • Gene Amplification
  • Haplotypes*
  • Heterozygote
  • Hot Temperature
  • Humans
  • Indicator Dilution Techniques*
  • Leukocytes / immunology
  • Nucleic Acid Amplification Techniques*
  • Nucleic Acid Hybridization
  • Oligonucleotide Array Sequence Analysis
  • Polymerase Chain Reaction
  • Polymorphism, Single Nucleotide
  • Reference Standards
  • Sequence Analysis, DNA
  • Templates, Genetic
  • Time Factors

Substances

  • Antigens
  • DNA
  • DNA-Directed DNA Polymerase