Heated oligonucleotide ligation assay (HOLA): an affordable single nucleotide polymorphism assay

J Med Entomol. 2006 Mar;43(2):238-47. doi: 10.1603/0022-2585(2006)043[0238:holaha]2.0.co;2.

Abstract

Most single nucleotide polymorphism (SNP) detection requires expensive equipment and reagents. The oligonucleotide ligation assay (OLA) is an inexpensive SNP assay that detects ligation between a biotinylated "allele-specific detector" and a 3' fluorescein-labeled "reporter" oligonucleotide. No ligation occurs unless the 3' detector nucleotide is complementary to the SNP nucleotide. The original OLA used chemical denaturation and neutralization. Heated OLA (HOLA) instead uses a thermal stable ligase and cycles of denaturing and hybridization for ligation and SNP detection. The cost per genotype is approximately US$1.25 with two-allele SNPs or approximately US$1.75 with three-allele SNPs. We illustrate the development of HOLA for SNP detection in the Early Trypsin and Abundant Trypsin loci in the mosquito Aedes aegypti (L.) and at the a-glycerophosphate dehydrogenase locus in the mosquito Anopheles gambiae s.s.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Aedes / genetics*
  • Animals
  • Anopheles / genetics*
  • Base Sequence
  • Costs and Cost Analysis / economics
  • Gene Frequency / genetics
  • Genes, Insect / genetics
  • Genetics, Population / economics
  • Genetics, Population / methods*
  • Genotype
  • Glycerolphosphate Dehydrogenase / genetics
  • Hot Temperature
  • Mali
  • Mexico
  • Molecular Biology / economics
  • Molecular Biology / methods*
  • Molecular Sequence Data
  • Oligonucleotides / chemistry
  • Oligonucleotides / metabolism
  • Polymorphism, Single Nucleotide / genetics*
  • Polymorphism, Single-Stranded Conformational

Substances

  • Oligonucleotides
  • Glycerolphosphate Dehydrogenase