Heated Oligonucleotide Ligation Assay (HOLA): An Affordable Single Nucleotide Polymorphism Assay

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 ≈US$1.25 with two-allele SNPs or ≈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 α -glycerophosphate dehydrogenase locus in the mosquito Anopheles gambiae s.s.