Black W C, Gorrochotegui-Escalante N, Duteau N M
Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523-1682, USA.
J Med Entomol. 2006 Mar;43(2):238-47. doi: 10.1603/0022-2585(2006)043[0238:holaha]2.0.co;2.
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.
大多数单核苷酸多态性（SNP）检测需要昂贵的设备和试剂。寡核苷酸连接分析（OLA）是一种低成本的SNP检测方法，它检测生物素化的“等位基因特异性检测器”与3'荧光素标记的“报告”寡核苷酸之间的连接。除非3'检测器核苷酸与SNP核苷酸互补，否则不会发生连接。最初的OLA使用化学变性和中和。加热OLA（HOLA）则使用热稳定连接酶以及变性和杂交循环进行连接和SNP检测。对于双等位基因SNP，每个基因型的成本约为1.25美元；对于三等位基因SNP，每个基因型的成本约为1.75美元。我们展示了HOLA在埃及伊蚊（L.）早期胰蛋白酶和丰富胰蛋白酶基因座以及冈比亚按蚊（冈比亚按蚊指名亚种）α-甘油磷酸脱氢酶基因座SNP检测中的开发过程。
