RESUMO
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.
Assuntos
Aedes/genética , Anopheles/genética , Genética Populacional/métodos , Biologia Molecular/métodos , Polimorfismo de Nucleotídeo Único/genética , Animais , Sequência de Bases , Custos e Análise de Custo/economia , Frequência do Gene/genética , Genes de Insetos/genética , Genética Populacional/economia , Genótipo , Glicerolfosfato Desidrogenase/genética , Temperatura Alta , Mali , México , Biologia Molecular/economia , Dados de Sequência Molecular , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Polimorfismo Conformacional de Fita SimplesRESUMO
Many applications in insect genetics require repeated analyses on individual organisms. These include population genetic and genomics, linkage mapping, molecular systematics and map-based positional cloning. However, using the polymerase chain reaction, a limited number of analyses are possible with DNA isolated from whole bodies or parts of small or preserved specimens. We describe the optimization of a new technique, Multiple Displacement Amplification, for 100-400-fold amplification of whole mosquito genomes. We demonstrate that MDA amplifies genomic DNA directly from an adult leg or from organisms as small as a first instar mosquito larva. Genetic polymorphisms revealed at individual loci are the same whether using the original genomic DNA or MDA DNA as a template.
Assuntos
Aedes/genética , Genes de Insetos/genética , Reação em Cadeia da Polimerase/métodos , Animais , DNA/análise , DNA/genética , Feminino , Técnicas Genéticas , Genoma , Polimorfismo Genético , Polimorfismo Conformacional de Fita SimplesRESUMO
A population genetic analysis of gene flow was conducted among 10 Aedes aegypti collections from seven cities along the northeastern coast of Mexico. Four collections were made from Monterrey to examine local patterns of gene flow. Markers included 60 random amplified polymorphic DNA (RAPD) loci amplified by the polymerase chain reaction and single strand conformation polymorphism analysis of variation in a 387-basepair region of the NADH dehydrogenase subunit 4 from the mitochondrial DNA (mtDNA). Seven mitochondrial haplotypes were detected and phylogenetic analysis identified two well-supported clades. Regression analysis of geographic distances and pairwise FST estimated from RAPD markers indicated that populations are isolated by distance and that free gene flow occurs among collections within 90-250 km. Isolation by distance was not detected using mtDNA haplotypes. The Nuevo Laredo collection had unique RAPD and mtDNA haplotype frequencies and reduced heterozygosity suggesting that few mosquitoes established this population.
Assuntos
Aedes/genética , Dengue/transmissão , Frequência do Gene/genética , Variação Genética/genética , Insetos Vetores/genética , Febre Amarela/transmissão , Aedes/química , Animais , Primers do DNA/química , DNA Mitocondrial/química , DNA Mitocondrial/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Feminino , Haplótipos , Humanos , Insetos Vetores/química , México , Filogenia , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Técnica de Amplificação ao Acaso de DNA Polimórfico , Análise de Regressão , Análise de Sequência de DNARESUMO
Field trials of the predacious copepod Mesocyclops longisetus Thiubaud were conducted in northeastern Mexico to determine the effectiveness of this species to control larval Aedes aegypti (L.) populations and to survive and reproduce in nature. Groups of 200, 50, and 50 ovigerous M. longisetus females were inoculated into 200-liter metal drums, discarded tires, and cemetery flower vases, respectively, which are 3 of the more important Aedes breeding sites in this area. Larvae were sampled at 15-d intervals, and total surviving cyclops were collected at the end of the study, 120 d later. Community participation was solicited through a simple training program on copepod rescue before drum cleaning and facilitated by the addition of a drum marker to remind residents of copepod presence. Results showed good cooperation and after 4 mo all peridomestic drums, still supported variable numbers of cyclopoids. Average of larvae reduction was 37.5% for drums, 67.5% for flower vases, and 40.9% for tires. This study shows difficulties of using cyclopoids for tires and vases in areas where prolonged dry seasons desiccated these habitats and reduced copepod survival.
