RESUMEN
Three DNA extraction protocols were compared for their ability to yield DNA from the leaves of herbarium specimens of nine species from nine genera of the Papilionoideae. We tested two protocols that use classic procedures for lysis and purification with cetyl trimethylammonium bromide (CTAB); a third protocol used a Nucleospin Plant kit. DNA obtained from all three procedures was quantified and tested by PCR. Test results indicated the superiority of one of the CTAB protocols. We made some modifications, developing a protocol that produced high-quality DNA from all nine species. The modification involved the use of a lower EDTA concentration (20 mM instead of 50 mM) and a higher beta-mercaptoethanol concentration (1% instead of 0.4%) in the extraction buffer. The modified protocol avoids the necessity for a second DNA precipitation step. This new CTAB protocol includes the use of 1.4 M NaCl, 20 mM EDTA and 1% beta-mercaptoethanol in the extraction; DNA precipitation time is reduced. A reduction in contaminating metabolites (such as PCR inhibitors) in the sample mixtures and lower costs for reagents are characteristics of this modified protocol; the cost of analysis per sample was lowered, compared to previous options. The quality of DNA was suitable for PCR amplification. This is a practical alternative to more difficult, time-consuming and expensive protocols.
Asunto(s)
ADN de Plantas/aislamiento & purificación , Fabaceae/genética , Genoma de Planta/genética , Biología Molecular/economía , Biología Molecular/métodos , Núcleo Celular/genética , Cloroplastos/genética , ADN Intergénico/genética , ADN de Plantas/genética , Electroforesis en Gel de AgarRESUMEN
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.
Asunto(s)
Aedes/genética , Anopheles/genética , Genética de Población/métodos , Biología Molecular/métodos , Polimorfismo de Nucleótido Simple/genética , Animales , Secuencia de Bases , Costos y Análisis de Costo/economía , Frecuencia de los Genes/genética , Genes de Insecto/genética , Genética de Población/economía , Genotipo , Glicerolfosfato Deshidrogenasa/genética , Calor , Malí , México , Biología Molecular/economía , Datos de Secuencia Molecular , Oligonucleótidos/química , Oligonucleótidos/metabolismo , Polimorfismo Conformacional Retorcido-SimpleRESUMEN
The limited resources of developing countries are forcing them to search for different options to keep up with the accelerating pace of research into genetic medicine. In Mexico, one such option is the Mexican Network of Molecular Biomedicine (MNMB). With the Internet as a means of communication and a source of information, the MNMB aims to provide a program based on cooperation, high-quality service and patient care.