RESUMEN
We had been unsuccessful to amplify desired nucleotide sequences from various environmental DNA samples by using the inverse polymerase chain reaction (IPCR) technique, most probably because the copy numbers of target DNA sequences had been quite low. To enrich the target DNA sequences prior to IPCR, a rolling-circle amplification was used with a site-specific primer containing locked nucleic acids (LNAs). This pre-amplified IPCR (PAI-PCR) method increased the sensitivity of PCR almost 10,000 times compared with the standard IPCR in model experiments using Escherichia coli. We then applied the PAI-PCR method to isolate glycosyl hydrolase genes from DNAs extracted from vermiform appendixes of horses and termite guts. The flanking sequences of the target genes were amplified and cloned successfully using PAI-PCR, whereas standard IPCR resulted in no amplification.
Asunto(s)
Técnicas de Amplificación de Ácido Nucleico/métodos , Reacción en Cadena de la Polimerasa/métodos , Animales , Cartilla de ADN , ADN Bacteriano/genética , Escherichia coli/genética , Tracto Gastrointestinal/enzimología , Genes , Glicósido Hidrolasas/genética , Caballos , Isópteros , Datos de Secuencia Molecular , Oligonucleótidos , Sensibilidad y EspecificidadRESUMEN
We reevaluated the bias toward a 1:1 ratio of products in multitemplate PCR used in ecological studies and showed that the template reannealing at the annealing step would not cause the bias; however, the preferential homoduplex formation during temperature decrease from denaturation to annealing step would cause the bias.
Asunto(s)
Sesgo , Reacción en Cadena de la Polimerasa/métodos , Moldes Genéticos , Actinomycetales/genética , Desnaturalización de Ácido Nucleico , Hibridación de Ácido Nucleico , Pseudomonas fluorescens/genética , ARN Ribosómico 16S/genética , TemperaturaRESUMEN
Fluorescence resonance energy transfer (FRET) is a simple procedure for detecting specific DNA sequences, and is therefore used in many fields. However, the cost is relatively high, because FRET-based methods usually require fluorescent probes. We have designed a cost-effective way of using FRET, and developed a novel approach for the genotyping of single nucleotide polymorphisms (SNPs) and allele frequency estimation. The key feature of this method is that it uses a DNA-binding fluorogenic molecule, SYBR Green I, as an energy donor for FRET. In this method, single base extension is performed with dideoxynucleotides labeled with an orange dye and a red dye in the presence of SYBR Green I. The dyes incorporated into the extended products accept energy from SYBR Green I and emit fluorescence. We have validated the method with ten SNPs, which were successfully discriminated by end-point measurements of orange and red fluorescence intensity in a microplate fluorescence reader. Using a mixture of homozygous samples, we also confirmed the potential of this method for estimation of allele frequency. Application of this strategy to large-scale studies will reduce the time and cost of genotyping a vast number of SNPs.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Frecuencia de los Genes , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ADN/métodos , Benzotiazoles , Diaminas , Transferencia Resonante de Energía de Fluorescencia/economía , Colorantes Fluorescentes/química , Genotipo , Humanos , Compuestos Orgánicos/química , Reacción en Cadena de la Polimerasa , Quinolinas , Análisis de Secuencia de ADN/economía , TemperaturaRESUMEN
Fluorescence resonance energy transfer (FRET) is one of the most powerful and promising tools for single nucleotide polymorphism (SNP) genotyping. However, the present methods using FRET require expensive reagents such as fluorescently labeled oligonucleotides. Here, we describe a novel and cost-effective method for SNP genotyping using FRET. The technique is based on allele-specific primer extension using mononucleotides labeled with a green dye and a red dye. When the target DNA contains the sequence complementary to the primer, extension of the primer incorporates the green and red dye-labeled nucleotides into the strand, and red fluorescence is emitted by FRET. In contrast, when the 3' end nucleotide of the primer is not complementary to the target DNA, there is no extension of the primer, or FRET signal. Therefore, discrimination among genotypes is achieved by measuring the intensity of red fluorescence after the extension reaction. We have validated this method with 11 SNPs, which were successfully determined by end-point measurements of fluorescence intensity. The new strategy is simple and cost-effective, because all steps of the preparation consist of simple additions of solutions and incubation, and the dye-labeled mononucleotides are applicable to all SNP analyses. This method will be suitable for large-scale genotyping.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Nucleótidos/genética , Polimorfismo de Nucleótido Simple/genética , Alelos , Carbocianinas/química , ADN/química , ADN/genética , Nucleótidos de Desoxicitosina/química , Nucleótidos de Desoxicitosina/genética , Nucleótidos de Desoxiuracil/química , Nucleótidos de Desoxiuracil/genética , Fluoresceínas/química , Colorantes Fluorescentes/química , Frecuencia de los Genes , Genotipo , Nucleótidos/química , Reacción en Cadena de la Polimerasa/métodos , Receptor de Serotonina 5-HT2A/genética , Reproducibilidad de los ResultadosRESUMEN
A two-membered coculture of strains KYM-7 and KYM-8, identified as Cellulomonas cellulans and Agrobacterium tumefaciens, respectively, produced a large amount of an extracellular polysaccharide, designated APK-78, from starch. Each strain in pure culture produced only very little amount of polysaccharide from starch; the coexistence of the two strains from the early stage of cultivation was indispensable for a large amount of polysaccharide to be produced. The polysaccharide APK-78 was acidic and composed of glucose, galactose, succinic acid, and pyruvic acid with a molar ratio of 8.1:1.0:1.7:1.0, indicating that it is a succinoglycan type of polysaccharide.