RESUMEN
The isolation and multiple genotyping of long individual DNA fragments are needed to obtain haplotype information for diploid organisms. Limiting dilution of sample DNA followed by multiple displacement amplification is a useful technique but is restricted to short (<5 kb) DNA fragments. In the current study, a novel modification was applied to overcome these problems. A limited amount of cellular DNA was carefully released from intact cells into a mildly heated alkaline agarose solution and mixed thoroughly. The solution was then gently aliquoted and allowed to solidify while maintaining the integrity of the diluted DNA. Exogenously provided Phi29 DNA polymerase was used to perform consistent genomic amplification with random hexameric oligonucleotides within the agarose gels. Simple heat melting of the gel allowed recovery of the amplified materials in a solution of the polymerase chain reaction (PCR)-ready form. The haplotypes of seven SNPs spanning 240 kb of the DNA surrounding the human ATM gene region on chromosome 11 were determined for 10 individuals, demonstrating the feasibility of this new method.
Asunto(s)
ADN/análisis , ADN/química , Geles , Técnicas de Amplificación de Ácido Nucleico/métodos , Sefarosa , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/genética , Línea Celular , Cromosomas Humanos/genética , ADN/genética , ADN/aislamiento & purificación , ADN Ligasas/genética , Proteínas de Unión al ADN/genética , Genómica , Genotipo , Haplotipos , Humanos , Peso Molecular , Péptido Hidrolasas/genética , Reacción en Cadena de la Polimerasa , Polimorfismo de Nucleótido Simple , Proteínas Serina-Treonina Quinasas/genética , Temperatura , Factores de Tiempo , Proteínas Supresoras de Tumor/genéticaRESUMEN
A visible sensor array system for simultaneous multiple SNP genotyping has been developed using a new plastic base with specific surface chemistry. Discrimination of SNP alleles is carried out by an allele-specific extension reaction using immobilized oligonucleotide primers. The 3'-ends of oligonucleotide primers are modified with a locked nucleic acid to enhance their efficiency in allelic discrimination. Biotin-dUTPs included in the reaction mixture are selectively incorporated into extending primer sequences and are utilized as tags for alkaline phosphatase-mediated precipitation of colored chemical substrates onto the surface of the plastic base. The visible precipitates allow immediate inspection of typing results by the naked eye and easy recording by a digital camera equipped on a commercial mobile phone. Up to four individuals can be analyzed on a single sensor array and multiple sensor arrays can be handled in a single operation. All of the reactions can be performed within one hour using conventional laboratory instruments. This visible genotype sensor array is suitable for "focused genomics" that follows "comprehensive genomics".