RESUMEN
BACKGROUND: To investigate relationships between phenotypes and genotypes is not simple. We propose a phenotype-to-genotype screening strategy and pooled DNA system. As a pilot study of this strategy, we used arbitrarily primed polymerase chain reaction (AP-PCR) in combination with single-stranded DNA conformation polymorphism (SSCP) to screen for genetic polymorphisms associated with longevity. METHODS: Study subjects were separated into 3 age groups, individuals aged >100 years, 90-99 years and 60-69 years. Genomic DNAs were prepared from each individual, pooled to represent the 5 study groups, and then the pooled genomic DNAs were subjected to AP-PCR-SSCP analysis. RESULTS: We found 1 SNP more frequently in senior citizens with longevity. The genotype frequency of the 82133G>A polymorphism of human chromosome 3 clone RP11-61K12 (AC011199) differed significantly (P=0.0189, Fisher's exact test) between older subjects (>90 years) and younger subjects (<70 years). It is noteworthy that the strategy we describe herein was useful for identifying an SNP that showed statistically significant differences in its distribution across the subject groups. CONCLUSIONS: The pooled DNA strategy and quantitative genotype discrimination can also be applied to screening for the relationship between phenotype and genotype more effectively.
Asunto(s)
ADN de Cadena Simple/genética , Longevidad/genética , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo Genético , Adulto , Anciano , Anciano de 80 o más Años , ADN de Cadena Simple/química , Genotipo , Humanos , Persona de Mediana Edad , Conformación de Ácido Nucleico , Fenotipo , Proyectos PilotoAsunto(s)
Germinoma/enzimología , Isoenzimas/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Neoplasias Testiculares/enzimología , Adulto , Carcinoma Embrionario/complicaciones , Carcinoma Embrionario/enzimología , Tumor del Seno Endodérmico/complicaciones , Tumor del Seno Endodérmico/enzimología , Germinoma/complicaciones , Humanos , Isoenzimas/sangre , Isoenzimas/genética , L-Lactato Deshidrogenasa/sangre , L-Lactato Deshidrogenasa/genética , Masculino , Neoplasias del Mediastino/complicaciones , Neoplasias del Mediastino/enzimología , Metilación , Mutación , Rabdomiosarcoma/complicaciones , Rabdomiosarcoma/enzimología , Teratoma/complicaciones , Teratoma/enzimología , Neoplasias Testiculares/complicacionesAsunto(s)
Biomarcadores de Tumor/análisis , ADN Mitocondrial/metabolismo , Neoplasias/diagnóstico , Línea Celular Tumoral , Neoplasias Colorrectales/diagnóstico , Metilación de ADN , Humanos , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-Simple , Neoplasias Gástricas/diagnósticoRESUMEN
BACKGROUND: We developed a rapid, precise, and accurate microarray-based method that uses a three-dimensional platform for detection of mutations. METHODS: We used the PamChip microarray to detect mutations in codons 12 and 13 of K-ras in 15 cell lines and 81 gastric or colorectal cancer tissues. Fluorescein isothiocyanate-labeled PCR products were analyzed with the microarray. We confirmed the microarray results with PCR-single-strand conformation polymorphism (SSCP) analysis and DNA sequencing. RESULTS: We could correctly identify wild-type, heterozygous, and homozygous mutant genotypes with the PamChip microarray in <3.5 h. The array data were consistent with those of PCR-SSCP analysis and DNA sequencing. All 15 cell lines and 80 of 81 clinical cancer specimens (98.8%; 95% confidence interval, 96.4-100%) were genotyped accurately with the microarray, a rate better than that of direct DNA sequencing (38.9%) or SSCP (93.8%). Only one clinical specimen was misdiagnosed as homozygous for the wild-type allele. Densitometric analysis of SSCP bands indicated that the content of the mutant allele in the specimen was approximately 16%. The PamChip microarray could detect mutant alleles representing more than 25% of the SSCP band proportions. Therefore, the limit for detection of mutant alleles by the PamChip microarray was estimated to be 16-25% of the total DNA. CONCLUSIONS: The PamChip microarray is a novel three-dimensional microarray system and can be used to analyze K-ras mutations quickly and accurately. The mutation detection rate was nearly 100% and was similar to that of PCR-SSCP together with sequencing analysis, but the microarray analysis was faster and easier.