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Fluorescent signatures for variable DNA sequences.
Rice, John E; Reis, Arthur H; Rice, Lisa M; Carver-Brown, Rachel K; Wangh, Lawrence J.
Afiliación
  • Rice JE; Department of Biology, Brandeis University, Waltham, MA 02454, USA.
Nucleic Acids Res ; 40(21): e164, 2012 Nov.
Article en En | MEDLINE | ID: mdl-22879378
ABSTRACT
Life abounds with genetic variations writ in sequences that are often only a few hundred nucleotides long. Rapid detection of these variations for identification of genetic diseases, pathogens and organisms has become the mainstay of molecular science and medicine. This report describes a new, highly informative closed-tube polymerase chain reaction (PCR) strategy for analysis of both known and unknown sequence variations. It combines efficient quantitative amplification of single-stranded DNA targets through LATE-PCR with sets of Lights-On/Lights-Off probes that hybridize to their target sequences over a broad temperature range. Contiguous pairs of Lights-On/Lights-Off probes of the same fluorescent color are used to scan hundreds of nucleotides for the presence of mutations. Sets of probes in different colors can be combined in the same tube to analyze even longer single-stranded targets. Each set of hybridized Lights-On/Lights-Off probes generates a composite fluorescent contour, which is mathematically converted to a sequence-specific fluorescent signature. The versatility and broad utility of this new technology is illustrated in this report by characterization of variant sequences in three different DNA targets the rpoB gene of Mycobacterium tuberculosis, a sequence in the mitochondrial cytochrome C oxidase subunit 1 gene of nematodes and the V3 hypervariable region of the bacterial 16 s ribosomal RNA gene. We anticipate widespread use of these technologies for diagnostics, species identification and basic research.
Asunto(s)

Texto completo: 1 Colección: 01-internacional Banco de datos: MEDLINE Asunto principal: Variación Genética / ADN / Sondas de Ácido Nucleico / Reacción en Cadena de la Polimerasa / Colorantes Fluorescentes Tipo de estudio: Prognostic_studies Idioma: En Revista: Nucleic Acids Res Año: 2012 Tipo del documento: Article País de afiliación: Estados Unidos

Texto completo: 1 Colección: 01-internacional Banco de datos: MEDLINE Asunto principal: Variación Genética / ADN / Sondas de Ácido Nucleico / Reacción en Cadena de la Polimerasa / Colorantes Fluorescentes Tipo de estudio: Prognostic_studies Idioma: En Revista: Nucleic Acids Res Año: 2012 Tipo del documento: Article País de afiliación: Estados Unidos