Fluorescent signatures for variable DNA sequences

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, highl...

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Autores principales: Rice, John E., Reis, Arthur H., Rice, Lisa M., Carver-Brown, Rachel K., Wangh, Lawrence J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3505974/
https://www.ncbi.nlm.nih.gov/pubmed/22879378
http://dx.doi.org/10.1093/nar/gks731
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author Rice, John E.
Reis, Arthur H.
Rice, Lisa M.
Carver-Brown, Rachel K.
Wangh, Lawrence J.
author_facet Rice, John E.
Reis, Arthur H.
Rice, Lisa M.
Carver-Brown, Rachel K.
Wangh, Lawrence J.
author_sort Rice, John E.
collection PubMed
description 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.
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spelling pubmed-35059742012-11-26 Fluorescent signatures for variable DNA sequences Rice, John E. Reis, Arthur H. Rice, Lisa M. Carver-Brown, Rachel K. Wangh, Lawrence J. Nucleic Acids Res Methods Online 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. Oxford University Press 2012-11 2012-08-08 /pmc/articles/PMC3505974/ /pubmed/22879378 http://dx.doi.org/10.1093/nar/gks731 Text en © The Author(s) 2012. Published by Oxford University Press. http://creativecommons.org/licenses/by-nc/3.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Methods Online
Rice, John E.
Reis, Arthur H.
Rice, Lisa M.
Carver-Brown, Rachel K.
Wangh, Lawrence J.
Fluorescent signatures for variable DNA sequences
title Fluorescent signatures for variable DNA sequences
title_full Fluorescent signatures for variable DNA sequences
title_fullStr Fluorescent signatures for variable DNA sequences
title_full_unstemmed Fluorescent signatures for variable DNA sequences
title_short Fluorescent signatures for variable DNA sequences
title_sort fluorescent signatures for variable dna sequences
topic Methods Online
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3505974/
https://www.ncbi.nlm.nih.gov/pubmed/22879378
http://dx.doi.org/10.1093/nar/gks731
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