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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...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2012
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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. |
format | Online Article Text |
id | pubmed-3505974 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2012 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
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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