| Fluorescence energy transfer dye-labeled primers for DNA sequencing and analysis. | |
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MedLine Citation:
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PMID: 7753809 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Fluorescent dye-labeled DNA primers have been developed that exploit fluorescence energy transfer (ET) to optimize the absorption and emission properties of the label. These primers carry a fluorescein derivative at the 5' end as a common donor and other fluorescein and rhodamine derivatives attached to a modified thymidine residue within the primer sequence as acceptors. Adjustment of the donor-acceptor spacing through the placement of the modified thymidine in the primer sequence allowed generation of four primers, all having strong absorption at a common excitation wavelength (488 nm) and fluorescence emission maxima of 525, 555, 580, and 605 nm. The ET efficiency of these primers ranges from 65% to 97%, and they exhibit similar electrophoretic mobilities by gel electrophoresis. With argon-ion laser excitation, the fluorescence of the ET primers and of the DNA sequencing fragments generated with ET primers is 2- to 6-fold greater than that of the corresponding primers or fragments labeled with single dyes. The higher fluorescence intensity of the ET primers allows DNA sequencing with one-fourth of the DNA template typically required when using T7 DNA polymerase. With single-stranded M13mp18 DNA as the template, a typical sequencing reaction with ET primers on a commercial sequencer provided DNA sequences with 99.8% accuracy in the first 500 bases. ET primers should be generally useful in the development of other multiplex DNA sequencing and analysis methods. |
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Authors:
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J Ju; C Ruan; C W Fuller; A N Glazer; R A Mathies |
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Publication Detail:
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Type: Comparative Study; Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S. |
Journal Detail:
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Title: Proceedings of the National Academy of Sciences of the United States of America Volume: 92 ISSN: 0027-8424 ISO Abbreviation: Proc. Natl. Acad. Sci. U.S.A. Publication Date: 1995 May |
Date Detail:
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Created Date: 1995-06-16 Completed Date: 1995-06-16 Revised Date: 2010-09-13 |
Medline Journal Info:
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Nlm Unique ID: 7505876 Medline TA: Proc Natl Acad Sci U S A Country: UNITED STATES |
Other Details:
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Languages: eng Pagination: 4347-51 Citation Subset: IM |
Affiliation:
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Department of Chemistry, University of California, Berkeley 94720, USA. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Bacteriophage M13 Bacteriophage T7 Base Sequence* DNA / chemistry* DNA Primers* / chemical synthesis, chemistry DNA, Viral / chemistry* DNA-Directed DNA Polymerase Energy Transfer Indicators and Reagents Molecular Sequence Data Spectrometry, Fluorescence Templates, Genetic |
| Chemical | |
Reg. No./Substance:
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0/DNA Primers; 0/DNA, Viral; 0/Indicators and Reagents; 9007-49-2/DNA; EC 2.7.7.-/bacteriophage T7 induced DNA polymerase; EC 2.7.7.7/DNA-Directed DNA Polymerase |
| Comments/Corrections | |
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