King, P; Goodbourn, S
(1992)
A method for sequence-specific deletion mutagenesis.
NUCLEIC ACIDS RESEARCH, 20 (5).
1039 - 1044 (6).
ISSN 0305-1048
https://doi.org/10.1093/nar/20.5.1039
SGUL Authors: Goodbourn, Stephen Edward
Abstract
We describe a novel procedure for the construction of deletion mutants. Existing exonuclease-based protocols are efficient at producing randomly positioned deletions over large regions of DNA, but are of limited use in targetted mutagenesis due to their inherent sequence-specificity. We have taken advantage of the Exonuclease Ill-resistant nature of α-thlo-dNTPs, incorporated into the target DNA template by a primer extension reaction, to generate base-specific α-thio-dNTP terminated products. Following removal of the 5′ overhanging strands, the products can be cloned to generate a nested set of deletions with single base-pair increments. We demonstrate the utility of this technique by isolating multiple deletions over a 40bp region of the human β-interferon promoter.
Item Type: | Article |
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Additional Information: | PubMed ID: 1549464 |
Keywords: | Base Sequence, Cloning, Molecular, Deoxyribonucleotides, Humans, Interferon-beta, Molecular Sequence Data, Mutagenesis, Site-Directed, Promoter Regions, Genetic, Thionucleotides, Science & Technology, Life Sciences & Biomedicine, Biochemistry & Molecular Biology, BETA-INTERFERON GENE, KINETOPLAST DNA FRAGMENT, EXONUCLEASE-III, NUCLEOTIDE, EXPRESSION, NUCLEASES, PROMOTER, ELEMENT |
SGUL Research Institute / Research Centre: | Academic Structure > Infection and Immunity Research Institute (INII) |
Journal or Publication Title: | NUCLEIC ACIDS RESEARCH |
ISSN: | 0305-1048 |
Related URLs: | |
Web of Science ID: | WOS:A1992HK00100011 |
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URI: | https://openaccess.sgul.ac.uk/id/eprint/102392 |
Publisher's version: | https://doi.org/10.1093/nar/20.5.1039 |
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