Reliable method for generating double-stranded DNA vectors containing site-specific base modifications
Cells of all living organisms are continuously exposed to physical and chemical agents that damage DNA and alter the integrity of their genomes. Despite the relatively high efficiency of the different repair pathways, some lesions remain in DNA when it is replicated or transcribed. Lesion bypass by...
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Published in | BioTechniques Vol. 37; no. 5; pp. 760 - 766 |
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Main Authors | , |
Format | Journal Article |
Language | English |
Published |
England
Future Science Ltd
01.11.2004
Eaton Publishing |
Subjects | |
Online Access | Get full text |
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Summary: | Cells of all living organisms are continuously exposed to physical and chemical agents that damage DNA and alter the integrity of their genomes. Despite the relatively high efficiency of the different repair pathways, some lesions remain in DNA when it is replicated or transcribed. Lesion bypass by DNA and RNA polymerases has been the subject of numerous investigations. However, knowledge of the in vivo mechanism of transcription lesion bypass is very limited because no robust methodology is available. Here we describe a protocol based on the synthesis of a complementary strand of a circular, single-stranded DNA molecule, which allows for the production of large amounts of double-stranded DNA containing a lesion at a specific position in a transcribed sequence. Such constructs can subsequently be used for lesion bypass studies in vivo by RNA polymerase and to ascertain how these events can be affected by the genetic background of the cells. |
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ISSN: | 0736-6205 1940-9818 |
DOI: | 10.2144/04375ST01 |