Efficient integration of artificial transposons into plasmid targets in vitro: a useful tool for DNA mapping, sequencing and genetic analysis (original) (raw)
Journal Article
,
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine
Baltimore, MD 21205, USA
Search for other works by this author on:
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine
Baltimore, MD 21205, USA
*To whom correspondence should be addressed
Search for other works by this author on:
Revision received:
21 July 1994
Published:
11 September 1994
Cite
Scott E. Devine, Jef D. Boeke, Efficient integration of artificial transposons into plasmid targets in vitro: a useful tool for DNA mapping, sequencing and genetic analysis, Nucleic Acids Research, Volume 22, Issue 18, 11 September 1994, Pages 3765–3772, https://doi.org/10.1093/nar/22.18.3765
Close
Navbar Search Filter Mobile Enter search term Search
Abstract
We have developed efficient methods for creating artificial transposons and inserting these transposons into plasmid targets in vitro, primarily for the purpose of DNA mapping and sequencing. A novel plasmid has been engineered to convert virtually any DNA sequence, or combination of sequences, into an artificial transposon; hence, custom transposons containing any desired feature can be easily designed and constructed. Such transposons are then efficiently inserted into plasmid targets, in vitro, using the integrase activity present in yeast Ty1 virus-like particles. A single in vitro integration reaction, which resembles a simple restriction digestion in the complexity of the reaction, gives rise to thousands of recoverable insertion events within DNA target molecules; this frequency approaches one insertion per phosphodiester bond in typical plasmids. Importantly, transposon insertions are recovered from all regions of DNA inserts carried on plasmid targets, indicating that integration is a random or nearly-random process. Because of its versatility, this technology offers a generalized method of generating recombinant DNA molecules of a desired structure. We have adapted this system for DNA sequencing by developing a customized artificial transposon to insert new primer binding sites into internal regions of DNA inserts carried on cloning vectors. Transposon insertions have been generated throughout several different yeast and human DNA inserts carried on plasmids, allowing the efficient recovery of sequence information from these inserts. Our results demonstrate the overall utility of this method for both small and large-scale DNA sequencing, as well as general DNA restructuring, and indicate that it could be adapted for use with a number of additional applications including functional genetic analysis.
This content is only available as a PDF.
© 1994 Oxford University Press
I agree to the terms and conditions. You must accept the terms and conditions.
Submit a comment
Name
Affiliations
Comment title
Comment
You have entered an invalid code
Thank you for submitting a comment on this article. Your comment will be reviewed and published at the journal's discretion. Please check for further notifications by email.
Citations
Views
Altmetric
Metrics
Total Views 201
43 Pageviews
158 PDF Downloads
Since 2/1/2017
Month: | Total Views: |
---|---|
February 2017 | 5 |
March 2017 | 2 |
April 2017 | 2 |
May 2017 | 3 |
June 2017 | 1 |
August 2017 | 5 |
September 2017 | 3 |
October 2017 | 3 |
November 2017 | 7 |
December 2017 | 21 |
January 2018 | 8 |
February 2018 | 8 |
March 2018 | 17 |
April 2018 | 21 |
May 2018 | 5 |
June 2018 | 1 |
July 2018 | 2 |
September 2018 | 1 |
June 2019 | 1 |
August 2019 | 2 |
September 2019 | 1 |
November 2019 | 1 |
March 2020 | 2 |
July 2020 | 1 |
December 2020 | 1 |
April 2021 | 2 |
May 2021 | 1 |
June 2021 | 1 |
August 2021 | 2 |
December 2021 | 1 |
February 2022 | 3 |
May 2022 | 1 |
August 2022 | 2 |
September 2022 | 2 |
October 2022 | 2 |
November 2022 | 5 |
February 2023 | 2 |
March 2023 | 2 |
May 2023 | 3 |
June 2023 | 1 |
July 2023 | 2 |
September 2023 | 2 |
November 2023 | 2 |
December 2023 | 1 |
January 2024 | 1 |
March 2024 | 3 |
April 2024 | 6 |
May 2024 | 4 |
June 2024 | 3 |
July 2024 | 7 |
August 2024 | 4 |
September 2024 | 10 |
October 2024 | 2 |
Citations
72 Web of Science
×
Email alerts
Citing articles via
More from Oxford Academic