Ligation-independent cloning of PCR products (LIC-PCR)

Clicks: 3
ID: 296201
1990
Free PDF
Article Quality & Performance Metrics
Overall Quality
Not rated
Combines reader engagement with the AI quality analysis. This article has not been analysed, so there is no overall score — reader engagement is measured and shown alongside.
AI Quality Assessment
Not analyzed
Readership in this journal
Popular

Ranked #497 of 1,216 articles by views in Nucleic Acids Research

Most read Least read

Bar heights use a square-root scale. Only the 120 most-read articles are drawn; the journal has 1,216 in total.

Mint this article as an NFT
Not yet minted

Create a permanent, verifiable on-chain record of this article on the Scimatic Network. The NFT is held in your Journament account, and you can withdraw it to your own wallet at any time.

5 SUSD one-off · no wallet required
Sign in to mint
Abstract
A new procedure has been developed for the efficient cloning of complex PCR mixtures, resulting in libraries exclusively consisting of recombinant clones. Recombinants are generated between PCR products and a PCR-amplified plasmid vector. The procedure does not require the use of restriction enzymes, T4 DNA ligase or alkaline phosphatase. The 5′-ends of the primers used to generate the cloneable PCR fragments contain an additional 12 nucleotide (nt) sequence lacking dCMP. As a result, the amplification products include 12-nt sequences lacking dGMP at their 3′-ends. The 3′-terminal sequence can be removed by the action of the (3′→5′) exonuclease activity of T4 DNA polymerase in the presence of dGTP, leading to fragments with 5′-extendlng single-stranded (ss) tails of a defined sequence and length. Similarly, the entire plasmid vector is amplified with primers homologous to sequences In the multiple cloning site. The vector oligos have additional 12-nt tails complementary to the tails used for fragment amplification, permitting the creation of ss-ends with T4 DNA polymerase in the presence of dCTP. Circularization can occur between vector molecules and PCR fragments as mediated by the 12-nt cohesive ends, but not in mixtures lacking insert fragments. The resulting circular recombinant molecules do not require in vitro ligation for efficient bacterial transformation. We have applied the procedure for the cloning of inter-ALU fragments from hybrid cell-lines and human cosmid clones.
Reference Key
openalex_W2094476648 Use this key to autocite in the manuscript while using SciMatic Manuscript Manager or Thesis Manager
Authors Charalampos Aslanidis, Pieter J. de Jong
Journal Nucleic Acids Research
Year 1990
DOI
10.1093/nar/18.20.6069
URL
https://doi.org/10.1093/nar/18.20.6069
Keywords Keywords not found

Citations

No citations found. To add a citation, contact the admin at info@scimatic.org

Comments

No comments yet. Be the first to comment on this article.