In:
BioTechniques, Future Science Ltd, Vol. 32, No. 3 ( 2002-03), p. 516-520
Abstract:
A universal restriction site-free cloning method has been developed to precisely insert a DNA fragment into a vector at any desired location without altering any nucleotide(s) in either the DNA fragment or the vector. The technique employs two pairs of chimeric primers, each containing a ribonucleotide. One pair of primers is used to amplify a target DNA fragment and another is used to prepare a linear vector. The ribonucleotide is used as a specific site for cleavage promoted by rare-earth metal ions such as La 3+ or Lu 3+ . Therefore, blunt-ended PCR products can be converted into a dsDNA with single-stranded 3′ overhangs for efficient ligation. The primers are designed so that both the target DNA fragment and vector PCR products create defined 3′ overhangs to permit the formation of a seamless plasmid during the subsequent ligation. This method has been used successfully to clone the E. coli gene coding for peptidyl-tRNA hydrolase.
Type of Medium:
Online Resource
ISSN:
0736-6205
,
1940-9818
Language:
English
Publisher:
Future Science Ltd
Publication Date:
2002
detail.hit.zdb_id:
1496354-1
SSG:
12
Permalink