A universal method for directional cloning of PCR products based on asymmetric PCR.
Biotechnol Appl Biochem
; 52(Pt 1): 41-4, 2009 Jan.
Article
in En
| MEDLINE
| ID: mdl-18352859
ABSTRACT
We have developed a novel protocol for directional cloning of PCR products into any vector. The target sequence is amplified in two parallel asymmetric PCRs using specially but simply designed primers. Two single-stranded products are produced and they are annealed to form a double-stranded DNA fragment bearing overhangs at both ends that correspond to the restriction overhangs of certain restriction enzymes. The fragment can then be cloned into a certain vector previously treated with the corresponding enzymes without restriction of the inserted fragment. Compared with previously published protocols, the procedure described in this paper is highly efficient and it is independent of the restriction sites of the insert and is therefore applicable to molecular biology and biotechnology studies.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Polymerase Chain Reaction
/
Cloning, Molecular
/
DNA Primers
Limits:
Animals
Language:
En
Journal:
Biotechnol Appl Biochem
Journal subject:
BIOQUIMICA
/
BIOTECNOLOGIA
Year:
2009
Document type:
Article