The Flp double cross system a simple efficient procedure for cloning DNA fragments

BMC Biotechnology

Paul D Sadowski¹ 

[1] Department of Medical Genetics and Microbiology, University of Toronto, Toronto, M5S 1A8 Canada

关键词: FRT site;    LacZ;    PCR;    Site-Specific Recombinase;    Flp;    Cloning;   

Others  :  1160961
DOI  :  10.1186/1472-6750-3-9

 received in 2003-06-13, accepted in 2003-07-18,  发布年份 2003

【 摘 要 】

Background

While conventional cloning methods using restriction enzymes and polynucleotide ligase are adequate for most DNAs, fragments made by the polymerase chain reaction are difficult to clone because the amplifying DNA polymerase tends to add untemplated nucleotides to the 3'-termini of the amplified strands. Conservative site-specific recombinases offer an efficient alternative to conventional cloning methods.

Results

In this paper I describe the use of the Flp site-specific recombinase for cloning PCR-amplified fragments. A DNA fragment is amplified with primers that contain at their ends inverted target sequences for Flp. Flp readily recombines these fragments in vitro into a vector that also contains two inverted Flp target sequences surrounding the α-complementing region of the lacZ gene of E. coli. The recombinants are conveniently detected as white colonies by the familiar blue/white screening test for lacZ activity. A useful feature of the system is that both orientations of the inserted DNA are usually obtained. If the recipient vector is cut between the two inverted Flp targets, Flp "heals" the double-strand break by inserting a linear fragment flanked by Flp targets.

Conclusion

This system ("The Flp Double Cross System") should be useful for cloning multiple PCR fragments into many sites in several vectors. It has certain advantages over other available recombinase-based cloning procedures.

【 授权许可】

   
2003 Sadowski; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.

【 图 表 】

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