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


CONCLUSIONS
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.
Sadowski
Paul D
PD
Department of Medical Genetics and Microbiology, University of Toronto, Toronto, M5S 1A8 Canada. p.sadowski@utoronto.ca
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D002880:Chromosomes, Human, Pair 11 / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004254:DNA Nucleotidyltransferases / Q000235:genetics*; D004269:DNA, Bacterial / Q000032:analysis*; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006801:Humans; D007438:Introns / Q000235:genetics; D051656:KCNQ Potassium Channels; D051657:KCNQ1 Potassium Channel; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D015221:Potassium Channels / Q000235:genetics; D024642:Potassium Channels, Voltage-Gated; D001616:beta-Galactosidase / Q000235:genetics
D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial; D004274:DNA, Recombinant; D051656:KCNQ Potassium Channels; D051657:KCNQ1 Potassium Channel; C496909:KCNQ1 protein, human; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase; D001616:beta-Galactosidase