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

Area of Science:

• Molecular Biology
• Biotechnology
• Genetic Engineering

Background:

• Conventional cloning methods using restriction enzymes and ligase are often inadequate for cloning DNA fragments generated by polymerase chain reaction (PCR).
• DNA polymerases used in PCR can introduce untemplated nucleotides at the 3'-termini of amplified DNA strands, complicating direct cloning.
• Conservative site-specific recombinases present an efficient alternative for cloning challenging DNA fragments.

Purpose of the Study:

• To describe and evaluate the use of the Flp site-specific recombinase for cloning PCR-amplified DNA fragments.
• To present a novel cloning system that simplifies the insertion of PCR products into vectors.

Main Methods:

• DNA fragments were amplified using primers containing inverted Flp target sequences.
• The Flp recombinase was used to mediate recombination of PCR fragments into a vector containing two inverted Flp target sequences.
• The vector contained the alpha-complementing region of the lacZ gene for blue/white screening.

Main Results:

• The Flp recombinase efficiently recombined PCR fragments into the target vector in vitro.
• Recombinants were easily identified as white colonies using the blue/white screening assay for lacZ activity.
• The system typically yielded both orientations of the inserted DNA fragment.
• Flp recombinase could also 'heal' double-strand breaks in the vector by inserting flanked fragments.

Conclusions:

• The Flp Double Cross System provides a robust and convenient method for cloning PCR-amplified fragments.
• This system is advantageous for cloning multiple PCR fragments into various vectors and sites.
• It offers benefits over existing recombinase-based cloning procedures.