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Fusion Cloning, Phusion Cloning, Pfusion Cloning and Confusion PCR Cloning

In-Fusion PCR Cloning: Clontech,

Cold Fusion Cloning Kit: SBI

Fast Seamless Cloning Kit: Dogene

CloneEZ Kit: Genescript

GENEART Seamless Cloning and Assembly Kit: Invitrogen


SLIC uses the 3′ ->5′ exo-nuclease activity of T4 DNA polymerase to generate ssDNA overhangs in insert and vector which are required for the fusion of vector and insert fragments by single strand annealing with or without the addition of RecA.

Exonuclease III induced ligase-free directional subcloning uses 3′ ->5′ exonuclease activity of Exonuclease III to generate ssDNA overhangs which facilitate cloning.

Ribocloning uses Rnase A to cleave at single rC or rU bases that were introduced by PCR into vector and insert and subsequent heating to generate ssDNA overhangs for cloning.

Enzyme-free cloning creates complementary ssDNA overhangs by PCR with tailed primer sets and post-PCR denaturation-hybridization reactions.

In-Fusion PCR Cloning promotes PCR cloning by the In-Fusion enzyme, a poxvirus DNA polymerase with 3′ ->5’exonuclease activity.

Confusion PCR Cloning

50–200 ng linear vector
appropriate amount of insert DNA in a 1:1 to 10:1 molar ratio of insert to vector
1 ul 10X Confusion buffer (500 mM Tris–HCl, pH 7.5 at 25 oC, 100 mM MgCl2, 10 mM ATP, 10 mM DTT)
1 ul Confusion enzyme mix (contains purified Redα, Redβ and Redgam proteins)
ddH2O to a total volume of 10 ul.

Incubate at 37 oC for 1 h.
Transform homemade Top10 competent cells


Redα, a 5′ to 3′ exonuclease, A highly processive enzyme that acts in the 5´ to 3´ direction, catalyzing the removal of 5´ mononucleotides from duplex DNA. The preferred substrate is 5´-phosphorylated double stranded DNA, although it will also degrade single-stranded and non-phosphorylated substrates at a greatly reduced rate. Lambda Exonuclease is unable to initiate DNA digestion at nicks or gaps.

Redβ,  ssDNA binding protein and annealase.

RecJf may not work:  RecJf is a 5′ -> 3′ exonuclease. When DNA containing a 22 base 5′ extension is used as a substrate for RecJf, the resulting products are a mixture of DNA fragments that have blunt-ends, 5′ extensions (1-5 nucleotides) and recessed 5′ ends (1-8 nucleotides). RecJf does not require a 5′ phosphate.

The RecJ exonuclease from Escherichia coli degrades single-stranded DNA (ssDNA) in the 5′–3′ direction and participates in homologous recombination and mismatch repair.  RecJ required single-stranded tails of 7 nt or greater for robust binding; addition of Mg2+ confirmed that substrates with 5′ tails of 6 nt or less were poor substrates for RecJ exonuclease. RecJ is a processive exonuclease, degrading ∼1000 nt after a single binding event to single-strand DNA, and releases mononucleotide products. RecJ is capable of degrading a single-stranded tail up to a double-stranded junction, although products in such reactions were heterogeneous and RecJ showed a limited ability to penetrate the duplex region. RecJ exonuclease is equally potent on 5′ phosphorylated and unphosphorylated ends.

SSB such as RecA may be used to replace Redβ.

These two endonucleases  may also work:

Exonuclease I (E. coli): 3′ -> 5′ single strand exonuclease

Exonuclease III (E. coli): 3’→5′ exonuclease

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