Construction of recombinant DNA by exonuclease recession

Abstract

We describe a new exonuclease-based method for joining and/or constructing two or more DNA molecules. DNA fragments containing ends complementary to those of a vector or another independent molecules were generated by the polymerase chain reaction. The 3' ends of these molecules as well as the vector DNA were then recessed by exonuclease activity and annealed in an orientation-determined manner via their complementary single-stranded regions. This recombinant DNA can be transformed directly into bacteria without a further ligase-dependent reaction. Using this approach, we have constructed recombinant DNA molecules rapidly, efficiently and directionally. This method can effectively replace conventional protocols for PCR cloning, PCR SOEing, DNA subcloning and sitedirected mutagenesis.

The terminal 5′ phosphate and proximate phosphorothioate promote ligation‐independent cloning

“…To date, these cloning techniques include the restriction enzyme cloning, 1 blunt-end cloning, 2 TA cloning, 3 classical ligation-independent cloning (LIC), [4][5][6][7][8][9][10][11] hybridization cloning, 12,13 and in vivo cloning. [14][15][16] In fact, the last two methods are also LIC because they are ligase-free cloning techniques.…”

“…[14][15][16] In fact, the last two methods are also LIC because they are ligase-free cloning techniques. All these methods require the specific enzymatic treatment of target gene and vector, 1,[5][6][7][8][9] the use of primers carrying modified bases, 4,[9][10][11] or some specific bacterial strains, [14][15][16] except for hybridization cloning. Among them, the LIC being easy to carry out can be easily adopted for high-throughout cloning.…”

“…The early LIC was based on the 3 0 exonuclease or T4 DNA polymerase. [5][6][7] However, both enzymes had some limitation on the selection of inserting sites of target gene. Nisson et al developed a new LIC method, using the dU-carrying primer, uracil-DNA glycosylase, and Taq DNA polymerase.…”

BR22, a 26 kDa thyroid transcription factor‐1 associated protein (TAP26), is expressed in human lung cells

“…Two bipartite primers, 59 primer TCTGACGGATCCATGGCG-CCGGTGAGGCGGT and 39 primer ATGGTGGGATCC-ACATTTTTCTTGTATTTTTTGAAGAA (TAP26 sequences are in bold) were used for reverse transcriptase (RT)-PCR amplification of the full-length TAP26 cDNA using human lung polyA RNA (BD Biosciences) as the template. Utilising the bipartite primers in T4 DNA polymerase exonuclease recession method [20], the PCR-amplified TAP26 fragment can be directly in-frame fused to GST in vector pGEX-VH [21] without the introduction of extra unnecessary amino acid residues. The pGEX-VH vector contains a six-histidine stretch that is designed at the end of fusion protein, which can be purified to homogeneity as a full-length protein (GST-TAP26) by performing sequential GST-and Ni-beads affinity chromatography.…”

Restriction-ligation-free (RLF) cloning: a high-throughput cloning method by in vivo homologous recombination of PCR products

“…Numerous gene cloning methods, including sticky-end cloning (Scharf et al, 1986), blunt-end cloning (Costa et al, 1994), TA cloning (Holton and Graham, 1991;Marchuk et al, 1991), ligation independent cloning (LIC) (Aslanidis and de Jong, 1990;Shuldiner et al, 1990;Hsiao, 1993;Yang et al, 1993;Kaluz and Flint, 1994;Tillett and Neilan, 1999), and site-specific recombination systems (Hartley et al, 2000), have been developed over the years. These cloning approaches are widely used and have proven to be highly efficacious in most instances; however, they present several limitations because of the extensive enzymatic treatment of the required PCR products or vectors.…”