Molecular Biology. A Practical Guide - Velikov V.A. 2013

Gene Cloning
DNA Ligation of Vector and Gene

The restriction fragments of the vector and Gene DNA must be covalently joined into a single double-stranded circular DNA molecule, and the vector

carrying the "foreign DNA" (or transgene) must be introduced into an E. coli Cell to propagate the resulting clone. Other Bacteria, as well as brewer's Yeast Saccharomyces cerevisiae, are also used as host Cells. Unlike E. coli, the cells of these eukaryotic microorganisms allow for post-translational Processing of the eukaryotic protein (protein maturation). Microbiological manipulations involving bacteria and yeast are similar.

Ligation ("joining") of DNA fragments is performed using the DNA ligase enzyme. To do this, the plasmid and gene DNAs digested with the same restriction enzyme are mixed, ligase buffer, T4 DNA ligase, and ATP are added, and the mixture is incubated at 16°C from 2 hours for routine ligation to 12 hours for ligation aimed at constructing gene libraries.

Such "in vitro recombination" yields recombinant (hybrid) DNA.

T4 DNA ligase catalyzes The formation of a phosphodiester bond between the 3'-OH group and the 5'-phosphate on adjacent (closely positioned, "annealed") complementary "sticky ends" in the presence of ATP and Mg²⁺ ions:

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Some restriction Enzymes have different recognition sites but generate identical sticky ends. Following such ligation, the resulting DNA will not be cleaved by either of the two restriction enzymes used. However, the hybrid site can be cleaved by a third restriction enzyme. For instance, the SauIIIA enzyme will cleave the site generated by the ligation of BamHI and BglII restriction DNA fragments (see App. 3).

The standard Procedure for ligating DNA Restriction fragments, widely used in constructing artificial genetic programs, is presented below.

Materials and Equipment

T4 DNA ligase, pBluescript II SK+ phagemid DNA digested with the BamHI restriction enzyme, pSUP106:nptI-sacB-sacR plasmid DNA hydrolyzed with BamHI, or a small plasmid fragment.

Solutions

- 10x T4 DNA ligase buffer: 400 mM Tris-HCl, pH 7.8; 100 mM MgCl₂; 100 mM DTT; 5 mM ATP.

- 2YT medium (Topic 1)

- Kanamycin. 100 mg/mL solution in Water.

Procedure

1. Mix the restricted plasmid and gene DNAs at a ratio ranging from 1:2 to 1:10. The molar amount of the insert DNA should exceed the number of

vector DNA molecules to "compete" for the vector in order to increase the yield of recombinants. An optimal concentration ratio exists between the vector and the insert. Using excessively low insert concentrations will promote vector self-ligation, whereas excessively high concentrations will predominantly lead to the formation of concatemers. The total DNA concentration in the reaction mixture should not exceed 10 µg/mL.

2. Add 2 µL of 10x ligase buffer ^a and 1–2 units ^b of T4 DNA ligase to the mixture, and bring the volume up to 20 µL with deionized water.

3. Incubate the mixture at 16°C for 2 to 12 h ^c.

4. Precipitate the ligation mixture with alcohol.

5. Perform Transformation of E. coli cells with clone Selection on kanamycin.

6. Analyze the selected clones for the presence of recombinant DNA using Electrophoresis and restriction Digestion (screening by PCR is also feasible).

Notes

^a T4 DNA ligase performs satisfactorily in many restriction buffers upon The addition of 0.5 mM ATP, retaining 75–100% of its activity. Consequently, the ligation reaction can be set up immediately following restriction. In this case, restriction activity is inhibited by pre-heating the sample.

^b Blunt-end ligation requires 5 units of DNA ligase. Additionally, for such ligations, 2 µL of 50% PEG-4000 or 1 µL of 100 mM spermidine is added to the ligation mixture to increase viscosity and reduce fluid layer fluctuations.

^v In the new vectors of the TOPO^TM TA Cloning® Kit series (InvitrogenTM, Life Technologies), DNA topoisomerase I is used for DNA ligation. These vectors are very convenient for cloning PCR products without The Need for prior restriction digestion or PolyA tailing. The ligation time is 5-10 minutes.