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

Gene Cloning
Preparation of Vector DNA and Target Gene

The circular vector DNA is cleaved with a restriction endonuclease at a unique site—meaning a single site per DNA molecule—located within the polylinker. The polylinker, or multiple cloning site, contains recognition sequences for several restriction Enzymes (Appendix 5). The target Gene DNA is likewise "excised" from the chromosome using the same restriction enzyme to generate complementary sticky ends.

In the case of PCR product cloning, polyadenine (polyA) sticky ends are enzymatically added to the product using a specialized enzyme, followed by ligation into the pAL-TA^TM PolyT vector (Evrogen). A novel enzymatic method for assembling overlapping DNA fragments (Gibson assembly, 2009) allows the ligation of unrestricted DNA, including PCR products.

Exercises 7.1–7.3 provide a straightforward example illustrating the cloning (or rather re-cloning) workflow of the levansucrase gene sacB from the hay bacillus Bacillus subtilis into the pBluescript II vector using the recombinant plasmid pSUP106::nptI-sacB-sacR (Velikov et al., Biotechnology, 2004, Vol. 3).

Materials and Equipment

BamHI restriction enzyme, pBluescript II SK+ phagemid DNA (1 mg/mL) or an E. coli strain harboring this phagemid, pSUP106::nptI-sacB-sacR plasmid DNA (1 mg/mL) or an E. coli strain harboring this plasmid, and thick filter paper.

Solutions

- 10× BamHI restriction buffer (Topic 4).

Procedure

Vector Preparation

1. Cultivate biomass of the host E. coli XL1-Blue strain containing the pBluescript II SK+ vector in 500 mL of nutrient medium.

2. Perform preparative plasmid isolation as described in Topic 4.

3. Digest 2 µg of vector DNA with the BamHI restriction enzyme (Topic 5).

4. Perform agarose gel Electrophoresis to verify complete Digestion after 1 hour of incubation. Only a single band of approximately 3 kb should be visible on the gel. The presence of multiple bands indicates incomplete Hydrolysis, in which case the reaction should be continued for an additional hour.

5. Precipitate the hydrolyzed DNA with ethanol and dissolve it in 20 µL of deionized Water. Treatment of the vector with calf intestinal alkaline phosphatase (CIP) to prevent self-ligation is optional, as Selection will be performed using the cassette's kanamycin resistance marker.

Gene Preparation

1. Excise the nptI-sacB-sacR cassette at the BamHI site from the recombinant plasmid. To do this, digest 1 µg of the pSUP106::nptI-sacB-sacR plasmid following the protocol described in Topic 5.

2. Perform agarose gel electrophoresis of the DNA.

3. Under UV light ^a, excise the agarose strip containing the DNA fragment corresponding in size to the cassette (also referred to as the construct) using a scalpel. This is the lower 3.8 kb band on the gel; the upper 9.6 kb band corresponds to the pSUP106 vector.

4. Extract the cassette DNA from the excised gel slice using a commercial DNA Extraction Kit (Fermentas^TM, Thermo Scientific Life Science Research) or any other established method ^b. An acceptable DNA yield (up to 50%) is achieved by centrifuging the gel slice placed inside a small pouch made of dense filter paper, set within a punctured microcentrifuge tube nested inside another intact tube. The agarose remains on the filter, while the DNA solution passes into the bottom tube.

Notes

^a UV exposure to the DNA must be minimized to prevent The formation of thymine dimers, which can interfere with molecular cloning.

^b Instead of separating the large and small fragments prior to ligation, both can be cloned together, and Plasmids containing the DNA insert of the correct size can be screened subsequently. In this simple case, only Two Types of recombinant plasmids will be generated: the desired 6.8 kb construct and a larger 12.6 kb by-product.

^c A higher percentage of DNA recovery from the gel slice can be achieved through enzymatic digestion of the agarose using agarase, electroelution of the DNA, or The Use of low-melting-point agarose. In the latter case, the gel is melted by heating, cold TBE buffer is added, agarose flakes are pelleted by centrifugation, and the DNA is precipitated with alcohol.