Steps in Gene Cloning (With Experiment) | Molecular Biology

In gene cloning, the major steps are cut­ting and joining lengths of DNA using re­striction endonuclease and ligase. Several steps are involved in gene cloning.

1. Preparation of cloning vector:

Before cloning the fragment of interest, the plasmid (cloning vector) is to be restric­tion digested.

Plasmids are small extra chromosomal, self-replicating, circular double stranded DNA molecules, which carry genes for antibiotic resistance, metabolism of unusual substrates etc. From such plasmids having multiple clon­ing sites, artificial plasmids can be prepared using restriction endonuclease recognition sites for cloning and insert.

Restriction endonucleases digest double stranded DNA after recognising specific nu­cleotide sequences like palindromic sequences where they cleave two phospho diester bonds, one within each strand of DNA. Based on the site at which the enzyme cuts, the re­striction digested plasmid will have cohesive end or blunt end.

2. Preparation of insert:

Insert is prepared from sheared DNA fragmens, PCR amplified DNA fragments or restriction digested DNA fragments.

3. Ligation:

Ligate is the restriction digested vector and fragment using DNA ligase to generate the desired clone.

DNA ligation. Catalyse formation of phosphodiester bond between juxtaposed 5′ and 3′ hydroxyl terminus in a duplex DNA. This is carried out at 12°-15°C in order to maintain good balance between annealing of ends and activity of enzyme. Ligation of blunt end is carried out at 24°C, since annealing is not a delerrent factor in such activities.

4. Transformation:

Treat the ligated DNA with recipient cells that have been rendered more susceptible to DNA uptake which takes place through the channels or pores in the cell membrane and permits replication of plasmids.

5. Screening of clones:

Grow transformed cells on an antibiotic containing medium or media which are defi­cient in some nutrients.

Competent cells that have taken up plasmids alone will grow on these media.

6. Confirmation of clones:

1. Check for retardation in mobility (since recombinant vector DNA is heavier than vector DNA).

2. Release insert from the vector, using spe­cific restriction endonucleases.

3. PCR amplification using primers that are specific to the insert.

4. Southern blotting.

5. Dot blot.

6. Reporter based detection.

Experiment:

Principle:

Using restriction endonucleases and ligase cutting and joining lengths of DNA is done.

Requirements:

1. EcoRI restriction enzyme.

2. Assay buffer for EcoRI restriction en­zyme.

3. T, DNA ligase.

4. Assay buffer for T₄ DNA ligase.

5. Vector (pUC 18).

6. GFP gene.

7. Competent cells.

8. 1.5 ml tubes.

9. Media.

10. Dialysis tubing.

11. Tris EDTA solution.

12. 3M sodium acetate.

13. Phenol.

14. TE.

15. Ethanol.

16. Chloroform.

17. 10x TAE solution.

18. U.V. transilluminator.

19. Electrophoresis unit.

20. Pipettes.

21. Tips.

22. Petri plates.

23. Beakers.

24. Conical flasks.

25. Distilled water.

26. Agarose.

27. Incubator.

Procedure:

1. Quantify the vector and insert DNA on a 1% agarose gel.

Cast a 1% agarose gel and load 2 µl of vector and insert DNA along with known quantity of DNA.

2. Digest vector and insert DNA with Eco RI. Digest 2 µg of vector and 2 µg of insert DNA as follows:

DNA (2 µg) = X µl

Restriction assay buffer = 20 µl

Enzyme (EcoRI) = 20 units.

Sterile distilled water to make up 200 pi and incubate for 1 hour at 37°C.

3. Precipitate both vector and insert DNA separately.

a. 200 µl of restriction digestion reac­tion mix + 20 µl (1/10 volume) of 3(M) sodium acetate (pH 5.2). Mix well and add 550 µl of 100% etha­nol (2.5 volume).

b. Mix well and centrifuge at 10,000 rpm for 10 minutes at 4°C.

c. Discard supernatant and dry DNA pellet at 37°C for 10 minutes.

4. Resuspend DNA in T.E. buffer.

Add 50 µl of T.E. (10 mM TrisCl, 1(M) EDTA, pH 8.0) and suspend well.

5. Electroelute the vector and insert DNA separately. Cast 1% agarose gel in TAE buffer and load vector and insert DNA in different channels. Run the gel in TAE buffer at constant voltage (80 volt). Cut the gel piece containing the insert DNA band precisely and electroelute the DNA.

6. Check amount of electroeluted vector and insert DNA separately on 1% agarose gel with known quantity of DNA and quantify the unknown DNA.

7. Set up the ligation reaction between vectors and insert DNA as follows:

Vector DNA (50 ng): X µl.

Insert (5 ng): Y µl.

Ligase assay buffer: 1 µl

T.DNA ligase: 3 units.

Water to make final volume: 10 µl.

8. Do transformation of E. coli strain DH 5 CC competent cells with 5 µl of ligation mix following standard transformation protocol.

9. Recombinants are checked by exposing the transformation plates to U.V. radia­tion. Green fluorescent (protein) emis­sion is a positive result.