Gene cloning

Habeeb Al-nhab.Ishaq Al-hammadMajed Al- roogy

Definition

• The terms recombinant DNA technology, DNA cloning, molecular cloning, or gene cloning all refer to the same process:

• the transfer of a DNA fragment of interest from one organism to a self-replicating genetic element such as a bacterial plasmid (cloning vector). The DNA of interest can then be propagated in a foreign host cell. This technology has been around since the 1970s, and it has become a common practice in molecular biology labs today."

Principle

• number of different processes that can be used to produce genetically identical copies of a biological entity. The copied material, which has the same genetic makeup as the original, is referred to as a clone.

• Task: Produce insulin by gene cloning.

Requirement for gene cloning

• Identification of interest gene structure like insulin.

• Identification the pathway of synthesis• Identification and purification of enzyme

responsible for production (insulin).• Identification the sequence of amino acid in

each enzyme.(like amino acid sequence of insulin).

Steps for Using Bacteria and Plasmids to Clone Genes

Step 1: Isolation of two kinds of DNA. foreign DNA containing the gene of interest

are isolated (the foreign DNA is insulin) Bacterial plasmids (plasmid is from E. coli )

Plasmid usually carry only one or a few genesampR that confers antibiotic resistance to ampicillin. lacZ that codes for beta-galactosidase, the enzyme that catalyzes the hydrolysis of lactose

cont…

Step 2: Treatment of plasmid and foreign DNA with the same restriction enzyme

• The restriction enzyme cuts plasmid DNA at the restriction site

• The foreign DNA is cut into thousands of fragments by the same restriction enzyme; one of the fragments contains the gene of interest.

• restriction enzyme cuts, it produces sticky ends on both the foreign DNA fragments and the plasmid

Cont…

Step 3:Addition of DNA ligase.• formation of covalent bonds, joining the two

Sticky ends ( of the plasmid will base pair with complementary sticky ends of foreign DNA fragments).

• Result (two probability):• Plasmid acquired new gene (gene of interest

instead of lacZ gene or gene of ampR ) formation recombinant DNA.

• Plasmid stay as initial.

Cont…

Step 4:Introduction of recombinant plasmid into bacterial cells.

• Bacteria chosen must be sensitive to plasmid of E.coli.

• This process (insertion of recombinant plasmid into bacteria cell) done by electroporation (DNA gun).

• Some bacteria will take up the plasmid DNA by transformation.

Cont…

Gene gun Microinjection

Cont…

Step 5: Production of multiple gene copies by gene cloning.

• Bacteria with the recombinant plasmid are allowed to reproduce, cloning the inserted gene in the process.

Cont…

Step 6:Final screening for transformed cells.• culture on X-gal, • a modified sugar added to the culture

medium, turns blue when hydrolyzed by beta-galactosidase. It is used as an indicator that cells have been transformed by plasmids containing the foreign insert.

Cont…

• Since the foreign DNA insert disrupts the lacZ gene, bacterial colonies that have successfully acquired the foreign DNA fragment will be white. Those bacterial colonies lacking the DNA insert will have a complete lacZ gene that produces beta-galactosidase and will turn blue in the presence of X-gal

Application of recombinant DNA technology

Use purified protein to make antibodies for medical purpose and make vaccines for the treatment of disease.

Diagnose human genetic disorders and infection disease condition.

Use in forensic application such as DNA fingerprinting.

mutate gene and study function of altered protein produced.

Cont…

Scale-up production,isolation,and purification of therapeutic protein (i.e.,insulin,human growth hormone,and clot-dissolving protein used to heart attacks) for use in humans as recombinant DNA products.

Express protein and study protein structure and function in vivo; isolate and purify protein to study protein structure and function in vivo.

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