introduction of Gene Cloning

Cloning is a valuable tool in molecular biology because it allows the multiplication of particular genes or proteins that are being studied.

Cloning vectors
Plasmid, Lambda (phage) and cosmid


Features of the useful vector
-Vector can be replicated in a host cell
-Vector and ligated DNA can be replicated in the host cell
-When the vector is in the host cell it gives a novel phenotype or at least easy to be identified


 Basic Skills Needed to Carry Out a simple Gene Cloning Experiment

1- preparation of pure samples of DNA-  amplification

2-Cutting DNA molecules

3-Analysis of DNA fragment sizes

4-Joining DNA molecules together

5-Introduction of DNA into host cells

6-Identification of cells that contain recombinant DNA molecules


 Why the Polymerase Chain Reaction (PCR) is Also Important

• In a PCR experiment, a single segment of a DNA molecule is copied many times, resulting in an amplified DNA fragment

• The experiment is designed so that the segment of DNA that is amplified is one that carries the gene of interest
  

Steps in gene cloning

 the cloning of any DNA fragment:

 (1) select the host organism and cloning vector.

(2) Preparation of vector DNA.

(3) Preparation of DNA to be cloned.(4)Treatment of plasmid and required DNA with the same restriction enzyme

(5) making the recombinant DNA by Mixture of foreign DNA with chopped plasmids and
Addition of DNA ligase.

(6) Introduction of recombinant DNA into host cell.

(7) Production of multiple gene copies & selection process for transformed cells.

(8) examination of clones with required DNA inserts and biological properties.



DNA modifying enzymes

• Methylation/Acetylation enzymes

• Nuclease enzymes (ex. endonucleases)

• Ligation enzymes (Ligase)

Restriction enzyme

Definition
Is a nuclease that cleaves duplex DNA at a particular short sequence (restriction site)
In a symmetrical shape (palindrome)

Mechanism of restricition
By breaking the phosphodiester bond in the DNA backbone creating a free
two 3’ end and 5’ end

Types of restriction products:
Sticky ends producing type
Blunt end producing type

Features:
Cut in a symmetric way
Restrict the DNA regardless its source

Categories: Gene cloning