By –AKSHAY PAREEKM.Sc. Biotech. III semDept. of BiotechnologyNIET, NIMS University

• Clones

– Genetically identical molecules, cells, or organisms all derived from a single ancestor

• Cloning

– The production of identical copies of molecules, cells, or organisms from a single ancestor

Why cloning -

• DNA clones are used to find genes, map them, and transfer them between species

• Cloning technology is used to find carriers of genetic disorders, perform gene therapy, and create disease-resistant plants

Cloning

Cell based

PCR based

Requirements

• A way to cut DNA at specific sites

• A carrier molecule to hold DNA for cloning

• A place where the DNA can be copied (cloned)

1 •Extraction

2 • Isolation

3 •Ligation

4 • Insertion

5 •Selection

6 •Storation

Isolation of desired gene

• Using restriction endonuclease

• Mostly RE II used

Gene of interest

Ligation

Overall process of cloning

Insertion (Transformation)

Vectors used –

1. Plasmid

2. Bacteriophage

3. Cosmid

4. YACs

5. BACs

Techniques for Transformation

Transformation using CaCl2

Electroporation

Microinjection

Selection

• Blue White Screening -

–Only plasmids with functional lacZ gene can grow on XgallacZ functional => polylinker intact => nothing inserted, no clone lacZ(-) => white colonies polylinkerdisrupted => successful insertion & recombination!

A Revolution in Cloning :Polymerase Chain Reaction

Requirement

1. Target DNA

2. Two Primers

3. Four dNTPs

4. DNA Polymerase

Natural habitat of Thermus aquaticus

• Thermostable DNA Polymerase is used like-Taq DNA Polymerase from Thermus aquaticusPfu DNA Polymerase from Pyrococcus furiosus

Steps in PCR

Denaturation AnnealingRenaturation

Application of Cloning

• Development of methods for cloning higherplants and animals represents a significantadvance in genetic technology

– Improving crops

– Producing domestic animals

• Cloned plants and animals are used in research, agriculture, and medicine

• Single base changes then detected by one or more of following:

-dot blot technique

-Restriction enzyme analysis (RFLP).

-direct sequencing of DNA.

• PCR to detect HIV

• Crucial forensic evidence may be present invery small quantities often too little materialfor direct DNA analysis.

• PCR also possible on extensively degradedDNA examples include DNA from single driedblood spot, saliva (on cigarette butt), semen,tissue from under fingernails, hair roots.