part iii. recombinant dna technology • cloning strategies...

Chapter 5Part III. Recombinant DNA technology

• Cloning strategies• Polymerase chain reaction (PCR)• Applications

Recombinant DNA technology

• Characterization of the genes• Large production of proteins• Mutants

(Gene cloning, molecular cloning, genetic engineering)

Methodology for transferring genetic information (genes) from one organism to another

Tools of recombinant DNA technology

• Restriction endonucleases - cut DNA at specific sites

• DNA ligase or other DNA modifying enzymes• Cloning vectors - DNA molecules that can be

replicated• Reporter genes• Model organisms

Restriction enzymes

• Bacteria’s “immune system” for protection from infection by foreign DNA

• Three types– Type I and Type III : Both the endonuclease

and the methylase activity; Remote recognition site

– Type II : Only endonuclease activity; Specific and predictable recognition;

• Cohesive (sticky) or blunt ends

Palindromic restriction sites

Restriction map

Restriction map

Restriction-fragment length polymorphism (RFLP)

Inheritance of RFLPs

Cloning vectors

• Plasmids– Replication origin, selectable marker & polylinker– Stringent control (low copy number) or relaxed

control (medium to high copy number)• Viral vectors

– Bacteriophage λ, cosmid & M13 : Bacteria– Baculoviruses: Insects– Retroviruses, lentiviruses & adenoviruses:

Mammalian cells• Yeast artificial chromosome (YAC) and bacterial

artificial chromosome (BAC)

Plasmid cloning vector

Polylinker(Multiple Cloning

Site)

Replication origin

ampR

lacZα

Construction of recombinant DNA

Cloning in λ phages

Cloning strategies

• DNA ligase• PCR • Terminal transferase• Adaptor• Topoisomerase• Recombinase

Cloning using DNA ligase

Cloning using terminal transferase

Cloning using synthetic adaptor

Reporter genes

• Selectable markers– Antibiotic resistance – Nutritional markers

• LacZ (β-galactosidase)• Luciferase• Green fluorescence protein (GFP)

Insertional gene inactivation

(Replica plating)

β-Galactosidase as a reporter

GFP as a reporter

Tsien at UCSD

GFP

Southern blotDetection of specific DNA sequence

Colony (in situ) hybridization

Identification of the clones containing a DNA of interest

Model organisms

Polymerase Chain Reaction (PCR)

Technique for the exponential amplification of a specific DNA segment

• Template DNA• Two oligonucleotide primers• Heat stable DNA polymerase (Taq, Pfu etc)

Three step process• Template denaturation• Primer annealing (hybridization)• Primer extension (polymerization)

Denaturing

Annealing

Extension

Heat stable DNA polymerasedNTPs

268,435,45630

8,19215

262,14420

8,388,60825

1,073,741,82432

25610

165

44

23

02

01

Number of the target DNA molecules

Cycle number

Exponential amplification

After 4 cycles

After 32 cycles

Application of PCR

• Clinical applications - Diagnosis of infectious diseases and rare mutations

• Forensics – DNA fingerprinting• Molecular archeology – Evolutionary study

• Asymmetric PCR – DNA sequencing• Site-directed mutagenesis

Site-directed mutagenesis

Applications of the recombinant DNA technology

• Recombinant proteins–Research–Medical purposes

• Genetically altered organisms– Transgenic– Knockout

• Gene therapy

Recombinant proteins

Genetically modified organisms (GMOs)

• Bacteria – Bioremidation, biomining, biofuel etc

• Plants – Resistace to pests, herbicides or harsh environmental conditions; improved shelflife; increased nutritional value

• Animals – Transgenic or cloned animals

GloFish Giant mouse Dolly, the cloned sheep

Insect-resistant cotton Golden rice

Approaches for gene therapy

• ex vivo – Treatment of cells with a vector outside of the body

• in situ – Direct application of the vector to affected tissues

• in vivo – Direct injection of the vector into the blood stream