Part III. Recombinant DNA technology • Cloning strategies...

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Chapter 5 Part III. Recombinant DNA technology • Cloning strategies • Polymerase chain reaction (PCR) • Applications

Transcript of Part III. Recombinant DNA technology • Cloning strategies...

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Chapter 5Part III. Recombinant DNA technology

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

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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

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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

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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

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Palindromic restriction sites

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Restriction map

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Restriction map

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Restriction-fragment length polymorphism (RFLP)

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Inheritance of RFLPs

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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)

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Plasmid cloning vector

Polylinker(Multiple Cloning

Site)

Replication origin

ampR

lacZα

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Construction of recombinant DNA

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Cloning in λ phages

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Cloning strategies

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

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Cloning using DNA ligase

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Cloning using terminal transferase

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Cloning using synthetic adaptor

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Reporter genes

• Selectable markers– Antibiotic resistance – Nutritional markers

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

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Insertional gene inactivation

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(Replica plating)

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β-Galactosidase as a reporter

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GFP as a reporter

Tsien at UCSD

GFP

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Southern blotDetection of specific DNA sequence

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Colony (in situ) hybridization

Identification of the clones containing a DNA of interest

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Model organisms

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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)

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

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Denaturing

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Annealing

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Extension

Heat stable DNA polymerasedNTPs

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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

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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

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Site-directed mutagenesis

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Applications of the recombinant DNA technology

• Recombinant proteins–Research–Medical purposes

• Genetically altered organisms– Transgenic– Knockout

• Gene therapy

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Recombinant proteins

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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

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GloFish Giant mouse Dolly, the cloned sheep

Insect-resistant cotton Golden rice

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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

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