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Chapter 23 (Part 1). Recombinant DNA Technology. Cloning Vector. Required features Origin of replication Selectable marker Screenable marker for recombinant molecules Cloning sites. AAGATGC GAATTC GTACA. AAGATGC GAATTC GTACA. DNA methylase. DNA methylase. *. *. AAGATGC GAATTC GTACA.

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chapter 23 part 1

Chapter 23 (Part 1)

Recombinant DNA Technology

cloning vector
Cloning Vector

Required features

  • Origin of replication
  • Selectable marker
  • Screenable marker for recombinant molecules
  • Cloning sites
restriction modification system

AAGATGCGAATTCGTACA

AAGATGCGAATTCGTACA

DNA methylase

DNA methylase

*

*

AAGATGCGAATTCGTACA

AAGATGCGAATTCGTACA

Restriction endonuclease

Restriction endonuclease

*

*

AAGATGCG AATTCGTACA

AAGATGCGAATTCGTACA

Restriction Modification System
slide4

5’ ATGCGAATTCCGGTT 3’

3’ TACGCTTAAGGCCTT 5’

EcoR1

5’-ATGCG-3’ 5’-AATTCCGGTT-3’

3’-TACGCTTAA-5’ 3’-GGCCTT-5’

5’ ATGCGATATCCGGTT 3’

3’ TACGCTATAGGCCTT 5’

EcoRV

5’-ATGCGAT-3’ 5’-ATCCGGTT-3’

3’-TACGCTA-5’ 3’-TAGGCCTT-5’

Sticky-end cutter

Blunt-end cutter

transformation
Transformation
  • All of the previous steps were performed in vitro.
  • We have generated a very small amount of a recombinant plasmid
  • Need to amplify in bacteria to get enough to work with.
  • Transformation – process to mobilize DNA into bacterial host
  • Select for transformed bacteria on specific antibiotic that corresponds to the antibiotic resistance gene present on the plasmid
how to produce a recombinant protein
How to produce a recombinant protein

0.1 to 1% of cellular protein

10 to 70% of cellular protein

dna hydridization screening for specific gene
DNA hydridization screening for specific gene
  • Requires that you know something about the gene sequence
  • Can get sequence information form purified protein
slide12

Product name 

Protein type

Application

Company

Adagen (Adenosine deaminase )

An enzyme

Severe combined immunodeficiency disease (SCID)

Enzon

Genotropin (Recombinant growth hormone)

A hormone

Growth hormone deficiency (GHD) in children

Pharmacia & Upjohn

Humalog (Recombinant human insulin)

A hormone

Diabetes

Eli Lilly

Nabi-HB (Anti-Hepatitis B)

 An antibody

Hepatitis-B

Nabi

Novo Seven (Recombinant coagulation factor VIIa)

A modified factor

Hemophillia patients with inhibitors

Novo Nordisk

Ontak (Diphtheria toxin-interleukin-2)

A fusion protein

Cutaneous T-cell lymphoma (CTCL)

Ligand Pharmaceuticals

Roferon-A (Recombinant interferon alfa-2a)

A modifier

Hairy cell leukemia or AIDS-related Kaposi's sarcoma

Hoffmann-La Roche

genetic modification of higher organisms
Genetic Modification of Higher Organisms
  • Can introduce gene into animals and plants
  • These modified organism are powerful research tools to study the effect of a specific gene product on metabolism, development etc….
  • Has also been used to develop improved agricultural products
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