1. Gene Technology
Chapter 16

2. Outline
Restriction Endonucleases
Host / Vector Systems
DNA Libraries
Genetic Engineering Experiment
Working With Gene Clones
Biotechnology
Medical Applications
Agricultural Applications
Risk and Regulation

3. Restriction Endonucleases
Restriction endonucleases recognize specific nucleotide sequences, and cleave DNA creating DNA fragments.
Type I - simple cuts
Type II - dyad symmetry
allows physical mapping
allows recombinant molecules

4. Restriction Endonucleases
Each restriction endonuclease has a specific recognition sequence and can cut DNA from any source into fragments.
Because of complementarity, single-stranded ends can pair with each other.
sticky ends
fragments joined together with DNA ligase

5. Restriction Endonucleases

7. Host / Vector Systems
DNA propagation in a host cell requires a vector that can enter the host and replicate.
most flexible and common host is E. coli
two most commonly used vectors are plasmids and phages
viruses and artificial chromosomes also being probed for use

8. Plasmid and Phage Vectors

9. Using Vectors to Transfer Genes
Chimeras
One of first recombinant genomes was a bacterial plasmid into which an amphibian ribosomal RNA gene was inserted.
Viruses can also be used as vectors to insert foreign DNA into host cells.

10. Early Genetic Engineering

12. DNA Libraries
A collection of DNA from a specific source in a form that can be propagated in a host
genomic library - representation of the entire genome in a vector
cDNA library is limited to expressed genes isolated by reverse transcriptase
isolated from retroviruses

13. DNA Libraries

15. Genetic Engineering Experiment
Four stages
DNA cleavage
restriction endonuclease cleaves source DNA into fragments
production of recombinant DNA
DNA fragments inserted into plasmids or viral vectors
cloning

16. Genetic Engineering Experiment
Screening
clones with DNA fragment of interest identified from clone library
preliminary screening - eliminate any clones without a vector and clones with vectors that do not contain DNA
employ vector with gene for antibiotic resistance and lac Z’ gene
expose to growth medium

17. Genetic Engineering Experiment
Secondary screening (gene of interest)
hybridization - cloned genes form base pairs with complementary sequences on another nucleic acid (probe)
grow on agar then transfer to filter pressed on colonies
treat filter with radioactive probe, and perform autoradiography

18. Genetic Engineering - Stages

19. Genetic Engineering - Stages

20. Working With Gene Clones
Polymerase chain reaction
used to copy specific gene sequences
three basic steps
denaturation
annealing of primers
primer extension

21. Polymerase Chain Reaction

22. Working With Gene Clones
Identifying DNA: Southern blotting
sample DNA cleaved into restriction fragments, and spread apart by gel electrophoresis
gel blotted with sheet of nitrocellulose
probe of purified, single-stranded DNA poured over sheet
if radioactive probe used, band of radioactivity appears where probe hybridized with complementary fragment

24. Working With Gene Clones
Restriction fragment length polymorphisms (RFLP’s) can be used to identify a particular individual.

25. Working With Gene Clones
DNA fingerprinting
Because two individuals rarely produce identical RFLP analyses, DNA fingerprints can be used in criminal investigations.

27. Biotechnology
Medical applications
pharmaceuticals
introduction of protein-encoding genes
atrial peptides - high blood pressure and kidney failure
tissue plasminogen activator - dissolving blood clots
gene therapy
add working copies of single defective gene

28. Medical Applications
Piggyback vaccines
produce subunit vaccines against viruses
herpes
hepatitis
DNA vaccine
cellular immune response

29. Subunit Herpes Vaccine

30. Agricultural Applications
Ti plasmid has been early successful vector.
nitrogen fixation
introduce genes that allow crops to fix nitrogen
reduce need for fertilizer
herbicide resistance
insert genes encoding for proteins making crops resistant to herbicide
widespread herbicide use possible

31. Ti Plasmid

33. Agricultural Applications
insect resistance
insert genes encoding proteins harmful to insects
Real promise - produce genetically modified plants with traits benefiting consumers
iron deficiency in developing countries
transgenic rice
increasing milk production
bovine somatotropin

34. Transgenic Rice

35. Bovine Somatotropin

36. Risk and Regulation
Questions
How do we measure the potential risks of genetically modified crops ?
Is eating genetically modified food dangerous ?
Are genetically modified crops harmful to the environment ?
Should we label genetically modified foods ?

37. Summary
Restriction Endonucleases
Host / Vector Systems
DNA Libraries
Genetic Engineering Experiment
Working With Gene Clones
Biotechnology
Medical Applications
Agricultural Applications
Risk and Regulation