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

6

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

11

Early Genetic Exp.

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

14

cDNA

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 librarypreliminary 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 stepsdenaturationannealing of primersprimer 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 nitrocelluloseprobe of purified, single-stranded DNA

poured over sheet if radioactive probe used, band of

radioactivity appears where probe hybridized with complementary fragment

23

Southern Blot

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.

26

RFLPs

27

Biotechnology

• Medical applications– pharmaceuticals

introduction of protein-encoding genesatrial 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

32

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

38