Gene Technology Chapter 16

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

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

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

Restriction Endonucleases

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

Plasmid and Phage Vectors

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.

Early Genetic Engineering

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

DNA Libraries

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

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

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

Genetic Engineering - Stages

Genetic Engineering - Stages

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

Polymerase Chain Reaction

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

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

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

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

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

Subunit Herpes Vaccine

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

Ti Plasmid

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

Transgenic Rice

Bovine Somatotropin

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 ?

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