Chapter 16 Gene Technology

Focus of Chapter u An introduction to the methods and developments in: u Recombinant DNA u Genetic Engineering u Biotechnology

Methods to Know 1. Bacteria Transformation 2. Restriction Enzymes 3. cDNA 4. DNA Sequencing 5. PCR 6. RFLP

Recombinant DNA u DNA in which genes from different sources are linked. u Ex: the “green” mice

Genetic Engineering u The direct manipulation of genes for practical purposes. u Ex: Using E. coli to produce human insulin.

Biotechnology u The use of living organisms or their components to perform practical tasks. u Ex: the use of bacteria to digest oil spills.

Plasmids u Used extensively in Biotechnology and Recombinant DNA. u Serve as a “vehicle” for transporting genes.

Steps for Plasmid Use 1. Get the DNA for the trait. 2. Insert DNA into the plasmid. 3. Bacterial Transformation. 4. Identification of the new trait.

Restrictive Enzymes u Cut DNA at specific nucleotide sequences called “restriction sites”. u Used to "cut and splice" DNA. u Obtained from bacteria. u Ex. EcoRI and Hind III

Insertion u Placing foreign DNA into a plasmid. u Open plasmid with enzymes to create “sticky ends”. u Splice the new DNA and plasmid together.

Transformation u Placing the plasmid into a bacterial cell. u Reminder - our lab.

Methods u Temperature shock & salt treatment u Electric current u Injection

Identification u Screening the altered cells for the desired gene. u Ex: Antibiotic sensitivity or the expression of a “new” trait (color, glowing etc.).

Example Applications 1. Insulin 2. Human Growth Hormone 3. Other Proteins

Comment u Gene can’t be above a certain size or a plasmid won’t work. Use other tools like YACS and BACS (artificial chromosomes). u mRNA must not need splicing to remove introns.

DNA Sources 1. Organism - use a section of their chromosome. 2. cDNA - Complementary DNA - created copy of DNA from the mRNA transcript to avoid introns. Uses reverse transcriptase.

DNA Sequencing: Sanger Method u Uses dideoxynucleotides. u Build new DNA from single strand DNA.

DNA Sequencers

Application u DNA sequence is read base by base. u By sequencing overlapping pieces of chromosomes, the entire genome of an organism can be read. (chromosome walking)

PCR u Polymerase Chain Reaction u Method for making many copies of a specific segment of DNA. u Also called “DNA Amplification”.

PCR - Method 1. Separate strands by heating (denature the DNA). 2. Cool slightly. 3. Build new strand from primers and nucleotides. 4. Repeat.

Importance - PCR u Can amplify any DNA with as little as one original copy. u Very useful in a variety of techniques and tests.

RFLP Analysis u Restriction Fragment Length Polymorphisms. u Method for detecting minor differences in DNA structure between individuals. u Common in DNA fingerprinting

Method 1. Digest DNA with restrictive enzymes. 2. Separate pieces by Gel Electrophoresis 3. Identify sequences with probes.

Result u Patterns of DNA markers or DNA fingerprint u Markers are inherited in a Mendelian pattern and can show relationshiops (Pedigree studies).

DNA Technology: Applications 1. Basic Research 2. Medical 3. Forensics 4. Agricultural

Basic Research 1. DNA and protein studies 2. Evolution 3. Gene structure and control mechanisms.

Medical Uses 1. Diagnosis of Diseases 2. Gene Therapy 3. Vaccines 4. Pharmaceutical Products

DNA Microarray

Forensic Uses u DNA fingerprints for crime solving – used in every TV crime show u DNA identification records – standard for the military

Comments u Links suspect bodily to the crime scene, but doesn’t prove they committed the crime. u Results take MUCH longer than on TV shows. u Analysis of old evidence is reversing some sentences.

Agricultural Uses 1. Animals u Increased milk production u Increased feed utilization u Increased meat production

Injecting DNA into egg

PharmAnimals

Agricultural Uses 2. Plants u Herbicide resistance u Retard spoilage of fruits u Insect resistance – BT corn u Nitrogen-Fixation ability

Golden Rice

Genetically Modified Organism or GMO u Produced by direct genetic manipulation, not traditional breeding practices. u FDA just approved sale of GMO animal products for human consumption. u Bioethics concerns

Future Of DNA Technology u Cloning of higher animals. u Stem Cells - growth of replacement tissues and organs. u Gene therapy to correct DNA defects. u?u?

Gene Therapy

Summary u Know the basics of some of the DNA technology techniques. u Know: u Bacterial transformation lab u How Gel electrophoresis works u Restriction enzymes

Summary u Watch the news for DNA technology discoveries. Be able to discuss one recent event.