Chapter 16Gene Technology

Focus of Chapter

An introduction to the methods and developments in: Recombinant DNA Genetic Engineering Biotechnology

Methods to Know

1. Bacteria Transformation2. Restriction Enzymes3. cDNA4. DNA Sequencing5. PCR6. RFLP

Recombinant DNA

DNA in which genes from different sources are linked.

Ex: the “green” mice

Genetic Engineering

The direct manipulation of genes for practical purposes.

Ex: Using E. coli to produce human insulin.

Biotechnology

The use of living organisms or their components to perform practical tasks.

Ex: the use of bacteria to digest oil spills.

Plasmids

Used extensively in Biotechnology and Recombinant DNA.

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

Cut DNA at specific nucleotide sequences called “restriction sites”.

Used to "cut and splice" DNA.

Obtained from bacteria. Ex. EcoRI and Hind III

Insertion

Placing foreign DNA into a plasmid.

Open plasmid with enzymes to create “sticky ends”.

Splice the new DNA and plasmid together.

Transformation

Placing the plasmid into a bacterial cell.

Reminder - our lab.

Methods

Temperature shock & salt treatment

Electric current Injection

Identification

Screening the altered cells for the desired gene.

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 Gene can’t be above a certain

size or a plasmid won’t work. Use other tools like YACS and BACS (artificial chromosomes).

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

Uses dideoxynucleotides. Build new DNA from single

strand DNA.

DNA Sequencers

Application

DNA sequence is read base by base.

By sequencing overlapping pieces of chromosomes, the entire genome of an organism can be read. (chromosome walking)

PCR

Polymerase Chain Reaction Method for making many

copies of a specific segment of DNA.

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

Can amplify any DNA with as little as one original copy.

Very useful in a variety of techniques and tests.

RFLP Analysis

Restriction Fragment Length Polymorphisms.

Method for detecting minor differences in DNA structure between individuals.

Common in DNA fingerprinting

Method

1. Digest DNA with restrictive enzymes.

2. Separate pieces by Gel Electrophoresis

3. Identify sequences with probes.

Result

Patterns of DNA markers or DNA fingerprint

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

DNA fingerprints for crime solving – used in every TV crime show

DNA identification records – standard for the military

Comments

Links suspect bodily to the crime scene, but doesn’t prove they committed the crime.

Results take MUCH longer than on TV shows.

Analysis of old evidence is reversing some sentences.

Agricultural Uses

1. Animals Increased milk production Increased feed utilization Increased meat production

Injecting DNA into egg

PharmAnimals

Agricultural Uses

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

Golden Rice

Genetically Modified Organism or GMO

Produced by direct genetic manipulation, not traditional breeding practices.

FDA just approved sale of GMO animal products for human consumption.

Bioethics concerns

Future Of DNA Technology

Cloning of higher animals. Stem Cells - growth of

replacement tissues and organs. Gene therapy to correct DNA

defects. ?

Gene Therapy

Summary Know the basics of some of

the DNA technology techniques.

Know: Bacterial transformation lab How Gel electrophoresis works Restriction enzymes

Summary

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