Chapter 20Biotechnology

Focus of Chapter

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

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.

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

Plasmids

A small ring of independently replicating DNA separate from main chromosomes.

Found in Prokaryotes and yeast

Plasmids

Used extensively in Biotechnology and Recombinant DNA.

Serve as a “vehicle” for transporting genes.

Comment – other “vehicles” are used in other methods

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.

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.

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 (12 kb) or a plasmid won’t work.

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.

Reverse Transcriptase

Reverse transcribes mRNA mRNA DNA

Other Vehicles

BACs – Bacteria Artificial Chromosome – handle inserts of 100-300 kb

YACs – Yeast Artificial Chromosome – have a centromere and telomomere, handle inserts >300 kb

Nucleic Acid Probes

Used to find a specific DNA sequence in a mixture of DNA pieces

How Used

1. DNA is denatured to produce pieces.

2. Piece of complement DNA is added as a “probe”. The probe has been “labeled”.

3. Look for where the probe goes in the DNA sample. (DNA Microarray)

Probe Diagram

DNA Microarray

Used to separate pieces into wells

Well that is “marked” will hold the correct piece of DNA

DNA Microarray

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.

Gel Electrophoresis

Used to separate mixtures of DNA or proteins according to size.

Smaller pieces will move further.

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.

RFLP - Results

Patterns of DNA markers or DNA fingerprint

Markers are inherited and can show relationships (Pedigree studies).

Boyfriend

didn’t do it

End of Part 1

Cloning of Organisms

Reproducing an organism by asexual means.

Commonly done in plants. Shows the concept of

“Totipotency” – that a single cell can develop into a new organism.

Cloning in Plants

Cloning in Animals

Has been done by nuclear transplantation.

Examples – Dolly Many other vertebrates have

now been cloned.

Cloning steps

1. Somatic cell is taken from donor parent and nucleus is removed.

2. Unfertilized egg is taken from surrogate parent and the nucleus is removed and replaced with the somatic nucleus.

3. The egg is now grown in vitro until it is an embryo

Cloning continued

4. Embryo is placed in the womb of the surrogate mother.

5. Embryo develops. 6. The new clone is born. (Identical to

the donor mother)

Therapeutic cloning

Goal is to produce a group of certain cells not an organism.

Start with embryonic stem cells.

Known as stem cell research

Dolly Picture

Stem Cells

Stem Cell – an unspecialized cell that reproduces itself or differentiates into other cells.

Types of Stem Cells

Embryonic – from an embryo. Adult – found in various

tissues of the adult body.

Research

Trying to use stem cells to replace damaged cells or body parts.

If can use own stem cells, avoids tissue rejection problems.

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

Gene Therapy

Forensic Uses

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

DNA identification records – standard for the military

Comments

Links suspect bodily fluids 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

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. ?

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.