Chapter 20 DNA Technology &

Genomics

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.

Restriction 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

• 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 restriction enzymes to create “sticky ends”.

• Splice the new DNA and plasmid together.

Transformation

• Placing the plasmid into a bacterial cell.

• Reminder - our lab – need to know for AP testing purposes.

Transformation 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.

– Why?

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.

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

• HACs – Human Artificial Chromosome

Nucleic Acid Probes

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

How Used

• DNA is denatured and cut to produce single stranded pieces.

• Piece of compliment DNA is added as a “probe”. The probe has been “labeled”.

• Look for where the probe goes in the DNA sample.

Probe DiagramNuNu

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

• Technique will be covered in lab.

• Used to separate mixtures of DNA or proteins.

RFLP Analysis

• Restriction Fragment Length Polymophisms.

• Method for detecting minor differences in DNA sequence between individuals.

• Common in DNA fingerprinting

Method

1. Digest DNA with restriction enzymes.

2. Separate pieces by Gel Electrophoresis

3. Identify sequences with probes.

RFLP - Results

• Patterns of DNA markers or DNA fingerprint

• Markers are inherited in a Mendelian pattern and can show relationships (Pedigree studies).

DNA Sequencing—Old Method

• Uses dideoxynucleotides which stop DNA Polymerase at a known point in DNA replication.

• Builds new DNA from single strand DNA.

• Produces fragments of different lengths.

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)

Cloning of Organisms

• Reproducing an organisms 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.

Dolly Picture

Stem Cells

• Stem Cell – an unspecialized cell that can reproduce itself or differentiate into other cells.

Types of Stem Cells

• Embryonic – from an embryo.

• Adult – found in various tissues of the adult body.

Research

• Attempting to use stem cells to replace damaged cells or body parts.

• If can use own stem cells, avoids tissue rejection problems. Ex. – grow a new bladder.

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 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 has 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 (pGlo)

– How Gel electrophoresis works

– Restriction enzymes