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