Download Topic 12 DNA Technology

Topic 12 DNA Technology
1. Selective Breeding vs. Genetic Engineering
2. Polymerase Chain Reaction, Gel
Electrophoresis & DNA Sequencing
3. HGP & DNA Profiling in Forensics
4. Stem Cells & Cloning
5. DNA Probes, PGD & Gene Therapy
Topic 12: Lesson 1
Resources:
1. Read Chapter 11.1-2A pp. 218-221
2. Kahn Academy – Restriction Enzymes
3. Lab – Restriction Enzymes
SELECTIVE BREEDING VS. GENETIC
ENGINEERING
Selective Breeding = Artificial Selection
• Humans have been using artificial selection for
thousands of years
Artificial Selection in Pets & Livestock
• Selective breeding takes many generations to
achieve desired results, but can be achieved
without much technology
Genetic Engineering
• Directly inserting, removing, or changing an
organism’s DNA using technology
• Genetically Modified Organisms (GMOs)
contain DNA from another species, or the
same species
NOT Examples of GMOs
• Grafting
• Hybrid animals
Example of GMO: Bt Corn
• European corn borer lays eggs in corn plants and
the larvae feed on the corn kernels and leaves
• Scientists inserted a gene from a soil bacteria,
Bacillus thuringiensis, that is deadly to the corn
borer in the corn
Transgenic organism
• When an organism receives genes from
another species
Restriction Enzymes
Steps to Make a Transgenic Organism
1. Obtain source DNA (gene)
2. Obtain a plasmid (circular DNA) from bacteria
3. Use restriction enzymes to cut out the gene of
interest and to cut the plasmid to receive the
gene
4. Mix source DNA gene with plasmid to form
recombinant DNA
5. DNA ligase will seal the phosphodiester bonds
6. Insert recombinant DNA into host cell
Transgenic Plant with DNA from Bacterium
Xenotransplantation
• When humans get animal parts
Viruses & Vaccines
• Viruses may be alive, dead, weakened, or
genetically engineered
• Genetically engineered vaccines only contain
genetic information that encodes proteins that
allow them to be recognized by the body’s
immune system
• Viruses have many promising uses in the field of
biotechnology as vectors
– Guinea pig pacemaker
– Tumor killer viruses
– Helping endangered animals survive outbreaks
Topic 12: Lesson 2
Resources
1. Read Chapter 11.2B-C pp. 223-4
2. Virtual Lab – PCR
3. DNA Learning Center – PCR
4. MIT Open Courseware – PCR
5. Kahn Academy – PCR
6. Virtual Lab – Gel Electrophoresis
7. Kahn Academy – Gel Electrophoresis
8. Kahn Academy – DNA Sequencing
9. AK Lectures – Sanger DNA Sequencing
POLYMERASE CHAIN REACTION, GEL
ELECTROPHORESIS & DNA SEQUENCING
PCR Amplifies Genes
95°C
54°C
72°C
PCR = Copying DNA by Alternating
Temperatures
• One gene can be copied millions of times within 1-2 hours
• A three step process is repeated for 30-40 times
• A thermal cycler machine controls temperature changes
STEPS OF PCR
1. Denaturation (95°C; 201.2°F) – the double stranded DNA
opens into two pieces of single stranded DNA
2. Annealing (54°C; 129.2°F) – primers pair up with the
single stranded DNA template; DNA (Taq) polymerase
begins to copy the template
3. Extension (72°C; 161.6°F) – DNA (Taq) polymerase is at
its optimum temperature and completes copying the
double stranded DNA
Uses of PCR
• First step in each of the following practices
– Establish paternity
– DNA fingerprinting
– Study human evolution
– Diagnose genetic diseases
– Clone DNA of dead organisms
Gel Electrophoresis
Sorting Molecules by Size
DNA Sequencing
• Finding the Order of Nucleotides in DNA
Using Gel Electro for DNA Sequencing
Different People Produce Different
Bands on Gels
• SNP/RFLP
• STR
Paternity Testing
Topic 12: Lesson 3
Resources
1. Read Chapter 11.2B pp. 222-3
2. Bozeman Science – DNA Fingerprinting
3. Naked Scientists – How Does DNA Fingerprinting Work?
4. Activity – Who Killed Sharon?
HUMAN GENOME PROJECT & DNA
PROFILING IN FORENSICS
Human Genome Project
• Aim to sequence the 3.2 billion base pairs
• Completed in 2003
• Found
– 25,000 genes
– 400,000 proteins
– 1.5% of DNA codes for protein
Finding Variation in DNA
• Each human’s DNA is 99.9% identical
• Large variation is found in between the genes
in non-coding regions
FBI Using DNA Fingerprinting
• The FBI uses 13 different STRs to determine
the matching identity of a person
Topic 12: Lesson 4
Resources
1. Read Chapter 11.3A-B pp. 226-228
2. Worksheet – Stem Cells & Cloning
3. NOVA – Stem Cell Video
4. NOVA - Stem Cell Poll
5. Biomed @ Brown University – detailed information on stem cells
6. Module 6 – Contrasting Cases of Animal Modifications
STEM CELLS & CLONING
Development of the Embryo
Zygote
Blastula
Morula
Gastrula
3 Germ Layers
What is a Stem Cell?
Plasticity of Stem Cells
Embryonic Stem Cells
• Totipotent – give rise to all
cell types in the body
– Derived from zygote cell or
morula (first 8 cells)
Adult Stem Cells
• Multipotent – give rise to a
small number of cells
– Derived from adult human
– Much less controversial;
being used in clinical trials
Blastocyst Stem Cells
• Pluripotent– give rise to many
but not all types of cells in the
body
– Derived from Inner Cell Mass (ICM)
Gene Cloning
• DNA/Molecular/Recombinant/Gene – DNA from one
organism is combined with a vector and then put into a
host cell
1.
2.
3.
4.
mRNA from eukaryotic cell is copied into cDNA
cDNA will be spliced into a vector plasmid with a restriction enzyme
The recombinant vector (cDNA + vector) is inserted into a host cell
Once in the host cell, the recombinant vector will replicate before
cell division
5. The cell will make the proteins encoded in the cDNA
Reproductive Cloning (SCNT)
Therapeutic Cloning
• Stem cells are extracted from a 5 day old embryo;
the embryo is destroyed but the stem cells can
transform into any of the 220 human cell types
Topic 12: Lesson 5
1. Read Chapter 11.4 pp. 229-31
2. PGD Video – Genetics & IVF Institute
3. What would you do? Alzheimer’s Disease
4. Module 4 – Max’s Case
DNA PROBES, PGD & GENE THERAPY
Probes Look for Genes
• DNA probes detect specific nucleotide sequences
in DNA
• DNA probes are ssDNA strands that are
complementary to a certain gene
–
–
–
–
Labeled with a fluorescent tag of radioactive isotope
Target sequence DNA is heated so it separates
DNA probe binds to region that it is complementary to
Scientists detect radioactivity or use a fluorescent
light to determine if the gene is present
FISH
Preimplantation Genetic Diagnosis
• If two carriers of a particular disease want to be
sure that they have a baby that is disease free,
PGD can reduce the odds
1. in vitro fertilization occurs (IVF)
2. Morula is formed and one of the cells is selected
for diagnosis
3. PCR amplifies the region of DNA where the
suspected allele may be
4. DNA probe determines if the alleles are present
of the recessive condition
5. Embryo can be discarded or implanted back into
the woman’s body if it is considered a healthy cell
Testing 1 of 8
• What bioethical issues
present themselves
when PGD is applied?
• Who should do PGD?
– Mothers over 35
– Repeated miscarriages
– Previous pregnancy
with chromosomal
abnormality
– X-linked carrier mothers
– Unexplained infertility
– IVF failures
Genotyping Using Gel Electro
Fixing Defective Genes
• Most genetic diseases do not have a cure, but
gene therapy could provide new treatment
options
• Gene therapy corrects defective genes with
genes from another human
– Swaps for the bad copy, reverses mutation, or
turns off the gene
• A vector (vehicle) that can deliver a gene to
all of the cells that are “infected”
– Viruses and plasmids are the two most commonly
used vectors
Treating Cystic Fibrosis with the Cold Virus
• Viruses can act as a
vector for CF patients
• The virus is introduced
to the lung cells with a
normally functioning
gene
• The CF gene is replaced
by the normal gene
RNA Interference
• RNAi can prevent translation of mRNA silencing
the gene
• RNAi bonds to a complementary mRNA strand to
make a double stranded and nonfunctional mRNA