chapter 20 dna technology p 365 animation
TRANSCRIPT
Chapter 20
DNA Technology
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Plasmid
Recombinant DNA Ligase seals backbone
Ligase seals backbone
Only some bacteria get a plamid
Only some bacteria get a plamid
Antibiotic added to screen out those without plasmid
Antibiotic added to screen out those without plasmid
Only one plasmid has the FROG gene
Only one plasmid has the FROG gene
Frog rRNA labelled with radioactive tracer is used to find which plasmid has the frog gene
Frog rRNA labelled with radioactive tracer is used to find which plasmid has the frog gene
Bacteria with frog gene can now be cloned
Bacteria with frog gene can now be cloned
Penicillin link
The plasmids are reinserted into the bacteria, but only some take up the plasmid -they must be SCREENED OUT
The plasmids are reinserted into the bacteria, but only some take up the plasmid -they must be SCREENED OUT
DNA probe (with radioactive tag) complementary to frog gene
Animation
Plasmid has 2 genesPlasmid has 2 genes
Bacteria that take up the plasmid will gain antibiotic resistance
Bacteria that take up the plasmid will gain antibiotic resistance Bacteria that
have also taken up the Human gene will lose the lactase gene
Bacteria that have also taken up the Human gene will lose the lactase gene
Blue colonies have the lactase gene
Blue colonies have the lactase gene
White colonies DON’T have the lactase gene - but have the human gene
White colonies DON’T have the lactase gene - but have the human gene
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Animation of removing introns to put eukaryotic gene in prokaryote
Cloning AnimationAnother cloning animation
Pearson Lab 6A simulationTurn in Lab Quiz 1 tomorrow.
Five Stages in Genetic Engineering
1. Isolate and Cleave DNA
2. Produce Recombinant DNA
3. Introduction of Vector into target cell
4. Clone Cells
5. Screen Target Cells• (Clone the screened cell once the target cell is
chosen)• Usually genetically modified bacteria are
crippled so that it cannot survive outside lab
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Eukaryotic Genes must be modified to work in bacteria
Expression Vector is created
1. A Promotor must be inserted
2. Introns must be removed
Eukaryotic Genes must be modified to work in bacteria
Expression Vector is created
1. A Promotor must be inserted
2. Introns must be removed
Genes can be found using tagged cDNA
Genes can be found using tagged cDNA
All the genes of an organism represents a Genomic Library and may be stored in a series of vectors such as viruses of bacteria
All the genes of an organism represents a Genomic Library and may be stored in a series of vectors such as viruses of bacteria
Plucky is an albino Xenopus laevis frog expressing green fluorescent protein (GFP) in her eye. GFP is a jellyfish protein that fluoresces bright green when illuminated by blue light.
Pioneering genetic engineering on mammals have been been done at UH
THE FIRST MOUSE CLONES The clones (two brown mice at bottom) are genetic duplicates of the mouse at top right, which donated its cumulous cells. They are the result of a technique perfected at the University of Hawaii in 1998
WILBUR WANNABES The first litter of cloned pigs, born in 2000 in Virginia, demonstrate that cloning could be used to generate organs for human transplant in the near future
Dr. Severino Antinori, an Italian embryologist, fires up the press in 2001 after announcing plans to clone the first human to help infertile couples have children. He claimed one of his patients was carrying a clone, but he failed to confirm his tale or produce the child. His comments launched a debate over the ethics of cloning human beings; countries like Britain and South Korea have since made it illegal to clone people, while the U.S. Congress has yet to ban the process.
Not to be outdone, Chinese researchers are perfecting cloning techniques in the hope of using the procedure to preserve the country's beloved panda species. For practice, they began with more common species, including goats like Yangyang (above). Cloning remains a tricky process; only 2%-5% of the eggs that start out as clones develop into live animals. The good news is that once they survive past the first year, clones like Yangyang, celebrating her sixth birthday, are relatively healthy.
Review Random Fertilization
Review Random FertilizationStop Three-Parent Babies
Scientists: Regulate Fertility Clinics To Prevent Babies with New Genes
By Robin Eisner
N E W Y O R K, May 18 — Scientists are calling for the immediate regulation of fertility clinics to prevent the birth of any future gene-altered babies, the first of which was reported earlier this year. STORY HIGHLIGHTS Fertility Method Creates Gene-Altered Babies Extra Genes From Mitochondria Social and Safety Consequences of Technology
In March, a team of fertility specialists at the Institute for Reproductive Medicine and Science of St. Barnabas, in West Orange, N.J., reported "the first case of human … genetic modification resulting in normal healthy children."
Fertility Method Creates Gene-Altered Babies
The group used a method that extracted cellular material from a donor woman's egg cell and transferred it into an infertile woman's egg. This material allowed the woman's egg to become fertile.
The donor egg contained DNA from mitochondria, little organs inside the cell that create the energy to do life's work. The group believes that problems with the mitochondria prevented the infertile women from becoming pregnant.
Mitochondria contain only about 0.03 percent of a cell's DNA, but that's enough that they can make copies of themselves when the cells divide. The other 99.97 percent of a cell's DNA comes from the nucleus and the 23 pairs of chromosomes.
The group says that transferring this mitochondrial DNA into the recipient eggs resulted in the birth of 30 babies, the first of which was born in 1997.
Extra Genes From Mitochondria
In March, the group reported for the first time in the medical journal Human Reproduction that genetic tests on two babies showed they had DNA from three parents: Two babies born with this method actually had mitochondrial genes from the donor mom, as well as chromosomal genes from the mother and father.
This extra-parental mitochondrial DNA could be transferred to the next generation.
Scientists in the latest issue of the journal Science are calling for the regulation of fertility clinics to prevent this practice from continuing.
"No research or clinical application involving humans should proceed that have the direct or indirect potential to cause inheritable genetic modification in either the public or private sector," unless it is reviewed by already existing federal regulators or a new body, wrote Mark S. Frankel and Audrey Chapman.
Both authors preside over public policy programs at the American Association for the Advance of Science, which publishes Science.
The two authors warn that efforts to modify genes transmitted to future generations could bring about both a medical and social revolution.
Social and Safety Consequences of Technology
"The dilemma is that inheritable genetic modification techniques developed for normal therapeutic purposes are also likely to be suitable for genetic alterations intended to improve what are already 'normal' genes," they write.
They warn that in a market economy the division between the haves and have-nots would increase if those who could pay could add "inherited advantage to the benefits of nurture and education already enjoyed by the affluent."
Safety concerns are also paramount, the authors say. It remains unclear how future generations with such genetic changes would fare.
"We have little experience and no evidence of long-term safety of inheritable genetic modification, whether intended or inadvertent," they write.
"There has not even been public consideration of how one would proceed in determining safety across generations. We should begin establishing an oversight process now so that we can make informed and reasoned choices about the future."
Geneticists had taken the luciferase gene from a firefly and inserted it into a tobacco plant. This meant that when the plant was fed with luciferin the result was a plant that glows in the dark!
Link to PCR Animation on Web
Animation
Different people have different DNA
-DNA when cut by enzymes will leave different size fragments
-which will separate into different electrophoresis patterns
-a DNA fingerprint
Different people have different DNA
-DNA when cut by enzymes will leave different size fragments
-which will separate into different electrophoresis patterns
-a DNA fingerprint
Link to DNA Fingerprint Lab
Link to DNA Fingerprint Lab
If there is only a small sample of DNA available- more copies can be made by PCR -polymerase chain reaction (p371)
If there is only a small sample of DNA available- more copies can be made by PCR -polymerase chain reaction (p371)
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Differences in DNA sequences on homologous chromosomes result in different restriction fragment length patterns RFLP
- these may be sorted by length using gel electrophoresis
Differences in DNA sequences on homologous chromosomes result in different restriction fragment length patterns RFLP
- these may be sorted by length using gel electrophoresis
Long frag
ments
Short
Specific genes (or fragments), how many places they show up plus the DNA fragments that they can be found is detected using Southern Blotting
1-Cut 2-Separate
4-Heat/Tag
3-Move fragment to permanent substrate
Animation
Different restriction fragments are found at different frequencies within different
people
For example fragment A may be found in 50% of the population, fragment B might be found in 10% of the population and fragment C might be found in 3% of the population
RFLP = restriction fragment length polymorphism
• If a person has fragment A, B and C in his DNA fingerprint and the fragments are found in these percentages in the population A= 50%, B= 10% and C=3%
What is the chance that someone else has the same 3 fragments in their fingerprint?
RFLP analysis identifies the presence of a specific gene by looking for an associated RFLP marker (recognition site) near the allele.
Animation
Certain restriction fragments can be looked for in a person’s fingerprint by adding a radioactive or dye labeled DNA probe that is complementary to the DNA of the fragment
Why is a DNA fingerprint NOT the same as a real fingerprint?
•ANSWER= A x B x C = .5 x .10 x .03 = .0015 or .15% or 1 out of 667 people
•Testing more fragments gives a smaller %
One to one relationship
Statistical relationship
OJ Simpson Trial• Odds of seeing 3 albino deer at the
same time:
85 million to 1• Odds of the blood on the glove not
being from R. Goldman, N. Brown-Simpson, and O.J. Simpson:
21.5 billion to 1
Pearson Lab 6B Electrophoresis simulationTurn in Lab Quiz 2 tomorrow.
Mapping Genomes3 steps
• Genetic (linkage) mapping (this was covered earlier –remember in the fly lab, the black body with vestigial wings genes were mapped using crossing over frequencies)
• Physical mapping
• DNA sequencing
Scientists have now sequenced the entire Human Genome
opening up the Human Genetic Library for research
Scientists have now sequenced the entire Human Genome
opening up the Human Genetic Library for research
Matching overlapping sequences allow scientist to put all the fragments in order
Section 13-2
Figure 13-7 DNA SequencingSequencing of DNA is accomplished by copying one side one base at a time using a modified base with a dye molecule.
Dye molecules replace -OH group – stops replication
DNA Microarray Assay of Gene Expression Levels. Genetic testing (genetic screening) allows the genetic diagnosis of vulnerabilities to inherited diseases, and can also be used to determine a person's ancestry
Slide 36Animation link
animation
Ti plasmids in bacteria can cause tumors in plants
Ti plasmids in bacteria can cause tumors in plants
If the tumor causing gene is removed from the plasmid and a useful gene spliced in - the Ti plasmid can be used as a VECTOR to move genes into plants
If the tumor causing gene is removed from the plasmid and a useful gene spliced in - the Ti plasmid can be used as a VECTOR to move genes into plants
Ti plasmid uses Agrobacterium tumefaciens to transduce its genetic material to plants
Cannot be used on grain plants
Practical Uses of DNA Technology
Diagnosis of disease
Human gene therapy
Pharmaceutical products (vaccines)
Forensics
Animal husbandry (transgenic organisms)
Genetic engineering in plants
Ethical concerns?
15.9
A genetically modified tobacco plant designed to resist a virus
Enzymes that eat holes in the gut of the caterpillar inserted into the plant.
Enzymes that eat holes in the gut of the caterpillar inserted into the plant.
Nodules in Legume plantNodules in Legume plant
Nitrogen fixing bacteria live in the nodules and provide the plant with a source of nitrogen
Nitrogen is needed to make proteins and DNA.
Scientists are trying to insert the enzymes to fix nitrogen into other plants to save on costly nitrogen fertilizers.
Nitrogen fixing bacteria live in the nodules and provide the plant with a source of nitrogen
Nitrogen is needed to make proteins and DNA.
Scientists are trying to insert the enzymes to fix nitrogen into other plants to save on costly nitrogen fertilizers.
Grain and Cereal Plants cannot use the Ti plasmid as a vector, but genes can be inserted using a DNA gun
Grain and Cereal Plants cannot use the Ti plasmid as a vector, but genes can be inserted using a DNA gun
A DNA gun can be used to shoot genes into cells or cell parts like chloroplasts
Growth hormones can increase yield and in cows, increase milk production
Growth hormones can increase yield and in cows, increase milk production
Genes for Human INSULIN might be inserted into plasmids
Genes for Human INSULIN might be inserted into plasmids
Bacteria can now produce Human insulin
Bacteria can now produce Human insulin
AIDS is an virus containing RNA
-the information for surface Antigens is located here
AIDS is an virus containing RNA
-the information for surface Antigens is located here
Proteins or ANTIGENS on the surface
Proteins or ANTIGENS on the surface
Cowpox viruses can be made with AIDS proteins (ANTIGENS) on its surface
Cowpox viruses can be made with AIDS proteins (ANTIGENS) on its surface
Humans will make ANTIBODIES to attack the AIDS ANTIGENS
Humans will make ANTIBODIES to attack the AIDS ANTIGENS
AIDS antigen gene
AIDS antigen gene
Vaccine animation
Inactivated cold viruses can be used as a VECTOR to move the genes for ANTIGENS from other viruses into a person.
For example this virus might carry genes for the herpes surface protein. If this was injected into a person, he would make ANTIBODIES for the herpes proteins -thus giving him immunity to herpes.
Inactivated cold viruses can be used as a VECTOR to move the genes for ANTIGENS from other viruses into a person.
For example this virus might carry genes for the herpes surface protein. If this was injected into a person, he would make ANTIBODIES for the herpes proteins -thus giving him immunity to herpes.
ADA: The First Gene Therapy Trial
• A four-year old girl became the first gene therapy patient on September 14, 1990 at the NIH Clinical Center. She has adenosine deaminase (ADA) deficiency, a genetic disease which leaves her defenseless against infections (SCIDS). White blood cells were taken from her, and the normal genes for making adenosine deaminase were inserted into them. The corrected cells were reinjected into her. Dr. W. French Anderson helped develop this landmark clinical trial when he worked at the National Heart, Lung, and Blood Institute
Cystic Fibrosis
• CF causes the body to produce thick, sticky mucus that clogs the lungs, leads to infection, and blocks the pancreas, which stops digestive enzymes from reaching the intestine where they are required in order to digest food.
Cystic Fibrosis: A Single Gene Disease
• Mutations in a single gene - the Cystic Fibrosis Transmembrane Regulator (CFTR) gene - causes CF. (autosomal recessive)
• In normal cells, the CFTR protein acts as a channel that allows cells to release chloride and other ions. But in people with CF, this protein is defective and the cells do not release the chloride. The result is an improper salt balance in the cells and thick, sticky mucus.
Gene Therapy Research Offers Promise of a Cure for Cystic Fibrosis
• In 1993, the first experimental gene therapy treatment was given to a patient with CF. Researchers modified a common cold virus to act as a delivery vehicle - or "vector"- carrying the normal genes to the CFTR cells in the airways of the lung.
Leber congenital amaurosis (LCA) is an inherited retinal disease that causes severe visual impairment in infancy or early childhood. Current research on a gene transfer therapy may offer hope to people with a form of this disease
Genetic Engineering may have environmental risks
Can you name some?
TIL Tumor Infiltrating Lymphocytes have the ability to find and slow the growth of tumors
TIL Tumor Infiltrating Lymphocytes have the ability to find and slow the growth of tumors
By genetically adding the TNF gene to these cell will increase their effectiveness at destroying tumor cells
By genetically adding the TNF gene to these cell will increase their effectiveness at destroying tumor cells
Altering a person’s genes to combat disease is called
Gene Therapy
Altering a person’s genes to combat disease is called
Gene Therapy
Changing only the genes in body cells like lymphocytes is called somatic cell gene therapy
Changing only the genes in body cells like lymphocytes is called somatic cell gene therapy
Chapter 20
DNA Technologyand Genomics
• The principal problem with inserting an unmodified mammalian gene into the bacterial chromosome, and then getting that gene expressed, is that A. prokaryotes use a different genetic code from that
of eukaryotes. B. bacteria translate polycistronic messages only. C. bacteria cannot remove eukaryotic introns. D. bacterial RNA polymerase cannot make RNA
complementary to mammalian DNA. E. bacterial DNA is not found in a membrane-
enclosed nucleus and is therefore incompatible with mammalian DNA.
• Which of the following statements is consistent with the results below? *
A. B is the child ofA and C.
B. C is the child ofA and B.
C. D is the child ofB and C.
D. A is the child ofB and C.
E. A is the child ofC and D.
• Which of the following statements is most likely true?
A. D is the child ofA and C.
B. D is the child ofA and B.
C. D is the child ofB and C.
D. A is the child ofC and D.
E. B is the child ofA and C.
• Which of the following are probably siblings?
A. A and B
B. A and C
C. A and D
D. C and D
E. B and D
• The segment of DNA shown in the figure below has restriction sites I and II, which create restriction fragments A, B, and C. Which of the gels producedby electrophoresis shown below would represent the separation and identity of these fragments?
• This restriction fragment contains a gene whose recessive allele is lethal. The normal allele has restriction sites for the restriction enzyme PSTI at sites I and II. The recessive allele lacks restriction site I. An individual who had a sister with the lethal trait is being tested to determine if he is a carrier of that allele. Indicate which of these band patterns would be produced on a gel if he is a carrier (heterozygous for the gene)?