Chapter 20 DNA Technology
Plasmid Recombinant DNA Ligase seals backbone
Only some bacteria get a plamid Antibiotic added to screen out those without plasmid Only one 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 Penicillin link
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
P 367 Plasmid has 2 genes Bacteria that take up the plasmid will gain antibiotic resistance Bacteria that have also taken up the Human gene will lose the lactase gene Blue colonies have the lactase gene White colonies DON’T have the lactase gene - but have the human gene
Cloning Animation Another cloning animation Animation of removing introns to put eukaryotic gene in prokaryote
Pearson Lab 6A simulation Turn in Lab Quiz 1 tomorrow.
Five Stages in Genetic Engineering • Isolate and Cleave DNA • Produce Recombinant DNA • Introduction of Vector into target cell • Clone Cells • 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
Eukaryotic Genes must be modified to work in bacteria Expression Vector is created 1. A Promotor must be inserted 2. Introns must be removed
All the genes of an organism represents a Genomic Library and may be stored in a series of vectors such as viruses of bacteria
Pioneering genetic engineering on mammals have been been done at UH 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.
Link to Cloning Mimi 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.
Stop 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." Review Random Fertilization
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!
Animation Link to PCR Animation on Web
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 If there is only a small sample of DNA available- more copies can be made by PCR -polymerase chain reaction (p371) Link to DNA Fingerprint Lab
P 373 Differences in DNA sequences on homologous chromosomes result in different restriction fragment length patterns RFLP - these may be sorted by length using gel electrophoresis
Short Long fragments
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 3-Move fragment to permanent substrate 4-Heat/Tag 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
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 % 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?
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 simulation Turn 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
Matching overlapping sequences allow scientist to put all the fragments in order Scientists have now sequenced the entire Human Genome opening up the Human Genetic Library for research
Sequencing of DNA is accomplished by copying one side one base at a time using a modified base with a dye molecule. Figure 13-7 DNA Sequencing Section 13-2 Dye molecules replace -OH group – stops replication