- Slides: 61
Genetic Engineering • 1. Genetic engineering is a faster and more reliable method for increasing the frequency of a specific allele in a population by cutting fragments of DNA from one organism and inserting the fragments into a host organism of the same or a different species.
• (It is a way to increase the frequency of a specific allele by putting pieces of DNA from one organism into another)
Genetic Engineering • 2. You also may hear genetic engineering referred to as recombinant DNA technology. • 3. Recombinant DNA is made by connecting or recombining, fragments of DNA from different sources.
Transgenic organisms contain recombinant DNA • 4. Plants and animals that contain functional recombinant DNA from an organism of a different type are known as transgenic organisms because they contain foreign DNA.
5. Transgenic organisms contain recombinant DNA • The first step of the process is to isolate the foreign DNA fragment that will be inserted. • The second step is to attach the DNA fragment to a carrier. • The third step is the transfer into the host organism.
Restriction enzymes cleave (cut) DNA • To isolate a DNA fragment, small pieces of DNA must be cut from a chromosome. • 6. Restriction enzymes are bacterial proteins that have the ability to cut both strands of the DNA molecule at a specific nucleotide sequence.
Cut Cleavage Restriction enzymes cleave (cut) DNA Insertion Sticky ends
Vectors transfer DNA • 7. A vector is the means by which DNA from another species can be carried into the host cell. • Vectors may be biological or mechanical.
Vectors transfer DNA • 8. Biological vectors include viruses and bacterial plasmids. • 9. A plasmid, is a small ring of DNA found in a bacterial cell.
Vectors transfer DNA • 10. Two types of mechanical vectors carry foreign DNA into a cell’s nucleus
Vectors transfer DNA • One, a micropipette, is inserted into a cell; . the other is a microscopic metal bullet coated with DNA that is shot into the cell from a gene gun
Insertion into a vector • If a plasmid and foreign DNA have been cleaved with the same restriction enzyme, the ends of each will match and they will join together, reconnecting the plasmid ring. • The foreign DNA is recombined into a plasmid or viral DNA with the help of a second enzyme.
Gene cloning • After the foreign DNA has been inserted into the plasmid, the recombined DNA is transferred into a bacterial cell. • 11. An advantage to using bacterial cells to clone DNA is that they reproduce quickly; therefore, millions of bacteria are produced and each bacterium contains hundreds of recombinant DNA molecules.
Gene cloning • 12. Clones are genetically identical copies. • Plasmids also can be used to deliver genes to animal or plant cells, which incorporate the recombinant DNA.
Gene cloning • 13. Each time the host cell divides it copies the recombinant DNA along with its own.
Gene cloning Foreign DNA (gene for human growth hormone) Cleavage sites Plasmid Recombined plasmid Recombined DNA Bacterial chromosome E. coli Human growth hormone
Cloning of animals • Although their techniques are inefficient, scientists are coming closer to perfecting the process of cloning animals.
What animals have been cloned? • Scientists have been cloning animals for many years. • In 1952, the first animal, a tadpole, was cloned. • Dolly the Sheep was cloned in 1997. • www. ornl. gov/sci/techresources/Human_Genome/elsi/cloning. shtml#animals. Q
• Before the creation of Dolly, the first mammal cloned from the cell of an adult animal, clones were created from embryonic cells. • Since Dolly, researchers have cloned a number of large and small animals including sheep, goats, cows, mice, pigs, cats, and rabbits. All these clones were created using nuclear transfer technology. • Hundreds of cloned animals exist today, but the number of different species is limited. Attempts at cloning certain species have been unsuccessful.
• The most productive and useful plants and animals can be cloned to help humans. • The drawback is using older DNA because it will have accumulated damage over the years. The clone will likely show signs of age early.
In the Star Wars movies, all of the storm troopers were clones of this one indidual who was a good soldier. This is science fiction, but the application is the same.
Polymerase chain reaction • 14. A method called polymerase chain reaction (PCR) has been developed in order to replicate DNA outside living organisms, • This method uses heat to separate DNA strands from each other.
Genetic markers are a known location of a gene, which is used as a reference point to describe the location of other genes.
Polymerase chain reaction • The machine repeatedly replicates the DNA, making millions of copies in less than a day.
Sequencing DNA • In DNA sequencing, millions of copies of a double-stranded DNA fragment are cloned using PCR. Then, the strands are separated from each other. • The single-stranded fragments are placed in four different test tubes, one for each DNA base.
Sequencing DNA • Each tube contains four normal nucleotides (A, C, G, T) and an enzyme that can catalyze the synthesis of a complementary strand. • One nucleotide in each tube is tagged with a different fluorescent color. • The reactions produce complementary strands of varying lengths.
Sequencing DNA • 15. These strands are separated according to size by 16. gel electrophoresis producing a pattern of fluorescent bands in the gel. • The bands are visualized using a laser scanner or UV light.
Gel Electrophoresis • Restriction enzymes are the perfect tools for cutting DNA. However, once the DNA is cut, a scientist needs to determine exactly what fragments have been formed. .
Restriction enzymes • Either one or several restriction enzymes is added to a sample of DNA. The restriction enzymes cut the DNA into fragments. DNA fragments
The gel • With a consistency that is firmer than dessert gelatin, the gel is molded so that small wells form at one end. Gel • DNA fragments are placed into small wells at the end of a firm block of gel.
Power source An electric field • The gel is placed in a solution and an electric field is applied. One end of the gel is positive and the other end is negative. Negative end Positive end
The fragments move Completed gel Shorter fragments Longer fragments • The negatively charged DNA fragments travel toward the positive end.
The fragments move • 18. The smaller the fragment, the faster it moves through the gel. • The smallest fragments move the farthest from the well.
Making a Gel (summarized)(#17) • 1. Restriction enzymes cut DNA into fragments • 2. DNA fragments are placed into small wells at the end of a firm block of gel which glows under UV light. • 3. One end of the gel is made + (pos. ) and the other – (neg. ) • 4. Neg. charged DNA fragments move toward the + end.
Applications of DNA Technology • The main areas proposed for recombinant bacteria are in industry, medicine, and agriculture. Recombinant DNA in industry • Many species of bacteria have been engineered to produce chemical compounds used by humans.
19. Recombinant DNA in industry • Scientists have modified the bacterium E. coli to produce the expensive indigo dye that is used to color denim blue jeans.
19. Industrial Applications of DNA Technology The production of • cheese • paper • laundry detergents • sewage treatment
20. Recombinant DNA in medicine • Pharmaceutical companies already are producing molecules made by recombinant DNA to treat human diseases. • Recombinant bacteria are used in the production of human growth hormone to treat pituitary dwarfism and insulin to treat diabetes.
20. Recombinant DNA in medicine • Scientists can study diseases and the role specific genes play in an organism by using transgenic animals
21. Transgenic animals • . An animal that contains recombinant DNA from other organisms inserted into them is called a transgenic organism.
Transgenic animals • Mouse chromosomes also are similar to human chromosomes. • Scientists know the locations of many genes on mouse chromosomes.
Transgenic animals • On the same farm in Scotland that produced the cloned sheep Dolly, a transgenic sheep was produced that contained the corrected human gene for hemophilia A. • This human gene inserted into the sheep chromosomes allows the production of the clotting protein in the sheep’s milk.
22. Recombinant DNA in agriculture • Recombinant DNA technology has been highly utilized in the agricultural and food industries. • Crops have been developed that are better tasting, stay fresh longer, and are protected from disease and insect infestations.
Recombinant DNA in agriculture The Most Common Genetically Modified (GM) Crops Millions of hectares 150 140 7% 100 50 0 72 36% Soybeans Corn 34 25 16% 11% Canola Cotton
23. Mapping and Sequencing the Human Genome • In 1990, scientists in the United States organized the Human Genome Project (HGP). It is an international effort to completely map and sequence the human genome, the approximately 35 000 -40 000 genes on the 46 human chromosomes. • The human genome map shows the sequence of the genes on the 46 chromosomes.
Linkage Maps show the positions of known genes on a chromosome.
Crossing over and linkage maps.
Mapping and Sequencing the Human Genome • In February of 2001, the HGP published its working draft of the 3 billion base pairs of DNA in most human cells. • The sequence of chromosomes 21 and 22 was finished by May 2000. • It was completed in 2003, but the data is still being analyzed.
24. Applications of the Human Genome Project • Improved techniques for • prenatal diagnosis of human disorders, • use of gene therapy, and • development of new methods of crime detection
Diagnosis of genetic disorders • One of the most important benefits of the HGP has been the diagnosis of genetic disorders.
Diagnosis of genetic disorders • The DNA of people with and without a genetic disorder is compared to find differences that are associated with the disorder. Once it is clearly understood where a gene is located and that a mutation in the gene causes the disorder, a diagnosis can be made for an individual, even before birth.
Gene therapy • Individuals who inherit a serious genetic disorder may now have hope—gene therapy. 25. Gene therapy is the insertion of normal genes into human cells to correct genetic disorders. Much research is being done, but the FDA has not approved any therapy for sale.
DNA fingerprinting • DNA fingerprinting can be used to convict or acquit individuals of criminal offenses because every person is genetically unique. • 26. DNA fingerprinting works because no two individuals (except identical twins) have the same DNA sequences, and because all cells (except gametes) of an individual have the same DNA.
• To identify individuals, forensic scientists scan 13 DNA regions, or loci, that vary from person to person and use the data to create a DNA profile of that individual (sometimes called a DNA fingerprint). There is an extremely small chance that another person has the same DNA profile for a particular set of 13 regions. (Human genome project info)
• Human Genome Project--DNA fingerprinting
DNA fingerprinting • In a forensic application of DNA fingerprinting, a small DNA sample is obtained from a suspect and from blood, hair, skin, or semen found at the crime scene. • The DNA, which includes the unique non-coding segments, is cut into fragments with restriction enzymes.
DNA fingerprinting • The fragments are separated by gel electrophoresis, then further analyzed. If the samples match, the suspect most likely is guilty.