Manipulating DNA

Selective Breeding Man has selectively bred plants and animals for desired traits

Mutations of Pointsettias induced by radiation

New DNA Technology Now DNA can be manipulated to produce new combinations not found in nature How far should we go? We need to set bioethical limits

Restriction Endonucleases In 1980, geneticists spliced the human gene that encodes interferon into a bacterial cell’s genome. Marked beginning of genetic engineering. Through natural selection, some bacteria contain enzymes, restriction endonucleases, that fragment viral DNA. Recognize specific nucleotide sequences, and cleave the DNA. They recognize palindromes Methylate their own DNA for protection

Restriction Endonucleases Sequences recognized by restriction endonucleases are typically four to six nucleotides long, and are often palindromes. Restriction enzymes can bind to and cleave both strands of DNA. Raven - Johnson - Biology: 6th Ed. - All Rights Reserved - McGraw Hill Companies

Restriction Endonucleases There are hundreds of bacterial restriction endonucleases, each with a specific recognition sequence. Given restriction endonuclease can cut DNA from any source into fragments. Complementarity, single-stranded ends can pair with each other. Sticky Ends Fragments joined together with DNA ligase.

Using RE’s to Manipulate Genes Chimeras One of first recombinant genomes produced by genetic engineering was a bacterial plasmid into which an amphibian ribosomal RNA gene was inserted. Viruses can also be used as vectors to insert foreign DNA into host cells.

Genetic Engineering Experiment Four Stages DNA Cleavage Restriction endonuclease cleaves source DNA into fragments. Production of Recombinant DNA DNA fragments inserted into plasmids or viral vectors. Cloning Screening

Genetic Engineering Experiment Screening Clones with DNA fragment of interest are identified from the clone library. Preliminary Screening - Eliminate any clones without a vector and clones with vectors that do not contain DNA. Employ vector with gene for antibiotic resistance and lac Z’ gene. Expose to growth medium.

Genetic Engineering Experiment Secondary Screening (Gene of Interest) Hybridization - Cloned genes form base pairs with complementary sequences on another nucleic acid (probe). Grow on agar and then transfer to a filter pressed on colonies. Treat filter with radioactive probe, and perform autoradiography.

Working With Gene Clones Polymerase Chain Reaction (PCR) Used to copy specific gene sequences. Three Basic Steps Denaturation Annealing of Primers Primer Extension

Restriction Fragment Length Polymorphism (RFLP) Analysis DNA is cut into small pieces using restriction enzymes Many copies are made using PCR Radioactive probes attached to mark ends An electric current is used to separate pieces by size in a gel Larger pieces are slower and don’t move as far down the gel http://www.sumanasinc.com/webcontent/animations/content/gelelectrophoresis.html

DNA Fingerprinting DNA is unique for every individual Comparing the sequences of small pieces of DNA can be used: Find relationships Narrow crime suspects Identify organisms

DNA Sequence Technology Genome Sequencing Due to technological advances, sequencing of entire genomes is now practical.

DNA Sequence Technology Human Genome Project Human genome contains around 3.2 billion bases and about 30,000 genes. Human genes are quite fragmented. Fully one-third of genome is composed of noncoding repeated sequences. Genes are not evenly distributed over the genome.

DNA Sequence Technology Proteonomics Cataloging and analyzing every protein in the human body. Each gene’s nucleotide sequence specifies an amino acid sequence folded in a certain way.

Biochips Gene Microarrays (Biochips) are small squares of glass covered with millions of strands of DNA. Scanning beam used to build nucleotides. Look at Single Nucleotide Polymorphisms (SNP’s). Cancer Screening Personal Privacy Issues

Medical Applications Pharmaceuticals Introduction of protein-encoding genes. Atrial peptides - High blood pressure and kidney failure. Tissue plasminogen activator - Dissolving blood clots.

Medical Applications Gene Therapy Add working copies of single defective gene. Leber's congenital amaurosis X-linked SCID Adrenoleukodystrophy chronic lymphocytic leukemia acute lymphocytic leukemia multiple myeloma Parkinson's disease These recent clinical successes have led to a renewed interest in gene therapy

Medical Applications Piggyback Vaccines Produce subunit vaccines against viruses. Herpes Hepatitis DNA Vaccine Cellular immune response.

Agricultural Applications Ti plasmid has been early successful vector. Herbicide Resistance Insert genes encoding for proteins making crops resistant to herbicide. Widespread herbicide use possible. Nitrogen Fixation Introduce genes that allow crops to fix nitrogen. Reduce need for fertilizer.

Agricultural Applications Insect Resistance Insert genes encoding proteins harmful to insects. Real Promise - Produce genetically modified plants with traits benefiting consumers. Iron Deficiency in developing countries Transgenic Rice Increasing milk production Bovine Somatotrophin

Cloning Copyright © McGraw-Hill Companies Permission required for reproduction or display Raven - Johnson - Biology: 6th Ed. - All Rights Reserved - McGraw Hill Companies

Tools needed in Gene Cloning Restriction enzymes Cuts the desired DNA strands Bacterial plasmid cloning vector used to move DNA to another organism Host DNA Donor gene(s) Produces a transgenic organism with recombinant DNA

Clones Copies of one organism

Clones Produced by taking cuttings from one plant Grafting Budding Rooted cuttings

Clones Produced for many years by dividing an embryo to form identical quadruplets Cows Pigs

A New Type of Clone The nucleus of a donor cell The ova (egg) of another organism A surrogate mother

How it’s done…

FDA says cloned animals safe to eat Food producers, including Tyson Foods Inc. and Dean Foods Co., said they won't use cloned foods anytime soon. Bloomberg News / January 16, 2008 WASHINGTON - Cloned cows, pigs, goats, and their offspring are safe to enter the US food supply, regulators said over protests from lawmakers, consumer groups, and worried eaters.

Maryann Mott for National Geographic News April 4, 2006 Meet Royal Blue Boon Too, the first commercially cloned horse in the United States. The cost of the frisky foal: U.S. $150,000. That may be a bargain, considering that the original Royal Blue Boon, a 26-year-old American Quarter Horse now past breeding age, has earned more than $380,000 as a competition and show horse. . Champion Horse Cloned by Texas Breeder

Some problems to Overcome: Many casualties Birth defects Failures Premature aging Improve technique

Stem Cells Embryonic Stem Cells Tissue Specific Stem Cells Cells produced very early in development- each capable of developing into a healthy individual. Can develop into any tissue. May provide ability to restore damaged tissue. Tissue Specific Stem Cells As development proceeds, tissue-specific stem cells persist.

Controversial topics… Scientists hope to use cloned embryos to harvest precious "stem cells" — cells that, in theory, can ultimately grow into any kind of cell in the body. These cells could be used to generate new organs or cell clusters to treat patients with failing organs or degenerative diseases In the case of transplant patients, organs made from exact copies of an individual's own cells and would not be rejected. This research is still in its earliest phase of development. All of the embryos generated actually died — none grew beyond the six-cell stage Controversial: The thinking is that by allowing any kind of cloning of human embryos, it is then not such an enormous leap to apply the same techniques to making copies of people.

Ethics and Regulation Questions How do we measure the Potential Risks of Genetically-Modified Crops? Is Eating Genetically Modified Food Dangerous? Are Genetically Modified Crops harmful to the Environment? Should We Label Genetically Modified Foods?

Copyright © McGraw-Hill Companies Permission required for reproduction or display