1. Biotechnology Spring 2013 - Althoff Reference: Mader & Windelspecht Ch. 14) Lec 21 DNA Cloning Recombinant DNA PCR Analysis Biotechnology

2. By definition, __________ is the production of genetically __________ copies of DNA, cells, or organisms through some __________ means. Ex.: when a plant sends up an underground stem or a root sends up a new shoot, the resulting plant is a clone of the original plant. Ex.: members of a bacteria colony on petri dish are clones because the all came from the division of a single original cell DNA Cloning

3. Reasons to clone: a) determine the difference in base _________ between a normal gene and a mutated gene b) use the genes to genetically _______ organisms in a beneficial way When cloned genes are used to modify a human, the process is called ______________ Otherwise, organisms are called ___________ organisms (trans = across, genic = producing). Value of transgenic organisms – produce a product desired by humans DNA Cloning

4. Recombinant DNA = ________ Contains DNA from ___________ different sources, such as a human cell and a bacterial cell. Recombinant DNA Technology

5. To make rDNA, one needs a _________ (i.e., a carrier) by which the rDNA will be introduced into a host cell Example of a vector: ________ = small accessory rings of DNA found in ________ that were first discovered in E. coli. The ring in not part of the main bacterial chromosome…and replicates on its own. Need 2 enzymes to introduce foreign DNA into vector DNA: 1) a restriction enzyme which cleaves DNA 2) DNA ligase (ligo = bind)…a ‘sealer’ Recombinant DNA Technology

6. 1 2

7. ________ Developed by Kary Mullis in 1985. Mullis earned Nobel Prize for Chemistry in 1993 Widely used in research labs to ____________ of a segment of DNA quickly in a test tube Process mimics DNA replication in the cell with the exception that the PCR is very specific—it amplifies (aka makes copies of) only a targeted DNA sequence Polymearse Chain Reaction

8. Requires the use of a) DNA polymerase b) supply of nucleotides for the new strands of DNA The DNA polymerase used is from the bacterium Thermus aquaticus—which lives in hot springs. It can withstand high temperatures used to separate the double-stranded DNA Referred to as a chain reaction because the targed DNA is repeatedly replicated as long as the process continues. PCR

9. Polymearse Chain Reaction

10. DNA amplified (i.e., copies made) enables one to decipher evolutionary history of organisms by looking at mitochrondrial DNA. It can also be used to ID a corpse to match to DNA found on the bristles of a toothbrush. Early analysis done using a process called ____ ________________ a) a process that separates DNA fragments according to their size b) separation process leaves a distinctive band that identifies a person, for example Analyzing DNA

12. Gel electrophoresis still used widely today…including in your Genetics course (if you continue on in Biology) More advanced (and requiring much more expensive equipment) is the STR Profiling method = short tandem repeat profiling. STR does __ require use of restriction enzymes Newest method to produce DNA profiles or “fingerprints” accomplished by fluorescently labeling DNA fragments. Involves a laser exciting the fluorescent DNA and a detector recording the amount of emissions for each DNA fragment Analyzing DNA…con’t

13. DNA “Fingerprint”

14. Transgenic bacteria, plants, and animals often now called GMOs = genetically modified organisms. GMO – bacteria: promote health of plants including one that helps plant roots be protected from some insect species. Some used to decompose oil !!!! Biotechnology Products

15. Gene Therapy Current use and much future potential for application to _____ many conditions harmful to humans