1. Very Basic Biotechnology Supplemental instruction Designed by Pyeongsug Kim ©2010 sibio@att.netsibio@att.net Fall 2010 For Dr. Wright’s Bio 7/27 Class

2. Biotechnology review Define: recombinant DNA technology; genetic engineering; gene cloning How do restriction enzymes work? How are they used for cloning? What is an expression vector? How does the polymerase chain reaction (PCR) work? What is a probe? What are some possible uses for probes?

3. Designed by Pyeongsug Kim, ©2010 www.science-i.comwww.science-i.comBiotechnology -The use of microbiological and biochemical techniques to solve practical problems and produce more useful(desirable) products. Picture from http://www.plantsci.cam.ac.uk/Haseloff/teaching/PlantBiotech/index.htmlhttp://www.plantsci.cam.ac.uk/Haseloff/teaching/PlantBiotech/index.html Genetic engineering -Alternating an organism’s genetic engineering using in vitro techniques.

4. A procedure performed in vitro (Latin: within the glass) is performed not in a living organism but in a controlled environment, such as in a test tube or Petri dish. Designed by Pyeongsug Kim, ©2010 www.science-i.comwww.science-i.com From http://en.wikipedia.org/wiki/In_vitrohttp://en.wikipedia.org/wiki/In_vitro Human embryos photographed developing in vitro

5. Picture from http://employees.csbsju.edu/hjakubowski/classes/ch331/dna/plasmid.gifhttp://employees.csbsju.edu/hjakubowski/classes/ch331/dna/plasmid.gif Using microbes(usually bacteria) -Protein production -DNA production -Gene regulation research -”DNA library” -Why microbes? Grow fast (even single-celled eukaryotes like yeast) Don’t take up much space Economical source of genetically-engineered material Have the same DNA as all living organisms.(DNA or RNA) Designed by Pyeongsug Kim, ©2010 www.science-i.comwww.science-i.com

6. Recombinant DNA technology : DNA molecule created by joining DNA fragments from two different sources (two different organisms). Picture from http://citnews.unl.edu/hscroptechnology/html/printLesson.shtml?lessonID=990631835http://citnews.unl.edu/hscroptechnology/html/printLesson.shtml?lessonID=990631835 Designed by Pyeongsug Kim, ©2010 www.science-i.comwww.science-i.com

7. ________________ -naturally occurring bacterial enzyme -recognizes and cut a specific nucleotide sequence of DNA. -used to cut DNA at specific points during production of recombinant DNA. -ECoRI ~ restriction enzyme from E.Coli(RY13) -can be joined to fragments to other source. For recombinant DNA, first, the DNA needs to be cut! And glue it! Restriction enzymes Picture from http://employees.csbsju.edu/hjakubowski/classes/ch331/dna/plasmid.gifhttp://employees.csbsju.edu/hjakubowski/classes/ch331/dna/plasmid.gif Designed by Pyeongsug Kim, ©2010 www.science-i.comwww.science-i.com Originally the bacterial enzyme that stops viral reproduction by cleaving viral DNA.

8. Expression vectors Picture from http://www.artes-biotechnology.com/service/hansenula.jsphttp://www.artes-biotechnology.com/service/hansenula.jsp _______________ -Generally a plasmid -used to introduce a specific gene into a target cell. -facilitate transcription and translation of cloned DNA. Designed by Pyeongsug Kim, ©2010 www.science-i.comwww.science-i.com

9. Picture from http://www.artes-biotechnology.com/service/hansenula.jsphttp://www.artes-biotechnology.com/service/hansenula.jsp Gene cloning -Fragments can be “cloned” into “expression vectors” to be inserted into microbes -the cloned cDNA is incorporated into an expression vector or plasmid and transferred into bacterial or yeast cells.  The cells multiply (cloning) The interesting DNA needs to be cloned. Designed by Pyeongsug Kim, ©2010 www.science-i.comwww.science-i.com

10. Cloning step -Isolate DNA. -Use a restriction enzyme to generate fragment. -Generate a recombinant molecule. -Introduce recombinant molecule. -Introduce recombinant into new host. Designed by Pyeongsug Kim, ©2010 www.science-i.comwww.science-i.com

11. To express the protein, the plasmid must be placed inside a cell, either prokaryote or eukaryote. Designed by Pyeongsug Kim, ©2010 www.science-i.comwww.science-i.com

12. Is there my interesting DNA? ___________ -Single-stranded piece of DNA tagged with a detectable marker its complement -used to detect its complement. -Analysis of DNA within the cell (germline) DNA Probes A lineage of cells from which gametes are derived. Also known as germ track Picture from http://citnews.unl.edu/hscroptechnology/html/printLesson.shtml?lessonID=990631835http://citnews.unl.edu/hscroptechnology/html/printLesson.shtml?lessonID=990631835 Designed by Pyeongsug Kim, ©2010 www.science-i.comwww.science-i.com

13. ______________________ -Technique used to exponentially amplify specific regions of a DNA molecule. (PCR)Polymerase chain reaction Picture from http://blogs.newamericamedia.org/photo-of-the-day/103/pcr http://nobelprize.org/nobel_prizes/chemistry/laureates/1993/illpres/pcr.htmlhttp://blogs.newamericamedia.org/photo-of-the-day/103/pcrhttp://nobelprize.org/nobel_prizes/chemistry/laureates/1993/illpres/pcr.html Designed by Pyeongsug Kim, ©2010 www.science-i.comwww.science-i.com

14. ______________________ -Can be used to detect the presence of DNA sequence. -Diagnostics, forensic analysis Polymerase chain reaction (PCR) Designed by Pyeongsug Kim, ©2010 www.science-i.comwww.science-i.com