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Transcript
Biotechnology 6.1 - Biotechnological Tools & Techniques Biotechnology • the use of living organisms in industrial, agricultural, medical, and other technological applications • there are many biotechnological tools and techniques used to gain knowledge of and to manipulate DNA Restriction Enzymes • first restriction endonuclease (enzyme) isolated in 1970 • produced by bacteria • function as an “immune system” against invading viruses by cutting up the viral DNA or RNA Restriction Enzymes • restriction enzymes cut double-stranded DNA at a specific recognition site • recognition sites are always palindromic: (same sequence when read from the 5’ to 3’ direction on either strand) Restriction Enzymes Image from: http://barleyworld.org/css430_09/lecture%208-09/notes8-09.htm Blunt Ends vs. Sticky Ends Image from: http://schoolworkhelper.net/2010/07/restriction-endonucleases-or-restriction-enzymes/ DNA Ligase • DNA ligase can be used to form the phosphodiester linkages between fragments of DNA cleaved by restriction enzymes • See simple animation: http://www.dnalc.org/resources/animations/restriction.html Methylases Image on next slide: http://www.uic.edu/classes/bios/bios100/lectures/techniques.htm • enzymes that protect bacterial DNA from being cleaved by restriction enzymes • add methyl groups (-CH3) to one of the bases in the recognition site, preventing digestion by restriction enzymes Plasmids • circular pieces of non-chromosomal DNA found in bacteria cells • artificial plasmids have been engineered to contain an area with many recognition sites (and none in other areas) Plasmids • DNA can be inserted into plasmids by using restriction enzymes and DNA ligase • can then introduce these genes into bacterial cells: transformation Plasmids • bacteria are able to express foreign genes inserted into plasmids, such as the gene for insulin • See narrated animation: http://www.sinauer.com/cooper5e/animation0402.html Gel Electrophoresis • a method of separating molecules based on size • DNA migrates through the gel towards a positive electrode • smaller fragments move faster through the gel, causing separation by size • DNA fragments can be seen by using a stain such as ethidium bromide Gel Electrophoresis • Excellent interactive animation of gel electrophoresis: http://learn.genetics.utah.edu/content/labs/gel/ • Slideshow with focus on scientists and history of gel electrophoresis: http://www.dnalc.org/resources/animations/gelelectroph oresis.html