6.1 - Biotechnological Tools & Techniques 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

Blunt Ends vs. Sticky Ends Image from: http://schoolworkhelper

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 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/gelelectrophoresis.html