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 EnzymesImage from: http://barleyworld.org/css430_09/lecture%208-09/notes8-09.htm

Blunt Ends vs. Sticky EndsImage 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

MethylasesImage 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/gelelectrophoresis.html