8.1 - Manipulating & Cloning DNA Genetic Technologies 8.1 - Manipulating & Cloning DNA Image from: http://www.forbes.com/fdc/welcome_mjx.shtml

Insulin E. coli and safflower plants have both been used to produce human insulin How? Image from: http://www.license.umn.edu/Products/Implantable-Microvalve-Device-for-Controlled-Insulin-Delivery-in-Type-1-Diabetes-Patients__Z02003.aspx

Genetic Engineering human insulin gene can be introduced to plasmids (recombinant DNA) plasmids can be introduced to bacteria cells How? Image from: http://en.wikipedia.org/wiki/Plasmid

Restriction Enzymes are produced by bacteria to function as an “immune system” against invading viruses by cutting up the viral DNA or RNA

Restriction Enzymes restriction enzymes cut 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/

Image from: https://bsp.med.harvard.edu/node/41 What’s inaccurate about this image? Recognition sequence is not palindromic.

DNA Ligase DNA ligase can be used to attach restriction fragments See simple animation: http://www.dnalc.org/resources/animations/restriction.html

Plasmids circular pieces of non-chromosomal DNA found in bacteria cells Image from: http://en.wikipedia.org/wiki/Plasmid

Plasmids as Vectors genes can be inserted into plasmids using restriction enzymes and DNA ligase can then introduce these genes into host bacterial cells copy number of plasmids determines how much protein will be produced

Image from: http://www.abfrontier.com/cs/gene.do

Restriction Maps Image from: http://www.ruf.rice.edu/~bioslabs/bios111/bios111_day1.htm

Constructing Restriction Maps Try Tutorial 1: p.370-373 Sample Problem 1: Plasmid X undigested Plasmid X digested with EcoRI Plasmid X digested with BamHI Plasmid X digested with EcoRI and BamHI 1400 bp 600 bp 800 bp 100 bp 700 bp

Practice #1 (p.372) Plasmid Z uncut Plasmid Z cut with EcoRI Plasmid Z cut with PstI Plasmid Z cut with EcoRI and PstI 1500 bp 500 bp 1000 bp 300 bp 700 bp

Practice #2 (p.372) Plasmid W uncut Plasmid W cut with HindIII Plasmid W cut with BamHI Plasmid W cut with HindIII and BamHI 1000 bp 450 bp 550 bp 400 bp 600 bp 150 bp 250 bp 300 bp

Practice #3 (p.373) EcoRI BamHI SmaI EcoRI + BamHI EcoRI + SmaI 800 bp 1500 bp 250 bp 700 bp 1350 bp 900 bp 1400 bp 200 bp 300 bp 500 bp 1050 bp 350 bp 450 bp 150 bp 550 bp 600 bp 750 bp

Do you remember this? Image from: http://en.wikipedia.org/wiki/Griffith's_experiment

Transformation successful introduction of DNA from another source bacterial cells can be made competent by placing in CaCl2 solution (stabilizes phospholipid bilayer) and then rapid heating & re-cooling animation: http://www.dnalc.org/resources/animations/transformation1.html