Genetic Technologies Manipulating & Cloning DNA

Insulin E. coli and safflower plants have both been used to produce human insulin How?

Genetic Engineering human insulin gene can be introduced to plasmids (recombinant DNA) plasmids can be introduced to bacteria cells How?

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

Blunt Ends vs. Sticky Ends

DNA Ligase DNA ligase can be used to attach restriction fragments See simple animation:

Plasmids circular pieces of non-chromosomal DNA found in bacteria cells

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

Restriction Maps

Constructing Restriction Maps Try Tutorial 1: p 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 600 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 bp500 bp 1000 bp 1500 bp300 bp 500 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 bp450 bp 550 bp 400 bp 600 bp 150 bp 250 bp 300 bp

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

Do you remember this?

Transformation successful introduction of DNA from another source bacterial cells can be made competent by placing in CaCl 2 solution

Technique Animation that shows engineering of plasmids with characteristics of 2 anti- biotic strains of E. coli & their introduction to E. coli cells: s/transformation1.html

Transformation of E. coli An excellent explanation of how a desired gene (e.g., human insulin gene) is introduced to plasmids with 2 anti- biotic resitant genes: level/biology/genetic-engineering/revise- it/transferring-the-genehttp:// level/biology/genetic-engineering/revise- it/transferring-the-gene

Hybridization