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Genetic Technologies 8.1 - Manipulating & Cloning DNA.
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Transcript of Genetic Technologies 8.1 - Manipulating & Cloning DNA.
Genetic Technologies
8.1 - 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:http://www.dnalc.org/resources/animations/restriction.html
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.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 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 bp 500 bp
1000 bp
1500 bp 300 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 bp 450 bp
550 bp
400 bp
600 bp
150 bp
250 bp
300 bp
300 bp
Practice #3 (p.373)
EcoRI BamHI SmaI EcoRI + 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
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 CaCl2 solution
Technique
• Animation that shows engineering of plasmids with characteristics of 2 anti-biotic strains of E. coli & their introduction to E. coli cells:
http://www.dnalc.org/resources/animations/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:
• http://www.s-cool.co.uk/a-level/biology/genetic-engineering/revise-it/transferring-the-gene
Hybridization