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