Lecture notes
LT8 DNA Cloning
- Institution
- University College London (UCL)
DNA Cloning techniques
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By: connieburns • 6 year ago
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DNA Cloning & DNA LibrariesGenetic Modification
Potential Applications
- Protein production: insulin, growth hormone
- Genetic research: transformed bacteria and knockout mice
- Food Production: increased resistance to antibiotics, increased growth
- Gene Therapy: correction of genetic defects – including cancer
Recombinant Protein Production
In 1972, Paul Berg’s group combined DNA from
the SV40 virus with DNA from the lambda virus
to produce the first recombinant DNA
1. EcoR1 to chop up both sets of extracted DNA
2. Mixed the resulting fragment sets together
3. Used DNA ligase to join random DNA
fragments together
- Many different combinations possible…
Clonal Growth of Bacteria
One bacteria produces two copies of itself – normally by fission, all genetic copy
replicated
Individual clones amplify into colonies of bacteria
Cloning can refer to the multiple copies of bacteria cells or sequences of DNA
produced during clonal growth
Bacteria and Plasmids
Contain additional non-chromosomal bacterial DNA which are also replicated during
clonal growth
Usually have specific or special functions – antibiotic resistance, nitrogen fixation,
virulence etc.
F(ertility) Plasmid: 99,159 bp, contains genes that regulate DNA replication, also
transposable elements that allow plasmid to integrate into host chromosome
Tra region: involved in sex pilus, synthesis of surface structure (conjugation
transfer genes)
R(esistance) plasmids are conjugative and carry multiple resistance genes (eg. R100)
Plasmids can be transferred between bacteria – basis of transferred antibiotic
resistance in bacteria (horizontal gene transfer)
Normally low (1%) transmission of plasmids between bacteris
, Increased (made competent) via heatshock, electroporation or certain chemicals
(eg. CaCl2)
Transgenic Organisms/ Genetically Modified Organisms (GMOs)
- To study the function of a gene that has been mutated in vitro – ideally to generate
an organism in which the normal gene has been replaced by the altered one
- In many organisms, gene replacement can be accomplished by homologous
recombination between introduced mutant and chromosomal DNA
- Gene knockout: DNA can be deleted entirely
- Gene Addition: A mutant gene can be added without any alteration to the genome
- In sexually reproducing organisms, these alterations are usually made in the germ
line (n reproductive cells)
First Transgenic experiments
Stanley Cohen and Herbert Boyer (1973) inserted frog (xenopus) DNA into
bacterial plasmids to create the first transgenic organism
1. Digest both sets of DNA with EcoR1 to produce complementary sticky ends on
both types of DNA fragment
2. Combine the 2 DNA fragment sets and ligate – forms recombinant DNA molecule
serves as a vector
3. Transfer vector into bacteria
4. Analyse clones of bacteria (colonies) for frog DNA
Selection of different outcomes
Bayer and Cohen started with a plasmid that
carried tetracycline0resistance
Possible recombinant DNA outcomes include:
a) Plasmid without ability to replicate in
bacteria
b) Plasmid without antibiotic resistance
c) Plasmid without xenopus DNA
Plasmid with all 3 included produce colonies that are antibiotic resistant and contain
frog DNA
Modern Plasmid Cloning Vector Properties
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