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Change in genetic structure of populations due to selective breeding by humans.
- Limited by the genes available
using lving organisms or other biological systems to manufacture products.
- Use genetic engineering
Recombinant DNA Technology:
Techniques used to alter the genetic material of an organism by adding new DNA fragments or altering existing DNA
- Using recombinant DNA tecchnology to insert DNA from one organims to another resulting in a:
- transgenic organism
Recombinant DNA and Genetic Engineering:
Stages of genetic engineering:
- 1.) Find the gene of interest.
- 2.) Isolate the gene of interest
- 3.) Produce recombinant plasmid
- 4.) Get bacteria to take up plasmid
- 5.) Screen for clone of interest
Step 1. Find the gene of interest:
- Know the sequeince of the gene
- Know the lenght between restriction sites that surround the gene of interest
Restriction site: a particular base sequence found on DNA
a particular base sequence found on DNA
Step 2: Isolate the gene of interest
Cut DNA with a restriction endonuclease
Restriction endonuclease: an enzyme that cleaves DNA at a restriction site, usually within or near palindrome sequence.
- Two ways:
- 1.) know sequence - use probe that has a radioactive or fluorescnet tag.
- 2.) know length -use gel electophoresis
an enzyme that cleaves DNA at a restriction site, usually within or near a palindrome sequence.
3.) Produce a recombinant plasmid
Vector: carriers recombinant DNA into a host cell
Plasmid: a small fragment of extrachromosal DNA, usually circular, that replicates independently of the main chromosome.
- 1.) Origin of replication site
- 2.) Marger gene (antibiotic resistant)
- 3.) Promoter / Terminator regions
- -Cut plasmid w the same restriciton endonuclease
- -Ligate to create recombinant plasmid
Step 4: Get bacteria to take up plasmid
- The uptake of DNA directly from the environment into bacterial cell.
- 1.) increase membrane permeability
- 2.) mix bacteria w the vector
- 3.) further increase permeabilty w heat and salt
- 4.) allow bacteria to replicate
Why E. Coli?
- Rapid growth and reproduction
- small organism
- simple genome
- asexual reproduction
- circular DNA
Increase membrane permeability: Incubate w/ CaCl2
- -bacteria membrane and plasmid both negatively charged.
- -CaCL2 cover bacteria membrane with Ca++ and negatively charged plasimd is attracted to the cell
- -Heat shock (produces pores)
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