Chapter 17: biotechnology

Recominant DNA = one DNA molecule from two different sources

  • Ability to isolate/manipulate DNA

Restriction endonucleases = enzyme that cleave (Cut) DNA

  • Used by bacteria against viruses

  • Able to cut DNA into specific fragments

3 types of restriction enzymes

Type 1 and 3 = cleave with less precision NOT used in manipulating DNA.

Type 2 = Recognize DNA sequences

  • sites are palindromes (read foward and reverse)

  • Cut at specific cite

  • Left with sticky ends

  • Think of velcroe

  • blunt cut (straight across)

Gel Electrophoresis

  • Negative charge to Positive charge

  • DNA can be cut from gel

  • Gel made of agarose or polyacrylamide

DNA ligase joins DNA molecule

  • Amp resistance gene→Restriction enzyme cuts recombinant plasmid→Foreign DNA and ligase added→yields the DNA inserted gene

DNA ligase

  • Joins two fragments = stable DNA molecule

  • Catalyzes (speeds up) formation of phosphodiester bond between phosphate and hydroxyl groups

  • enzyme joins okazaki fragments on lag strand as well

DNA Libraries

  • Collection of DNA molecules

  • DNA of interested ——> inserted in cloning vectors

  • consist of plasmids or artificial chromosomes

PCR = polymerase chain reaction —> developed in 1993

  1. Denaturation

  2. Annealing of primers

  3. Extend (With Taq Polymerase) = Thermus Aquaticus

Decrease amount of DNA

DNA fingerprinting etc

Detect genetic defects

analyze mitochondrial DNA

use as diagnostic tool (identify pathogenic microorganism)

Reverse transcriptase = RT can use RNA as a template to make DNA

Recap = DNA —> RNA —> protein (a1 = transcription) (a2 = translation)

BUT RNA (through reverse transcriptase) can make DNA

  • use this to make DNA from RNA (complimentary DNA)

  • only looking at genes creating proteins (not introns etc)

cDNA libraries

  • used to construct libraries that rep genes expressed at the time in the tissue

Reverse transcription PCR

  • PCR performed on cDNA

  • creation of recombinant DNA (contains only exons of genes)

Quantitative RT-PCR

  • measured in real time

  • Quantiated by using DNA-binding dyes or binding probes

RNA has own genome

DNA fingerprinting = identify individual based on small amount of tissue/body fluids

  • uses short tandem repeats (STR’S)

  • population is polymorphic for markers

  • pcr primers flank region known to contain an STR

Direct editing of genome

  • discover multiple proteins = easy to change gene’s sequence in cell

  • cas9 can replace mutated DNA with new proper DNA

  • Creation of transgenic animals

  • used for many genes at once

  • Two main ways

    • TALE = transcription activator-like effector

    • CRISPR = clustered regulary interspaced short palindromic repeats)/Cas9 system

      • Cas9 can cut DNA

      • DNA can be repaired

      • DNA with mutation can be replaced to normal gene

      • can also identify gene location, activate, or repress gene expression

PAM

cues from extra strands of DNA

simple to complex

nucleotide or a whole gene

used for a variety of problems

Transgenic organism

  • Transgene = gene from other species

  • genetic modified organisms

Knock-out and knock-in mice

  • Knock out mice = gene targeted and inactivated

  • Knock in mice = altering genetic sequence to add foreign gene material (create new gene)

    • “Helps study human genome (human into mice = see what gene does”

Medical Applications (picture on slide 29)

  • Human insulin

    • Harvested from pigs (while ago) (inserted into bacteria)

    • Genetic engineering used to make high concentrations of insulin

    • Bacteria don’t posttranslationally process proteins

      • Accomodations are made

    • “other examples = production interferon, human growth hormone, sub unit vaccines”

      • Gene for surface marker without dealing with virus itself.

Stem Cell Therapy

  • Embryonic stem cells are pluripotent

  • Goal = use stem cells = replace damaged tissues

    • limited success

  • Bone marrow = Successful

  • Expensive

  • Ethical/political challenges

Genetic Engineering (plants “Ti plasmid”)

  • Ti = Tumor-inducing

  • Obtained by Agrobacterium tumefaciens.

  • Gene of interest = engineered to Ti plasmid

  • Ti plasmid integrates into plant DNA

  • grow plants containing the transgene

Agriculture applications

  • herbicide resistance

  • broadleaf plants= engineered to be resistant to glyphosate

    • wont be weeded

    • Glyphosate breaks down in environment

  • Testing glyphosate resistance

Bt crops

  • Insecticidal protens tranferred into crop plants (pest resistance)

Stacked crops

  • Glyphosate resistant and Bt-Producing

Golden Rice

  • Rice genetically modified = produces beta carotene

  • Converted to vitamin A in body

    • New biochemical pathway

Social issues with transgenic crops

  • no documentations on adverse effect on human health.

  • Possible loss of biodiversity

  • Movement of genes to other relatives

  • Biopharming = use plants to develop pharmaseuticals

    • potential hazards

    • Transgenic live stock

      • Enviropig = genetically modified (breaks down phosphorus in food supply) = decrease phosphorus in waste by 70%

Environmental biotechbology

  • Biofuels derived from biomass from fixed carbon sources (crop plants or algae)

  • Renewable; more environemntally friendly

  • Microalgae cultured in large open ponds

  • Species of algae = chosen for production of lipids —> create biofuels. (biodiesel)

  • enhance genes to work better