DNA and Proteins

• DNA extraction

  1. Obtain cell/tissue sample and add detergents to physically break down the nuclear membrane exposing the chromosomes

  2. Centrifuge (device) spins the cells at high speeds to separate the nucleus from other smaller organelles.

  3. Add ethanol to solution for DNA to precipitate.

  4. Remove DNA from solution by spooling it around rod and wash any impurities.
• Gel Electrophoresis

  1. Restriction enzymes cleave DNA into smaller segments of various sizes

  2. DNA segments are loaded into a well in a porous gel (e.g. Agarose). The gel floats in a buffer solution within a chamber between two electrodes (one negative and one positive)

  3. When an electric current is passed through the chamber, DNA fragments will move towards the positively charged cathode

  4. Smaller DNA segments will move faster and farther than larger segments, hence segments will be separated
• Gel Electrophoresis in DNA profiling

  1. Once the DNA fragments are separated; it is transferred to a membrane and the addition of a fluorescent probe that is complimentary to the VNTR

  2. The fluorescent probes will target the STRs to help produce a DNA profile

  3. This DNA profile will be x-rayed to view the unique patterns of the DNA fragments

  4. This is used in forensics to match different DNA profiles to a suspect of a crime of a DNA sample left at the scene
• Gene Therapy (with CRISPR)

  1. Therapeutic gene is inserted into a viral vector.
  2. The vector is inserted into the host cell.
  3. The vector migrates to the nucleus where it will incorporate the therapeutic gene into the genome.
• Transgenic Organisms

  1. Plasmid is removed from a bacterium and the t-DNA is cut out by a restriction enzyme
  2. The same restriction enzyme cuts the foreign DNA
  3. The foreign DNA is inserted into the plasmid of the t-DNA, to make recombinant DNA.
  4. The plasmid is reinserted back into the bacterium.
  5. The bacterium carries the recombinant DNA into the chromosome of an organism.
  6. The cells are grown via mitosis.
  7. A new trait is expressed.
• Microinjection

  1. The gene is inserted into the pronucleus of an embryo.
  2. The gene divides via mitosis.
  3. The gene is now in every cell.
• CRISPR (Bacteria Defence System Process)

  • When a bacterium is infected by viruses, it will capture a sequence of the virus DNA and store it in a DNA archive called CRISPR.
  • When the virus attacks again, the bacterium quickly makes an RNA copy from CRISPR and makes a protein called CAS9.
  • CAS9 can identify the virus's DNA by comparing it to the CRISPR found in the bacteria and once activated, it will cut out the virus DNA, making it useless which will protect the bacteria against the attack.
• PCR (Polymerase Chain Reaction)

  1. Denaturation - DNA will be heated to high temperature to separate the weak H-Bonds that exist between complementary base pairs, which will expose the base sequence on the nucleotides (it denatures it).
  2. Annealing - DNA will be cooled so H-Bonds will be reformed which will allow the strands to rejoin again by primers (based off complementary base paring)
  3. DNA Synthesis - Taq Polymerase will elongate a growing DNA strand to make two new double-stranded molecules, which can be repeated to make as many copies.
• Bacterial Transformation

  1. DNA is extracted from a target cell or a recombinant DNA plasmid (e.g. E.coli)
  2. One fragment that contains gene of interest is removed and inserted into the DNA of bacterial cells.
  3. Bacteria replicates in a laboratory cultures (put it in a solution so it can survive) copying the human gene to make proteins.
  4. The proteins are extracted from bacterial cells.
• CRISPR (Laboratory Process)

  • Scientists will create a guide-RNA, which will be complementary to the 'gene of interest'.
  • guide-RNA will help locate the gene for CAS9, which will act as molecular scissors to cut the gene out.
  • It can also insert a new gene, to give it a new trait.