DNA TECH

DNA TECHNOLOGY & GENETIC ENGINEERING

BIOTECHNOLOGY

  • the technical application of biological knowledge for human purposes 

  1. GENETIC ENGINEERING

  • a general term that we use to refer to processes that involves the manipulation of the genetic makeup of cells or whole organisms

  1. RECOMBINANT DNA TECHNOLOGY

  • getting two DNAs from two different organisms and recombining them to form a new organism

  • a technique which involves taking the DNA apart, analyzing its structure, and recombining it in new ways

DNA SEQUENCING

  • a process of determining the nucleotide sequence of fragments of DNA

  • STEP 0. DNA Extraction and creation of millions of identical copies

  • STEP 1: The sequence is mixed with primer, enzymes (DNA polymerase), free bases (A, C, G, T), and terminator bases.

    • This is done to produce short DNA sequences ending with fluorescent labels (terminator bases).

  • STEP 2: Gel electrophoresis

    • The short DNA sequences are separated according to size using electricity and are analyzed by a laser.

  • STEP 3: Analyze the fluorescent intensity

    • The graphic display of fluorescent labels represents the complementary strand of DNA.

    • A (green), C (blue), G (yellow), and T (red)

RECOMBINANT DNA TECHNOLOGY

  • explores applications of cutting, splicing, and copying DNA

  • to transfer (and the genes that it contains) DNA from one organism into another

  • Vector: bacteria

    • carrier: plasmid — bacterial DNA

  • Human insulin

TOOLS FOR MANIPULATING DNA

  1. RESTRICTION ENZYMES

  • cut DNA at specific sites; make their cut in a palindromic nucleotide sequence

  • Palindrome

  • a sequence of letters or words that reads the same backward as forward, such as the word, “racecar.”

  • CTTAAG, GAATTC

  1. DNA LIGASE

  • join fragments of DNA

  1. PLASMIDS

  • small circular pieces of DNA to which desired genes can be added and inserted into bacteria for amplification

  1. DNA is isolated from bacterial and human cells.

  2. Both DNAs are cut with the same restriction enzyme.

  3. DNAs are mixed. Human fragments line up with the plasmid by base pairing of exposed single-strand regions.

  4. DNA ligase is added to connect the human and plasmid DNA together.

  5. Plasmids are absorbed by bacteria.

  6. Bacteria containing the recombinant plasmids of interest are selected and cloned.

POLYMERASE CHAIN REACTION (PCR)

  • to rapidly amplify/make millions of copies of a small fragment of DNA very quickly

  • involves repeated heating and cooling cycles

STEPS IN PCR

  1. Denaturation (94-96 °C)

  • Heating the reaction strongly for a minute to separate or denature the DNA strands.

    • This provides the single-stranded template for the next step.

  1. Annealing (50-65 °C)

  • Cooling the reaction for 45 seconds so primers can bind to their complementary sequences on the single-stranded template DNA.

  1. Extension (72 °C)

  • Raising the reaction temperature for 2 minutes so Taq polymerase extends the primers, synthesizing new strands of DNA

  1. Cooling (5 °C)

  • Find the temperature of amplicon solution to ensure proper bonding of newly synthesized strands of DNA.

[PCR is not a useful technique for copying whole genes and the proteins they produce, because the copies of small segments of DNA produced by PCR lack the regulatory genes and proteins required to activate genes.]

Denaturation (94-96) - annealing (50-65) - extension (72) - cooling (5)

DNA FINGERPRINTING

  • used to identifying the source of a fragment of DNA

  • commonly used for the positive identification of suspects in a crimical investigation and paternity testing

GEL ELECTROPHORESIS

  • the separation and analysis of DNA according to their size and charge

  • uses electricity and a gel

  • DNA fragments are negatively charged, so they move towards the positive electrode

  • small fragments move faster

STEPS OF DNA FINGERPRINTING

  1. Prepare the DNA sample (blood, semen, skin, and hair follicles)

  2. Increase the amount of DNA sample by using PCR.

  3. Separate DNA fragments through gel electrophoresis.

  4. Analyze the results by comparing the samples.

CREATING TRANSGENIC ORGANISMS

  • Zebra danio

  • turned into GloFish

    • genetically engineered with a gene from sea coral that causes the fish to glow in the presence of environmental toxins

    • the gene was inserted into the embryo of the fish

  • first GMO available as a pet

TRANSGENIC ORGANISMS

  • genetically engineered so that they carry one or more foreign genes from a different species

  • GMOS but not all GMOs are transgenic

  • uses recombinant DNA technique

  1. TRANSGENES

  • an organism containing genes from an organism that is not naturally compatible

  1. CISGENES

  • an organism containing only its own genes or those of a sexually compatible organism

THREE TRANSGENIC ORGANISMS

  1. TRANSGENIC BACTERIA

  • manufacture human proteins, hormones, and enzymes

  • manufacture vaccines

  • clean up oil spills

  1. TRANSGENIC PLANTS

  • more vitamins

  • better pest resistance

  • manufacture vaccines

  1. TRANSGENIC ANIMALS

  • for food production

    • the gene for bovine growth hormone (bGH) has been inserted into cows, pigs, and sheep in order to create faster-growing and larger animals

  • study a specific human disease like Alzheimer’s disease

  • gene pharming

GENE THERAPY

  • the insertion of human genes into human cells to treat or correct disease

  • get the gene into enough living cells to produce enough of the missing protein to prevent the disease

  • makes use of transporters (vectors) called RETROVIRUSES

    • RETROVIRUSES: splice their own RNA-based genetic code permanently into the DNA of the cells they infect; does not negatively affect our DNA

  • success with severe combined immunodeficiency disease (SCID)

    • an inherited disorder, lack enzyme, adenosine deaminase

    • deficiency of B and T cells

    • highly susceptible to infections

  • attempts to introduce the gene for ADA into the patient’s T cells and reintroduce the genetically modified T cells in to the patient

Research targets:

  1. cystic fibrosis

  2. cancer

CREATING mRNA VACCINES

VACCINES

  • protect individuals against disease by stimulating the body’s immune system to recognize and fight off specific pathogens, like viruses or bacteria

  • 1796

    • Dr. Edward Jenner created the world’s first successful vaccine.

    • Found out that people with infected with cowpox were immune to smallpox

  • 1971

    • The measles vaccine (1963) is combined with recently developed vaccines against mumps (1967) and rubella (1969) into a single vaccination (MMR)