Recombinant DNA and Genetic Engineering

Genetic engineering is the ultimate manipulation of nature. We now have the technology to cure disease and create life. Are we too busy wondering what we can do with this knowledge that we aren't stopping to ask if we should..?

  1. Recombination in Nature–and in the Laboratory
    1. For approximately 3.5 billion years, mutation, crossing over, random gene mixing at fertilization and hybridizations between species have contributed to the diversity of life on earth.
    2. Through artificial selection of animals and plants, humans have been manipulating the genetic character of many species for thousands of years.
    3. Today, we can "engineer" genetic changes through recombinant DNA technology.
      1. DNA from different species can be cut, spliced together, and inserted into bacteria which then multiply the DNA necessary for protein production.
      2. Genetic engineering has great promise for agriculture, medicine, and industry, but it has also raised ecological, social, and ethical questions.
    4. Plasmids, Restriction Enzymes, and the New Technology
      1. Bacterial cells have a single chromosome, a circular piece of DNA.
      2. Some bacteria also have plasmids, which are circular DNA, or RNA, molecules that carry only a few genes and can replicate independently of the single "main" chromosome.
      3. Many bacteria can transfer plasmid genes to other bacteria, transforming the recipient’s chromosome into a recombinant DNA molecule.
      4. Viruses can also participate in gene transfers.
    5. Producing Restriction Fragments
      1. Bacteria possess restriction enzymes whose usual function is to cut apart foreign DNA molecules.
      2. Each enzyme cuts only at specific base sequences, allowing researchers to produce DNA fragments with specific genes.
        1. Many times the "sticky ends" that result from the cut can be used to pair up with another DNA fragment cut by the same enzyme.
        2. DNA ligase can be used to splice together cut plasmids and chromosome fragments.
      3. Recombinant plasmids have pieces of DNA from another organism inserted into them.
      4. The collection of DNA fragments that have been incorporated into plasmids is a DNA library.
  2. Working with DNA Fragments
    1. Amplification Procedures
      1. By cloning: bacteria and yeasts are hosts for DNA library replications, which yield cloned DNA.
      2. By PCR: the polymerase chain reaction uses several steps to split DNA into two strands, portions of which are then copied and reassembled into millions of double-stranded forms.

    2. Sorting Out Fragments of DNA
      1. Gel electrophoresis can be used to separate restriction fragments according to their size using electrical current.
      2. After the DNA fragments have been sorted out according to length, researchers can determine the nucleotide sequence of each using techniques such as the Sanger method.
  3. Use of DNA Probes
    1. Cells that might have taken up a particular gene of interest can be identified by nucleic acid hybridization techniques using a radioactive DNA probe.
    2. A plasmid with antibiotic-resistant genes is selected as the cloning vector so that only the bacterial colonies with the desired genes will survive in the antibiotic-laced growth medium.
    3. A replica plate of the colonies is made and the transformed colonies identified and allowed to grow.
  4. Use of cDNA
    1. Even after a desired gene has been isolated and amplified, it may not be translated into functional protein by the bacteria because introns (noncoding regions) are still present.
    2. Researchers minimize this problem by using cDNA, (copied DNA) which is made from "mature" transcripts by reverse transcription.
      1. Reverse transcriptase can be used to produce a single strand of DNA from an mRNA template.
      2. Other enzymes then convert it to a double-stranded form known as cDNA.
  5. Bacteria, Plants, and the New Technology
    1. Genetic Engineered Bacteria
      1. One of the most notable accomplishments of genetic engineering is the production of human insulin from bacteria, replacing the often troublesome and limited source from cattle and pigs.
      2. The bacteria used are harmless and have been modified to not survive outside the laboratory.
      3. Even after a harmful gene (called "ice-forming") was removed from a bacterium that lives on the leaves of strawberries, the public objected to experiments; years of litigation were needed before the experiments proceeded.
    2. Genetic Engineered Plants
      1. Botanists are searching the world for "wild" genes that will put more diversity into the monoculture crops we now raise.
      2. Whole plants can be grown from cultured cells.
        1. Mutations result in new varieties.
        2. Disease resistance can be identified in a plant and passed on to others by hybridization.
      3. An early experiment showed that replacing the tumor genes with desirable genes could modify a plasmid from a bacterium that normally causes tumors in plants.
      4. Genetically modified crop plants could increase production by naturally resisting insect pests.
  6. Genetic Engineering of Animals
    1. Supermice and Biotech Barnyards
      1. The rat gene for somatotropin production was introduced into mouse eggs; the mice subsequently expressed the rat gene by growing larger than normal.
      2. In research into the genetic basis of Alzheimer disease, human genes are being inserted into mouse embryos, which then serve as models for study.
      3. Barnyard animals may be engineered to produce needed substances such as CFTR and TPA from goats and human collagen from cattle.
    2. Applying the New Technology to Humans
      1. Researchers have embarked on the Human Genome Project to map all of the human chromosomes by sequencing the approximately 3 billion nucleotides.
      2. This project could enhance the field of genetic therapy, the transfer of modified genes into the body to correct a genetic defect or boost resistance to disease.

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