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Chapter 12: Gene Technology: DNA Sequencing

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Specialization: Eukaryotic Cells and Differentiation

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Designer Genes

Let's assume that we have an interesting DNA fragment which we want to study or use. A single fragment is fragile, and our investigations (which may involve cutting it apart even more) could destroy the sample. Before we can study it, we need to make copies.

DNA is primarily information. The nucleotide components are cheap. All we need is a method of copying the information without making an error. The early methods of cloning DNA involved using the more or less natural process of having viruses inject DNA into bacteria.

First, the cells of a colony of similar bacteria are treated to make them permeable to DNA strands. Then copies of the desired DNA strands (taken from other cells) are mixed with the bacteria medium. The bacterial cells absorb the DNA through their cell walls. The bacterial restriction enzymes cut the foreign DNA, but they also cut the bacterial DNA, and if we are lucky, the foreign fragment is incorporated into the bacterial DNA in such a way that the bacteria remains alive and functioning. The bacterial DNA ligase then "cements" the new DNA (now called recombinant DNA) in place. The bacterial DNA ring, the plasmid, now replicates, and the recombinant DNA segment replicates as well. As the bacterial colony grows, the number of copies of the recombinant DNA strand also grows.

Now we need a way to sort the bacteria for the strands with our interesting DNA segment. One way to tag the segment is to make a resistance gene part of the introduced segment. Bacteria with the new genetic material will have resistance to a particular antibiotic. We can then treat the bacteria colony with the antibiotic, and only the bacteria with our new DNA segment will survive. Another possibility is to create RNA segment which is complimentary to our DNA segment. If we make the RNA radioactive and introduce it into bacterial cells, it will bind with our DNA segment and "tag" it, making it possible to identify and isolate the bacteria carrying the new DNA segment. Either we have a pure colony which will continue to breed copies of the new DNA--but this method is slow.

A much faster way of creating specific DNA segments is to use a polymerase chain reaction, or PCR. The desired DNA single-strand sequence is placed in a medium with lots of nucleotides and DNA ligase, which immediately creates a double-helix version of the strand. The mix is heated, separating the strands, then cooled, and each strand is paired again from the pool of available individual nucleotides. Each heating-cooling cycle doubles the number of identical DNA strands, so after about 20 cycles, we have over a million copies of the strand to examine.

DNA Sequencing

Now let's suppose that our goal is to determine the actual sequence of the DNA. First, we isolate our sequence of interest by using the appropriate restriction enzyme, and clone it several times. Then we add a special molecule called dideoxyadenosine triphosphate (ddATP) to a solution containing our DNA. This particular molecule finds the adenosine (A) molecules and cuts the DNA at each one, then binds to it so that we have a set of strands of different lengths, all ending in A.

DNA Sequence

Using this technique, researchers have been able to map the entire E. coli genome, and have completed mapping all 100000 or so genes coded by the 3 billion pairs of nucleotides found in human chromosomes.

Use the Gel Electrophoresis simulation at the University of Utah to work through the lab process. You may need to play a bit to get the materials in exactly the right position (for example, you have to put the spoon properly in the top of the flask) for the animation to register your actions and continue.

Where do we go from here?

Obviously, genetic engineering holds many possibilities for application. Here are some possibilities:

  • Identify and isolate genes which control growth to produce bigger and better animals.
  • Identify, isolate, and transfer genes which provide immunity to disease in one organism to another organism which otherwise has no immunity (often done for plants).
  • "Teach" bacteria or isolated mammalian cells to manufacture complex organic molecules which some individuals lack (this is how we make insulin for diabetics now).
  • "Teach" bacteria to eat waste products or pollutants (we did this to clean up the Exxon Valdez oil spill).
  • Of course, we also need to create safety restrictions to prevent "experimental" genes from getting lose in the general population and spreading characteristics which are undesirable there. The new technology raises many safety and ethical questions which geneticists cannot answer in isolation from the rest of the human community.

    Practical Genetics

    Now that we have the genome for a species, what can we do with it? Studying DNA usually requires that we take it apart, so before geneticists can work with a particular DNA strand or gene, they need to clone that strand. That way, destructive techniques won't ruin the only available copy of the sequence.

    The most common method of gene cloning is the polymerase chain reaction (PCR). This reaction occurs so quickly under the right conditions that a researcher can get two million copies of the gene in under two hours.

    Use the Polymerase Chain Reaction animation at the Gene Almanac to make DNA copies with the PCR process.

    • What is "denaturing"? How does it affect the DNA?
    • What is the "annealing" step?
    • What happens in "extension"?
    • How many cycles does it take for the PCR process to produce over 30000 copies?

    The Human Genome

    The blueprint for a normal human being is carried on 22 chromosome pairs and a gender-specific chromosome set. When these chromosomes are extracted from blood samples or other tissues, the chromosomes can be separated into their pairs, and laid out in a specific pattern , usually larger to smaller, which makes a karyotype, or chromosomal map. By studying the genetic map--especially when the genetic material is defective--we have been able to identify specific locations of the genes that program for various traits. We have also been able to identify which genes cause certain types of disease or defects if something goes wrong in the DNA sequence.

    Genetic research in humans is limited by serious ethical considerations. We cannot breed humans for specific traits. We cannot purposely inject humans with different chemicals or expose them to radiation to see how genetic mutations affect offspring. So one of the most effective ways of studying human genetics is to map pedigrees of diseases or deformities which appear to be inherited, such as hemophilia. Since not all birth defects are inherited (many are caused by nutrition deficiencies or exposure to disease during pregnancy), even mapping pedigrees has been difficult.

    Visit the Human Genome Project page at the US National Institute of Health site.

    • What is the project? What were its goals? When did it begin? Who has access to the information it uncovered?

    Ethical Considerations

    Once we know what "normal" genes look like, gene modification is a practical step, but one that raises serious concerns. In one sense, we've been modifying genes deliberately by cross-breeding plants and animals for the traits we desire — disease resistance, higher yield, lack of inherited defects, curly ears!

    What are some of the ethical and societal issues involved in gene modification of plants, animals, or people?