What Is the Most Logical Sequence of Steps for Splicing Foreign DNA?

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It wasn't that long ago that genetic engineering was the stuff of science fiction -- making one organism grow with characteristics of another. Since the 1970s, though, genetic manipulation techniques have advanced to the point where splicing foreign DNA into an organism is almost routine. For example, genes for pest resistance can be spliced into corn, genes for making human insulin can be put in bacteria and genes for mimicking human cancers can be put into laboratory mice. The details of the procedure are too complex to describe in a short article, with many options at each step, but the conceptual outline of the logical sequence of steps is fairly straightforward.

    Incubate the plasmid DNA and the DNA of interest with a restriction enzyme. The restriction enzyme will detect a specific sequence of DNA bases and cut the DNA apart at that point. Restriction enzymes are derived from some bacteria's defense mechanism against virus. They're molecules that will snip DNA where they detect a given pattern of bases.

    Incubate the cut-apart plasmid and the genomic DNA fragments with DNA ligase. With most restriction enzymes, the circular plasmid and the genomic DNA fragments will have complementary "sticky ends" that will grab on to each other. DNA ligase will then finish gluing the pieces together. The result is a bunch of circular plasmids that include portions of the genomic DNA.

    Insert the plasmids into bacteria and culture the bacteria to grow colonies of organisms impregnated with modified DNA. If your plasmid has an antibiotic-resistant gene that the host bacteria lacks, you can automatically screen for successfully modified bacteria by culturing the bacteria on antibiotic-infused growth medium. There are several methods for inserting the plasmids into the bacteria, such as using a microneedle, applying an electric field to open up little holes in the bacteria's membrane, or just putting the bacteria and plasmids together in the same solution and letting the bacteria absorb them naturally.

    Sample cells from the different colonies of modified bacteria. Wash the sampled cells with a detergent solution to break down the bacterial membranes and extract the DNA, then heat it or expose it to sodium hydroxide to separate the strands. This exposes the base sequence of the DNA to analysis.

    Incubate the DNA with a fluorescent probe. Shine an ultraviolet light on the incubated DNA and observe for fluorescence. The probe consists of a short sequence of DNA that matches the genomic DNA you've inserted. Where the probe matches up with the DNA you're looking for, it will glow when illuminated.

    Isolate the bacteria from the colonies containing the gene you're looking to insert. Duplicate your DNA by either letting the bacterial colonies grow, or extract the DNA as you did before and duplicate it in a polymerase chain reaction machine.


About the Author

First published in 1998, Richard Gaughan has contributed to publications such as "Photonics Spectra," "The Scientist" and other magazines. He is the author of "Accidental Genius: The World's Greatest By-Chance Discoveries." Gaughan holds a Bachelor of Science in physics from the University of Chicago.

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