Structural Biochemistry/DNA recombinant techniques/DNA Cataloging

Scientists now have techniques that allow genes and DNA sequences of interest to be stored, through the use of cDNA and phages. DNA cataloging is a process used for recording and storing specific samples of DNA. The basic process is to take a sample of DNA, ligate it to a plasmid, inserting the plasmid into a bacteria, selecting for bacteria of interest, and storing the bacteria in a large freezer.

Sample of DNA

The sample of DNA is usually synthesized by PCR from a single DNA strand that contains within it three things: the gene of interest, a restriction enzyme binding site on each end, and a "test" gene. The "test" gene usually codes for a pigment or a pigment inhibitor so later in the process it will be easy to detect which bacterial have the gene of interest.

Ligation to a Plasmid

A Plasmid is a circular piece of DNA naturally found in many bacterial cells. The useful features of plasmids to scientists are that they can be absorbed through the bacterial membrane. Once absorbed, the bacteria will begin to express the genes on the plasmids. To ligate a gene of interest to a plasmid, often an artificially produced plasmid will be used. This plasmid will have some sort of antibiotic resistance (usually to Ampicillin) and a specific restriction enzyme (the same one used in the DNA amplified through PCR). when the plasmids and the DNA of interest are mixed, many of the "sticky ends" of the plasmid and the DNA will connect. DNA ligase and ATP will need to be added to the solution to covalently link the strands to each other.

Unfortunately, sometimes the plasmid can simply reconnect with itself where it was broken, which is why there is a test gene in the DNA sequence of interest. We will know (later) which plasmids have the gene, and which do not.

Insertion of Plasmid to Bacteria

Once the plasmid is put back together, it can be inserted into a bacteria. The common method of doing this is through "heat-shock" therapy. (note: the bacteria must have the capability of surviving the "heat-shock" often labeled as having the "heat-shock" genes) Heat-Shocking the bacteria is rapidly heating the bacteria up to a temperature higher than the current temperature of the bacteria. While the mechanism is not clearly understood, the effect is that some of the bacteria will absorb the plasmid into its cytoplasm.

Selecting for Bacteria of Interest

Some of the bacteria in the solution will not have taken up the plasmid, even still, some of the bacterial may have take up a plasmid whose "sticky ends" have simply recombined with itself. The way to determine which bacteria we want and which we do not want is to use the test gene and the antibiotic resistance gene on the ideal plasmid. The way to do this is to culture the bacteria on an agar petri dish containing the antibiotic, which will kill any bacteria without some form of the plasmid. Next is to examine which colonies are expressing the "test gene" on the DNA of interest that was ligated to the plasmid. Whichever colonies live on the antibiotic plant and exhibit the "test gene" are taken and cultured separately in their own agar dish.

Storing the DNA

The most common method of storage is to use a large freezer which keeps the bacteria of interest frozen at around minus seventy(70) degrees C.

References

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