Colony blots and Southern blots are similar in that they both use a DNA probe to detect specific fragments. Colony blots are used to locate a specific sequence in colonies growing on an agar plate, whereas Southern blots are used to locate a sequence in restriction fragments that have been separated by gel electrophoresis.
A DNA probe can be obtained in a number of ways. In some cases, a similar gene from another organism has already been cloned, and so can be used as a probe. For example, the Shiga toxin gene of Shigella dysenteriae can be used to probe for similar toxin genes, such as the one encoded by E. coli O157:H7. The double-stranded DNA is labeled with a detectable marker and then denatured to create the single-stranded probe. Because the probe originated from a double-stranded DNA molecule, it contains each of the two strands of complementary DNA. Although these two probe strands can anneal to each other, the concentration of probe molecules ensures that sufficient numbers will remain single-stranded until they can hybridize to the DNA of interest. ■ Shiga toxin, p. 613 ■ E. coli O157:H7, p. 615
Another way to obtain a probe is to synthesize a short sequence of nucleotides, an oligonucleotide. Rapid and efficient DNA synthesis machines now make this a relatively simple task. Synthesizing the appropriate probe, however, requires at least some advance knowledge about the sequence of nucleotides that are to be located. In many cases, relevant information about the sequence can be determined by searching a computerized database of known sequences. Alternatively, the protein encoded by the gene can be isolated and then a portion of its amino acid sequence determined. Knowing the amino acid sequence makes it possible to deduce the potential nucleotide sequences using the genetic code. When using probes based on amino acid sequences, several different oligonucleotides may need to be synthesized, because the redundancy of the genetic code means that several slightly different sequences can encode the same short stretch of amino acids. ■ genetic code, p. 175
To do a colony blot, a plate of colonies is replica-plated onto a nylon membrane, creating a pattern of colonies identical to that of the original plate (see figure 9.7). The membrane is then treated to immobilize the DNA. Next, the membrane is soaked in an alkaline solution, which simultaneously lyses the cells and denatures their DNA. A liquid solution containing the labeled single-stranded probe is then added to the membrane and incubated under conditions that allow the probe to hybridize to complementary sequences on the filter. Any probe that has not bound is then washed off. If probe labeled with a radioactive isotope is employed, autoradiography is used to determine the location of the hybridized probe. The dark spots that appear on the film can then be correlated with a specific colony on the original plate. Other methods are used to detect probes that have been labeled with a fluorescent molecule or other marker. ■ replica plating, p. 200
Steps of a Southern Blot
A Southern blot differs from a colony blot in that the DNA must first be digested into fragments that are then separated using gel electrophoresis.
Digestion and Gel Electrophoresis of DNA The first step of the Southern blot technique is the isolation and restriction enzyme digestion of the DNA to be studied (see figure 9.8). The DNA fragments are then separated according to size using gel electrophoresis. This technique uses a slab of gel that has the consistency of very firm gelatin and is made of agarose, a highly purified form of agar. The digested DNA samples are put into slots or wells in the gel. As a means to eventually determine the size of the various fragments in the restriction digest, a size standard is routinely put into a well in the same gel. The size standard is simply a series of DNA fragments of known sizes that can be used as a basis for later comparison.
The gel is then placed in an electric field. DNA is negatively charged, and the fragments are attracted to and migrate toward the positively charged electrode. As the DNA moves in the electric field, the gel acts as a sieve, impeding the large fragments while allowing the smaller ones to pass through more quickly. Because of the sieve-like effect of the gel, the restriction fragments can be separated according to their size.
The DNA is not directly visible on the gel; to view it the gel must first be immersed in a solution containing a dye, ethidium bromide, that binds to nucleic acid. Ethidium bromide-stained DNA is fluorescent when viewed with UV light. Each fluorescent band represents a specific-sized fragment of DNA (figure 9.18). Staining the gel, however, only confirms that different-sized bands are present; it gives no information to suggest which band has the nucleotide sequence of interest. ■ ethidium bromide, p. 195
Figure 9.18 Bands of DNA on an Ethidium Bromide-Stained Agarose Gel The stained DNA is visible because it fluoresces when viewed with UV light. Each band represents a specific-sized fragment of DNA.
Transferring the DNA to a Solid Membrane Support In order to immobilize the DNA on a solid support, the DNA fragments are transferred in-place to a nylon membrane. To do this, the gel is first soaked in an alkaline solution, which denatures the DNA. Then, by placing the membrane directly on the gel in a special set-up, the DNA transfers to the membrane in the same relative position as it occupied on the gel.
Detecting Specific DNA Sequences A liquid solution containing the labeled probe is added to the membrane and incubated under conditions that allow the probe to hybridize to complementary sequences. Any probe that has not bound is then washed off, and the location of the bound probe is detected. If the probe was labeled with a radioisotope, autoradiography is used for detection; the dark bands that appear on the film correspond to specific bands on the original gel.
Techniques Used in Fluorescence in situ Hybridization
Sample preparation is a critical aspect of fluorescence in situ hybridization (FISH). The sample must first be treated with chemicals to preserve the shape of the cells, inactivate enzymes that might otherwise degrade the nucleic acid, and make the cells more permeable so that the labeled probe molecules can readily enter. The methods used depend largely on the type of organisms being detected. The specimen is then applied to a glass slide that has been coated with a material that facilitates attachment of the cells.
Once the specimen has been prepared, a solution containing the fluorescently-labeled probe is applied and incubated under conditions that allow hybridization to occur. Then, unbound probe is washed off. Finally, the specimen is viewed using a fluorescence microscope.
Sophisticated techniques are used to construct nucleotide microarrays. One method employs a robotic device to deliver microscopic droplets, each containing a different DNA sequence, onto exact positions of a solid support. Another synthesizes oligonucleotides, one nucleotide at a time, directly on the solid
9.8 Techniques Used in DNA Sequencing 237
support. Although the methods used to produce a microarray are expensive, each array can be used multiple times.
The sample of nucleic acid to be analyzed is first labeled with a fluorescent marker. Then, the labeled DNA or RNA is added to the microarray and incubated using conditions that facilitate hybridization. Unbound material is then washed off and the location of the fluorescent label detected. Note that separate samples can be analyzed simultaneously by using different fluorescent labels.
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