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Figure 9.14. Apparatus for Southern blot.
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hydroxide, glyoxal, or formaldehyde instead of sodium hydroxide, because NaOH would hydrolyze RNA at the 20 -hydroxyl position. The RNA is not rmly bound to nitrocellulose until after the baking step at 80  C, so that no rinsing step is used prior to this step. Gels with RNA are not prestained for photography prior to the transfer step, because the stain interferes with the transfer to nitrocellulose. Finally, special precautions are required to prevent the contamination of the samples with RNase, including the treatment of glassware and solutions, and the wearing of gloves during the entire procedure. 9.5.5. The Western Blot18 This procedure is similar in principle to Southern and Northern blots, but it is designed for the transfer of proteins from gels onto nitrocellulose membranes. An electrophoretic technique is often used to speed the transfer by $ 10-fold, and the apparatus used for such Western transfers is shown in Figure 9.15. The rapid electrophoretic process ensures that complete transfer occurs with minimal diffusional zone broadening. 9.5.6. Detection of DNA Fragments on Membranes with DNA Probes DNA probes take advantage of the speci city of base pair hydrogen-bonding interactions over oligonucleotide lengths of 20 to >1000 base pairs. The DNA probe is an oligo- or polynucleotide to which a detectable label has been attached.19 Hybridization of the probe to the target nucleic acid on the nitrocellulose or nylon membrane occurs through A-T and G-C hydrogen bonding. This interaction is relatively weak for individual base pairs, since only two (A-T) or three (G-C) hydrogen bonds exist. However, if hybridization occurs over a number of consecutive base pairs
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Figure 9.15. Apparatus for western transfer of proteins to nitrocellulose.
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Figure 9.16. Heteroduplex between target DNA and a labeled DNA probe.
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(> 5), the process is exothermic at room temperatures. The oligonucleotide sequence and length of the DNA probe determines the selectivity of detection of a target species. For example, statistical considerations have shown that a 17base probe may be used to locate a single, unique segment in a randomly ordered, six-billion base DNA sequence, such as the human genome.20 Of course, DNA does not possess a statistically random sequence, and longer probes are generally used. Hybridization on the membrane is used to form a heteroduplex, as shown in Figure 9.16. Heteroduplexes may be dissociated at low ionic strength or at high temperatures. The temperature at which the double-to-single-strand transition occurs is called the melting temperature, Tm . Probes with high G C content have higher Tm values, because a greater number of hydrogen bonds are present in the heteroduplex. The DNA probes may be labeled with single atoms (i.e., radiolabeled), functional groups such as uorophores, or side chains attached to enzymes, to allow the detection of bands in a separation pattern that contain the sequence of interest. The label attached to the DNA probe must not interfere with the hydrogen bonding of the probe to the target sequence. The hybridization step is carried out by immersing the nitrocellulose or nylon membrane in a solution containing excess DNA probe, and incubating at a temperature just below the Tm for the heteroduplex. Excess probe is then removed by rinsing, and the membrane is examined for the location of the label. The DNA probes are often labeled by the nick-translation method.21 This involves the combined activities of three enzymes: DNaseI, 50 ,30 -exonuclease and
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Figure 9.17. Labeling DNA probes by the nick-translation method.
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50 ,30 -polymerase. The latter two enzymes are present in the E. coli DNA polymerase complex. As shown in Figure 9.17, DNaseI randomly introduces singlestranded breaks, or nicks, by the hydrolysis of double-stranded DNA to create free 30 -hydroxyl groups. The exonuclease then removes one or more of the bases from the 50 -phosphoryl side of this nicked region, while the polymerase catalyzes the incorporation of labeled nucleotides onto the 30 -hydroxyl side, to ll the gap with labeled bases. The labeled nucleotides that are incorporated are complementary, that is, the resulting strand has the same sequence as the original strand. As the reaction proceeds, the nick shifts by one base in the 30 -direction; this process is allowed to continue until 30 60% of the total bases have been replaced by labeled nucleotides. Puri cation of the labeled probe species is unnecessary if the original DNA sample contained only the sequence of interest. By adjusting the DNaseI concentration, it is possible to produce short probes with many labels incorporated, or long probes with few labels. The nick-translation method was originally used to incorporate radiolabeled nucleotides into DNA probes. More recent work has shown that nucleotides labeled with biotin can also be incorporated using this method, to yield probes of equivalent sensitivity. Uracil and adenine nucleotides that have been labeled with biotin are shown in Figure 9.18. By itself, biotin is not a detectable label. It is used as a label because a subsequent irreversible reaction with the proteins avidin or streptavidin (K > 1012 M 1 ) can be used for ultrasensitive detection, if these proteins have been labeled with an enzyme and an activity stain is used. Conjugates of these proteins with alkaline