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10:3 2. The C Reaction: The C reaction uses hydrazine, piperazine, and hydroxide to selectively remove dC residues from single-stranded DNA, as shown in Reaction 10.4. Hydrazine attacks cytosine at the C4 and C6 positions, opening the pyrimidine ring. This product then cyclizes into a new ve-membered ring. The hydrazine will not attack thymidine residues if 1 M NaCl is present. Further reaction with hydrazine releases the ring, leaving the sugar residue in the DNA backbone as a hydrazone. Piperidine will react with this hydrazone, and in the presence of hydroxide, the sugar residue is removed, leaving two fragments that are phosphorylated as in the G reaction.
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TABLE 10.4. Labeled Fragments Expected from the Using the Maxam Gilbert Sequencing Method G
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P-ACTGTAGC Cleavage C
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Both the G and C reactions are speci c for these bases. The two other reactions needed for sequencing result in cleavage at both G and A residues, and at both C and T residues, respectively. 3. The G A Reaction: In this reaction, acid is used to weaken the glycosidic bond by protonating (rather than methylating as in the G reaction) at the N7 position of both A and G. This protonated form is susceptible to piperidine displacement, which then occurs as shown in Reaction 10.3. 4. The C T Reaction: The C T reaction is performed under identical conditions to the C reaction, except that the 1 M NaCl used in the C reaction is absent. Under these conditions, cleavage will occur at both C and T residues. For example, the fragments expected for the cleavage of the 50 labelled fragment 32P-ACTGTAGC in each reaction are shown in Table 10.4. It can be seen from this table that all fragments resulting from the G reaction will also be present in the G A reaction product, and, similarly, that all fragments resulting from the C reaction are also present in the C T reaction product. These product mixtures are electrophoresed in separate lanes on a polyacrylamide
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Figure 10.10. Autoradiogram of DNA sequencing gel obtained after Maxam Gilbert sequencing reactions of 32P-ACTGTAGC.
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slab, are separated according to size, and autoradiography then reveals the pattern shown in Figure 10.10. To read the sequence from the autoradiogram, we recall that the smallest fragments migrate the furthest from the cathode towards the anode. Furthermore, all of the lanes must have an identical band closest to the origin that results from the unreacted strand if this is absent, then the reactions have been allowed to proceed
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Figure 10.11. Autoradiograms of three 1-m sequencing gels, A 16%T, B 6%T, and C 4%T. Each gel has 16 lanes, containing the four reaction products, in order (left to right) G, G A, C T, C, for each if four DNA samples. The arrows indicate crossover points from one gel to the next. [Reprinted, with permission, from R. F Barker, in Nucleic Acids Sequencing: A Practical Approach , C. J. Howe and E. S. Ward, Eds., Oxford University Press, New York, 1989. # IRL Press at Oxford University Press 1989.]
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too far toward completion. Since the smallest fragment is known to be the 32P label, and since this band occurs in the G A lane, we know that the base closest to the label must be A. The second furthest migrating band is observed in both the C T and the C lanes, so that the second base is a C. The third band occurs only in the C T lane, and is therefore a T residue. We have now sequenced the rst three bases, as 32P-ACT. Continuing in this manner, the entire sequence of the fragment is easily read from the sequencing gel. Polyacrylamide gels as long as 1 m have been used to enable the sequence of a 600-base DNA fragment to be read from a single gel.12 More often, however, several gels are employed. As shown in Figure 10.11, for example, a gel with up to 20%T is used for bases 0 150, a 6%T gel for bases 140 300, and a 4%T gel is used to sequence bases 290 600 . It is necessary to include a region of overlap from one gel to the next, to ensure that no fragments remain undetected. 10.9. IMMUNOELECTROPHORESIS13 Antigen antibody binding interactions can be induced after an electrophoretic separation pattern is blotted from the gel onto a membrane, in a technique called immunoblotting, and will also occur directly in the gels used for electrophoresis. Immunoelectrophoretic methods are used for the identi cation and quantitation of antigens. The immunoblotting technique follows after the Western blotting of the proteins onto a membrane. The membrane with bound proteins is incubated in a solution containing a polyclonal antiserum or a monoclonal antibody, and is then rinsed. This primary binding step identi es the antigen of interest through the high selectivity of the binding reaction. Subsequent labeling steps, for example, with an enzyme-labeled anti-IgG followed by an activity stain, allow the detection of the antibody selectively bound to the antigen on the membrane. For the primary step, monoclonal antibodies are favored over polyclonals, because of the single epitope selectivity which reduces interferences that result in the detection of nonanalyte proteins. If polyclonal antiserum is used, all antibodies present must be considered. A good antiserum contains 6 7 mg/mL immunoglobulins, and, of these, 20 30% are so-called speci c antibodies directed against the immunogen. The remaining 70 80% of the antibodies are irrelevant to the antigen of interest, and are directed against environmental immunogens such as microorganisms and foodstuffs as well as against impurities in the injected immunogen. The af nity or avidity of the primary binding interaction is often signi cantly lower in immunoelectrophoretic methods than in typical immunoassays such as ELISA. This result is of antigen denaturation in the gel, and is particularly noticeable with SDS PAGE separations. Figure 10.12 shows examples of protein detection with polyclonal and monoclonal antibodies.14 Note the large number of visible bands in the polyclonal immunoblot, and the fact that the monoclonal appears to interact with a different antigen epitope than most of the antibodies in the polyclonal mixture, since the one band