Sequencing gel, figure

When gel electrophoresis is performed on each reaction mixture, a band corresponding to each position of chain termination appears. The sequence of the newly formed strand, which is complementary to that of the template DNA, can be read direcdy from the sequencing gel (Figure 13.28). A variation on this method is to use a single reaction mixture with a different fluorescent label on each of the four ddNTPs. Each fluorescent label can be detected by its characteristic spectrum, requiring only a single gel electrophoresis experiment. 

FIGURE 12.V A photograph of the antoradiogram from an actual sequencing gel. 

Figure 5-49 A DNA sequencing gel obtained using a segment of DNA from salmon sperm selected by suitable oligonucleotide primers, amplified by PCR, and sequenced with a 35S label in the primer. Four samples were used, one with each of the four dideoxy chain terminators (A, G, C, T, A, C, G, T from left to right). After electrophoresis the shorter fragments are at the lower end of the gel. The sequence of the strand complementary to the template strand whose sequence is being determined is read from the bottom of the gel. Here it starts CTATGATAC. Reproduced by permission of Amersham Pharmacia Biotech, Limited.

Autoradiogram of a DNA sequencing gel. From Zyskind and Bernstein, Recombinant DNA Laboratory Manual (1989), Academic Press (San Diego, CA), Figure 7.4. 

Molecular Conformation of sPP gel. Figure 27 shows the DD/MAS 13C NMR spectrum of sPP gel. This spectrum was obtained by a single-pulse sequence (tt /2—FIDdd-tt)ii with the repetition time ty more than 5 times the longitudinal relaxation time Tic. Hence, this spectrum reflects the thermal equilibrium state of the gel. For comparison, the spectrum of the bulk ttgg crystal of this sample... 

The principle of this method is shown in Figure 3.7. and Figure 3.8. shows a diagram of a sequencing gel (from Barnes, 1978). 

Figure 3.10. shows an example of a sequencing gel prepared using the method described. Inspection of the band pattern shows the remarkable clarity and lack of artifacts produced and also the sharpness and definition of the bands. The method, however, is not... 

In the Forward reaction, which is analogous to the Maat and Smith procedure, the incubation is carried out in the presence of all four dNTPs and an adjusted concentration of one ddNTP. Under these conditions the exposed 3 -ends, formed by nicking, are elongated in the 5 - 3 direction until polymerization is halted by the incorporation of the dd-nucleotide present in the reaction mixture. The samples generated by the four Backward and four Forward reactions are finally denatured and electrophoresed on a denaturing polyacrylamide gel. The principle of the method is shown in Fig. 3.13. (modified from Seif et al. 1980) and a sequencing gel in Figure 3.14. 

Figure 5.8. shows two examples of sequencing gels. Autoradiograph (a) shows a short electrophoretic run in which the sequence of the first 29 nucleotides from the labelled 5 -end can be deduced. The sequencing procedures used were those originally proposed by Maxam and Gilbert (1977). The discrimination between the G s... 

Figure 10.10. Autoradiogram of DNA sequencing gel obtained after Maxam-Gilbert sequencing reactions of 32P-ACTGTAGC.

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.]...

A higher resolution gel. Figure 8, shows an interesting effect of this ethidiiim titration. The first bands to appear are those due to platination of the 5 -(dG)p site the (dG) sequence shows little detectable platination at -aie lowest level of ethidium. As the amount of ethidium in the reaction mixture is increased, the (dG)g site shows more platination, and platination at the (dG) site appears relatively to decrease. 

The same reaction is done with each of the dideoxynucleotides. The DNA fragments are then separated by gel electrophoresis on a DNA sequencing gel. The four reactions are placed in four wells, side by side, on the gel. Following electrophoresis, the DNA sequence can be read directly from the gel, as shown in Figure 24.26. 

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