Sequencing gel

Fig. 6. DNA sequence analysis, (a) Simplified methodology for dideoxy sequencing. A primer, 5 -TCTA, hybridized to the template, is used to initiate synthesis by DNA polymerase, (b) Stmcture of 2, 3 -dideoxy CTP. When no 3 -OH functionaUty is available to support addition of another nucleotide to the growing chain, synthesis terminates once this residue is incorporated into the synthetic reaction, (c) Representation of a DNA sequencing gel and the sequence, read from bottom to the top of the gel, gives sequence information in the conventional 5 to 3 direction.

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

Fig. 3 Autoradiograms of the denaturing sequencing gel for photoreactions of duplexes 9/10, 11/12, 13/14, and 15/16. Lanes 1-4, ODN 9 lane 5, ODN 11 lane 6, ODN 13 lanes 7 and 8, ODN 15 ODNs in lanes 3-7 were photoirradiated all ODNs except in lane 3 were heated with piperidine lanes 1 and 8, Maxam-Gilbert G+A sequencing reactions for ODNs 9 and 15, respectively. Partial base sequences of oligomers were shown on the side. dCNBPU was located opposite to the A (shown with a box)...

Following visualization (d), the sequence of the fragments in the individual lanes is simply read from bottom to top (e) to directly obtain the nucleotide sequence. A detail from such a sequencing gel and the corresponding protein sequence are shown in Fig. 2. 

Backfill the needle by using a sequencing gel loading tips to load the injectable. These tips fit inside the back of the needle, and the injectable will flow to the tip by capillary action. It is highly recommended to spin injectable before loading it, in order to remove any particles that could clog the needle. 

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. 

Answer If dCTP is omitted from the reaction mixture, when the first G residue is encountered in the template, ddCTP is added and polymerization halts. Only one band will appear in the sequencing gel. 

In these procedures the DNA fragment to be sequenced is initially labelled, at either the 3 - or 5 -ends with [32P] and the DNA chain is cleaved by a set of base-specific reactions to yield families of end-labelled fragments which are separated from each other by electrophoresis on sequencing gels, in exactly the same way as the primed-synthesis fragments are identified. 

Fig. 2.3. Diagram of a plus and minus sequencing gel with its interpretation. The dotted bands represent umeproducible artifact bands (from Barrell et al., 1976).

Fig. 2.4. Autoradiograph of a plus and minus sequencing gel. A sequence of 30 nucleotides from positions 280-310 is written alongside (from Brown and Smith,...

Fig. 2.7. Autoradiograph of a plus and minus DNA sequencing gel after single-site ribosubstitution with rCTP using an Alul primer on X174 viral strand DNA as template (courtesy of Dr. N.L. Brown).

Load 3-5 /xl onto thin 8% sequencing gel (below). Electro-phorese at constant 30 mA ( 1200 V) until the slow blue marker dye (xylene cyanol FF) reaches the bottom of the gel. 

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