Chain-termination sequencing method

The use of exonuclease III for preparing single-stranded DNA for use as a template in the chain terminator sequencing method (Smith, 1979, 1980)... 

Among DNA-based drugs, only antisense DNA is currently analyzed by capillary electrophoresis. Antisense DNA therapy requires reliable and convenient methods for sequencing short single-stranded oligonucleotides. A method for phosphorothioate antisense DNA sequencing by capillary electrophoresis coupled to UV detection has been developed based on a modified chain-termination sequencing method.Capillary gel electrophoresis alone or in combination with HPLC is also used for pharmacokinetics study of antisense... 

Fig. 3.17. Two examples of chain-termination sequencing gels using single-stranded templates prepared by the exonuclease III method of Smith (1979). In (a) a small MboI fragment was employed as the primer and in (b) a Hpall fragment. The numbered bands refer to the published human mitochondrial cytochrome oxidase II gene sequence. (Courtesy of Barrel et al., 1979).

In summary there is no doubt that this represents a powerful and convenient sequencing strategy of general applicability. Where the decision is to use a primed-synthesis method and the phage M-13 cloning approach (described in the next chapter) is to be avoided for any reason, the exolll procedure is likely to be the method of choice. A summarized procedure for chain terminator sequencing is given at the end of Chapter 4. 

The most common method is called chain-termination sequencing or the dideoxy method, and is described in Chap. 16. 

Determining the Base Sequence of DNA The Chain-Terminating (Dideoxynucleotide) Method... 

FIGURE 12.3 The chain termination or dideoxy method of DNA sequencing, (a) DNA polymerase reaction, (b) Structure of dideoxynucleotide. (c) Four reaction mixtures with nucleoside triphosphates plus one dideoxynucleoside triphosphate, (d) Electro-phoretogram. Note that the nucleotide sequence as read from the bottom to the top of the gel is the order of nucleotide addition carried out by DNA polymerase. 

The result of sequence determination of an oligonucleotide as performed by the Sanger dideoxy chain termination method is displayed at right. 

Edman degradation (Section 26.6) A method for N-terminal sequencing of peptide chains by treatment with Af-phenylisothiocyanate. 

Antibody-based detection methods include immuno-cytochemistry, which gives qualitative data but has very good spatial resolution. Radioimmunoassays provide a quantitative measure of release or content. One of the major limitations of all antibody-based methods is the potential for cross-reactivity among the many peptides. For example, some of the most sensitive gastrin antisera also detect CCK, since the peptides share a common COOH-terminal tetrapeptide sequence. Methods for detection of the mRNAs encoding neuropeptides include Northern blots, which provide quantitative data and information on splice variants, but lack fine anatomical resolution. The more commonly used polymerase chain reaction, which can be quantitative but often is used in a more qualitative manner, provides great sensitivity. Alternatively, in situ hybridization preserves anatomical relationships and can be used to obtain both qualitative and quantitative data. 

This method was developed by Sanger and his colleagues and is also referred to as the chain termination method. The procedure requires a single-stranded DNA template and a short primer complementary to the 3 end of the region of DNA to be sequenced. A complementary copy of the DNA strand is produced... 

Figure 13.20 The dideoxy (chain termination) method of DNA sequencing. A...

An unusual method for chain termination is utilized in the biosynthesis of myxothiazol and melithiazol. The domains in the terminal modules of these synthetases are arranged in the following order C-A-MOx-A-PCP-TE (MOx = monooxygenase domain). In the termination modules of these synthetases, the MOx domain likely catalyzes hydroxylation of the ct-carhon of the C-terminal glycine residue of the requisite peptidyl-5-PCP. The resulting intermediate then undergoes spontaneous conversion into PCP-bound glyoxylic acid and a terminal amide. This sequence yields the final product in the case of myxothiazol, whereas the terminal amide is transformed to a methyl ester to complete melithiazol biosynthesis. 

The Maxam-Gilbert method doesn t require synthesis of a primer and it sometimes works well for sequences that are difficult to obtain with the Sanger -Coulson procedure. The two methods may both be used to provide additional certainty about a sequence. The Maxam-Gilbert method is very convenient for sequencing small oligonucleotides which often react poorly with the polymerase used for the chain termination method. The method usually requires that a restriction map be prepared. 

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