Sanger method

Decau.se its longer half-life and lower energy make it more convenient to handle, is replacing "" P as the radioactive tracer of choice in. sequencing by the Sanger method. "" S-ct-labeled deoxynucleotide analogs provide die. source for incorporating radioactivity into DNA. 

For MAP, the corresponding acid is prepared by the Sangers method (25) and esterified by methanol or placed in reaction with diazomethane. 

The AT-terminal amino acid can be ascertained by the Sanger method. Enzymic cleavage using either chymotrypsin or trypsin will break the peptide into smaller fragments. The fragments obtained will be different, depending on the enzyme and its specificity. The smaller fragments are then each sequenced by the Edman technique. C-Terminal residues in the smaller... 

DNA Sequencing The following DNA fragment was sequenced by the Sanger method. The red asterisk indicates a fluorescent label. 

The dansyl chloride method provides an alternative to the Sanger method for N-terminal amino acid determination. Because it is considerably more sensitive than the Sanger method, it has become the method of choice. The reaction is... 

By the primary structure of a peptide or protein, we mean its amino acid sequence. Complete hydrolysis gives the amino acid content. The N-terminal amino acid can be identified by the Sanger method, using 2,4-dinitrofluorobenzene. The Edman degradation uses phenyl isothiocyanate to clip off one amino acid at a time from the N-terminus. Other reagents selectively cleave peptide chains at certain amino acid links. A... 

The Sanger method for N-terminus determination is a less common alternative to the Edman degradation. In the Sanger method, the peptide is treated with the Sanger reagent, 2,4-dinitrofluorobenzene, and then hydrolyzed by reaction with 6 M aqueous HC1. The N-terminal amino acid is recovered as its 2,4-dinitrophenyl derivative and identified. 

Another sequencing approach based on mass spectrometry makes use of the sequencing products from the Sanger method, but mass spectrometry replaces the standard electrophoretic method. [166,181]... 

Waldschmidt-Leitz and Reicheneder (1961) identified an NH2-terminal glutamic acid residue in a wheat protein bound by way of its 7-carboxyl group. They inferred this from the evolution of carbon dioxide during reaction with ninhydrin. In view of the fact that ninhydrin is able to split many peptide bonds (Dowmont and Fruton, 1952 Yanari, 1956) its use in this way is open to some question however, since glutamic acid could be identified as the NH2-terminal residue by the Sanger method and yet was not released by aminopeptidase, it seems probable that a 7-glutamyl link is present. 

The Sanger end-group determination is sometimes used as an alternative to the Edman degradation. In the Sanger method, a peptide is allowed to react witi> 2.4-dinitrolluorobenr ae. the peptide is hydrolyzed, and the N-terminal amino add is identified by separation as its iV-2,4-dinitrophenyl derivative. Propose a mechanism to account for the initial reaction between peptide and dinitrofluorobenzene. 

PCR is used to amplify some of the exons of the HMBS gene, which are then sequenced using the Sanger method. If no mutations are found, the remaining exons and the 5 intron are evaluated. If a mutation is found in an exon, it is resequenced in the reverse direction. 

Figure 5.1. The Sanger method for sequencing DNA. In the four columns are listed the oligonucleotides formed in the presence of radioactive dideoxonucleoside phosphates. The electrophoretic pattern shows the relative positions of the four types of oligonucleotides. Decreasing...

One sequencing technique is the Sanger method (developed by Fred Sanger) which uses dideoxynucleotides that stop chain elongation at the site of their incorporation. The four dideoxynucleotide substrates are labeled with... 

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