Sanger reaction

Reactions important in determining the amino acid sequence of a protein include several techniques for end-group analysis. There are enzymes (proteins themselves) that cleave the amino acid at the carboxy terminus of the polymer. The amino terminus can be found through the Sanger reaction. 

Nucleophilic aromatic substitution is much less common than electrophilic substitution but nevertheless does have certain uses. One such use is the reaction of proteins with 2,4-dinitrofluorobenzene, known as Sanger s reagent, to attach a "label" to the terminal NH2 group of the amino acid at one end of the protein chain. 

Sequencing of DNA is carried out by the Sanger dideoxy method, and small DNA segments can be synthesized in the laboratory by automated instruments. Small amounts of DNA can be amplified by factors of 106 using the polymerase chain reaction (PCR). 

Historically, the amidinate story begins with the discovery of N,N,N -tris(trimethylsilyl)benzamidine, PhC( = NSiMe3)[N(SiMe3)2], by Sanger. The compound was prepared by the reaction of benzonitrile with LiN(SiMe3)2 followed by treatment with chlorotrimethylsilane. The method was later... 

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

AP Ryle, F Sanger. Disulfide interchange reactions. Biochem J 535, 1955. 

The amino group of the N-terminal amino acid residue of a peptide will react with the FDNB reagent to form the characteristic yellow DNP derivative, which may be released from the peptide by either acid or enzymic hydrolysis of the peptide bond and subsequently identified. This is of historic interest because Dr F. Sanger first used this reaction in his work on the determination of the primary structure of the polypeptide hormone insulin and the reagent is often referred to as Sanger s reagent. 

In 1950 an alternative to the Sanger procedure for identifying N-terminal amino acids was reported by Edman—reaction with phenyl-isothiocyanate to give a phenylthiocarbamide labeled peptide. When this was heated in anhydrous HC1 in nitromethane, phenylthiohy-dantoin was split off, releasing the free a-NH2 group of the amino acid in position 2 in the sequence. While initially the FDNB method was probably the more popular, the quantitative precision which could be obtained by the Edman degradation has been successfully adapted to the automatic analysis of peptides in sequenators. 

The promoter region and all four exons of the apoCII gene are amplified in four separate PCR reactions. These PCR products are then purified from the amplification primers and the template is used for direct sequencing analysis using the method of Sanger et al. [83]. 

Number of Polypeptide Chains in a Multisubunit Protein A sample (660 mg) of an oligomeric protein of Mr 132,000 was treated with an excess of l-fluoro-2,4-dinitrobenzene (Sanger s reagent) under slightly alkaline conditions until the chemical reaction was complete. The peptide bonds of the protein were then completely hydrolyzed by heating it with concentrated HC1. The hydrolysate was found to contain 5.5 mg of the following compound ... 

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