METHODS FOR AUTOMATIC NUCLEOTIDE-SEQUENCE ANALYSIS. MULTICOMPONENT SPECTROPHOTOMETRIC ANALYSIS OF MIXTURES OF NUCLEIC ACID COMPONENTS BY A LEAST-SQUARES PROCEDURE

Abstract

A theoretical analysis of the errors in multicomponent spectrophotometric analysis of nucleo-side mixtures, by a least-squares procedure, has been made to obtain an expression for the error coefficient, relating the error in calculated concentration to the error in extinction measurements. The error coefficients, which depend only on the "library" of spectra used to fit the experimental curves, have been computed for a number of "libraries" containing the following nucleosides found in s-RNA: adenosine, guano-sine, cytidine,uridine, 5-ribosyluracil, 7-methyl-guanosine, 6-dimethyl-aminopurineriboside,6-methylaminopurineriboside and thymineriboside. The error coefficients have been used to determine the best conditions for maximum accuracy in the determination of the compositions of nucleoside mixtures. Experimental determinations of the compositions of nucleoside mixtures have been made and the errors found to be consistent with those predicted by the theoretical analysis. It has been demonstrated that, with certain precautions, the multicomponent spectrophotometric method described is suitable as a basis for automatic nucleotide-com-position analysis of oligonucleotides containing nine nucleotides. Used in conjunction with continuous chromatography and flow chemical techniques, this method can be applied to the study of the sequence of s-RNA.