Nucleotide sequence determination employing matched dideoxynucleotide

Abstract

A method of positively deciphering a DNA sequence of a sample by comparing data of two electropherograms obtained from separate electrophoresis of products from two complete sequence reactions of the same sample, whereby dideoxynucleotide terminators of different but matched concentration ratios are employed for the two reactions. The sequencing chemistry is chosen to result in terminator-concentration dependent in peak intensity in the electropherograms. By appropriately choosing the matched concentration ratios of the terminators used in the two sequence reactions, ambiguities in the discrimination of the peaks in the result of one sequence reaction can be resolved by comparing the result of the other sequence reaction. The sequence of the sample can therefore be deciphered to a high degree of accuracy. For fluorescence detection of electrophoresis of the sequence reactions, three ddNTP terminators and one fluor are included in the sequencing chemistry of each reaction. The position of the fourth terminator in the sequence is determined from the location of the gaps or tiny peaks in the background signal in the electropherogram which appear between the peaks produced by the three ddNTP terminators used. A dual channel electrophoresis device facilitates obtaining two matched electropherograms of two simultaneous sequence reactions which employ matched concentration ratios of ddNTP terminators. The device comprises a single radiation source and two parallel electrophoresis channels simultaneously operating under substantially similar conditions.