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Abstract

The sequence of a target nucleic acid polymer can be determined by (a) performing a first chain-extension sequencing reaction on the target nucleic acid polymer in a reaction mixture containing first and second chain-terminators to produce a first product mixture containing commonly-labeled polynucleotide fragments complementary to a first strand of the target nucleic acid polymer, each fragment in the mixture being terminated with the first or second chain-terminator; (b) performing a second chain extension sequencing reaction on the target nucleic acid polymer in a reaction mixture containing the first and a third chain-terminator to produce a second product mixture containing commonly-labeled polynucleotide fragments complementary to the first strand of the target nucleic acid polymer, each fragment in the mixture being terminated with the first or the third chain-terminator, said first, second and third chain-terminators each being different; and (c) evaluating the lengths of the polynucleotide fragments in the first and second product mixtures to determine the sequence of the target nucleic acid polymer. In the evaluation step, the first and second product mixtures can be evaluated in two separate lanes of a gel, in which case the labels employed in the two chain extension sequencing reactions can be the same. Alternatively, if the labels employed in the two chain extension reactions are different and spectroscopically distinguishable from one another, the first and second product mixtures can be combined before electrophoresis and the entire analysis can be performed in a single lane of a gel.

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First Claim