Polynucleotide sequencing method

A method for determining a sequence of one or more nucleotides in a target polynucleotide, the method comprising:

• contacting a target-specific primer with a polynucleotide sample under conditions effective for the primer to anneal specifically to a primer-complementary region in one or more target polynucleotides, to form one or more target-primer hybrid(s), wherein each target-specific primer contains (i) a target binding segment and (ii) a mobility-reducing moiety which does not bind the target,
• reacting said hybrid(s) with a primer extension reagent in the presence of at least one labeled 3′-nucleotide terminator complementary to a selected nucleotide base-type, under conditions effective to form one or more primer extension products which tenninate with at least one labeled 3′-nucleotide terminator that produces a detectable signal,
• separating electrophoretically the primer extension product(s) under denaturing conditions in a sieving matrix such that (i) smaller extension products migrate more rapidly than larger extension products and (ii) at least one detectable breakdown product from the labeled 3′-nucleotide terminator elutes before non-extended primer so as not to overlap in elution with said primer extension product(s), and
• detecting at least one detectable signal from said separated primer extension product(s) to determine a target sequence on the basis of the mobilities and detectable signals from the separated extension product(s).