摘要：

Nucleic acid sequencing-by-synthesis. Primed synthesis of a second strand complementary to a template strand in repeated sets of steps, each step comprising providing one or more of the possible nucleotide complementarity classes for incorporation into the synthesized strand, and each set of steps comprising providing all four possible nucleotide complementarity classes. Three of the four possible nucleotide complementarity classes may first be provided for incorporation into the synthesized strand, then separately the fourth nucleotide complementarity class alone. Also, a DNA molecule consisting of a stem portion and first and second loop portions, wherein the stem portion consists of a first strand and a second strand, wherein the first strand and second strand are equal in length, complementary and annealed together, wherein the first loop portion joins the 3′ end of the first strand to the 5′ end of the second strand and the second loop portion joins the 3′ end of the second strand to the 5′ end of the first strand so the DNA molecule has no free 5′ or 3′ ends, and uses thereof, especially in sequencing.

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