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Methods and means for nucleic acid sequencing

  • US 20060147935A1
  • Filed: 02/09/2004
  • Published: 07/06/2006
  • Est. Priority Date: 02/12/2003
  • Status: Abandoned Application
First Claim
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1. A method of determining sequence and/or base composition information for a nucleic acid, the method comprising:

  • (i) providing a nucleic acid comprising a first strand that comprises a nucleic acid template, wherein a free 3′

    end of a nucleic acid strand annealed to the first strand allows for elongation of a strand of nucleic acid complementary to the nucleic acid template by template sequence-dependent incorporation of nucleotides into the strand of nucleic acid complementary to the nucleic acid template by a template-dependent nucleic acid polymerase;

    (ii) performing a set of one or more steps, which set of one or more steps is cycled a desired number of times or performed in combination with other sets of one or more steps to elongate the strand of nucleic acid complementary to the nucleic acid template allowing for information indicative of base composition or sequence of the nucleic acid to be obtained, wherein a step comprises;

    (a) providing, in the presence of;

    the nucleic acid comprising a first strand that comprises a nucleic acid template, said free 3′

    end of a nucleic acid strand annealed to the first strand of the nucleic acid template, and a template-dependent nucleic acid polymerase;

    nucleotides selected from one, two, three or four nucleotide complementarity classes for template-dependent incorporation by the nucleic acid polymerase of the nucleotides into the strand of nucleic acid complementary to the nucleic acid template, wherein each of said nucleotides is a natural nucleotide or a nucleotide analog capable of template-dependent incorporation by a nucleic acid polymerase into a nucleic acid strand at a free 3′

    end of the nucleic acid strand, and within each said nucleotide complementarity class the nucleotides and nucleotide analogs are complementary to one of Adenosine (A), Cytosine (C), Thymine (T) and Guanine (G);

    and (b) removing or inactivating unincorporated nucleotides;

    and wherein within a set of steps nucleotides selected from all four nucleotide complementarity classes are provided and available for template-dependent incorporation, in at least one step nucleotides selected from more than one, optionally two, three or four, nucleotide complementarity classes are provided and available for template-dependent incorporation, and the nucleotides in at least one of the nucleotide complementarity classes, if incorporated into the strand of nucleic acid complementary to the nucleic acid template, allow further elongation of the strand of nucleic acid complementary to the nucleic acid template, and optionally no nucleotide complementarity class is provided in more than one step;

    and wherein if nucleotides selected from all four complementarity classes are provided in one step then the nucleotides in one, two or three of the nucleotide complementarity classes, if incorporated into the strand of nucleic acid complementary to the nucleic acid template, prevent further elongation of the strand of nucleic acid complementary to the nucleic acid template and all copies present if multiple copies are present;

    (iii) performing multiple sets of said steps, cycling sets of steps and/or performing sets of steps in combination with different sets of steps;

    (iv) determining the nature of and/or quantity of nucleotides incorporated into the strand of nucleic acid complementary to the nucleic acid template in at least one set of steps by determining the nature and/or quantity of nucleotides incorporated into the strand of nucleic acid complementary to the nucleic acid template in at least one step in each set for which the nature and/or quantity of nucleotides incorporated is determined for the set.

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