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Single molecule sequencing with two distinct chemistry steps

  • US 8,603,741 B2
  • Filed: 02/08/2011
  • Issued: 12/10/2013
  • Est. Priority Date: 02/18/2010
  • Status: Active Grant
First Claim
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1. A method for sequencing a nucleic acid template comprising:

  • providing a sequencing mixture comprising a polymerase enzyme, a template nucleic acid, a primer, a polymerase regent solution having the components for carrying out template directed synthesis of a growing nucleic acid strand, a plurality of types of nucleotide analogs;

    wherein each type of nucleotide analog has a labeled leaving group, each with a different label, that blocks further nucleotide incorporations at the 3′

    hydroxyl position of the incorporated nucleotide analog; and

    a selective cleaving activity which more effectively cleaves a leaving group from an incorporated nucleotide analog than from an unincorporated nucleotide analog;

    carrying out nucleic acid synthesis such that a plurality of nucleotide analogs are added sequentially whereby;

    i) a nucleotide analog is incorporated, then ii) the labeled leaving group on that nucleotide analog is cleaved by the selective cleaving activity, making the 3′

    hydroxyl group available for a subsequent nucleotide analog addition; and

    detecting light from the labels while the polymerase reaction is occurring to determine a sequence of the template nucleic acid.

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