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Methods and solutions for inhibiting undesired cleaving of labels

  • US 8,883,999 B2
  • Filed: 03/17/2009
  • Issued: 11/11/2014
  • Est. Priority Date: 03/19/2008
  • Status: Active Grant
First Claim
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1. A method of incorporating labeled nucleotides into nucleic acid, comprising:

  • a) providing i) a reaction chamber comprising a plurality of nucleic acid template molecules bound to a solid support, ii) a first solution comprising polymerase and a plurality of nucleotide analogues wherein each nucleotide analogue is labeled with a unique label and contains a removable chemical moiety capping the 3′

    -OH group, iii) a second solution comprising a cleaving agent, iv) a cleaving agent scavenger, wherein said cleaving agent scavenger is cystamine, and v) a wash solution;

    b) introducing said first solution into said reaction chamber under conditions wherein a first nucleotide analogue is incorporated by said polymerase;

    c) detecting the label of the incorporated nucleotide analogue;

    d) introducing said second solution into said reaction chamber under conditions such that the chemical moiety of the incorporated nucleotide analogue capping the 3′

    -OH group is removed by said cleaving agent leaving a 3′

    hydroxyl group to enable the next nucleotide incorporation;

    e) washing said reaction chamber with said wash solution under conditions where some leftover cleaving agent remains in said reaction chamber; and

    f) introducing said cleaving agent scavenger into said reaction chamber under conditions where cystamine reacts with said leftover cleaving agent.

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