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Massive parallel method for decoding DNA and RNA

  • US 6,664,079 B2
  • Filed: 10/05/2001
  • Issued: 12/16/2003
  • Est. Priority Date: 10/06/2000
  • Status: Expired due to Term
First Claim
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1. A method for sequencing a nucleic acid by detecting the identity of a nucleotide analogue after the nucleotide analogue is incorporated into a growing strand of DNA in a polymerase reaction, which comprises the following steps:

  • (i) attaching a 5′

    end of the nucleic acid to a solid surface;

    (ii) attaching a primer to the nucleic acid attached to the solid surface;

    (iii) adding a polymerase and one or more different nucleotide analogues to the nucleic acid to thereby incorporate a nucleotide analogue into the growing strand of DNA, wherein the incorporated nucleotide analogue terminates the polymerase reaction and wherein each different nucleotide analogue comprises (a) a base selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil, and their analogues;

    (b) a unique label attached through a cleavable linker to the base or to an analogue of the base;

    (c) a deoxyribose; and

    (d) a cleavable chemical group to cap an —

    OH group at a 3′

    -position of the deoxyribose;

    (iv) washing the solid surface to remove unincorporated nucleotide analogues;

    (v) detecting the unique label attached to the nucleotide analogue that has been incorporated into the growing strand of DNA, so as to thereby identify the incorporated nucleotide analogue;

    (vi) adding one or more chemical compounds to permanently cap any unreacted —

    OH group on the primer attached to the nucleic acid or on a primer extension strand formed by adding one or more nucleotides or nucleotide analogues to the primer;

    (vii) cleaving the cleavable linker between the nucleotide analogue that was incorporated into the growing strand of DNA and the unique label;

    (viii) cleaving the cleavable chemical group capping the —

    OH group at the 3′

    -position of the deoxyribose to uncap the —

    OH group, and washing the solid surface to remove cleaved compounds; and

    (ix) repeating steps (iii) through (viii) so as to detect the identity of a newly incorporated nucleotide analogue into the growing strand of DNA;

    wherein if the unique label is a dye, the order of steps (v) through (vii) is;

    (v), (vi), and (vii); and

    wherein if the unique label is a mass tag, the order of steps (v) through (vii) is;

    (vi), (vii), and (v).

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