Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators

  • US 7,883,869 B2
  • Filed: 11/30/2007
  • Issued: 02/08/2011
  • Est. Priority Date: 12/01/2006
  • Status: Active Grant
First Claim
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1. A method for determining the sequence of a DNA comprising performing the following steps for each residue of the DNA to be sequenced:

  • (a) contacting the DNA with a DNA polymerase in the presence of (i) a primer and (ii) DNA polymerization reagents, said reagents comprising four labeled nucleotide analogues under conditions permitting the DNA polymerase to catalyze DNA synthesis, wherein (1) the four labeled nucleotide analogues consist of an analogue of dGTP, an analogue of dCTP, an analogue of dTTP or dUTP, and an analogue of dATP, (2) each labeled nucleotide analogue comprises (i) a base selected from the group consisting of adenine, guanine, cytosine, thymine or uracil, and analogues thereof, (ii) a deoxyribose, (iii) a moiety cleavably linked to the 3′

    -oxygen of the deoxyribose and (iv) a unique label cleavably linked to the base, so that a nucleotide analogue complementary to the residue being sequenced is incorporated into the DNA by the DNA polymerase, and (3) each of the four labeled analogues has a unique label which is different than the unique labels of the other three analogues;

    (b) removing unbound nucleotide analogues;

    (c) again contacting the DNA with a DNA polymerase in the presence of (i) a primer and (ii) DNA polymerization reagents, said reagents comprising four reversible terminators under conditions permitting the DNA polymerase to catalyze DNA synthesis, wherein (1) the reversible terminators consist of an analogue of dGTP, an analogue of dCTP, an analogue of dTTP or dUTP, and an analogue of dATP, (2) each nucleotide analogue comprises (i) a base selected from the group consisting of adenine, guanine, cytosine, thymine or uracil, and analogues thereof, which base does not have a unique label bound thereto, (ii) a deoxyribose, and (iii) a moiety cleavably linked to the 3′

    -oxygen of the deoxyribose;

    (d) determining the identity of the nucleotide analogue incorporated in step (a) via determining the identity of the corresponding unique label, with the proviso that step (d) can either precede step (c) or follow step (c); and

    (e) following step (d), except with respect to the final DNA residue to be sequenced, cleaving from the incorporated nucleotide analogues the unique label, if applicable, and the moiety linked to the 3′

    -oxygen atom of the deoxyribose, thereby determining the sequence of the DNA.

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