Method for DNA- or RNA-sequencing
First Claim
1. A method for base sequencing of DNA or RNA comprising the following steps:
- (1) immobilizing DNA or RNA single strands on a planar surface;
(2) focussing a laser beam on a single, immobilized single strand;
(3) producing a DNA or RNA complementary strand of said immobilized, focussed single strand by adding a solution containing (i) a mixture of nucleotides of the bases adenine, cytosine, guanine and thymine for producing a DNA complementary strand or a mixture of nucleotides of the bases adenine, cytosine, guanine and uracil for producing a RNA complementary strand and (ii) a polymerase, wherein at least two of the four nucleotides of the bases adenine, cytosine, guanine, and thymine, or at least two of the four nucleotides of the bases adenine, cytosine, guanine, or uracil are partially or fully luminescence-tagged, said step (3) further comprising (a) detecting each insertion of a luminescence-tagged nucleotide into the complementary strand with a single-molecule detector, and (b) deleting the luminescence signal of the luminescence-tagged nucleotide after detection and prior to insertion of the respective next luminescence-tagged nucleotide, thereby base sequencing DNA or RNA.
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Abstract
The invention relates to methods for the base sequencing of deoxyribonucleic acid or ribonucleic acid, comprising the following steps: (1) immobilising DNA or RNA single strands on a planar support; (2) focussing a laser beam on a single, immobilised single strand; (3) producing DNA or RNA complimentary strand of said immobilised, focused single strand by adding a solution containing (i) at least one luminescence-tagged nucleotide on the bases of adenine, cytosine, guanine and thymine for producing a DNA or RNA complementary strand or at least one luminescence-tagged nucleotide of the bases adenine, cytosine, guanine and uracil for producing an RNA complementary strand and (ii) a polymerase, each insertion of a luminescence-tagged nucleotide into the complementary strand being detected with a single-molecule detector and the luminescence signal of the previous luminescence-tagged nucleotide being deleted before the next luminescence-tagged nucleotide is inserted.
261 Citations
15 Claims
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1. A method for base sequencing of DNA or RNA comprising the following steps:
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(1) immobilizing DNA or RNA single strands on a planar surface;
(2) focussing a laser beam on a single, immobilized single strand;
(3) producing a DNA or RNA complementary strand of said immobilized, focussed single strand by adding a solution containing (i) a mixture of nucleotides of the bases adenine, cytosine, guanine and thymine for producing a DNA complementary strand or a mixture of nucleotides of the bases adenine, cytosine, guanine and uracil for producing a RNA complementary strand and (ii) a polymerase, wherein at least two of the four nucleotides of the bases adenine, cytosine, guanine, and thymine, or at least two of the four nucleotides of the bases adenine, cytosine, guanine, or uracil are partially or fully luminescence-tagged, said step (3) further comprising (a) detecting each insertion of a luminescence-tagged nucleotide into the complementary strand with a single-molecule detector, and (b) deleting the luminescence signal of the luminescence-tagged nucleotide after detection and prior to insertion of the respective next luminescence-tagged nucleotide, thereby base sequencing DNA or RNA. - View Dependent Claims (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
(a) hybridizing a luminescence-tagged nucleotide oligomer to the single strand, (b) determining the position of the hybridized nucleotide-oligomer by scanning the surface with a laser beam, and (c) deleting the luminescence signal of the hybridized nucleotide-oligomer after determining the position of the hybridized nucleotide-oligomer. -
8. A method according to claim 1, wherein each insertion of a luminescence-tagged nucleotide into the complementary strand is detected in step (3a) by means of the fluorescence life time of the luminescence-tagged nucleotide.
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9. A method according to claim 1, wherein each insertion of a luminescence-tagged nucleotide into a complementary strand is detected in step (3a) by means of the color of the luminescence-tagged nucleotide.
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10. A method according to claim 1, wherein the single-molecule detector in step (3a) comprises an auto-correlation function.
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11. A method according to claim 1, wherein deletion of the luminescence signal in step (3b) occurs by cleavage of a luminescence-tagged nucleotide.
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12. A method according to claim 1, wherein deletion of the luminescence signal in step (3b) occurs by photo-bleaching the luminescence-tagged nucleotide.
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13. A method according to claim 1, wherein the deletion of the luminescence signal is deleted is controlled by the output of the laser pulse used for deleting the luminescence signal and the duration of the time period between detection and laser pulse, these parameters being matched to the production velocity of the complementary strand which is controlled by the polymerase activity and the nucleotide concentration.
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14. A method for base sequencing of DNA or RNA, wherein the complementary DNA or RNA strand produced according to claim 1 is disassociated from the immobilized DNA or RNA single strand by increasing the temperature, and the method according to one of the previous claims is repeated on the immobilized single strand.
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15. A method for base sequencing of DNA or RNA, wherein said method according to claim 1 is repeated on an additional or the same DNA or RNA single strand immobilized on the surface.
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2. A method for base sequencing of DNA or RNA comprising the following steps:
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(1) immobilizing DNA or RNA single strands on a planar surface;
(2) focussing a laser beam on a single, immobilized single strand;
(3) producing a DNA or RNA complementary strand of the immobilized, focussed single strand by sequential addition of solutions containing (i) one nucleotide of the bases adenine, cytosine, guanine and thymine for producing a DNA complementary strand or one nucleotide of the bases adenine, cytosine, guanine and uracil for producing a RNA complementary strand and (ii) a polymerase, wherein the nucleotide contained in the solution is luminescence-tagged, said step (3) further comprising (a) detecting each insertion of a luminescence-tagged nucleotide into the complementary strand with a single molecule-detector, (b) deleting the luminescence signal of the luminescence-tagged nucleotide after detection, and (c) rinsing the surface-immobilized DNA or RNA prior to the addition of each next solution, thereby base sequencing DNA or RNA.
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Specification