DNA sequencing by synthesis using Raman and infrared spectroscopy detection
First Claim
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1. A method for determining the sequence of consecutive nucleotide residues present in a single-stranded DNA comprising:
- (a) contacting the single-stranded DNA, having a primer hybridized to a portion thereof, with a DNA polymerase and four different nucleoside triphosphate (NTP) analogues under conditions permitting the DNA polymerase to catalyze incorporation onto the primer of a NTP analogue complementary to a nucleotide residue of the single-stranded DNA which is immediately 5′
to a nucleotide residue of the single-stranded DNA hybridized to the 3′
terminal nucleotide residue of the primer, so as to form a DNA extension product, wherein (i) each of the four NTP analogues has the structure;
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Abstract
This invention provides nucleoside triphosphate analogs having the structure:
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- wherein B is a base and is adenine, guanine, cytosine, uracil or thymine, wherein R″ is an OH or an H, and wherein R′ is azidomethyl, a hydrocarbyl, or a substituted hydrocarbyl, and which has a Raman spectroscopy peak with wavenumber from 2000 cm−1 to 2300 cm−1 or a Fourier transform-infrared spectroscopy spectroscopy peak with wavenumber from 2000 cm−1 to 2300 cm−1, and also to methods of DNA sequencing and SNP detection.
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Citations
20 Claims
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1. A method for determining the sequence of consecutive nucleotide residues present in a single-stranded DNA comprising:
(a) contacting the single-stranded DNA, having a primer hybridized to a portion thereof, with a DNA polymerase and four different nucleoside triphosphate (NTP) analogues under conditions permitting the DNA polymerase to catalyze incorporation onto the primer of a NTP analogue complementary to a nucleotide residue of the single-stranded DNA which is immediately 5′
to a nucleotide residue of the single-stranded DNA hybridized to the 3′
terminal nucleotide residue of the primer, so as to form a DNA extension product, wherein (i) each of the four NTP analogues has the structure;- View Dependent Claims (2, 3, 4, 11, 12, 13, 17)
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5. A method for determining the identity of a nucleotide residue within a stretch of consecutive nucleic acid residues in a single-stranded DNA comprising:
(a) contacting the single-stranded DNA, having a primer hybridized to a portion thereof such that the 3′
terminal nucleotide residue of the primer is hybridized to a nucleotide residue of the single-stranded DNA immediately 3′
to the nucleotide residue being identified, with a DNA polymerase and at least four nucleoside triphosphate (NTP) analogues under conditions permitting the DNA polymerase to catalyze incorporation into the primer of an NTP analogue complementary to the nucleotide residue of the single-stranded DNA being identified, so as to form a DNA extension product, wherein (i) each of the four NTP analogues has the structure;- View Dependent Claims (8, 14, 18)
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6. A method for determining the identity of a nucleotide residue within a stretch of consecutive nucleic acid residues in a single-stranded DNA comprising:
(a) contacting the single-stranded DNA, having a primer hybridized to a portion thereof such that the 3′
terminal nucleotide residue of the primer is hybridized to a nucleotide residue of the single-stranded DNA immediately 3′
to the nucleotide residue identified, with a DNA polymerase and at least one nucleoside triphosphate (NTP) analogue under conditions permitting the DNA polymerase to catalyze incorporation into the primer of the at least one NTP analogue if it is complementary to the nucleotide residue of the single-stranded DNA being identified, so as to form a DNA extension product, wherein (i) the at least one NTP analogue has the structure;- View Dependent Claims (9, 15, 19, 20)
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7. A method for determining the identity of a nucleotide residue within a stretch of consecutive nucleic acid residues in a single-stranded DNA comprising:
(a) contacting the single-stranded DNA with four different oligonucleotide probes, (1) wherein each of the oligonucleotide probes comprises (i) a portion that is complementary to a portion of consecutive nucleotides of the single stranded DNA immediately 3′
to the nucleotide residue being identified, and (ii) a 3′
terminal nucleotide residue analogue comprising on its sugar a 3′
-O—
R′
group wherein R′
(a) has the structure;- View Dependent Claims (10, 16)
Specification