Parallel primer extension approach to nucleic acid sequence analysis
First Claim
1. A method of analyzing the sequence of a polynucleotide of interest, comprising the steps of:
- a) annealing a polynucleotide of interest to free oligonucleotide primers having known sequences of N nucleotides in length to generate annealed primers;
b) subjecting the annealed primers to a single base extension reaction to extend the annealed primers by the addition of a terminating nucleotide;
c) observing the identity of each terminating nucleotide that has been added to the annealed primers.
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Abstract
A method of analyzing a polynucleotide of interest, comprising providing one or more sets of consecutive oligonucleotide primers differing within each set by one base at the growing end therof, annealing a single strand of the polynucleotide or a fragment of the polynucleotide to the oligonucleotide primers under hybridization conditions; subjecting the primers to single base extension reactions with a polymerase and terminating nucleotides, the terminating nucleotides being mutually distinguishable; and observing the location and identity of each terminating nucleotide to thereby analyze the sequence or a part of the nucleotide sequence of the polynucleotide of interest, is disclosed. An apparatus comprising a solid support to which is attached at defined locations thereon one or more sets of consecutive oligonucleotide primers differing within each set by one base at the growing end thereof is also described.
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Citations
21 Claims
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1. A method of analyzing the sequence of a polynucleotide of interest, comprising the steps of:
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a) annealing a polynucleotide of interest to free oligonucleotide primers having known sequences of N nucleotides in length to generate annealed primers;
b) subjecting the annealed primers to a single base extension reaction to extend the annealed primers by the addition of a terminating nucleotide;
c) observing the identity of each terminating nucleotide that has been added to the annealed primers. - View Dependent Claims (7, 11, 12, 13, 14, 15, 16, 18)
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2. A method of analyzing the sequence of a polynucleotide of interest, comprising the steps of:
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a) annealing a polynucleotide of interest to oligonucleotide primers having known sequences of N nucleotides in length under hybridization conditions, to generate annealed primers;
b) subjecting the annealed primers to a single base extension reaction which comprises providing to the annealed primers nucleotides corresponding to each of the four bases, to extend the annealed primers by the addition of a terminating nucleotide;
c) observing the identity and location of each terminating nucleotide that has been added to the annealed primers.
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3. A method of analyzing the sequence of a polynucleotide of interest, comprising the steps of:
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a) attaching an array of oligonucleotide primers having known sequences of N nucleotides in length to a solid support at known locations;
b) annealing the polynucleotide of interest to the array of oligonucleotide primers to generate annealed primers;
c) subjecting the annealed primers to a single base extension reaction to extend the annealed primers by the addition of a terminating nucleotide;
d) observing the identity and location of each terminating nucleotide within the array on the solid support.
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4. A method of analyzing the sequence of a polynucleotide of interest, comprising the steps of:
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a) attaching an array of oligonucleotide primers having known sequences of N nucleotides in length to a solid support at known locations;
b) annealing the polynucleotide of interest to the array of oligonucleotide primers to generate annealed primers;
c) subjecting the annealed primers to a single base extension reaction to extend the annealed primers by the addition of a terminating nucleotide;
d) selecting a starting annealed primer;
e) observing the identity and location of the terminating nucleotide which has been added to the starting annealed primer, to determine the next nucleotide in sequence;
f) selecting a second annealed primer which has the same nucleotide sequence as nucleotides 2 through N of the starting annealed primer nucleotide plus the next nucleotide in sequence as determined in step (e), and g) repeating steps (e) and (f), using the second annealed primer as the starting annealed primer for each repetition, to determine the sequence of the polynucleotide of interest.
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5. A method of analyzing the sequence of a polynucleotide of interest, comprising the steps of:
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a) attaching an array of oligonucleotide primers, having known sequences of N nucleotides in length to a solid support at defined locations;
b) annealing the polynucleotide of interest to the array of oligonucleotide primers under hybridization conditions, to generate annealed primers;
c) subjecting the annealed primers to a single base extension reaction which comprises providing to the annealed primers nucleotides corresponding to each of the four bases, to extend the annealed primers by the addition of a terminating nucleotide;
d) observing the identity and location of each terminating nucleotide within the array on the solid support. - View Dependent Claims (8, 9, 10, 17)
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6. A method of analyzing the sequence of a polynucleotide of interest, comprising the steps of:
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a) attaching an array of oligonucleotide primers, having known sequences of N nucleotides in length to a solid support at defined locations;
b) annealing the polynucleotide of interest to the array of oligonucleotide primers under hybridization conditions, to generate annealed primers;
c) subjecting the annealed primers to a single base extension reaction which comprises providing to the annealed primers nucleotides corresponding to each of the four bases, to extend the annealed primers by the addition of a terminating nucleotide;
d) selecting a starting annealed primer;
e) observing the identity and location of the terminating nucleotide which has been added to the starting annealed primer, to determine the next nucleotide in sequence;
f) selecting a second annealed primer which has the same nucleotide sequence as nucleotides 2 through N of the starting annealed primer nucleotide plus the next nucleotide in sequence as determined in step (e), and g) repeating steps (e) and (f), using the second annealed primer as the starting annealed primer for each repetition, to determine the sequence of the polynucleotide of interest.
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- 19. An apparatus for analyzing the sequence of a polynucleotide of interest, comprising a solid support having attached thereon at defined locations an array of oligonucleotide primers having known sequences.
Specification