Shot-gun sequencing and amplification without cloning
First Claim
1. A method of sequencing a nucleic acid template comprising:
- (a) providing a plurality of first primers, each first primer comprising (i) a region of different fixed nucleotide sequence from about 5 to 15 bases long and (ii) a region of randomized nucleotide sequence from about 2 to 11 bases long located 5′
to or 3′
to the region of fixed nucleotide sequence, and wherein the region of fixed nucleotide is identical in sequence in each primer within the first plurality;
(b) annealing the plurality of first primers to different locations on a nucleic acid template, wherein at least one primer from within the plurality of first primers anneals specifically to the template;
(c) extending the specifically annealed primer from step (b) with a mixture of dNTPs and ddNTPs to generate a series of nucleic acid fragments;
(d) determining the nucleotide sequence of a first region of the template from the series of nucleic acid fragments;
(e) providing a plurality of second primers, each second primer comprising (i) a region of fixed nucleotide sequence from about 5 to 15 bases long and (ii) a region of randomized nucleotide sequence from about 2 to 11 bases long located 5′
to or 3′
to the region affixed nucleotide sequence, and wherein the region of fixed nucleotide is identical in sequence in each primer within the second plurality;
(f) repeating steps (b)-(d) for the second plurality of primers to thereby determine the nucleotide sequence of a second region of the template; and
(g) assembling the first sequenced region and the second sequenced region of the template nucleic acid to form a first contig.
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Abstract
Disclosed is a method for sequencing and amplifying nucleic acid templates wherein a degenerate primer with a fixed sequence region and a random sequence region is utilized. By determining the statistical expectancy of the fixed sequence in the nucleic acid template, this determines the average length of a nucleic acid template that can be sequenced. During the annealing of such a primer with the nucleic acid template, the fixed sequence determines where the complete primer binds by binding to its complementary sequence on the nucleic acid template. The random sequence regions of the primers make it possible for the presence of a unique sequence adjacent to the fixed sequence to be present, thus providing a primer with full complementarity with the nucleic acid template. Thus, this procedure is able to provide a full-length primer with a fully complementary sequence capable of binding statistically once within an expected length of the nucleic acid template, even though the sequence of the template is unknown. The method can also be adopted for use in PCR amplification of a nucleic acid template.
61 Citations
21 Claims
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1. A method of sequencing a nucleic acid template comprising:
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(a) providing a plurality of first primers, each first primer comprising (i) a region of different fixed nucleotide sequence from about 5 to 15 bases long and (ii) a region of randomized nucleotide sequence from about 2 to 11 bases long located 5′
to or 3′
to the region of fixed nucleotide sequence, and wherein the region of fixed nucleotide is identical in sequence in each primer within the first plurality;
(b) annealing the plurality of first primers to different locations on a nucleic acid template, wherein at least one primer from within the plurality of first primers anneals specifically to the template;
(c) extending the specifically annealed primer from step (b) with a mixture of dNTPs and ddNTPs to generate a series of nucleic acid fragments;
(d) determining the nucleotide sequence of a first region of the template from the series of nucleic acid fragments;
(e) providing a plurality of second primers, each second primer comprising (i) a region of fixed nucleotide sequence from about 5 to 15 bases long and (ii) a region of randomized nucleotide sequence from about 2 to 11 bases long located 5′
to or 3′
to the region affixed nucleotide sequence, and wherein the region of fixed nucleotide is identical in sequence in each primer within the second plurality;
(f) repeating steps (b)-(d) for the second plurality of primers to thereby determine the nucleotide sequence of a second region of the template; and
(g) assembling the first sequenced region and the second sequenced region of the template nucleic acid to form a first contig. - View Dependent Claims (2, 3)
(h) repeating steps (e)-(g) to form a second contig;
(i) providing a plurality of third primers, each third primer comprising (i) a region of fixed nucleotide sequence from about 5 to 15 bases long and (ii) a region of randomized nucleotide sequence from about 2 to 11 bases long located 5′
to or 3′
to the region of fixed nucleotide sequence, and wherein the region of fixed nucleotide is identical in sequence in each primer within the third plurality;
(j) annealing the plurality of third primers to different locations on the nucleic acid template, wherein at least one primer from the third plurality anneals to the template near a terminus of one of the first or second contigs;
(k) extending the annealed third primer with a mixture of dNTPs and ddNTPs to generate a series of nucleic acid fragments; and
(l) determining the sequence of the template between the first and second contigs from the series of nucleic acid fragments.
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4. A method of sequencing a nucleic acid template comprising:
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(a) providing a plurality of first primers, each first primer comprising (i) a region of fixed nucleotide sequence from about 5 to 15 bases long and (ii) a region of randomized nucleotide sequence from about 2 to 11 bases long located 5′
to or 3′
to the region of fixed nucleotide sequence, and wherein the region of fixed nucleotide is identical in sequence in each primer within the first plurality;
(b) annealing the plurality of first primers to different locations on a nucleic add template wherein at least one primer from within the plurality of first primers anneals specifically to the template;
(c) adding a plurality of fixed-sequence primers, each fixed-sequence primer comprising (i) a region of fixed nucleotide sequence that is shorter than the region of fixed nucleotide sequence in the first plurality of primers and (ii) a region of randomized nucleotide sequence located 5′
to or 3′
to the region of fixed nucleotide sequence;
(d) annealing the plurality of fixed-sequence primers to different locations on the nucleic acid template, wherein at least one fixed-sequence primer anneals to the nucleic acid template; and
(e) amplifying the nucleic acid template with the annealed first and annealed fixed-sequence primers with a mixture of dNTPs to amplify copies of the nucleic acid template bounded by the annealed first and fixed-sequence primers;
(f) extending the specifically annealed primer from step (b) with a mixture of dNTPs and ddNTPs to generate a series of nucleic acid fragments; and
(g) determining the nucleotide sequence of a first region of the template from the series of nucleic acid fragments.
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5. A method of sequencing a nucleic acid template comprising:
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(a) providing a plurality of first primers, each first primer comprising (i) a region of fixed nucleotide sequence from about 5 to 15 bases long and (ii) a region of randomized nucleotide sequence from about 2 to 11 bases long located 5′
to or 3′
to the region of fixed nucleotide sequence, and wherein the region of fixed nucleotide is identical in sequence in each primer within the first plurality;
(b) annealing the plurality of first primers to different locations on a nucleic acid template, wherein at least one primer from within the plurality of first primers anneals specifically to the template, and further wherein a sequence corresponding to or complementary to the region of fixed nucleotide sequence of the first plurality of primers occurs within the nucleic add template at a frequency that is different than statistically predicted based on a random distribution of bases throughout the template;
(c) extending the specifically annealed primer from step (b) with a mixture of dNTPs and ddNTPs to generate a series of nucleic acid fragments; and
(d) determining the nucleotide sequence of a first region of the template from the series of nucleic acid fragments. - View Dependent Claims (6, 7, 8)
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9. A method of sequencing a nucleic acid template comprising:
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(a) providing a plurality of first primers, each first primer comprising (i) a region of fixed nucleotide sequence from about 5 to 15 bases long and (ii) a region of randomized nucleotide sequence from about 2 to 11 bases long located 5′
to or 3′
to the region of fixed nucleotide sequence, and wherein the region of fixed nucleotide is identical in sequence in each primer within the first plurality;
(a)(i) relaxing torsion, secondary structure, or tertiary structure in the template;
(b) annealing the plurality of first primers to different locations on a nucleic acid template, wherein at least one primer from within the plurality of first primers anneals specifically to the template;
(c) extending the specifically annealed primer from step (b) with a mixture of dNTPs and ddNTPs to generate a series of nucleic acid fragments; and
(d) determining the nucleotide sequence of a first region of the template from the series of nucleic acid fragments. - View Dependent Claims (10, 11)
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12. A method of sequencing a nucleic acid template comprising:
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(a) providing a plurality of first primers, each first primer comprising (i) a region of fixed nucleotide sequence from about 5 to 15 bases long, (ii) a region of randomized nucleotide sequence from about 2 to 11 bases long located 5′
to or 3′
to the region of fixed nucleotide sequence, and wherein the region of fixed nucleotide is identical in sequence in each primer within the first plurality, and (iii) a handle at an end of each first primer;
(b)annealing the plurality of first primers to different locations on a nucleic acid template, wherein at least one primer from within the plurality of first primers anneals specifically to the template;
(c) extending the specifically annealed primer from step (b) with a mixture of dNTPs and ddNTPs to generate a series of nucleic acid fragments; and
(d) determining the nucleotide sequence of a first region of the template from the series of nucleic acid fragments. - View Dependent Claims (13, 14, 15, 16, 17, 18, 19, 20, 21)
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Specification