INTERMITTENT DETECTION DURING ANALYTICAL REACTIONS
First Claim
1. A method of performing paired-end sequencing on a single template molecule, the method comprising:
- a) providing a double-stranded nucleic acid molecule comprising a first terminal portion, an intermediate portion, and a second terminal portion;
b) ligating a first linker to the first terminal portion of the nucleic acid molecule, wherein the first linker connects the 3′
terminus at the first terminal portion with the 5′
terminus at the first terminal portion;
c) ligating a second linker to the second terminal portion of the nucleic acid molecule, wherein the second linker connects the 3′
terminus at the second terminal portion with the 5′
terminus at the second terminal portion, and further wherein the nucleic acid molecule having both the first linker and the second linker ligated thereto is termed a template molecule; and
d) subjecting the template molecule to a sequencing process in which sequence reads are generated for the first terminal portion and the second terminal portion, but sequence reads are not generated for the intermediate portion.
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Accused Products
Abstract
Methods, devices, and systems for performing intermittent detection during analytical reactions are provided. Such methods facilitate collection of reaction data from disparate reaction times. Further, such methods are useful for reducing photo-induced damage of one or more reactants in an illuminated analytical reaction at a given reaction time. In preferred embodiments, the reaction mixture is subjected to at least one illuminated and non-illuminated period and allowed to proceed such that the time in which the reaction mixture is illuminated is less than a photo-induced damage threshold period.
99 Citations
38 Claims
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1. A method of performing paired-end sequencing on a single template molecule, the method comprising:
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a) providing a double-stranded nucleic acid molecule comprising a first terminal portion, an intermediate portion, and a second terminal portion; b) ligating a first linker to the first terminal portion of the nucleic acid molecule, wherein the first linker connects the 3′
terminus at the first terminal portion with the 5′
terminus at the first terminal portion;c) ligating a second linker to the second terminal portion of the nucleic acid molecule, wherein the second linker connects the 3′
terminus at the second terminal portion with the 5′
terminus at the second terminal portion, and further wherein the nucleic acid molecule having both the first linker and the second linker ligated thereto is termed a template molecule; andd) subjecting the template molecule to a sequencing process in which sequence reads are generated for the first terminal portion and the second terminal portion, but sequence reads are not generated for the intermediate portion. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
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20. A method of generating a nucleic acid construct for analytical reactions, the method comprising:
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a) providing a double-stranded nucleic acid molecule comprising a first terminal portion, an intermediate portion, and a second terminal portion; b) providing a first stem-loop linker hybridized to a second stem-loop linker; c) ligating the first stem-loop linker to the first terminal portion of the nucleic acid molecule, wherein the first stem-loop linker connects the 3′
terminus at the first terminal portion with the 5′
terminus at the first terminal portion; andd) ligating the second stem-loop linker to the second terminal portion of the nucleic acid molecule, wherein the second stem-loop linker connects the 3′
terminus at the second terminal portion with the 5′
terminus at the second terminal portion, thereby generating the nucleic acid construct. - View Dependent Claims (21, 22, 23)
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24-28. -28. (canceled)
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29. A machine-implemented method for transforming enzyme velocity data from one or more detection periods into a distribution of the distance x traveled by an enzyme during a time t, where time t occurs during a non-detection period, the machine-implemented method comprising:
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a) estimating a distribution of local rates p(v); b) making independent identically distributed draws of N=t/τ
corr velocities from from p(v);c) summing the velocities; d) recording the velocities summed in c) as an estimate of x/τ
corr; ande) repeating b-d M times.
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30-37. -37. (canceled)
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38. A composition comprising a single, optically resolvable polymerase enzyme in association with a single-stranded circular nucleic acid molecule, wherein the single-stranded circular nucleic acid molecule comprises first, second, third, and fourth regions, and further wherein the first region is complementary to the second region, and the third region is complementary to the fourth region, and further wherein the regions are ordered on the single-stranded circular nucleic acid molecule as follows:
- first region, third region, second region, fourth region.
Specification