Single molecule amplification and detection of DNA length
First Claim
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1. A method of determining whether a nucleic acid of interest in a sample comprises at least a given length, the method comprising:
- contacting the nucleic acid of interest in a reaction mixture with two or more different probes, wherein the probes each comprise a detectable marker;
flowing the nucleic acid of interest into a detection region; and
, detecting one or more detectable marker signals from the probes;
wherein coincident detection of signals from two or more of the different probes indicates the nucleic acid of interest is not fragmented between the probes, thereby determining the nucleic acid of interest in the sample has at least a given length.
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Abstract
Methods and systems for performing single molecule amplification for detection, quantification and statistical analysis of nucleic acids are provided. Methods and systems are provided for determining and quantifying lengths of nucleic acids of interest.
301 Citations
69 Claims
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1. A method of determining whether a nucleic acid of interest in a sample comprises at least a given length, the method comprising:
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contacting the nucleic acid of interest in a reaction mixture with two or more different probes, wherein the probes each comprise a detectable marker;
flowing the nucleic acid of interest into a detection region; and
,detecting one or more detectable marker signals from the probes;
wherein coincident detection of signals from two or more of the different probes indicates the nucleic acid of interest is not fragmented between the probes, thereby determining the nucleic acid of interest in the sample has at least a given length. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 31)
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28. A method of differentiating lengths of a nucleic acid of interest in a sample, the method comprising:
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a) contacting the nucleic acid of interest with a first primer pair;
b) contacting the nucleic acid of interest with a second primer pair comprising at least one primer complimentary to a sequence of the nucleic acid of interest or its compliment outside of a sequence defined by the first primer pair;
c) amplifying the nucleic acid of interest in a reaction mixture comprising a single copy of the nucleic acid of interest to produce first amplicons defined by the first primer pair or second amplicons defined by the second primer pair;
d) contacting the reaction mixture with a first probe complimentary to a sequence of the first amplicons or with a second probe complimentary to a sequence of the second amplicons, which probes comprise signals from detectable markers; and
,e) detecting one or more of the signals;
wherein detection of a signal from only one of the probes indicates a fragmented nucleic acid of interest or detecting signals from both probes indicates a nucleic acid that has a given length, thereby differentiating the length of the nucleic acid of interest. - View Dependent Claims (29)
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30. A method of differentiating lengths of nucleic acids of interest in a sample, the method comprising:
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a) contacting a nucleic acid of interest with a first primer pair;
b) contacting the nucleic acid of interest with a second primer pair comprising at least one primer complimentary to a sequence of the nucleic acid of interest or its compliment outside of a sequence defined by the first primer pair;
c) amplifying the nucleic acid of interest in a reaction mixture contained in a microchannel or microchamber with primer extensions initiated at the primers to produce first amplicons defined by the first primer pair or second amplicons defined by the second primer pair;
d) contacting the reaction mixture with a first probe complimentary to a sequence of the first amplicons or with a second probe complimentary to a sequence of the second amplicons, which probes comprise signals from detectable markers; and
,e) detecting one or more signals;
wherein detection of a signal from only one of the probes indicates a fragmented nucleic acid of interest or detecting signals from both probes indicates a nucleic acid that has a given length, thereby differentiating the length of the nucleic acid of interest.
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32. A method of quantifying a nucleic acid of interest in a sample, the method comprising:
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amplifying the nucleic acid of interest through a plurality of amplification cycles;
detecting signals associated with amplicons produced for two or more of the amplification cycles;
preparing a sample curve of a signal parameter versus a number of amplification cycles; and
,comparing one or more identifiable points from the sample curve to a standard curve of identifiable points versus concentration, thereby quantifying the nucleic acid of interest. - View Dependent Claims (33)
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34. A method of quantifying a nucleic acid of interest in a sample, the method comprising:
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amplifying the nucleic acid of interest through a plurality of amplification cycles in a reaction mixture defining two or more different amplicons of the nucleic acid of interest;
detecting from homogenous reaction mixtures different signals associated with each of the different amplicons after at least two of the plurality of amplification cycles;
preparing sample curves of the different signals versus numbers of amplification cycles; and
,comparing one or more identifiable points from the sample curves to one or more standard curves of identifiable points versus nucleic acid concentration, thereby quantifying one or more sequences of the nucleic acid of interest associated with one or more of the amplicons. - View Dependent Claims (35, 36, 37, 38)
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39. A method of quantifying a nucleic acid of interest in a sample, the method comprising:
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amplifying a plurality of nucleic acid of interest standard materials through a number of amplification cycles;
detecting signals associated with standard amplicons produced for standard materials having different known concentrations of the nucleic acid;
amplifying the sample nucleic acid of interest the number of amplification cycles;
detecting a signal associated with sample amplicons produced for the sample nucleic acid of interest; and
,comparing one or more standard amplicon signals to the sample amplicon signal to determine a concentration value for the nucleic acid of interest in the sample, thereby quantifying the nucleic acid of interest. - View Dependent Claims (40, 41)
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42. A system for differentiating the lengths of nucleic acids of interest in a sample, the system comprising:
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a microfluidic device comprising an amplification microchannel or microchamber containing a reaction mixture under conditions that provide one or more amplicons of the nucleic acid of interest;
a detector integral with or proximal to the microfluidic device, which detector is configured to detect the amplicons as one or more signals from a homogenous mixture; and
,a software system that interprets one or more coincidently detected signals to indicate lengths of one or more individual nucleic acid molecules of interest, thereby differentiating the lengths of the nucleic acids of interest. - View Dependent Claims (43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69)
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Specification