SINGLE MOLECULE AMPLIFICATON 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.
56 Citations
31 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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Specification