System and method for detecting a biological entity in a water sample
First Claim
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1. A method of monitoring water contamination, comprising,(a) combining nucleic acid molecules having one or more nucleic acid sequences and that are from at least one sample of water, with multiple oligonucleotide primers, wherein the primers comprise randomized nucleotide sequences;
- (b) amplifying the water sample nucleic acid molecules that hybridize to the randomized primers by subjecting the mixture of combined primers and sample nucleic acid molecules to a plurality of cycles of the polymerase chain reaction to generate an amplification product comprising a population of amplified nucleic acid molecules, wherein the primers are sufficiently randomized such that substantially all of the nucleic acid sequences of a biological entity in the water sample are represented among the amplified nucleic acid molecules;
(c) hybridizing the amplified nucleic acid molecules to an array of nucleic acid molecules having predetermined nucleic acid sequences, such that at least a portion of the amplification product hybridizes to the array, and wherein the array comprises redundancies comprising several distinct nucleic acid sequences from the same biological entity; and
(d) detecting the amplified nucleic acid molecules that hybridize to the array and relating the detected amplified nucleic acid molecules to at least one biological entity in the at least one sample of water.
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Abstract
The invention relates to the detection of a biological entity in a sample. More particularly, the invention relates to detection of specific pathogens from a possible presence of hundreds to thousands of distinct biological species. The invention provides new assays that can detect the presence of one or more biological entity in a sample out of a possible number of hundreds to thousands of distinct biological species. The method according to the invention for detecting a biological entity in a sample comprises randomly amplifying nucleic acids in the sample to produce labeled nucleic acids; hybridizing the labeled nucleic acids to an array of predetermined nucleic acids; and detecting the labeled nucleic acids that have hybridized to the array.
33 Citations
21 Claims
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1. A method of monitoring water contamination, comprising,
(a) combining nucleic acid molecules having one or more nucleic acid sequences and that are from at least one sample of water, with multiple oligonucleotide primers, wherein the primers comprise randomized nucleotide sequences; -
(b) amplifying the water sample nucleic acid molecules that hybridize to the randomized primers by subjecting the mixture of combined primers and sample nucleic acid molecules to a plurality of cycles of the polymerase chain reaction to generate an amplification product comprising a population of amplified nucleic acid molecules, wherein the primers are sufficiently randomized such that substantially all of the nucleic acid sequences of a biological entity in the water sample are represented among the amplified nucleic acid molecules; (c) hybridizing the amplified nucleic acid molecules to an array of nucleic acid molecules having predetermined nucleic acid sequences, such that at least a portion of the amplification product hybridizes to the array, and wherein the array comprises redundancies comprising several distinct nucleic acid sequences from the same biological entity; and (d) detecting the amplified nucleic acid molecules that hybridize to the array and relating the detected amplified nucleic acid molecules to at least one biological entity in the at least one sample of water. - View Dependent Claims (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21)
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2. A method monitoring water contamination, comprising,
(a) combining nucleic acid molecules having one or more nucleic acid sequences and that are from at least one sample of water, with multiple oligonucleotide primers, wherein the primers comprise randomized nucleotide sequences; -
(b) amplifying the water sample nucleic acid molecules that hybridize to the randomized primers by subjecting the mixture of combined primers and sample nucleic acid molecules to a plurality of cycles of the polymerase chain reaction to generate an amplification product comprising a population of amplified nucleic acid molecules, wherein the primers are sufficiently randomized such that substantially all of the nucleic acid sequences of a biological entity in the water sample are represented among the amplified nucleic acid molecules; (c) hybridizing the amplified nucleic acid molecules to an array of nucleic acid molecules having predetermined nucleic acid sequences, such that at least a portion of the amplification product hybridizes to the array, and wherein the array comprises positive controls, negative controls, and redundancies comprising several distinct nucleic acid sequences from the same biological entity, and wherein each of the predetermined nucleic acid sequences comprises a predetermined position on the array; and (d) detecting the amplified nucleic acid molecules that hybridize to the array and relating the detected amplified nucleic acid molecules to at least one biological entity in the at least one sample of water.
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Specification
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Current AssigneeLeidos, Inc. (Leidos Holdings, Inc.)
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Original AssigneeScience Applications International Corporation
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InventorsSchaudies, R. Paul, Robinson, Doreen A.
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Primary Examiner(s)Chunduru; Suryaprabha
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Application NumberUS10/271,076Time in Patent Office2,233 DaysField of Search435/6, 435/91.2, 536/24.33, 536/24.32US Class Current435/6.1CPC Class CodesC12Q 1/6837 using probe arrays or probe...Y10S 977/924 using nanostructure as supp...
