US6403319B1 — Analysis of sequence tags with hairpin primers

Claim 1

Independent Claim 135 words

A method of identifying nucleic acid fragments in nucleic acid samples, the method comprising (a) mixing one or more nucleic acid samples with one or more different hairpin primers, wherein each hairpin primer comprises a different primer sequence, (b) incubating the samples under conditions that promote amplification of nucleic acids in the samples, wherein amplified nucleic acid fragments are formed which have hairpin primer sequences at one or both ends, (c) incubating the samples under conditions that promote formation of hairpin structures by the hairpin primer sequences at the ends of the amplified fragments, (d) hybridizing each sample with a plurality of detector probes and covalently coupling the hairpin structures to the probes, wherein each probe has a different sequence, and (e) detecting, directly or indirectly, coupling of the amplified fragments to the detector probes.

Claim 2

Independent Claim 135 words

A method of identifying nucleic acid fragments in nucleic acid samples, the method comprising (a) mixing one or more nucleic acid samples with one or more different hairpin primers, wherein each hairpin primer comprises a different primer sequence, (b) incubating the samples under conditions that promote amplification of nucleic acids in the samples, wherein amplified nucleic acid fragments are formed which have hairpin primer sequences at one or both ends, (c) incubating the samples under conditions that promote formation of hairpin structures by the hairpin primer sequences at the ends of the amplified fragments, (d) hybridizing each sample with a plurality of detector probes and covalently coupling the hairpin structures to the probes, wherein each probe has a different sequence, and (e) detecting, directly or indirectly, coupling of the amplified fragments to the detector probes.

Claim 3

Dependent Claim 14 words

The method of claim 1 wherein the probes are all of the same length.

Claim 4

Dependent Claim 16 words

The method of claim 2 wherein the detector probes are six, seven, or eight nucleotides long.

Claim 5

Dependent Claim 13 words

The method of claim 1 wherein the probes all have similar hybrid stability.

Claim 6

Dependent Claim 18 words

The method of claim 1 wherein the amplified fragments are covalently coupled to the detector probes by ligation.

Claim 7

Dependent Claim 42 words

The method of claim 1 further comprising, prior to step (a), dividing the sample into a plurality of index samples, wherein a different hairpin primer is mixed with each index sample, wherein steps (a) through (e) are performed with each index sample.

Claim 8

Dependent Claim 49 words

The method of claim 6 further comprising, prior to step (b), dividing each index sample into a set of two or more of secondary index samples, and mixing each secondary index sample in each set of secondary index samples with a different set of one or more second primers.

Claim 9

Dependent Claim 91 words

The method of claim 6 further comprising, prior to step (a), dividing each index sample into a set of two or more of secondary index samples, prior to, simultaneous with, or following step (a), mixing each secondary index sample in each set of secondary index samples with a different set of one or more second primers, wherein mixing each index sample with one or more different hairpin primers is accomplished by mixing the one or more different hairpin primers with each secondary index sample in a set of secondary index samples.

Claim 10

Dependent Claim 28 words

The method of claim 1 further comprising, following step (b), separating the strands of the amplified fragments and proceeding with step (c) using only one of the strands.

Claim 11

Dependent Claim 20 words

The method of claim 9 wherein the strands are separated using a capture tag incorporated into one of the strands.

Claim 12

Dependent Claim 15 words

The method of claim 10 wherein the capture tag is incorporated into the hairpin primers.

Claim 13

Dependent Claim 18 words

The method of claim 9 wherein the strands are separated by selective digestion of one of the strands.

Claim 14

Dependent Claim 26 words

The method of claim 12 wherein the linkages between a plurality of nucleotides at the 5′ end of each hairpin primer are insensitive to nuclease digestion.

Claim 15

Dependent Claim 24 words

The method of claim 13 wherein the linkages between a plurality of nucleotides at the 5′ end of each hairpin primer are phosphorothioate linkages.

Claim 16

Dependent Claim 19 words

The method of claim 1 wherein the concentration of the various nucleic acid fragments in the samples are normalized.

Claim 17

Dependent Claim 79 words

The method of claim 15 wherein the strands of the nucleic acid fragments are separated and the concentration of the nucleic acid fragments is normalized by immobilizing one strand of the nucleic acid fragments, denaturing the nucleic acid fragments, renaturing the nucleic acid fragments for a time greater than the c 0 t ½ for abundant nucleic acid fragments and less than the c 0 t ½ for rare nucleic acid fragments, and collecting the un-renatured nucleic acid fragments.

Claim 18

Dependent Claim 20 words

The method of claim 1 further comprising, prior to step (e), amplifying the amplified fragments coupled to the detector probes.

Claim 19

Dependent Claim 14 words

The method of claim 1 wherein each detector probe is immobilized on a substrate.

Claim 20

Dependent Claim 17 words

The method of claim 18 wherein all of the detector probes are immobilized on the same substrate.

Claim 21

Dependent Claim 17 words

The method of claim 18 wherein all of the detector probes are immobilized on a different substrate.

Claim 22

Dependent Claim 10 words

The method of claim 20 wherein the substrates are beads.

Claim 23

Dependent Claim 43 words

The method of claim 18 wherein the detector probes are immobilized on a plurality of different substrates such that at least one detector probe is immobilized on one substrate and at least one other detector probe, respectively, is immobilized on a different substrate.

Claim 24

Dependent Claim 13 words

The method of claim 18 wherein the detector probes are in an array.

Claim 25

Dependent Claim 19 words

The method of claim 1 wherein each detector probe is associated with a capture tag, sorting tag, or both.

Claim 26

Dependent Claim 15 words

The method of claim 24 wherein the detector probes are captured via the capture tags.

Claim 27

Dependent Claim 15 words

The method of claim 24 wherein the detector probes are sorted via the sorting tags.

Claim 28

Dependent Claim 25 words

The method of claim 24 wherein the detector probes are associated with a plurality of different capture tags or a plurality of different sorting tags.

Claim 29

Dependent Claim 51 words

The method of claim 1 wherein the detector probes are in an array, wherein each detector probe is immobilized at a different location in the array, and wherein detecting coupling of amplified fragments to detector probes is accomplished by detecting the presence of amplified fragments at different locations in the arrays.

Claim 30

Dependent Claim 60 words

The method of claim 28 wherein the location, amount, or location and amount of amplified fragments in the arrays constitutes a pattern of amplified fragments in the arrays, the method further comprising comparing the pattern of amplified fragments in the arrays with the pattern of amplified fragments in arrays determined in a separate procedure using a second nucleic acid sample.

Claim 31

Dependent Claim 39 words

The method of claim 29 further comprising comparing the pattern of amplified fragments in the arrays with the pattern of amplified fragments in arrays determined in a plurality of separate procedures using a plurality of different nucleic acid samples.

Claim 32

Dependent Claim 20 words

The method of claim 1 further comprising, following covalent coupling in step (d), incubating the samples with T4 endonuclease VII.

Claim 33

Dependent Claim 14 words

The method of claim 1 wherein the nucleic acid fragments are amplified by PCR.

Claim 34

Dependent Claim 26 words

The method of claim 1 wherein each hairpin primer contains a label, wherein coupling of the amplified fragments to the probes is detected via the label.

Claim 35

Dependent Claim 34 words

The method of claim 33 wherein the label is detectable by nuclear magnetic resonance, electron paramagnetic resonance, surface enhanced raman scattering, surface plasmon resonance, fluorescence, phosphorescence, chemiluminescence, resonance raman, microwave, or a combination thereof.

Claim 36

Dependent Claim 37 words

The method of claim 1 wherein the presence of the amplified fragments is detected by rolling circle replication of an amplification target circle wherein replication is primed by primer sequences at the end of the amplified fragments.

Claim 37

Dependent Claim 53 words

The method of claim 1 wherein the pattern of the amount of amplified fragments coupled to different detector probes constitutes a catalog of nucleic acid fragments in the nucleic acid sample, wherein the pattern is compared to a predicted pattern based on probabilities of base mismatches of sequences hybridized to the detector probes.

Claim 38

Dependent Claim 41 words

The method of claim 1 wherein detecting coupling of the amplified fragments to the detector probes is accomplished by detecting mass labels associated with the coupled fragments, mass labels associated with the detector probes, or a combination thereof, by mass spectroscopy.

Claim 39

Dependent Claim 31 words

The method of claim 37 wherein the mass labels associated with the coupled fragments and mass labels associated with the detector probes are detected by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy.

Claim 40

Dependent Claim 32 words

The method of claim 37 wherein the composition of the mass labels associated with the coupled fragments and mass labels associated with the detector probes are determined by analyzing the fragmentation pattern.

Claim 41

Dependent Claim 22 words

The method of claim 37 wherein uncoupled fragments are washed away from the detector probes prior to detection of the coupled fragments.

Claim 42

Dependent Claim 53 words

The method of claim 37 wherein the hairpin primers, the detector probes, or both, contain a photocleavable nucleotide, wherein the method further comprises, following coupling of the amplified fragments to the detector probes, photocleavage of the photocleavable nucleotides, and detection of one or both strands of the coupled amplified fragment by mass spectroscopy.

Claim 43

Dependent Claim 47 words

The method of claim 37 further comprising, following coupling of the amplified fragments to the detector probes, incubation of the couple fragments and detector probes with one or more nucleic acid cleaving reagents, and detection of one or both strands of the coupled fragment by mass spectroscopy.

Claim 44

Dependent Claim 109 words

The method of claim 1 further comprising performing steps (a) through (c) on one or more control nucleic acid samples, wherein the hairpin primers used with the control nucleic acid samples contain a different label from the label of the hairpin primers used with the nucleic acid samples, mixing the control nucleic acid samples with corresponding nucleic acid samples and proceeding with step (d) by hybridizing the mixed samples with the detector probes, detecting coupling of both types of amplified fragments to different detector probes, and identifying differences in the pattern of coupling of amplified fragments to probes from the nucleic acid samples and the control nucleic acid samples.

Claim 45

Dependent Claim 124 words

The method of claim 1 further comprising performing steps (a) through (c) on one or more control nucleic acid samples, wherein the hairpin primers used with the nucleic acid samples contain a labile nucleotide in the loop, mixing the control nucleic acid samples with corresponding nucleic acid samples and proceeding with step (d) by hybridizing the mixed samples with the detector probes, detecting coupling of amplified fragments from both types of samples to different detector probes, treating the mixed nucleic acid samples to cleave the labile nucleotide, detecting coupling of amplified fragments from nucleic acid samples to different detector probes, and identifying differences in the pattern of coupling of amplified fragments to probes from the nucleic acid samples and the control nucleic acid samples.

Claim 46

Dependent Claim 81 words

The method of claim 1 wherein the detector probes are in an array, wherein each probe is immobilized at a different location in the array, wherein the location of amplified fragments in the array constitutes a pattern of coupling of amplified fragments to probes in the array, the method further comprising comparing the pattern of coupling of amplified fragments in the arrays with the pattern of amplified fragments in arrays determined in a separate procedure using a second nucleic acid sample.

Claim 47

Dependent Claim 37 words

The method of claim 1 wherein the pattern of the presence, amount, presence and amount, or absence of amplified fragments coupled to different detector probes constitutes a catalog of nucleic acid fragments in the nucleic acid sample.

Claim 48

Dependent Claim 29 words

The method of claim 46 further comprising preparing a second catalog of nucleic acid fragments in a second nucleic acid sample and comparing the first catalog and second catalog.

Claim 49

Dependent Claim 42 words

The method of claim 47 further comprising identifying or preparing nucleic acid fragments corresponding the nucleic acid fragments present at a threshold amount in the first nucleic acid sample but not present at the threshold amount in the second nucleic acid sample.

Claim 50

Dependent Claim 26 words

The method of claim 47 wherein the second nucleic acid sample is a sample from the same type of organism as the first nucleic acid sample.

Claim 51

Dependent Claim 26 words

The method of claim 47 wherein the second nucleic acid sample is a sample from the same type of tissue as the first nucleic acid sample.

Claim 52

Dependent Claim 24 words

The method of claim 47 wherein the second nucleic acid sample is a sample from the same organism as the first nucleic acid sample.

Claim 53

Dependent Claim 23 words

The method of claim 51 wherein the second nucleic acid sample is obtained at a different time than the first nucleic acid sample.

Claim 54

Dependent Claim 24 words

The method of claim 47 wherein the second nucleic acid sample is a sample from a different organism than the first nucleic acid sample.

Claim 55

Dependent Claim 26 words

The method of claim 47 wherein the second nucleic acid sample is a sample from a different type of tissue than the first nucleic acid sample.

Claim 56

Dependent Claim 26 words

The method of claim 47 wherein the second nucleic acid sample is a sample from a different species of organism than the first nucleic acid sample.

Claim 57

Dependent Claim 26 words

The method of claim 47 wherein the second nucleic acid sample is a sample from a different strain of organism than the first nucleic acid sample.

Claim 58

Dependent Claim 25 words

The method of claim 47 wherein the second nucleic acid sample is a sample from a different cellular compartment than the first nucleic acid sample.

Claim 59

Dependent Claim 34 words

The method of claim 47 further comprising identifying or preparing nucleic acid fragments corresponding the nucleic acid fragments present in the first nucleic acid sample but not present in the second nucleic acid sample.

Claim 60

Dependent Claim 14 words

The method of claim 58 further comprising using the nucleic acid fragments as probes.

Claim 61

Dependent Claim 42 words

The method of claim 59 wherein using the nucleic acid fragments as probes is accomplished by repeating steps (a) through (d) with a different nucleic acid sample, wherein the nucleic acid fragments are used as detector probes in steps (d) and (e).

Claim 62

Dependent Claim 21 words

The method of claim 1 further comprising determining the sequence of a portion of at least one of the amplified fragments.

Claim 63

Dependent Claim 43 words

The method of claim 61 wherein the portion of the amplified fragment corresponds to the sequence complementary to the primer sequence of the hairpin primer and the sequence adjacent to the sequence complementary to the primer sequence to which the detector probe hybridized.

Claim 64

Dependent Claim 42 words

The method of claim 62 further comprising detecting or amplifying a nucleic acid corresponding to a nucleic acid fragment in the nucleic acid sample using a probe or primer based on the determined sequence of the portion of the nucleic acid fragment.

Claim 65

Dependent Claim 30 words

The method of claim 1 wherein each hairpin primer or detector probe contains a label, wherein coupling of the amplified fragments to the detector probes is detected via the label.

Claim 66

Dependent Claim 40 words

The method of claim 64 wherein each hairpin primer contains a label, wherein detecting coupling of the amplified fragments to the detector probes is accomplished by separating coupled fragments from uncoupled fragments, and detecting the labels of the coupled fragments.

Claim 67

Dependent Claim 62 words

The method of claim 65 wherein each different hairpin primer contains a different label, wherein each detector probe is associated with a capture tag or a sorting tag, wherein separating coupled fragments from uncoupled fragments is accomplished by separating the detector probes from the uncoupled fragments using the capture tags or sorting tags, wherein the coupled fragments separate with the detector probes.

Claim 68

Dependent Claim 30 words

The method of claim 66 wherein the sorting tag is a fluorescent label, and wherein separating the detector probes from the uncoupled fragments is accomplished using a fluorescent label sorter.

Claim 69

Dependent Claim 42 words

The method of claim 64 wherein each detector probe contains a label, wherein detecting coupling of the amplified fragments to the detector probes is accomplished by separating coupled detector probes from uncoupled detector probes, and detecting the labels of the detector probes.

Claim 70

Dependent Claim 66 words

The method of claim 68 wherein each different detector probe contains a different label, wherein each amplified fragment is associated with a capture tag or a sorting tag, wherein separating coupled detector probes from uncoupled detector probes is accomplished by separating the amplified fragments from the uncoupled detector probes using the capture tags or sorting tags, wherein the coupled detector probes separate with the amplified fragments.

Claim 71

Dependent Claim 31 words

The method of claim 69 wherein the sorting tag is a fluorescent label, and wherein separating the amplified fragments from the uncoupled detector probes is accomplished using a fluorescent label sorter.

Claim 72

Dependent Claim 14 words

The method of claim 64 wherein the labels are fluorescent, phosphorescent, or chemiluminescent labels.

Claim 73

Dependent Claim 23 words

The method of claim 71 wherein at least two of the labels are distinguished temporally via different fluorescent, phosphorescent, or chemiluminescent emission lifetimes.

Claim 74

Dependent Claim 34 words

The method of claim 64 wherein the labels are detectable by nuclear magnetic resonance, electron paramagnetic resonance, surface enhanced raman scattering, surface plasmon resonance, fluorescence, phosphorescence, chemiluminescence, resonance raman, microwave, or a combination thereof.

Claim 75

Dependent Claim 34 words

The method of claim 73 wherein the label is detected using nuclear magnetic resonance, electron paramagnetic resonance, surface enhanced raman scattering, surface plasmon resonance, fluorescence, phosphorescence, chemiluminescence, resonance raman, microwave, or a combination thereof.

Claim 76

Dependent Claim 13 words

The method of claim 64 wherein the labels are beads comprising a label.

Claim 77

Dependent Claim 12 words

The method of claim 75 wherein the label is a molecular barcode.

Claim 78

Dependent Claim 11 words

The method of claim 64 wherein the labels are mass labels.

Claim 79

Dependent Claim 42 words

The method of claim 1 further comprising performing steps (a) through (e) on a control nucleic acid sample, identifying differences between the nucleic acid sample and the control nucleic acid sample in the pattern of amplified fragments coupled to different detector probes.

Claim 80

Dependent Claim 50 words

The method of claim 78 wherein the hairpin primers used with the control nucleic acid sample contain a different label from the label of the hairpin primers used with the nucleic acid sample, wherein the control nucleic acid samples are mixed with corresponding nucleic acid samples prior to step (d).

Claim 81

Dependent Claim 19 words

The method of claim 1 further comprising performing steps (a) through (e) on a plurality of nucleic acid samples.

Claim 82

Dependent Claim 42 words

The method of claim 80 further comprising performing steps (a) through (e) on a control nucleic acid sample, identifying differences between the nucleic acid samples and the control nucleic acid sample in the pattern of amplified fragments coupled to different detector probes.

Claim 83

Dependent Claim 25 words

The method of claim 80 further comprising identifying differences between the nucleic acid samples in the pattern of amplified fragments coupled to different detector probes.

Claim 84

Independent Claim 165 words

A method of identifying a nucleic acid sequence in a nucleic acid sample, the method comprising (a) mixing a nucleic acid sample with a hairpin primer and a second primer, wherein the hairpin primer and the second primer comprise primer sequences complementary to sequences flanking and on opposite strands of a nucleic acid sequence of interest, (b) incubating the nucleic acid sample under conditions that promote amplification of the nucleic acid sequence of interest, wherein a nucleic acid fragment is formed which comprises the nucleic acid sequence of interest flanked by sequences of the hairpin primer and the second primer, (c) incubating the nucleic acid sample under conditions that promote formation of a hairpin structure by the sequence of the hairpin primer, (d) hybridizing the nucleic acid sample with a plurality of detector probes and coupling the hairpin structure to a probe, wherein each probe has a different sequence, and (e) detecting, directly or indirectly, coupling of the nucleic acid fragment to a detector probe.

Claim 85

Independent Claim 165 words

A method of identifying a nucleic acid sequence in a nucleic acid sample, the method comprising (a) mixing a nucleic acid sample with a hairpin primer and a second primer, wherein the hairpin primer and the second primer comprise primer sequences complementary to sequences flanking and on opposite strands of a nucleic acid sequence of interest, (b) incubating the nucleic acid sample under conditions that promote amplification of the nucleic acid sequence of interest, wherein a nucleic acid fragment is formed which comprises the nucleic acid sequence of interest flanked by sequences of the hairpin primer and the second primer, (c) incubating the nucleic acid sample under conditions that promote formation of a hairpin structure by the sequence of the hairpin primer, (d) hybridizing the nucleic acid sample with a plurality of detector probes and coupling the hairpin structure to a probe, wherein each probe has a different sequence, and (e) detecting, directly or indirectly, coupling of the nucleic acid fragment to a detector probe.

Claim 86

Independent Claim 191 words

A method of identifying nucleic acid fragments in a nucleic acid sample, the method comprising (a) dividing the sample into a plurality of index samples, wherein the index samples are organized into sets of index samples wherein each set comprises a plurality of index samples, (b) mixing each index sample in a set of index samples with one or more different hairpin primers, wherein each hairpin primer comprises a different primer sequence, and mixing each index sample with one or more different second primers, (c) incubating the index samples under conditions that promote amplification of nucleic acids in the samples, wherein amplified nucleic acid fragments are formed which are flanked by hairpin primer sequences and second primer sequences, (d) incubating the index samples under conditions that promote formation of hairpin structures by the hairpin primer sequences at the ends of the amplified fragments, (e) hybridizing each index sample with a plurality of detector probes and coupling the hairpin structures to the probes, wherein in a given set of detector probes each probe has a different sequence, and (f) detecting, directly or indirectly, coupling of the amplified fragments to different detector probes.

Claim 87

Independent Claim 191 words

A method of identifying nucleic acid fragments in a nucleic acid sample, the method comprising (a) dividing the sample into a plurality of index samples, wherein the index samples are organized into sets of index samples wherein each set comprises a plurality of index samples, (b) mixing each index sample in a set of index samples with one or more different hairpin primers, wherein each hairpin primer comprises a different primer sequence, and mixing each index sample with one or more different second primers, (c) incubating the index samples under conditions that promote amplification of nucleic acids in the samples, wherein amplified nucleic acid fragments are formed which are flanked by hairpin primer sequences and second primer sequences, (d) incubating the index samples under conditions that promote formation of hairpin structures by the hairpin primer sequences at the ends of the amplified fragments, (e) hybridizing each index sample with a plurality of detector probes and coupling the hairpin structures to the probes, wherein in a given set of detector probes each probe has a different sequence, and (f) detecting, directly or indirectly, coupling of the amplified fragments to different detector probes.

Claim 88

Independent Claim 229 words

A method of comparing nucleic acid samples, the method comprising (a) comparing a catalog of nucleic acid fragments in a first nucleic acid sample with a catalog of nucleic acid fragments in a second nucleic acid sample, and (b) identifying or preparing nucleic acid fragments corresponding to the nucleic acid fragments present in the first nucleic acid sample but not present in the second nucleic acid sample; wherein the catalogs of nucleic acid fragments are each produced by (i) mixing the nucleic acid sample with one or more different hairpin primers, wherein each hairpin primer comprises a different primer sequence, (ii) incubating the sample under conditions that promote amplification of nucleic acids in the sample, wherein amplified nucleic acid fragments are formed which have hairpin primer sequences at one or both ends, (iii) incubating the sample under conditions that promote formation of hairpin structures by the hairpin primer sequences at the ends of the amplified fragments, (iv) hybridizing the sample with a plurality of detector probes and covalently coupling the hairpin structures to the probes, wherein each probe has a different sequence, and (v) detecting, directly or indirectly, coupling of the amplified fragments to the detector probes, wherein the pattern of the presence, amount, presence and amount, or absence of amplified fragments coupled to different detector probes constitutes the catalog of nucleic acid fragments in the nucleic acid sample.

Claim 89

Independent Claim 229 words

A method of comparing nucleic acid samples, the method comprising (a) comparing a catalog of nucleic acid fragments in a first nucleic acid sample with a catalog of nucleic acid fragments in a second nucleic acid sample, and (b) identifying or preparing nucleic acid fragments corresponding to the nucleic acid fragments present in the first nucleic acid sample but not present in the second nucleic acid sample; wherein the catalogs of nucleic acid fragments are each produced by (i) mixing the nucleic acid sample with one or more different hairpin primers, wherein each hairpin primer comprises a different primer sequence, (ii) incubating the sample under conditions that promote amplification of nucleic acids in the sample, wherein amplified nucleic acid fragments are formed which have hairpin primer sequences at one or both ends, (iii) incubating the sample under conditions that promote formation of hairpin structures by the hairpin primer sequences at the ends of the amplified fragments, (iv) hybridizing the sample with a plurality of detector probes and covalently coupling the hairpin structures to the probes, wherein each probe has a different sequence, and (v) detecting, directly or indirectly, coupling of the amplified fragments to the detector probes, wherein the pattern of the presence, amount, presence and amount, or absence of amplified fragments coupled to different detector probes constitutes the catalog of nucleic acid fragments in the nucleic acid sample.

Claim 90

Dependent Claim 53 words

The method of claim 85 wherein nucleic acid fragments present in the first nucleic acid sample but not present in the second nucleic acid sample are nucleic acid fragments present at a threshold amount in the first nucleic acid sample but not present at the threshold amount in the second nucleic acid sample.

Claim 91

Dependent Claim 36 words

The method of claim 85 wherein the pattern of the presence, amount, presence and amount, or absence of amplified fragments coupled to different detector probes is embodied by the sequences represented by the coupled amplified fragments.

Claim 92

Dependent Claim 38 words

The method of claim 87 wherein each represented sequence corresponds to sequence complementary to the primer sequence of the hairpin primer and sequence adjacent to the sequence complementary to the primer sequence to which the detector probe hybridized.

Claim 93

Dependent Claim 14 words

The method of claim 85 wherein the probes are all of the same length.

Claim 94

Dependent Claim 13 words

The method of claim 85 wherein the probes all have similar hybrid stability.

Claim 95

Independent Claim 251 words

A method of identifying nucleic acid fragments in a nucleic acid sample, the method comprising (a) incubating a nucleic acid sample with one or more nucleic acid cleaving reagents that collectively generate sticky ends having a plurality of different sequences to produce nucleic acid fragments with sticky ends, (b) dividing the sample into a plurality of index samples, (c) mixing a different adaptor-indexer with each index sample and covalently coupling the adaptor-indexers to the nucleic acid fragments, wherein each adaptor-indexer has a different sticky end, wherein each sticky end of the adaptor-indexes is compatible with a sticky end generated by the nucleic acid cleaving reagents, wherein each index sample has a different adaptor-indexer, (d) mixing each index sample with one or more different hairpin primers, wherein each hairpin primer comprises a different primer sequence, wherein each primer sequence is complementary to all or part of the sequence of at least one of the adaptor-indexers, (e) incubating the index samples under conditions that promote amplification of nucleic acids in the samples, wherein amplified nucleic acid fragments are formed which have hairpin primer sequences at one or both ends, (f) incubating the index samples under conditions that promote formation of hairpin structures by the hairpin primer sequences at the ends of the amplified fragments, (g) hybridizing each index sample with a plurality of detector probes and covalently coupling the hairpin structures to the probes, wherein each probe has a different sequence, and (h) detecting coupling of the amplified fragments to different detector probes.

Claim 96

Independent Claim 251 words

A method of identifying nucleic acid fragments in a nucleic acid sample, the method comprising (a) incubating a nucleic acid sample with one or more nucleic acid cleaving reagents that collectively generate sticky ends having a plurality of different sequences to produce nucleic acid fragments with sticky ends, (b) dividing the sample into a plurality of index samples, (c) mixing a different adaptor-indexer with each index sample and covalently coupling the adaptor-indexers to the nucleic acid fragments, wherein each adaptor-indexer has a different sticky end, wherein each sticky end of the adaptor-indexes is compatible with a sticky end generated by the nucleic acid cleaving reagents, wherein each index sample has a different adaptor-indexer, (d) mixing each index sample with one or more different hairpin primers, wherein each hairpin primer comprises a different primer sequence, wherein each primer sequence is complementary to all or part of the sequence of at least one of the adaptor-indexers, (e) incubating the index samples under conditions that promote amplification of nucleic acids in the samples, wherein amplified nucleic acid fragments are formed which have hairpin primer sequences at one or both ends, (f) incubating the index samples under conditions that promote formation of hairpin structures by the hairpin primer sequences at the ends of the amplified fragments, (g) hybridizing each index sample with a plurality of detector probes and covalently coupling the hairpin structures to the probes, wherein each probe has a different sequence, and (h) detecting coupling of the amplified fragments to different detector probes.

Claim 97

Dependent Claim 21 words

The method of claim 91 further comprising determining the sequence of a portion of at least one of the amplified fragments.

Claim 98

Dependent Claim 43 words

The method of claim 92 wherein the portion of the nucleic acid fragments corresponds to the sticky end sequence, the sequence adjacent to the sticky end sequence to which the detector probe hybridized, and the recognition sequence of the nucleic acid cleaving reagent.

Claim 99

Dependent Claim 35 words

The method of claim 93 wherein the portion includes a gap of known length but unknown sequence between the sequence adjacent to the sticky end and the recognition sequence of the nucleic acid cleaving reagent.

Claim 100

Dependent Claim 48 words

The method of claim 94 wherein the portion has the structure A-B-C-D wherein A is the recognition sequence of the nucleic acid cleaving reagent, B is the gap of unknown sequence, C is the sequence to which the detector probe hybridized, and D is the sticky end sequence.

Claim 101

Dependent Claim 49 words

The method of claim 91 wherein, for at least one of the hairpin primer sequences, the sticky end sequence is involved in the stem of the hairpin structure but none of the fragment sequence adjacent to the sticky end sequence is involved in the stem of the hairpin structure.

Claim 102

Dependent Claim 14 words

The method of claim 91 wherein the probes are all of the same length.

Claim 103

Dependent Claim 13 words

The method of claim 91 wherein the probes all have similar hybrid stability.

Claim 104

Dependent Claim 66 words

The method of claim 91 further comprising, prior to step (c), incubating the index samples with one or more second nucleic acid cleaving reagents, and mixing a second adaptor with each digested index sample and covalently coupling the second adaptor to the nucleic acid fragments, wherein the second adaptor has an end compatible with the end generated by one of the second nucleic acid cleaving reagents.

Claim 105

Dependent Claim 59 words

The method of claim 99 further comprising, prior to digestion with the second nucleic acid cleaving reagents, dividing each index sample into a set of two or more of secondary index samples, wherein each secondary index sample in each set of secondary index samples is digested with a different set of one or more second nucleic acid cleaving reagents.

Claim 106

Independent Claim 53 words

The method of 99 further comprising, simultaneous with step (d) mixing each secondary index sample in each set of secondary index samples with a different set of one or more second primers, wherein each secondary primer is complementary to all or part of the sequence of at least one of the second adaptors.

Claim 107

Independent Claim 53 words

The method of 99 further comprising, simultaneous with step (d) mixing each secondary index sample in each set of secondary index samples with a different set of one or more second primers, wherein each secondary primer is complementary to all or part of the sequence of at least one of the second adaptors.

Claim 108

Dependent Claim 28 words

The method of claim 91 further comprising, following step (e), separating the strands of the amplified fragments and proceeding with step (d) using only one of the strands.

Claim 109

Dependent Claim 21 words

The method of claim 91 further comprising, following covalent coupling in step (e), incubating the index samples with T4 endonuclease VII.

Claim 110

Dependent Claim 24 words

The method of claim 91 wherein each hairpin primer contains a label, wherein the presence of the amplified fragments is detected via the label.

Claim 111

Dependent Claim 98 words

The method of claim 91 further comprising performing steps (a) through (f) on a control nucleic acid sample to produce control index samples, wherein the hairpin primers used with the control nucleic acid sample contain a different label from the label of the hairpin primers used with the nucleic acid sample, mixing the control index samples with corresponding index samples and proceeding with step (g) by hybridizing the mixed samples with the detector probes, identifying differences between the nucleic acid sample and the control nucleic acid sample in the pattern of amplified fragments coupled to different detector probes.

Claim 112

Dependent Claim 234 words

A kit comprising a set of hairpin primers wherein each hairpin primer has a different primer sequence, and a plurality of detector probes, wherein each probe has a different sequence, wherein the detector probes are nucleic acid fragments prepared by (a) mixing one or more nucleic acid samples with one or more different hairpin primers, wherein each hairpin primer comprises a different primer sequence, (b) incubating the samples under conditions that promote amplification of nucleic acids in the samples, wherein amplified nucleic acid fragments are formed which have hairpin primer sequences at one or both ends, (c) incubating the samples under conditions that promote formation of hairpin structures by the hairpin primer sequences at the ends of the amplified fragments, (d) hybridizing each sample with a plurality of detector probes and covalently coupling the hairpin structures to the probes, wherein each probe has a different sequence, and (e) detecting, directly or indirectly, coupling of the amplified fragments to the detector probes, wherein the pattern of amplified fragments coupled to different detector probes constitutes a catalog of nucleic acid fragments in the nucleic acid sample, (f) preparing a second catalog of nucleic acid fragments in a second nucleic acid sample and comparing the first catalog and second catalog, and (g) preparing nucleic acid fragments corresponding the nucleic acid fragments present in the first nucleic acid sample but not present in the second nucleic acid sample.

Claim 113

Dependent Claim 234 words

A kit comprising a set of hairpin primers wherein each hairpin primer has a different primer sequence, and a plurality of detector probes, wherein each probe has a different sequence, wherein the detector probes are nucleic acid fragments prepared by (a) mixing one or more nucleic acid samples with one or more different hairpin primers, wherein each hairpin primer comprises a different primer sequence, (b) incubating the samples under conditions that promote amplification of nucleic acids in the samples, wherein amplified nucleic acid fragments are formed which have hairpin primer sequences at one or both ends, (c) incubating the samples under conditions that promote formation of hairpin structures by the hairpin primer sequences at the ends of the amplified fragments, (d) hybridizing each sample with a plurality of detector probes and covalently coupling the hairpin structures to the probes, wherein each probe has a different sequence, and (e) detecting, directly or indirectly, coupling of the amplified fragments to the detector probes, wherein the pattern of amplified fragments coupled to different detector probes constitutes a catalog of nucleic acid fragments in the nucleic acid sample, (f) preparing a second catalog of nucleic acid fragments in a second nucleic acid sample and comparing the first catalog and second catalog, and (g) preparing nucleic acid fragments corresponding the nucleic acid fragments present in the first nucleic acid sample but not present in the second nucleic acid sample.

Annuity Stage	Window Opens	Payment Deadline	Surcharge Period Until	Estimated Fee	Status
3.5 Years (Year 4)	2005-06-10	2005-12-10	2006-06-11	$2000.00	Window Closed / Lapsed
7.5 Years (Year 8)	2009-06-10	2009-12-10	2010-06-11	$3760.00	Window Closed / Lapsed
11.5 Years (Year 12)	2013-06-10	2013-12-10	2014-06-11	$7700.00	Window Closed / Lapsed

Analysis of sequence tags with hairpin primers

Abstract

Inventors

Bibliographic Details

Claims Analysis

References Cited (Backward Citations)

USPTO Annuity & Maintenance Windows

Assigned Categories & Tags

Assign Category Tag