US2006205006A1PendingUtilityA1
PCR method and related apparatus
Individually held — no corporate assignee on recordPriority: Mar 2, 2001Filed: May 12, 2006Published: Sep 14, 2006
Est. expiryMar 2, 2021(expired)· nominal 20-yr term from priority
C12Q 1/6851C12Q 1/6886C12Q 2600/158C12Q 2600/16C12Q 2600/166
53
PatentIndex Score
0
Cited by
0
References
0
Claims
Abstract
A method for balancing multiplexed PCR methods is provided. In the method, two or more sequential temporal PCR stages are used to effectively separate two or more PCR reactions in a single tube as an alternative to primer limiting to modulate the relative rate of production of a first amplicon by a first primer set and a second amplicon by a second primer set during the first and second amplification stages. Also provided are rapid RT-PCR methods that find particular use in intraoperative diagnoses and prognoses, for instance in diagnosing malignant esophageal adenocarcenoma by determining expression levels of carcinoembryonic antigen (CEA) in sentinel lymph nodes.
Claims
exact text as granted — not AI-modified1 - 66 . (canceled)
67 . An oligonucleotide consisting of one of SEQ ID NOS: 6, 7, 13, 14, 16, 17, 19, 20 and 23-35, and a derivative thereof.
68 - 77 . (canceled)
78 . The oligonucleotide of claim 67 , wherein the derivative is capable of hybridizing in a PCR reaction to the same target sequence as the oligonucleotides of SEQ ID NOS: 6, 7, 13, 14, 16, 17, 19, 20 and 23-35.
79 . The oligonucleotide of claim 78 , consisting of one of SEQ ID NOS: 6, 7, 13, 14, 16, 17, 19, 20 and 23-35.
80 . The oligonucleotide of claim 78 , consisting of SEQ ID NO: 6.
81 . The oligonucleotide of claim 78 , consisting of SEQ ID NO: 7.
82 . The oligonucleotide of claim 78 , consisting of SEQ ID NO: 13.
83 . The oligonucleotide of claim 78 , consisting of SEQ ID NO: 14.
84 . The oligonucleotide of claim 78 , consisting of SEQ ID NO: 16.
85 . The oligonucleotide of claim 78 , consisting of SEQ ID NO: 17.
86 . The oligonucleotide of claim 78 , consisting of SEQ ID NO: 19.
87 . The oligonucleotide of claim 78 , consisting of SEQ ID NO: 20.
88 . The oligonucleotide of claim 78 , consisting of SEQ ID NO: 23.
89 . The oligonucleotide of claim 78 , consisting of SEQ ID NO: 24.
90 . The oligonucleotide of claim 78 , consisting of SEQ ID NO: 25.
91 . The oligonucleotide of claim 78 , consisting of SEQ ID NO: 26.
92 . The oligonucleotide of claim 78 , consisting of SEQ ID NO: 27.
93 . The oligonucleotide of claim 78 , consisting of SEQ ID NO: 28.
94 . The oligonucleotide of claim 78 , consisting of SEQ ID NO: 29.
95 . The oligonucleotide of claim 78 , consisting of SEQ ID NO: 30.
96 . The oligonucleotide of claim 78 , consisting of SEQ ID NO: 31.
97 . The oligonucleotide of claim 78 , consisting of SEQ ID NO: 32.
98 . The oligonucleotide of claim 78 , consisting of SEQ ID NO: 33.
99 . The oligonucleotide of claim 78 , consisting of SEQ ID NO: 34.
100 . The oligonucleotide of claim 78 , consisting of SEQ ID NO: 35.
101 . A cartridge for use in a PCR system, the cartridge comprising one or more compartments containing a first PCR primer set and a second PCR primer set, wherein the first PCR primer set has a first effective Tm and the second PCR primer set has a second effective Tm that is different from the first effective Tm.
102 . The cartridge of claim 101 , further comprising a third compartment including one of reverse transcription reagents, cell lysis reagents and RNA purification reagents.
103 . The cartridge of claim 102 , comprising reverse transcriptase and a reverse transcriptase primer.
104 . The cartridge of claim 103 , comprising random hexanucleotide reverse transcriptase primers.
105 . The cartridge of claim 103 , comprising sequence-specific reverse transcriptase primers.
106 . The cartridge of claim 105 , wherein the sequence-specific reverse transcriptase primers are specific to one or more of β-glucuronidase mRNA, carcinoembryonic antigen mRNA, tyrosinase mRNA and 18S ribosomal RNA.
107 . The cartridge of claim 101 , comprising a PCR primer set for producing one or more of β-glucuronidase, carcinoembryonic antigen, tyrosinase mRNA and 18S ribosomal RNA-specific amplicon.
108 . The cartridge of claim 101 , further comprising an internal positive control nucleic acid.
109 . The cartridge of claim 108 , wherein the internal positive control nucleic acid comprises one or more uracil residues.
110 . The cartridge of claim 101 , further comprising a fluorescent reporter for indicating accumulation of a specific amplicon in a PCR reaction.
111 . The cartridge of claim 110 , wherein the fluorescent reporter is a probe for a fluorescent 5′ nuclease assay.
112 . The cartridge of claim 110 , wherein the fluorescent reporter is a molecular beacon probe.
113 . The cartridge of claim 101 , wherein the cartridge is disposable after a single use.
114 . The cartridge of claim 101 , comprising an oligonucleotide consisting of one of SEQ ID NOS: 6, 7, 13, 14, 16, 17, 19, 20 and 23-35 or a derivative thereof capable of hybridizing in a PCR reaction to the same target sequence as the oligonucleotides of SEQ ID NOS: 6, 7, 13, 14, 16, 17, 19, 20 and 23-35.
115 . The cartridge of claim 114 , comprising one or more oligonucleotides consisting of one of SEQ ID NOS: 6, 7, 13, 14, 16, 17, 19, 20 and 23-35.
116 . The cartridge of claim 101 , wherein the effective Tm of the first primer set and the effective Tm of the second primer set differ by at least 5° C.
117 . A kit comprising a first PCR primer set and a second PCR primer set, wherein the first PCR primer set has a first effective Tm and the second PCR primer set has a second effective Tm that is different from the first effective Tm.
118 . The kit of claim 117 , comprising a cartridge for use in a PCR system, the cartridge comprising one or more compartments containing the first PCR primer set and the second PCR primer set.
119 . The kit of claim 117 , further comprising one of reverse transcription reagents, cell lysis reagents and RNA purification reagents.
120 . The kit of claim 119 , comprising reverse transcriptase and a reverse transcriptase primer.
121 . The kit of claim 120 , comprising random hexanucleotide reverse transcriptase primers.
122 . The kit of claim 120 , comprising sequence-specific reverse transcriptase primers.
123 . The kit of claim 122 , wherein the sequence-specific reverse transcriptase primers are specific to one or more of β-glucuronidase mRNA, carcinoembryonic antigen mRNA, tyrosinase mRNA and 18S ribosomal RNA.
124 . The kit of claim 117 , comprising a PCR primer set for producing one or more of β-glucuronidase, carcinoembryonic antigen, tyrosinase mRNA and 18S ribosomal RNA-specific amplicon.
125 . The kit of claim 117 , further comprising an internal positive control nucleic acid.
126 . The kit of claim 125 , wherein the internal positive control nucleic acid comprises one or more uracil residues.
127 . The kit of claim 117 , further comprising a fluorescent reporter for indicating accumulation of a specific amplicon in a PCR reaction.
128 . The kit of claim 127 , wherein the fluorescent reporter is a probe for a fluorescent 5′ nuclease assay.
129 . The kit of claim 127 , wherein the fluorescent reporter is a molecular beacon probe.
130 . The kit of claim 117 , comprising an oligonucleotide consisting of one of SEQ ID NOS: 6, 7, 13, 14, 16, 17, 19, 20 and 23-35 or a derivative thereof capable of hybridizing in a PCR reaction to the same target sequence as the oligonucleotides of SEQ ID NOS: 6, 7, 13, 14, 16, 17, 19, 20 and 23-35.
131 . The kit of claim 130 , comprising one or more oligonucleotides consisting of one of SEQ ID NOS: 6, 7, 13, 14, 16, 17, 19, 20 and 23-35.
132 . The kit of claim 117 , wherein the effective Tm of the first primer set and the effective Tm of the second primer set differ by at least 5° C.Join the waitlist — get patent alerts
Track US2006205006A1 — get alerts on status changes and closely related new filings.
We store only your email — no account needed. See our privacy policy.