Patent US6013442A successfully refreshed from Google Patents.
US6013442A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Direct quantitation of low copy number RNA
Assignee: Wisconsin Alumni Research Foundation
•
Filing: 1997-08-05
•
Granted: 2000-01-11
•
Expires: 2017-08-05
Abstract
Abstract A direct quantitation of RNA contained in a sample is obtained by capillary electrophoresis of the RNA hybridized to a DNA probe of complementary sequence stabilized by the combination of a fluorophore terminally conjugated to the DNA probe and a dye intercalating the RNA-DNA hybrid so formed. The RNA is quantified by measuring the total fluorescence emitted by the electrophoresed hybrid upon excitation by a laser generated light beam.
Inventors
- 👤 Jill M. Kolesar
- 👤 Peter G. Allen
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-31 04:57
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816; C12Q1/6825
Claims Analysis
Total Claims: 11
Claim 1
Independent Claim
106 words
A method for direct quantitation of an RNA sequence present in a sample in low copy number comprising extracting the RNA from a biological specimen hybridizing said RNA to a fluorescent DNA probe having a complementary sequence to said RNA to form a RNA-DNA hybrid applying said RNA-DNA hybrid to a capillary electrophoresis column electrophoresing said hybrid in a band under an electric current applied to said capillary column in the presence of a dye capable of intercalating double stranded nucleic acids, and quantitatively detecting said RNA-DNA hybrid by measuring the total fluorescent intensity of light emitted from said hybrid band upon excitation by laser-induced fluorescence.
Claim 2
Independent Claim
106 words
A method for direct quantitation of an RNA sequence present in a sample in low copy number comprising extracting the RNA from a biological specimen hybridizing said RNA to a fluorescent DNA probe having a complementary sequence to said RNA to form a RNA-DNA hybrid applying said RNA-DNA hybrid to a capillary electrophoresis column electrophoresing said hybrid in a band under an electric current applied to said capillary column in the presence of a dye capable of intercalating double stranded nucleic acids, and quantitatively detecting said RNA-DNA hybrid by measuring the total fluorescent intensity of light emitted from said hybrid band upon excitation by laser-induced fluorescence.
Claim 3
Independent Claim
107 words
A method for direct quantitation of an RNA sequence present in a sample comprising extracting the RNA from a biological specimen hybridizing said RNA to a fluorescent DNA probe having a complementary sequence to said RNA to form an RNA-DNA hybrid loading said RNA-DNA hybrid onto a capillary electrophoresis column having a matrix containing a dye capable of intercalating double stranded nucleic acids applying an electric current through the matrix of said column to effect electrophoresis of said RNA-DNA hybrid in a hybrid band, and quantitatively detecting said RNA-DNA hybrid by measuring the total fluorescence of light emitted from said hybrid band upon excitation by laser-induced fluorescence.
Claim 4
Independent Claim
107 words
A method for direct quantitation of an RNA sequence present in a sample comprising extracting the RNA from a biological specimen hybridizing said RNA to a fluorescent DNA probe having a complementary sequence to said RNA to form an RNA-DNA hybrid loading said RNA-DNA hybrid onto a capillary electrophoresis column having a matrix containing a dye capable of intercalating double stranded nucleic acids applying an electric current through the matrix of said column to effect electrophoresis of said RNA-DNA hybrid in a hybrid band, and quantitatively detecting said RNA-DNA hybrid by measuring the total fluorescence of light emitted from said hybrid band upon excitation by laser-induced fluorescence.
Claim 5
Independent Claim
48 words
A method for stabilizing during capillary electrophoresis nucleic acid hybrids consisting of an RNA strand and a fluorophore terminally labelled DNA strand 15 to 40 base pairs in length comprising electrophoresing said nucleic acid hybrids in the presence of a dye capable of intercalating double stranded nucleic acids.
Claim 6
Independent Claim
48 words
A method for stabilizing during capillary electrophoresis nucleic acid hybrids consisting of an RNA strand and a fluorophore terminally labelled DNA strand 15 to 40 base pairs in length comprising electrophoresing said nucleic acid hybrids in the presence of a dye capable of intercalating double stranded nucleic acids.
Claim 7
Dependent Claim
81 words
A direct quantitative test kit of for an RNA sequence suspected of being present in a biological specimen utilizing capillary electrophoresis and laser-induced fluorscence comprising A DNA probe of 15 to 30 nucleotides terminally labeled with a fluorophore a the 5' end and having a sequence complementary to a target RNA sense sequence, and a dye capable of intercalating a RNA-DNA hybrid molecule of 15-30 base pairs, wherein said RNA sequence is an RNA strand conforming to Seq. I.D. Nos. 1-12.
Claim 8
Dependent Claim
81 words
A direct quantitative test kit of for an RNA sequence suspected of being present in a biological specimen utilizing capillary electrophoresis and laser-induced fluorscence comprising A DNA probe of 15 to 30 nucleotides terminally labeled with a fluorophore a the 5' end and having a sequence complementary to a target RNA sense sequence, and a dye capable of intercalating a RNA-DNA hybrid molecule of 15-30 base pairs, wherein said RNA sequence is an RNA strand conforming to Seq. I.D. Nos. 1-12.
Claim 9
Independent Claim
21 words
The method of claims 1 or 2 wherein said RNA sequence is a portion of the sequence encoding the HIV-1 genome.
Claim 10
Dependent Claim
24 words
The method of claim 1 or 2 wherein said RNA sequence is a 25 base sense strand segment conforming to Seq. I.D. No. 1.
Claim 11
Independent Claim
22 words
The method of claims 1, 2 or 3 wherein said dye is selected from the group consisting of thiazole orange and YoYo.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 2003-01-10 | 2003-07-12 | 2004-01-11 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2007-01-10 | 2007-07-12 | 2008-01-11 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2011-01-10 | 2011-07-12 | 2012-01-11 | $7700.00 | Window Closed / Lapsed |