US5846729A
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Assaying nucleotides in solution using a fluorescent intensity quenching effect
Assignee: LORNE PARK RESEARCH USA LLC
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Filing: 1997-07-01
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Granted: 1998-12-08
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Expires: 2017-07-01
Abstract
Abstract The invention provides a method for rapidly, economically and efficiently sequencing and assaying nucleotides in a liquid medium using laser induced fluorescence of antisense probes, including PNA probes. Fluorescent intensity of the resulting medium is inversely proportional to the hybridization efficiency of the probes with respect to the target sequence. The method is particularly advantageous in not requiring separation of unhybridized probes and hybridization complexes prior to detection. The method can be used to identify accessible regions in folded nucleotide sequences, to determine the number of mismatched pairs in a hybridization complex, and to map genomes.
Inventors
- 👤 Yuan Min Wu
- 👤 Eileen Xiao-Feng Nie
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-05 13:15
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6832; C12Q1/6876; C12Q1/6883; C12Q1/6886; C12Q2600/00; C12Q2600/156; Y; Y10; Y10S; Y10S435/00; Y10S435/81
Claims Analysis
Total Claims: 29
Claim 1
Independent Claim
157 words
A method for detecting at least one single stranded or double stranded nucleotide sequence in a liquid medium, said method comprising: providing said liquid medium comprising said at least one nucleotide sequence; adding to said liquid medium at least one antisense probe to form at least one hybridization complex with said at least one nucleotide sequence, wherein said at least one probe comprises at least one marker; and detecting said at least one nucleotide sequence by detecting at least one signal that is inversely proportional to an amount of said at least one hybridization complex in said liquid medium and proportional to an amount of said at least one probe unhybridized in said liquid medium, wherein said method is conducted without separating unhybridized probes from said at least one hybridization complex prior to said signal detecting, and without providing a signal quenching agent on said at least one probe or on said at least one nucleotide sequence.
Claim 2
Independent Claim
157 words
A method for detecting at least one single stranded or double stranded nucleotide sequence in a liquid medium, said method comprising: providing said liquid medium comprising said at least one nucleotide sequence; adding to said liquid medium at least one antisense probe to form at least one hybridization complex with said at least one nucleotide sequence, wherein said at least one probe comprises at least one marker; and detecting said at least one nucleotide sequence by detecting at least one signal that is inversely proportional to an amount of said at least one hybridization complex in said liquid medium and proportional to an amount of said at least one probe unhybridized in said liquid medium, wherein said method is conducted without separating unhybridized probes from said at least one hybridization complex prior to said signal detecting, and without providing a signal quenching agent on said at least one probe or on said at least one nucleotide sequence.
Claim 3
Dependent Claim
23 words
The method for detecting at least one nucleotide sequence according to claim 1, wherein said at least one probe has an uncharged backbone.
Claim 4
Dependent Claim
23 words
The method for detecting at least one nucleotide sequence according to claim 2, wherein said at least one probe comprises a PNA sequence.
Claim 5
Dependent Claim
47 words
The method for detecting at least one nucleotide sequence according to claim 1, wherein said at least one marker includes a fluorescent dye and said at least one signal is at least one fluorescent emission generated by irradiating said at least one marker with a light source.
Claim 6
Dependent Claim
41 words
The method for detecting at least one nucleotide sequence according to claim 4, wherein said light source is an argon ion laser which irradiates said at least one marker with light having a wavelength of about 200 to about 1000 nm.
Claim 7
Dependent Claim
30 words
The method for detecting at least one nucleotide sequence according to claim 5, wherein said at least one fluorescent emission is detected in a range of 200 to 1000 nm.
Claim 8
Dependent Claim
38 words
The method for detecting at least one nucleotide sequence according to claim 6, wherein said at least one signal is at least one fluorescent emission intensity generated by irradiating said at least one hybridization complex with a laser.
Claim 9
Dependent Claim
52 words
The method for detecting at least one nucleotide sequence according to claim 7, wherein a laser induced fluorescent intensity maximum of said liquid medium is compared with a laser induced fluorescent intensity maximum of a standard sample to detect whether said at least one nucleotide sequence is present in said liquid medium.
Claim 10
Dependent Claim
37 words
The method for detecting at least one nucleotide sequence according to claim 8, wherein a fluorescent intensity quenching effect correlated with probe to nucleotide sequence hybridization efficiency is analyzed to detect said at least one nucleotide sequence.
Claim 11
Dependent Claim
60 words
The method for detecting at least one nucleotide sequence according to claim 9, wherein a first said at least one probe is complementary to a first segment of a first nucleotide sequence, a second said at least one probe is complementary to a second segment of a second nucleotide sequence, and said first and second segments differ from each other.
Claim 12
Dependent Claim
29 words
The method for detecting at least one nucleotide sequence according to claim 10, wherein said first and second probes are completely complementary to said first and second segments, respectively.
Claim 13
Dependent Claim
25 words
The method for detecting at least one nucleotide sequence according to claim 11, wherein said first nucleotide sequence is complementary to said second nucleotide sequence.
Claim 14
Dependent Claim
31 words
The method for detecting at least one nucleotide sequence according to claim 12, wherein said first nucleotide sequence and said second nucleotide sequence are within opposing strands of double stranded DNA.
Claim 15
Dependent Claim
27 words
The method for detecting at least one nucleotide sequence according to claim 11, wherein said first nucleotide sequence and said second nucleotide sequence are in different genes.
Claim 16
Dependent Claim
88 words
The method for detecting at least one nucleotide sequence according to claim 14, wherein said first probe has a first marker which has a first fluorescent emission intensity at a first wavelength, said second probe has a second marker which has a second fluorescent emission intensity at a second wavelength, said first and second wavelengths are different, said first nucleotide sequence is detected by monitoring fluorescent emission intensity at said first wavelength and said second nucleotide sequence is detected by monitoring fluorescent emission intensity at said second wavelength.
Claim 17
Dependent Claim
88 words
The method for detecting at least one nucleotide sequence according to claim 11, wherein said first probe has a first marker which has a first fluorescent emission intensity at a first wavelength, said second probe has a second marker which has a second fluorescent emission intensity at a second wavelength, said first and second wavelengths are different, said first nucleotide sequence is detected by monitoring fluorescent emission intensity at said first wavelength and said second nucleotide sequence is detected by monitoring fluorescent emission intensity at said second wavelength.
Claim 18
Dependent Claim
48 words
The method for detecting at least one nucleotide sequence according to claim 16, wherein said first nucleotide sequence is a positive control expected to be present in said liquid medium being analyzed, and said second nucleotide sequence is detected by comparing said first intensity and said second intensity.
Claim 19
Dependent Claim
46 words
The method for detecting at least one nucleotide sequence according to claim 17, wherein the second nucleotide sequence is found only in a mutant genomic DNA and the first nucleotide sequence is found in said mutant genomic DNA and in a corresponding wild type genomic DNA.
Claim 20
Dependent Claim
29 words
The method for detecting at least one nucleotide sequence according to claim 18, wherein the first and second markers are selected from the group consisting of fluorescein and rhodamine.
Claim 21
Dependent Claim
74 words
The method for detecting at least one nucleotide sequence according to claim 17, wherein a third probe having a third marker which has a third fluorescent emission intensity at a third wavelength differing from the first and second wavelengths is added to said liquid medium as a negative control which is not expected to hybridize with any nucleotide sequence, and said second nucleotide sequence is detected by comparing the first, second and third intensities.
Claim 22
Dependent Claim
66 words
The method for detecting at least one nucleotide sequence according to claim 9, wherein prior to subjecting said liquid medium to hybridizing conditions, said liquid medium is divided into a plurality of equal portions with a different probe in each portion, and the laser induced fluorescent intensities of said portions are compared to determine which probe is most complementary to said at least one nucleotide sequence.
Claim 23
Dependent Claim
91 words
The method for detecting at least one nucleotide sequence according to claim 9, wherein at least two different types of probes differing by at least one base are fixed to different internal surfaces of a container in which said liquid medium is added, and after subjecting the liquid medium to hybridization conditions and fluorescence inducing radiation, fluorescent intensities emanating from the different surfaces of the container are compared to determine whether the sample contains a nucleotide sequence perfectly complementary to either or both types of said probes fixed to the surface.
Claim 24
Dependent Claim
34 words
The method for detecting at least one nucleotide sequence according to claim 1, further comprising determining a hybridization efficiency of said at least one probe with respect to said at least one nucleotide sequence.
Claim 25
Dependent Claim
52 words
The method for detecting at least one nucleotide sequence according to claim 1, wherein said at least one nucleotide sequence is mRNA, and antisense drug accessible segments of said mRNA are identified by comparing the fluorescent intensities of a plurality of different probes for a plurality of different segments of said mRNA.
Claim 26
Dependent Claim
51 words
The method for detecting at least one nucleotide sequence according to claim 1, further comprising comparing the fluorescent intensities of a plurality of different probes for a single segment of said at least one nucleotide sequence to determine a number of base mismatches between each of said probes and said segment.
Claim 27
Dependent Claim
30 words
The method for detecting at least one nucleotide sequence according to claim 1, further comprising detecting locations of a plurality of nucleotide sequences within a genome to map said genome.
Claim 28
Dependent Claim
36 words
The method for detecting at least one nucleotide sequence according to claim 1, wherein said at least one marker is one fluorophore, and said one fluorophore is the only fluorophore on said at least one probe.
Claim 29
Dependent Claim
25 words
The method for detecting at least one nucleotide sequence according to claim 1, wherein said method is conducted without using enzymes to digest said probe.
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| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
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| 3.5 Years (Year 4) | 2001-12-07 | 2002-06-08 | 2002-12-08 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2005-12-07 | 2006-06-08 | 2006-12-08 | $3760.00 | Window Closed / Lapsed |
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