CA2148140A1 - Homogeneous method for assay of double-stranded nucleic acids using fluorescent dyes and kit useful therein - Google Patents
Homogeneous method for assay of double-stranded nucleic acids using fluorescent dyes and kit useful thereinInfo
- Publication number
- CA2148140A1 CA2148140A1 CA002148140A CA2148140A CA2148140A1 CA 2148140 A1 CA2148140 A1 CA 2148140A1 CA 002148140 A CA002148140 A CA 002148140A CA 2148140 A CA2148140 A CA 2148140A CA 2148140 A1 CA2148140 A1 CA 2148140A1
- Authority
- CA
- Canada
- Prior art keywords
- carbon atoms
- double
- nucleic acid
- target nucleic
- alkyl
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/81—Packaged device or kit
Abstract
A highly sensitive homogeneous assay allows for quantitative detection of amplified nucleic acids.
This detection is achieved during or after amplification with a high affinity fluorescent dye which is from the class of unsymmetrical cyanine dyes having at least two positive charges and a binding constant (Kb) within the range of from about 1 x 104 to about 5 x 105 (molar-1). The reagents used for the assay can be contained in a kit designed for amplification such as by polymerase chain reaction.
This detection is achieved during or after amplification with a high affinity fluorescent dye which is from the class of unsymmetrical cyanine dyes having at least two positive charges and a binding constant (Kb) within the range of from about 1 x 104 to about 5 x 105 (molar-1). The reagents used for the assay can be contained in a kit designed for amplification such as by polymerase chain reaction.
Claims (20)
1. A homogeneous method for the detection of a double-stranded target nucleic acid, comprising the steps of:
A) amplifying a double-stranded target nucleic acid, B) contacting the resulting amplified double-stranded target nucleic acid with a fluorescent dye which, when bound to said double-stranded target nucleic acid, exhibits a detectable signal as compared to either: the signal generated when said dye is bound to a single strand of said double-stranded target nucleic acid, or the signal of the dye in its free form, and C) detecting or monitoring said detectable signal as a measure of the presence or amount of said double-stranded target nucleic acid, whereby said fluorescent dye is an unsymmetrical cyanine dye having a binding constant (Kb) with double-stranded nucleic acids of from about 1 x 104 to about 5 x 105 (molar-1), and is represented by the structure (I):
wherein X is -S-, -O-, -Se-, =CH- or -NR1-, Y is -CH=CH-, R is alkyl of 1 to 6 carbon atoms, R1 is hydrogen or alkyl of 1 to 6 carbon atoms, R2 and R6 are independently alkylene of 1 to 10 carbon atoms, R3, R4 and R5 are independently alkyl of 1 to 6 carbon atoms, Z1 comprises the carbon and hetero atoms necessary to complete a 5- or 6-membered heterocyclic ring, Z2 comprises the carbon and hetero atoms necessary to complete a 5- or 6-membered aromatic ring, Q is an acid anion, n is 0, 1 or 2, m, p and q are independently 0 or 1, provided that p and q are not the same, r is 1 or 2, and t is 0, 1 or 2, whereby said detecting or monitoring is carried out without hybridization of said amplified double-stranded target nucleic acid to a capture probe.
A) amplifying a double-stranded target nucleic acid, B) contacting the resulting amplified double-stranded target nucleic acid with a fluorescent dye which, when bound to said double-stranded target nucleic acid, exhibits a detectable signal as compared to either: the signal generated when said dye is bound to a single strand of said double-stranded target nucleic acid, or the signal of the dye in its free form, and C) detecting or monitoring said detectable signal as a measure of the presence or amount of said double-stranded target nucleic acid, whereby said fluorescent dye is an unsymmetrical cyanine dye having a binding constant (Kb) with double-stranded nucleic acids of from about 1 x 104 to about 5 x 105 (molar-1), and is represented by the structure (I):
wherein X is -S-, -O-, -Se-, =CH- or -NR1-, Y is -CH=CH-, R is alkyl of 1 to 6 carbon atoms, R1 is hydrogen or alkyl of 1 to 6 carbon atoms, R2 and R6 are independently alkylene of 1 to 10 carbon atoms, R3, R4 and R5 are independently alkyl of 1 to 6 carbon atoms, Z1 comprises the carbon and hetero atoms necessary to complete a 5- or 6-membered heterocyclic ring, Z2 comprises the carbon and hetero atoms necessary to complete a 5- or 6-membered aromatic ring, Q is an acid anion, n is 0, 1 or 2, m, p and q are independently 0 or 1, provided that p and q are not the same, r is 1 or 2, and t is 0, 1 or 2, whereby said detecting or monitoring is carried out without hybridization of said amplified double-stranded target nucleic acid to a capture probe.
2. The method of claim 1 wherein X is -S-or -O-, R is alkyl of 1 to 3 carbon atoms, R1 is hydrogen or alkyl of 1 to 3 carbon atoms, R2 is alkylene of 1 to 4 carbon atoms, R3, R4 and R5 are independently alkyl of 1 to 3 carbon atoms, Z1 comprises the carbon atoms necessary to complete a benzoxazolium or benzothiazolium ring, Z2 comprises the carbon atoms necessary to complete a benzo ring, n is 0 or 1, t is 0 or 1, and at least one but not both of p and q are 1.
3. The method of claim 2 wherein R is methyl or ethyl, R2 is an alkylene of 3 carbon atoms, each of R3, R4 and R5 is methyl, p is 0, q is 1, and t is 0.
4. The method of claim 1 wherein said fluorescent dye is Compound A, B, C, D, E, F, G or H.
5. The method of claim 1 wherein said amplification step A) is carried out using polymerase chain reaction.
6. A method for monitoring the amplification of a double-stranded target nucleic acid, comprising the steps of:
A) amplifying a double-stranded target nucleic acid in the presence of a fluorescent dye which, when bound to said double-stranded target nucleic acid, exhibits a detectable signal as compared to either: the signal generated when said dye is bound to a single strand of said double-stranded target nucleic acid, or the signal of the dye in its free form, and B) detecting or monitoring said detectable signal as a measure of the presence or amount of said double-stranded target nucleic acid during said amplification step, whereby said fluorescent dye is an unsymmetrical cyanine dye having a binding constant (Kb) with double-stranded nucleic acids of from about 1 x 104 to about 5 x 105 (molar-1), and is represented by the structure (I):
wherein X is -S-, -O-, -Se-, =CH- or -NR1-, Y is -CH=CH-, R is alkyl of 1 to 6 carbon atoms, R1 is hydrogen or alkyl of 1 to 6 carbon atoms, R2 and R6 are independently alkylene of 1 to 10 carbon atoms, R3, R4 and R5 are independently alkyl of 1 to 6 carbon atoms, Z1 comprises the carbon and hetero atoms necessary to complete a 5- or 6-membered heterocyclic ring, Z2 comprises the carbon and hetero atoms necessary to complete a 5- or 6-membered aromatic ring, Q is an acid anion, n is 0, 1 or 2, m, p and q are independently 0 or 1, provided that p and q are not the same, r is 1 or 2, and t is 0, 1 or 2, and whereby said detecting or monitoring is carried out without binding said amplified double-stranded target nucleic acid to a capture probe.
A) amplifying a double-stranded target nucleic acid in the presence of a fluorescent dye which, when bound to said double-stranded target nucleic acid, exhibits a detectable signal as compared to either: the signal generated when said dye is bound to a single strand of said double-stranded target nucleic acid, or the signal of the dye in its free form, and B) detecting or monitoring said detectable signal as a measure of the presence or amount of said double-stranded target nucleic acid during said amplification step, whereby said fluorescent dye is an unsymmetrical cyanine dye having a binding constant (Kb) with double-stranded nucleic acids of from about 1 x 104 to about 5 x 105 (molar-1), and is represented by the structure (I):
wherein X is -S-, -O-, -Se-, =CH- or -NR1-, Y is -CH=CH-, R is alkyl of 1 to 6 carbon atoms, R1 is hydrogen or alkyl of 1 to 6 carbon atoms, R2 and R6 are independently alkylene of 1 to 10 carbon atoms, R3, R4 and R5 are independently alkyl of 1 to 6 carbon atoms, Z1 comprises the carbon and hetero atoms necessary to complete a 5- or 6-membered heterocyclic ring, Z2 comprises the carbon and hetero atoms necessary to complete a 5- or 6-membered aromatic ring, Q is an acid anion, n is 0, 1 or 2, m, p and q are independently 0 or 1, provided that p and q are not the same, r is 1 or 2, and t is 0, 1 or 2, and whereby said detecting or monitoring is carried out without binding said amplified double-stranded target nucleic acid to a capture probe.
7. The method of claim 6 wherein X is -S-or -O-, R is alkyl of 1 to 3 carbon atoms, R1 is hydrogen or alkyl of 1 to 3 carbon atoms, R2 is alkylene of 1 to 4 carbon atoms, R3, R4 and R5 are independently alkyl of 1 to 3 carbon atoms, Z1 comprises the carbon atoms necessary to complete a benzoxazolium or benzothiazolium ring, Z2 comprises the carbon atoms necessary to complete a benzo ring, n is 0 or 1, t is 0 or 1, and at least one but not both of p and q are 1.
8. The method of claim 7 wherein R is methyl or ethyl, R2 is an alkylene of 3 carbon atoms, each of R3, R4 and R5 is methyl, p is 0, q is 1, and t is 0.
9. The method of claim 6 wherein said fluorescent dye is Compound A, B, C, D, E, F, G or H.
10. The method of claim 6 wherein said amplification step A) is carried out using polymerase chain reaction.
11. The method of claim 6 wherein said fluorescent dye is present at a concentration of at least about 10-9 molar.
12. The method of claim 6 wherein said detectable signal is an increase in emission intensity.
13. A test kit for homogeneous amplification and detection of a double-stranded target nucleic acid, comprising in the same or separate packaging:
1) a fluorescent dye which, when bound to a double-stranded target nucleic acid, exhibits a detectable signal as compared to either: the signal generated when said dye is bound to a single strand of said double-stranded target nucleic acid, or the signal of the dye in its free form, whereby said fluorescent dye is an unsymmetrical cyanine dye having a binding constant (Kb) with double-stranded nucleic acids of from about 1 x 104 to about 5 x 105 (molar-1), and is represented by the structure (I):
wherein X is -S-, -O-, -Se-, =CH- or -NR1-, Y is -CH=CH-, R is alkyl of 1 to 6 carbon atoms, R1 is hydrogen or alkyl of 1 to 6 carbon atoms, R2 and R6 are independently alkylene of 1 to 10 carbon atoms, R3, R4 and R5 are independently alkyl of 1 to 6 carbon atoms, Z1 comprises the carbon and hetero atoms necessary to complete a 5- or 6-membered heterocyclic ring, Z2 comprises the carbon and hetero atoms necessary to complete a 5- or 6-membered aromatic ring, Q is an acid anion, n is 0, 1 or 2, m, p and q are independently 0 or 1, provided that p and q are not the same, r is 1 or 2, and t is 0, 1 or 2, and 2) at least one amplification reagent.
1) a fluorescent dye which, when bound to a double-stranded target nucleic acid, exhibits a detectable signal as compared to either: the signal generated when said dye is bound to a single strand of said double-stranded target nucleic acid, or the signal of the dye in its free form, whereby said fluorescent dye is an unsymmetrical cyanine dye having a binding constant (Kb) with double-stranded nucleic acids of from about 1 x 104 to about 5 x 105 (molar-1), and is represented by the structure (I):
wherein X is -S-, -O-, -Se-, =CH- or -NR1-, Y is -CH=CH-, R is alkyl of 1 to 6 carbon atoms, R1 is hydrogen or alkyl of 1 to 6 carbon atoms, R2 and R6 are independently alkylene of 1 to 10 carbon atoms, R3, R4 and R5 are independently alkyl of 1 to 6 carbon atoms, Z1 comprises the carbon and hetero atoms necessary to complete a 5- or 6-membered heterocyclic ring, Z2 comprises the carbon and hetero atoms necessary to complete a 5- or 6-membered aromatic ring, Q is an acid anion, n is 0, 1 or 2, m, p and q are independently 0 or 1, provided that p and q are not the same, r is 1 or 2, and t is 0, 1 or 2, and 2) at least one amplification reagent.
14. The test kit of claim 13 wherein said amplification reagent is a PCR reagent, and said PCR
reagent and said fluorescent dye are in the same package.
reagent and said fluorescent dye are in the same package.
15. The test kit of claim 14 wherein said PCR reagent is a primer for the target nucleic acid, a thermostable DNA polymerase, a dNTP or a DNA polymerase cofactor.
16. The test kit of claim 15 wherein all of said PCR reagents are in the same buffered reaction mixture.
17. The test kit of claim 15 further containing a reaction vessel for carrying out amplification and detection of the target nucleic acid.
18. The test kit of claim 15 wherein X is -S- or -O-, R is alkyl of 1 to 3 carbon atoms, R1 is hydrogen or alkyl of 1 to 3 carbon atoms, R2 is alkylene of 1 to 4 carbon atoms, R3, R4 and R5 are independently alkyl of 1 to 3 carbon atoms, Z1 comprises the carbon atoms necessary to complete a benzoxazolium or benzothiazolium ring, Z2 comprises the carbon atoms necessary to complete a benzo ring, n is 0 or 1, t is 0 or 1, and at least one but not both of p and q are 1.
19. The test kit of claim 18 wherein R is methyl or ethyl, R2 is an alkylene of 3 carbon atoms, each of R3, R4 and R5 is methyl, p is 0, q is 1, and t is 0.
20. The test kit of claim 15 wherein said fluorescent dye is Compound A, B, C, D, E, F, G or H.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US23539694A | 1994-04-29 | 1994-04-29 | |
| US08/235,396 | 1994-04-29 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2148140A1 true CA2148140A1 (en) | 1995-10-30 |
| CA2148140C CA2148140C (en) | 2010-08-10 |
Family
ID=22885321
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA2148140A Expired - Lifetime CA2148140C (en) | 1994-04-29 | 1995-04-28 | Homogeneous method for assay of double-stranded nucleic acids using fluorescent dyes and kit useful therein |
Country Status (8)
| Country | Link |
|---|---|
| US (1) | US5563037A (en) |
| EP (1) | EP0684316B1 (en) |
| JP (1) | JP4260903B2 (en) |
| KR (1) | KR100388732B1 (en) |
| CN (1) | CN1100884C (en) |
| AT (1) | ATE280243T1 (en) |
| CA (1) | CA2148140C (en) |
| DE (1) | DE69533660T2 (en) |
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| US5312921A (en) * | 1990-03-14 | 1994-05-17 | Regents Of The University Of California | Dyes designed for high sensitivity detection of double-stranded DNA |
| ATE199157T1 (en) * | 1990-03-14 | 2001-02-15 | Univ California | MULTICHROMOPHORE FLUORESCENT PROBE |
| JP2985446B2 (en) * | 1990-10-31 | 1999-11-29 | 東ソー株式会社 | Methods for detecting and measuring nucleic acids |
| US5994056A (en) * | 1991-05-02 | 1999-11-30 | Roche Molecular Systems, Inc. | Homogeneous methods for nucleic acid amplification and detection |
| JPH0540097A (en) * | 1991-08-06 | 1993-02-19 | Hitachi Chem Co Ltd | Fluorescent labeling dye, organism derived substance labelled therewith and reagent containing them |
| US5316906A (en) * | 1991-08-23 | 1994-05-31 | Molecular Probes, Inc. | Enzymatic analysis using substrates that yield fluorescent precipitates |
| DE69219610T2 (en) * | 1991-09-16 | 1997-10-02 | Molecular Probes Inc | Dimeric asymmetrical cyanine dyes |
| US5321130A (en) * | 1992-02-10 | 1994-06-14 | Molecular Probes, Inc. | Unsymmetrical cyanine dyes with a cationic side chain |
-
1995
- 1995-04-28 DE DE69533660T patent/DE69533660T2/en not_active Expired - Lifetime
- 1995-04-28 EP EP95302921A patent/EP0684316B1/en not_active Expired - Lifetime
- 1995-04-28 CA CA2148140A patent/CA2148140C/en not_active Expired - Lifetime
- 1995-04-28 AT AT95302921T patent/ATE280243T1/en not_active IP Right Cessation
- 1995-04-29 KR KR1019950010417A patent/KR100388732B1/en not_active IP Right Cessation
- 1995-04-29 CN CN95106515A patent/CN1100884C/en not_active Expired - Lifetime
- 1995-05-01 JP JP10746895A patent/JP4260903B2/en not_active Expired - Fee Related
- 1995-12-21 US US08/576,861 patent/US5563037A/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| DE69533660D1 (en) | 2004-11-25 |
| US5563037A (en) | 1996-10-08 |
| CA2148140C (en) | 2010-08-10 |
| EP0684316B1 (en) | 2004-10-20 |
| CN1100884C (en) | 2003-02-05 |
| CN1114359A (en) | 1996-01-03 |
| EP0684316A1 (en) | 1995-11-29 |
| KR100388732B1 (en) | 2004-09-04 |
| DE69533660T2 (en) | 2005-11-10 |
| ATE280243T1 (en) | 2004-11-15 |
| JP4260903B2 (en) | 2009-04-30 |
| JPH0889299A (en) | 1996-04-09 |
| KR950032641A (en) | 1995-12-22 |
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