CA1271705A - Method for the identification of nucleic acids - Google Patents
Method for the identification of nucleic acidsInfo
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- CA1271705A CA1271705A CA000498834A CA498834A CA1271705A CA 1271705 A CA1271705 A CA 1271705A CA 000498834 A CA000498834 A CA 000498834A CA 498834 A CA498834 A CA 498834A CA 1271705 A CA1271705 A CA 1271705A
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- probe
- nucleic acid
- hybridization
- target nucleic
- affinity pair
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- 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
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- 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
- C12Q1/6813—Hybridisation assays
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Abstract
Abstract hybridization method for the identification of nucleic acids in solution is presented. At least two probes, a detector probe and a capturing probe are used in the method.
The detector probe is labeled with a detectable label and to the capturing probe a member of an affinity pair is attached.
The capturing probe; target nucleic acid: detector probe hybrid formed in the hybridization reaction is isolated by the aid of the other member of the affinity pair. Suitable affinity pairs are biotin-avidin or biotin-streptavidin homopolynucleotide, antigens-antibodies etc. The method is faster to perform than the traditional sandwich hybridization .
The detector probe is labeled with a detectable label and to the capturing probe a member of an affinity pair is attached.
The capturing probe; target nucleic acid: detector probe hybrid formed in the hybridization reaction is isolated by the aid of the other member of the affinity pair. Suitable affinity pairs are biotin-avidin or biotin-streptavidin homopolynucleotide, antigens-antibodies etc. The method is faster to perform than the traditional sandwich hybridization .
Description
The invention relates to a method for the identification of nucleic acids by means of hybridization in solution. The method according to the invention is characterized in that at least two probes are used in the hybridization reaction.
To the detector probe a label is attached, and to the other probe, the so-called capturing probe, there is attached a component having affinity for another component, by means of which the capturing probe : targe-t nucleic acid : detector probe hybrid resulting from the hybridization can be separated from the other components present in the hybridization mixture.
Various hybridization methods have been used for the identification of nucleic acids. Direct hybridization methods and sandwich hybridization methods can be mentioned as examples. In direct hybridization methods the nucleic acid specimen is either in solution or affixed to a solid carrier. The nucleic acid which is to be identified is demonstrated by using one labeled probe. In sandwich hybridization methods (US 4.486.539), two separate probes are used by means of which the nucleic acid to be identified is demonstrated in the sample solution. The detector probe is labeled with a label substance and the other probe is affixed to solid carrier.
It is an object of the present invention to perform a nucleic acid identification protocol faster -than with the traditional sandwich hybridization technique.
In meeting this and other objects, the inven-tion provides a method for identification of a target nucleic acid by hybridization in a solution phase, comprising the steps of:-a) combining in the solution phase, the target nucleic acid ,- ~' ~ ~7~7(~
,
To the detector probe a label is attached, and to the other probe, the so-called capturing probe, there is attached a component having affinity for another component, by means of which the capturing probe : targe-t nucleic acid : detector probe hybrid resulting from the hybridization can be separated from the other components present in the hybridization mixture.
Various hybridization methods have been used for the identification of nucleic acids. Direct hybridization methods and sandwich hybridization methods can be mentioned as examples. In direct hybridization methods the nucleic acid specimen is either in solution or affixed to a solid carrier. The nucleic acid which is to be identified is demonstrated by using one labeled probe. In sandwich hybridization methods (US 4.486.539), two separate probes are used by means of which the nucleic acid to be identified is demonstrated in the sample solution. The detector probe is labeled with a label substance and the other probe is affixed to solid carrier.
It is an object of the present invention to perform a nucleic acid identification protocol faster -than with the traditional sandwich hybridization technique.
In meeting this and other objects, the inven-tion provides a method for identification of a target nucleic acid by hybridization in a solution phase, comprising the steps of:-a) combining in the solution phase, the target nucleic acid ,- ~' ~ ~7~7(~
,
- 2 -with at least one detector probe and at leas-t one capturing probe, the probes being non-overlapping and substantially homologous to the target nucleic acid, the detector probe being labeled wi-th a detectable label and the capturing probe being attached to one member of an affinity pair;
wherein the target nucleic acid and probes are rendered single stranded if necessary;
b) allowing hybridization between the probes and the target nucleic acid to take place in the solution phase at a temperature ranging from 0C to 80C, c) isolating a capturing probe/target nucleic acid/detector probe hybrid formed in the hybridization step by means of the other member of the affinity pair affixed to a solid carrier; and d) measuring the amount of the isolated hybrid by means of the detectable label.
Since both the target nucleic acid taking part in the hybridization and the two probes are in the same solution phase, the hybridization reaction is considerably more rapid than in sandwich hybridization in which one probe is affixed to a solid carrier. In the method according to the invention an incubation time of one hour is sufficient. In the single-step sandwich hybridization (U.S. 4.486.539), suficient hybridization is not achieved in Eewer than 12 hours. When two-step sandwich hybridization (Dunn &
Hassell, Cell. Vol. 12 pp. 23-36, 1977) is carried out, a hybridization time of at least 24 hours is required.
At least two probes are advantageously used in the method according to the invention. The probes are nucleic acid 70~;
fragments at least partially homologous to the target nucleic acid. It is advantageous, but not necessary, that the probes be homologous to sites located relatively close to each other in the nucleic acid to be identified. The probes must be non overlapping to one another.
The probes can be prepared synthetically, semi-synthetical-ly, by recombinant-DNA techniques, or from nucleic acids isolated directly from nature. Probes are also commercially available from several sources. A probe may be bound to a suitable vector. It may contain vector parts or be completely devoid of vector parts.
The detector probe is labeled with a suitable label.
Various radioactive isotopes or radioactively labeled compounds may be used as the labels. The Label substance may also be fluorescent, luminescent, light-emitting, enzymatically or immunologically demonstrable, etc. Labels based on biotin and avidin or s-treptavidin, lanthanide chelates, ferritin and heme compounds, and immunologically demonstrable haptenes such as acetoxyacetylfluorene derivatives (WO 8302286) can be mentioned as examples.
Identification by mediation of proteins is also possible.
The method according to the invention is not dependent on the label used. A11 currently known label substances suitable for nuclelc acid hybridization, or those developed in the future, can be freely applied to the method.
--1.2717(t~
To the other probe, the so-called capturing probe a ~omp~nent having affin~ty for another c~mponent i8 atta~hed. Bistin -avidin or ~treptavidin, heavy-metal derivatives - thio groups, various homop~lynucleotides, such as poly dG - poly dC, poly dA - poly dT, and poly dA - poly U are sultable affinity pairs. But al60 other affinity pairs can be used, provided that the components have sufficiently strong affinity for one another. Suitable affinity pairs are found among ligands and conjugates used in immunological methods.
Before the hybridization reaction i8 carried out the 6pecimen is treated 80 as to release the target nucleic acids in single-stranded form into the hybridization solution. The hybridization is carried out in a hybridization mixture in which the target nucleic acids, the labeled probe and the capturing probe have, when necessary, been rendered single-stranded. Various suitable buffers can be used as the hybridization solutionæ. The hybridization takes place within the temperature range 0 - 80C, but it is advantageous to use a temperature of 65C. If the hybridization solution contains formamide (40-55 ~), a temperature of 37C can be used. An hour i6 sufficient a~ hybridization time.
When the hybridization has taken place, the solution is diluted, when necessary, to render the conditions advantageous for the affinity pair. Thereafter the mixture is contacted with the other member of the affinity pair.
Affinity chromatography columns, filters, plastic ~urfaces, glass surfaces etc. can be used to capture the capturing probe : target nucleic acid : detector probe hybrid.
The carrier material of the affinity column may be, for example, cellulose, latex, polyacrylamide, dextran or agarose. These materials can also be used as suspensions in a test tube. It is also advantageous to use test tubes having the other component of the affinity pair affixed to its inner ~27170~
surface. It i6 a prerequi6ite for the material selected that it i~ possible to affix to it a component having affinity for the component attached to the capturing probe.
If the pecimen contain~ the nucleic acid to be identified, a capturing probe : target nucleic acid : detector probe hybrid results from the hybridization. During fractionation this hybrid adheres to the carrier. The label of the fraction adhering to the carrier can be measured by conventional methods directly from the carrier or after elution from the eluted solution.
Other systems, for example phase extraction or magnetic fields, can also be used instead of affinity chromatography in the fractionationO
The method according to the invention is described in greater detail in the following examples. The method according to the invention is not dependent on the nucleic acid fragments used in the examples.
Example 1 Identification of the DNA of adenovirus from a cell lysate by the acid of homopolynucleotides The detector probe used is 1 5I-labeled recombinant phage mKTH 1206, which contains a Bgl II-fragment of the adenovirus genome from position 42 - 45.3 % on the gene map of the adenovirus type 2. The recombinant phage has been deposited in the culture collection Deutsche Sammlung von Mikro-organismen under the number DSM 2827. Its specific activity is 7 x 107 cpm/~ug DNA. The probe is described in greater detail in Ranki et al Gene 21 pp. 77-85, 1983.
The capturing probe used is the recombinant plasmid pKTH
i~:73L70~3 -1202, which ha~ been dep~ited in the ~ulture ~ollection Deut~he ~ammlung von Mikroorganismen u~der the number DSM
2824, and it comprises a BamHI D fragment of the adenoviru6 (map position 29 - 42 ~) cloned into the plasmid pBR322. The recombinant plasmid pKTH 1202 (DSM 2824) was fragmented by using the restriction enzyme Hae III, and a poly A tail was linked to the 3' ends of the fragments by u~ing terminal transferase enzyme. The length of the tail wa~ measured by 3H-A incorporation. The length was on average about 70 A-re6idues. Before being used the capturing probe was denatured by boiling.
The 6ample used con~isted of adeno~irus-infected A-549 cells.
The cells were incubated for 21 hour~ after the infection.
The cells were collected and lysed by using a 1 % sodium dodecyl sulfate solution. The solution contained about cells/ml. Its viscosity was lowered by sonication.
Identically treated non-infected A-549 cells were used as control6. Before hybridization the specimen was boiled for 5 minutes in 0.02 M NaOH, cooled to 0C, and neutralized with acetic acid.
For the test, 500,000 cpm detector probe, 50 ng capturing probe DNA and 10/ul specimen were combined in a test tube.
The volume was adjusted to 50 ~1, and the buffer solution used was 0.6 M sodium chloride, 0.06 M sodium citrate, 0.02 M
sodium, phosphate (pH 7.6) and 0.5 % sodium dDdecyl sulfate.
The mixture was incubated for 1 hour at 65C.
After the hybridization the solution was cooled to +20 C and run slowly through a chromatography column having 1 ml oligo dT celluloRe. The ~olution which passed through was recovered and re run through the column. Thereafter the column was washed with 20 ml of a solution which contained 0.15 M sodium chloride, 0.015 M sodium phosphate (pH 7.6) and 0.5 % sodium dodecyl 6ulfate. The attached DNA was finally detached by using 1 ml O.02 M NaOH. This solution was recovered and its radioactivity was determined.
1~ 7~ ~0~;
__ Result I activity (cpm) Target nucleio ~cid: infected cell6 Control ~ell~
Example 2 Identification of the DNA of Chlamydia trachomatis by the aid of biotin-fitreptavidin The detector probe used was I-labeled recombinant phage mKTH 1245, which contains two BamHI - SalI DNA fragments from the clone pKTH 1220, which together are linked to the M13mp8 vector. The clone pKTH 1220 has been deposited in the culture collection Deutsche Sammlung von Mikroorganismen under the number DSM 2825 and is described in Palva et al FEMS
M~crobiology Letters 23 pp. 83-89, 1984.
The capturing probe used was the recombinant plasmid pKTH
1250. This plasmid consi~ts of a 2.9 kb SalI - ClaI fragment from the plasmid pKTH 1220 (DSM 2825) and of the vector pAT
153. Biotin molecules were linked to the pKTH 1250 DNA
covalently by using the known nick-translation method (Rigby et al. J.Mol.Biol. 113, pp. 237-251, 1977) and the biotin-ll-UTP as the substrate (Bethesda Research Laboratories). The capturing probe DNA was boiled in a buffer comprising 10 mM
Tris-Cl pH 7.6, 1 mM EDTA, for 5 min before use.
The target nucleic acid was the recombinant plasmid pKTH 1220 ~DSM 2825). This plasmid contains about 10 kb of the DNA
7~
characteristic of Chlamydia trachomatis, linked to the vector pBR322. The plasmid serves as a model DNA, representing the genome DNA of the bacterium. Before use, the plasmid was boiled for 5 min in 0.02 M NaOH, whereafter the solution was neutralized with acetic acid.
Streptavidin, which was used as the affinity material, was fixed to CNBr-activated Sepharose (Pharmacia) according to Axen et al. Nature 214 pp. 1302-1304, 1967.
For the test, 500,000 cpm probe, 50 ng capturing probe DNA
and 10 ng target DNA were combined in a test tube. The volume was adjusted to 20 ~ul, and the buffer solution was the same as in Example 1. The control DNA was calf thymus DNA.
The mixture was incubated for 60 min at 65 C. Thereafter, 500~ul of a buffer solution having the composition 0.1 M
Tris-Cl pH 7.5, 0.1 M NaCl, 2 mM MgC12, 0.05 % Triton x-100*
was added. Finally the solution was fractionated in a 0.2 ml Streptavidin-Sepharose*column. The column was washed with 10 ml of the buffer mentioned above and 10 ml 0.015 M sodium chloride, 0.015 M sodium phosphate (pH 7.6), 0.5 % sodium dodecyl sulphate (50 C). Thus the biotinylated DNA adhered to the Streptavidin while the other DNA passed through the column. The radioactive probe adhered only as a result of hybrid formation. The captured radioactivity was determined by transferring the entire column into the counter tube of a gamma counter.
* (trade mark) 125I activity (cpm) Result: Target nucleic acid:
=
10 ng pkTH 1220 10 ng control DNA
~ 27l7~tj E~ample 3 Identification of the plasmid pBR322 D~A by the aid of an antigen-antibody ~air The detector probe used i~ a derivative of the plasmid pBR322 (commercially available from several ~ources) from which the P~tI - SalI (3613 - 651~ fragment ha~ been removed. The plasmid wa6 labeled with photobiotin using a known method (Forster et al. Nucleic Acids Res. 13, pp. 745-761, 1985~ and co~mercial reagents (Bresa, Adelaide, Australia).
The capturing probe used was DNA from a recombinant phage M13mpll into which the pBR322 PstI - SalI fragment had been introduced. The DNA had been sulfonated using a known method (Orgenics Ltd. Yavne, Israel).
The specimen was E.coli HB101 carrying the plasmid pBR322, the amount of which was increased using chloramphenicol amplification (Maniatis et al. Molecular cloning, A
laboratory manual, Cold Spring Harbor Laboratory 1982). The bacterial cells were lysed with lysozyme followed by boiling in NaOH as described in the publication Palva, J. Clin.
Microbiol. 18, pp. 92-100, 1983).
Antisulfone monoclonal antibodies were used to coat polystyrene microtiter wells by standard methods ~McKearn, in Hybridomas: A New Dimension in Biological Analyses, ed.
Kennett et al., Plenum Press 1980).
For the test 5 x 106 lysed E.coli cells (both with and without pBR322) were combined with 100 ng each of capturing DNA and detector probe in a 50 ~ul hybridization mixture. The conditions were as in Example 1 except that 5 % polyethylen glycol (PEG 6000) was added to the mixture and the sodium dodecyl sulfate concentration was 0.1 %. After hybridization the solution was diluted to 250 ~1 by the addition of 0.02 M
12717~
sodium phosphate (pH 7.6), whereafter the solution wa6 transferred to the antibdy-coated microtiter w~ll. This was followed by incubation for 2 h at 37C. The well wa3 then washed with a solution containing 0.15 M ~odium ~hloride, 0.02 M sodium phosphate pH 7.6 and 0.05 % triton X-100. The preæence of detector probe was visuali~ed by addition of streptavidin (Bethesda Research ~aboratories BRL~, washing, addition of biotinylated alkaline phosphatase (BRL), and washing as described by Leary et al. Proc. ~atl. Acad. Sci.
USA 80, pp. 4045-4049, 1983. Finally 250 ~1 of a 35 mg/ml paranitrophenyl phosphate (Sigma) solution in diethanolamine buffer (pH 10) was added. After 60 min the reaction was stopped and the absorbance was measured at 410 nm.
Result: A410 nm Target nucleic acid:
cells with pBR 322 Cells without pBR322 ~2 0.15
wherein the target nucleic acid and probes are rendered single stranded if necessary;
b) allowing hybridization between the probes and the target nucleic acid to take place in the solution phase at a temperature ranging from 0C to 80C, c) isolating a capturing probe/target nucleic acid/detector probe hybrid formed in the hybridization step by means of the other member of the affinity pair affixed to a solid carrier; and d) measuring the amount of the isolated hybrid by means of the detectable label.
Since both the target nucleic acid taking part in the hybridization and the two probes are in the same solution phase, the hybridization reaction is considerably more rapid than in sandwich hybridization in which one probe is affixed to a solid carrier. In the method according to the invention an incubation time of one hour is sufficient. In the single-step sandwich hybridization (U.S. 4.486.539), suficient hybridization is not achieved in Eewer than 12 hours. When two-step sandwich hybridization (Dunn &
Hassell, Cell. Vol. 12 pp. 23-36, 1977) is carried out, a hybridization time of at least 24 hours is required.
At least two probes are advantageously used in the method according to the invention. The probes are nucleic acid 70~;
fragments at least partially homologous to the target nucleic acid. It is advantageous, but not necessary, that the probes be homologous to sites located relatively close to each other in the nucleic acid to be identified. The probes must be non overlapping to one another.
The probes can be prepared synthetically, semi-synthetical-ly, by recombinant-DNA techniques, or from nucleic acids isolated directly from nature. Probes are also commercially available from several sources. A probe may be bound to a suitable vector. It may contain vector parts or be completely devoid of vector parts.
The detector probe is labeled with a suitable label.
Various radioactive isotopes or radioactively labeled compounds may be used as the labels. The Label substance may also be fluorescent, luminescent, light-emitting, enzymatically or immunologically demonstrable, etc. Labels based on biotin and avidin or s-treptavidin, lanthanide chelates, ferritin and heme compounds, and immunologically demonstrable haptenes such as acetoxyacetylfluorene derivatives (WO 8302286) can be mentioned as examples.
Identification by mediation of proteins is also possible.
The method according to the invention is not dependent on the label used. A11 currently known label substances suitable for nuclelc acid hybridization, or those developed in the future, can be freely applied to the method.
--1.2717(t~
To the other probe, the so-called capturing probe a ~omp~nent having affin~ty for another c~mponent i8 atta~hed. Bistin -avidin or ~treptavidin, heavy-metal derivatives - thio groups, various homop~lynucleotides, such as poly dG - poly dC, poly dA - poly dT, and poly dA - poly U are sultable affinity pairs. But al60 other affinity pairs can be used, provided that the components have sufficiently strong affinity for one another. Suitable affinity pairs are found among ligands and conjugates used in immunological methods.
Before the hybridization reaction i8 carried out the 6pecimen is treated 80 as to release the target nucleic acids in single-stranded form into the hybridization solution. The hybridization is carried out in a hybridization mixture in which the target nucleic acids, the labeled probe and the capturing probe have, when necessary, been rendered single-stranded. Various suitable buffers can be used as the hybridization solutionæ. The hybridization takes place within the temperature range 0 - 80C, but it is advantageous to use a temperature of 65C. If the hybridization solution contains formamide (40-55 ~), a temperature of 37C can be used. An hour i6 sufficient a~ hybridization time.
When the hybridization has taken place, the solution is diluted, when necessary, to render the conditions advantageous for the affinity pair. Thereafter the mixture is contacted with the other member of the affinity pair.
Affinity chromatography columns, filters, plastic ~urfaces, glass surfaces etc. can be used to capture the capturing probe : target nucleic acid : detector probe hybrid.
The carrier material of the affinity column may be, for example, cellulose, latex, polyacrylamide, dextran or agarose. These materials can also be used as suspensions in a test tube. It is also advantageous to use test tubes having the other component of the affinity pair affixed to its inner ~27170~
surface. It i6 a prerequi6ite for the material selected that it i~ possible to affix to it a component having affinity for the component attached to the capturing probe.
If the pecimen contain~ the nucleic acid to be identified, a capturing probe : target nucleic acid : detector probe hybrid results from the hybridization. During fractionation this hybrid adheres to the carrier. The label of the fraction adhering to the carrier can be measured by conventional methods directly from the carrier or after elution from the eluted solution.
Other systems, for example phase extraction or magnetic fields, can also be used instead of affinity chromatography in the fractionationO
The method according to the invention is described in greater detail in the following examples. The method according to the invention is not dependent on the nucleic acid fragments used in the examples.
Example 1 Identification of the DNA of adenovirus from a cell lysate by the acid of homopolynucleotides The detector probe used is 1 5I-labeled recombinant phage mKTH 1206, which contains a Bgl II-fragment of the adenovirus genome from position 42 - 45.3 % on the gene map of the adenovirus type 2. The recombinant phage has been deposited in the culture collection Deutsche Sammlung von Mikro-organismen under the number DSM 2827. Its specific activity is 7 x 107 cpm/~ug DNA. The probe is described in greater detail in Ranki et al Gene 21 pp. 77-85, 1983.
The capturing probe used is the recombinant plasmid pKTH
i~:73L70~3 -1202, which ha~ been dep~ited in the ~ulture ~ollection Deut~he ~ammlung von Mikroorganismen u~der the number DSM
2824, and it comprises a BamHI D fragment of the adenoviru6 (map position 29 - 42 ~) cloned into the plasmid pBR322. The recombinant plasmid pKTH 1202 (DSM 2824) was fragmented by using the restriction enzyme Hae III, and a poly A tail was linked to the 3' ends of the fragments by u~ing terminal transferase enzyme. The length of the tail wa~ measured by 3H-A incorporation. The length was on average about 70 A-re6idues. Before being used the capturing probe was denatured by boiling.
The 6ample used con~isted of adeno~irus-infected A-549 cells.
The cells were incubated for 21 hour~ after the infection.
The cells were collected and lysed by using a 1 % sodium dodecyl sulfate solution. The solution contained about cells/ml. Its viscosity was lowered by sonication.
Identically treated non-infected A-549 cells were used as control6. Before hybridization the specimen was boiled for 5 minutes in 0.02 M NaOH, cooled to 0C, and neutralized with acetic acid.
For the test, 500,000 cpm detector probe, 50 ng capturing probe DNA and 10/ul specimen were combined in a test tube.
The volume was adjusted to 50 ~1, and the buffer solution used was 0.6 M sodium chloride, 0.06 M sodium citrate, 0.02 M
sodium, phosphate (pH 7.6) and 0.5 % sodium dDdecyl sulfate.
The mixture was incubated for 1 hour at 65C.
After the hybridization the solution was cooled to +20 C and run slowly through a chromatography column having 1 ml oligo dT celluloRe. The ~olution which passed through was recovered and re run through the column. Thereafter the column was washed with 20 ml of a solution which contained 0.15 M sodium chloride, 0.015 M sodium phosphate (pH 7.6) and 0.5 % sodium dodecyl 6ulfate. The attached DNA was finally detached by using 1 ml O.02 M NaOH. This solution was recovered and its radioactivity was determined.
1~ 7~ ~0~;
__ Result I activity (cpm) Target nucleio ~cid: infected cell6 Control ~ell~
Example 2 Identification of the DNA of Chlamydia trachomatis by the aid of biotin-fitreptavidin The detector probe used was I-labeled recombinant phage mKTH 1245, which contains two BamHI - SalI DNA fragments from the clone pKTH 1220, which together are linked to the M13mp8 vector. The clone pKTH 1220 has been deposited in the culture collection Deutsche Sammlung von Mikroorganismen under the number DSM 2825 and is described in Palva et al FEMS
M~crobiology Letters 23 pp. 83-89, 1984.
The capturing probe used was the recombinant plasmid pKTH
1250. This plasmid consi~ts of a 2.9 kb SalI - ClaI fragment from the plasmid pKTH 1220 (DSM 2825) and of the vector pAT
153. Biotin molecules were linked to the pKTH 1250 DNA
covalently by using the known nick-translation method (Rigby et al. J.Mol.Biol. 113, pp. 237-251, 1977) and the biotin-ll-UTP as the substrate (Bethesda Research Laboratories). The capturing probe DNA was boiled in a buffer comprising 10 mM
Tris-Cl pH 7.6, 1 mM EDTA, for 5 min before use.
The target nucleic acid was the recombinant plasmid pKTH 1220 ~DSM 2825). This plasmid contains about 10 kb of the DNA
7~
characteristic of Chlamydia trachomatis, linked to the vector pBR322. The plasmid serves as a model DNA, representing the genome DNA of the bacterium. Before use, the plasmid was boiled for 5 min in 0.02 M NaOH, whereafter the solution was neutralized with acetic acid.
Streptavidin, which was used as the affinity material, was fixed to CNBr-activated Sepharose (Pharmacia) according to Axen et al. Nature 214 pp. 1302-1304, 1967.
For the test, 500,000 cpm probe, 50 ng capturing probe DNA
and 10 ng target DNA were combined in a test tube. The volume was adjusted to 20 ~ul, and the buffer solution was the same as in Example 1. The control DNA was calf thymus DNA.
The mixture was incubated for 60 min at 65 C. Thereafter, 500~ul of a buffer solution having the composition 0.1 M
Tris-Cl pH 7.5, 0.1 M NaCl, 2 mM MgC12, 0.05 % Triton x-100*
was added. Finally the solution was fractionated in a 0.2 ml Streptavidin-Sepharose*column. The column was washed with 10 ml of the buffer mentioned above and 10 ml 0.015 M sodium chloride, 0.015 M sodium phosphate (pH 7.6), 0.5 % sodium dodecyl sulphate (50 C). Thus the biotinylated DNA adhered to the Streptavidin while the other DNA passed through the column. The radioactive probe adhered only as a result of hybrid formation. The captured radioactivity was determined by transferring the entire column into the counter tube of a gamma counter.
* (trade mark) 125I activity (cpm) Result: Target nucleic acid:
=
10 ng pkTH 1220 10 ng control DNA
~ 27l7~tj E~ample 3 Identification of the plasmid pBR322 D~A by the aid of an antigen-antibody ~air The detector probe used i~ a derivative of the plasmid pBR322 (commercially available from several ~ources) from which the P~tI - SalI (3613 - 651~ fragment ha~ been removed. The plasmid wa6 labeled with photobiotin using a known method (Forster et al. Nucleic Acids Res. 13, pp. 745-761, 1985~ and co~mercial reagents (Bresa, Adelaide, Australia).
The capturing probe used was DNA from a recombinant phage M13mpll into which the pBR322 PstI - SalI fragment had been introduced. The DNA had been sulfonated using a known method (Orgenics Ltd. Yavne, Israel).
The specimen was E.coli HB101 carrying the plasmid pBR322, the amount of which was increased using chloramphenicol amplification (Maniatis et al. Molecular cloning, A
laboratory manual, Cold Spring Harbor Laboratory 1982). The bacterial cells were lysed with lysozyme followed by boiling in NaOH as described in the publication Palva, J. Clin.
Microbiol. 18, pp. 92-100, 1983).
Antisulfone monoclonal antibodies were used to coat polystyrene microtiter wells by standard methods ~McKearn, in Hybridomas: A New Dimension in Biological Analyses, ed.
Kennett et al., Plenum Press 1980).
For the test 5 x 106 lysed E.coli cells (both with and without pBR322) were combined with 100 ng each of capturing DNA and detector probe in a 50 ~ul hybridization mixture. The conditions were as in Example 1 except that 5 % polyethylen glycol (PEG 6000) was added to the mixture and the sodium dodecyl sulfate concentration was 0.1 %. After hybridization the solution was diluted to 250 ~1 by the addition of 0.02 M
12717~
sodium phosphate (pH 7.6), whereafter the solution wa6 transferred to the antibdy-coated microtiter w~ll. This was followed by incubation for 2 h at 37C. The well wa3 then washed with a solution containing 0.15 M ~odium ~hloride, 0.02 M sodium phosphate pH 7.6 and 0.05 % triton X-100. The preæence of detector probe was visuali~ed by addition of streptavidin (Bethesda Research ~aboratories BRL~, washing, addition of biotinylated alkaline phosphatase (BRL), and washing as described by Leary et al. Proc. ~atl. Acad. Sci.
USA 80, pp. 4045-4049, 1983. Finally 250 ~1 of a 35 mg/ml paranitrophenyl phosphate (Sigma) solution in diethanolamine buffer (pH 10) was added. After 60 min the reaction was stopped and the absorbance was measured at 410 nm.
Result: A410 nm Target nucleic acid:
cells with pBR 322 Cells without pBR322 ~2 0.15
Claims (8)
1. A method for identification of a target nucleic acid by hybridization in a solution phase, comprising the steps of:-a) combining, in said solution phase, the target nucleic acid with at least one detector probe and at least one capturing probe, the probes being non-overlapping and substantially homologous to the target nucleic acid, the detector probe being labeled with a detectable label and the capturing probe being attached to one member of an affinity pair, wherein the target nucleic acid and probes are rendered single stranded if necessary;
b) allowing hybridization between said probes and said target nucleic acid to take place in the solution phase at a temperature ranging from 0°C to 80°C, c) isolating a capturing probe/target nucleic acid/detector probe hybrid formed in said hybridization step by means of the other member of said affinity pair affixed to a solid carrier; and d) measuring the amount of the thus isolated hybrid by means of said detectable label.
b) allowing hybridization between said probes and said target nucleic acid to take place in the solution phase at a temperature ranging from 0°C to 80°C, c) isolating a capturing probe/target nucleic acid/detector probe hybrid formed in said hybridization step by means of the other member of said affinity pair affixed to a solid carrier; and d) measuring the amount of the thus isolated hybrid by means of said detectable label.
2. A method according to claim 1, wherein the affinity pair is biotin-steptavidin or biotin-avidin.
3. A method according to claim 1, wherein the affinity pair is a homopolynucleotide pair.
4. A method according to claim 1, wherein the affinity pair is poly dC- poly dG.
5. A method according to claim 1, wherein the affinity pair is poly dA - poly dT.
6. A method according to claim 1, wherein the affinity pair is poly A - poly U.
7. A method according to claim 1, wherein the affinity pair is a heavy-metal derivative - a thio group.
8. A method according to claim 1, wherein the affinity pair is an antigen - an antibody.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FI850004 | 1985-01-02 | ||
| FI850004A FI72146C (en) | 1985-01-02 | 1985-01-02 | Procedure for Identifying Nucleic Acids. |
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| Publication Number | Publication Date |
|---|---|
| CA1271705A true CA1271705A (en) | 1990-07-17 |
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ID=8520136
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000498834A Expired - Lifetime CA1271705A (en) | 1985-01-02 | 1985-12-31 | Method for the identification of nucleic acids |
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| JP (1) | JPH0669400B2 (en) |
| AT (1) | AT397514B (en) |
| AU (1) | AU561382B2 (en) |
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| CA (1) | CA1271705A (en) |
| CH (1) | CH666696A5 (en) |
| DE (1) | DE3546312A1 (en) |
| DK (1) | DK164932C (en) |
| FI (1) | FI72146C (en) |
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| HU (1) | HU196453B (en) |
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| RO (1) | RO94651B (en) |
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| ZA (1) | ZA859895B (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1303221C (en) * | 2003-01-27 | 2007-03-07 | 英科新创(厦门)科技有限公司 | Method for detecting target nucleic acid using affinity amplifying integrated signal group |
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| US5288609A (en) * | 1984-04-27 | 1994-02-22 | Enzo Diagnostics, Inc. | Capture sandwich hybridization method and composition |
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| US4868105A (en) * | 1985-12-11 | 1989-09-19 | Chiron Corporation | Solution phase nucleic acid sandwich assay |
| US4910300A (en) * | 1985-12-11 | 1990-03-20 | Chiron Corporation | Method for making nucleic acid probes |
| US4882269A (en) * | 1985-12-13 | 1989-11-21 | Princeton University | Amplified hybridization assay |
| FI76119C (en) * | 1986-02-27 | 1988-09-09 | Orion Yhtymae Oy | Quantitative determination of nucleic acid molecules and reagent packaging used in the process |
| AU622104B2 (en) * | 1987-03-11 | 1992-04-02 | Sangtec Molecular Diagnostics Ab | Method of assaying of nucleic acids, a reagent combination and kit therefore |
| EP0365595A4 (en) * | 1987-06-26 | 1990-06-05 | Du Pont | Affinity removal of contaminating sequences from recombinant cloned na using capture beads. |
| AU623090B2 (en) * | 1987-07-31 | 1992-05-07 | Gen-Probe Incorporated | Assay for polynucleotides employing oligonucleotides to eliminate undesirable cross reactions |
| EP0305145A3 (en) * | 1987-08-24 | 1990-05-02 | Ortho Diagnostic Systems Inc. | Methods and probes for detecting nucleic acids |
| EP0304845A3 (en) * | 1987-08-28 | 1991-03-06 | Profile Diagnostic Sciences Inc. | Method and kit for assaying gene expressions |
| CA1325761C (en) * | 1987-12-25 | 1994-01-04 | Takanori Oka | Method of detecting an intended nucleic acid in a sample |
| DE3800644A1 (en) * | 1988-01-12 | 1989-07-20 | Boehringer Mannheim Gmbh | PROCESS FOR HIGHLY SPECIFIC DETECTION OF NUCLEIC ACIDS IN SOLUTION |
| US5104791A (en) * | 1988-02-09 | 1992-04-14 | E. I. Du Pont De Nemours And Company | Particle counting nucleic acid hybridization assays |
| WO1989010979A1 (en) * | 1988-05-10 | 1989-11-16 | E.I. Du Pont De Nemours And Company | Process for rapid nucleic acid detection |
| US5185243A (en) * | 1988-08-25 | 1993-02-09 | Syntex (U.S.A.) Inc. | Method for detection of specific nucleic acid sequences |
| AU629845B2 (en) * | 1988-08-30 | 1992-10-15 | Abbott Laboratories | Detection and amplification of target nucleic acid sequences |
| US5858652A (en) * | 1988-08-30 | 1999-01-12 | Abbott Laboratories | Detection and amplification of target nucleic acid sequences |
| GB2225112A (en) * | 1988-11-22 | 1990-05-23 | Ici Plc | Hybridisation probes |
| US5082935A (en) * | 1988-12-15 | 1992-01-21 | Amoco Corporation | Diagnostic reagents made by attaching cytidine containing nucleic acid probes to amino functionalized solid supports by bisulfite mediated transamination |
| WO1990008841A1 (en) * | 1989-02-06 | 1990-08-09 | Gene-Trak Systems | Probes and methods for the detection of listeria |
| JP3046838B2 (en) * | 1989-03-10 | 2000-05-29 | バイシス・インコーポレーテツド | Prevent interference of the affinity capture method |
| EP0463036B1 (en) | 1989-03-10 | 1995-02-22 | Amoco Corporation | Immobilized oligonucleotide probes and uses therefor |
| US5334501A (en) * | 1989-07-11 | 1994-08-02 | Microprobe Corporation | Quantification of bacteria using a nucleic acid hybridization assay |
| JPH05501052A (en) * | 1989-07-11 | 1993-03-04 | マイクロプローブ・コーポレーション | Quantification of bacteria using a nucleic acid hybridization assay |
| GB8924989D0 (en) * | 1989-11-06 | 1989-12-28 | Scotgen Ltd | Method and device for the detection of antibiotic resistance |
| US5580970A (en) * | 1989-12-01 | 1996-12-03 | Amoco Corporation | Detection of HPV transcripts |
| AU7429591A (en) * | 1990-04-18 | 1991-10-24 | Gene-Trak Systems | Nucleic acid probes for the detection of giardia lamblia |
| EP0529070A1 (en) * | 1991-02-27 | 1993-03-03 | Amoco Corporation | Methods for improving the sensitivity of hybridization assays |
| WO1993020234A1 (en) * | 1992-03-31 | 1993-10-14 | E.I. Du Pont De Nemours And Company | A rapid, high capacity nucleic acid based assay |
| US7713528B1 (en) | 1993-02-18 | 2010-05-11 | Enzo Therapeutics, Inc. | Method for in vivo delivery of active compounds using reagent conjugate |
| US5853993A (en) * | 1996-10-21 | 1998-12-29 | Hewlett-Packard Company | Signal enhancement method and kit |
| GB0016833D0 (en) | 2000-07-07 | 2000-08-30 | Lee Helen | Improved dipstick assays (2) |
| US7465540B2 (en) | 2000-09-21 | 2008-12-16 | Luminex Corporation | Multiple reporter read-out for bioassays |
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| FI63596C (en) * | 1981-10-16 | 1983-07-11 | Orion Yhtymae Oy | MICROBIA DIAGNOSIS FOERFARANDE SOM GRUNDAR SIG PAO SKIKTSHYBRIDISERING AV NUCLEINSYROR OCH VID FOERFARANDET ANVAENDA KOMBINATIONER AV REAGENSER |
| FR2518755B1 (en) * | 1981-12-23 | 1986-04-11 | Pasteur Institut | PROBE CONTAINING MODIFIED NUCLEIC ACID RECOGNIZABLE BY SPECIFIC ANTIBODIES AND USE OF THE SAME TO DETECT AND CHARACTERIZE A HOMOLOGATED DNA SEQUENCE |
| GB8306426D0 (en) * | 1983-03-09 | 1983-04-13 | Malcolm A D B | Detecting polynucleotide sequence |
| CA1228811A (en) * | 1983-05-05 | 1987-11-03 | Robert G. Pergolizzi | Assay method utilizing polynucleotide sequences |
| NO843838L (en) * | 1983-09-26 | 1985-03-27 | Ortho Diagnostic Systems Inc | PROCEDURE FOR DETECTING NUCLEIC ACID |
| ZA849596B (en) * | 1983-12-12 | 1985-07-31 | Miles Lab | Hybridization assay with immobilization of hybrids by anti-hybrid binding |
| FR2558172B1 (en) * | 1984-01-16 | 1986-06-13 | Inst Nat Sante Rech Med | PROBE CONTAINING MODIFIED NUCLEIC ACID AND RECOGNITION BY SPECIFIC ANTIBODIES AND USE OF SUCH PROBE AND THESE SPECIFIC ANTIBODIES TO DETECT AND CHARACTERIZE A HOMOLOGATED DNA SEQUENCE |
| CA1223222A (en) * | 1984-02-22 | 1987-06-23 | Nanibhushan Dattagupta | Immobilized nucleic acid-containing probes |
| FR2560298B1 (en) * | 1984-02-28 | 1988-07-15 | Mors | METHOD AND DEVICE FOR ADJUSTING THE PRECISION OF THE PRESSURE DROP OF A FLUID INITIALLY AT HIGH PRESSURE, SUCH AS A HYDRAULIC FLUID FOR EXAMPLE SUPPLYING A HYDRAULIC GROUP, AND APPLICATION OF THE METHOD TO THE DEVICE IN A HYDRAULIC GROUP OR PLANT INTEGRATED OR NOT, ESPECIALLY IN A MACHINE TABLE OR ANY TOOL SUPPORT DEVICE |
| NZ211453A (en) * | 1984-03-22 | 1989-01-06 | Biotechnology Research Enterpr | Aryl azides, their preparation and use in the detection, localisation and isolation of polynucleotides |
| US4766062A (en) * | 1984-05-07 | 1988-08-23 | Allied Corporation | Displacement polynucleotide assay method and polynucleotide complex reagent therefor |
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- 1985-12-17 CH CH5378/85A patent/CH666696A5/en not_active IP Right Cessation
- 1985-12-20 GB GB08531414A patent/GB2169403B/en not_active Expired
- 1985-12-24 BE BE0/216059A patent/BE903937A/en not_active IP Right Cessation
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- 1985-12-27 JP JP60299797A patent/JPH0669400B2/en not_active Expired - Lifetime
- 1985-12-29 HU HU855030A patent/HU196453B/en unknown
- 1985-12-30 FR FR858519394A patent/FR2575493B1/en not_active Expired - Lifetime
- 1985-12-30 AT AT0376785A patent/AT397514B/en not_active IP Right Cessation
- 1985-12-30 DE DE19853546312 patent/DE3546312A1/en active Granted
- 1985-12-30 IT IT23408/85A patent/IT1201514B/en active
- 1985-12-30 RO RO121637A patent/RO94651B/en unknown
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- 1985-12-31 NL NLAANVRAGE8503597,A patent/NL189427C/en not_active IP Right Cessation
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- 1985-12-31 IL IL77489A patent/IL77489A/en not_active IP Right Cessation
- 1985-12-31 IE IE333385A patent/IE58290B1/en not_active IP Right Cessation
- 1985-12-31 CA CA000498834A patent/CA1271705A/en not_active Expired - Lifetime
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1986
- 1986-01-02 SE SE8600011A patent/SE463212B/en not_active IP Right Cessation
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1303221C (en) * | 2003-01-27 | 2007-03-07 | 英科新创(厦门)科技有限公司 | Method for detecting target nucleic acid using affinity amplifying integrated signal group |
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