GB2169403A - Identification of nucleic acids - Google Patents

Identification of nucleic acids Download PDF

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Publication number
GB2169403A
GB2169403A GB08531414A GB8531414A GB2169403A GB 2169403 A GB2169403 A GB 2169403A GB 08531414 A GB08531414 A GB 08531414A GB 8531414 A GB8531414 A GB 8531414A GB 2169403 A GB2169403 A GB 2169403A
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nucleic acid
affinity pair
probes
probe
hybridization
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GB2169403B (en
GB8531414D0 (en
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Hans Erik Soderlund
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Orion Oyj
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Orion Yhtyma Oy
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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
    • 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

Abstract

A 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 labelled 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 homopolynucleotides, antigens-antibodies etc. The method is faster to perform than the traditional sandwich hybridization.

Description

SPECIFICATION A method for the identification of nucleic acids The invention relates to a method for the identification of nucleic acids by means of hybridization in solution.
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.
A new hybridization method in which two different probes are used, and both probes are in the solution phase has been developed. 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.
According to the present invention there is provided a method for the identification of nucleic acids, wherein at least two probes which are in the same solution phase are used in a hybridization method, the two probes being at least a detector probe labeled with a detactable label and a capturing probe having one member of an affinity pair attached, the capturing probe:target nucleic acid:detector probe hybrid formed in the hybridization reaction being isolated by means of the other member of the affinity pair.
In the method according to the invention an incubation time of around one hour is usually sufficient. In the single-step sandwich hybridization (US 4.486.539), sufficient hybridization is not achieved in fewer 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 preferably nucleic acid fragments sufficiently homologous to the targert nucleic acid.
It is advantageous, but not necessary, that the probes are homologous to sites located relatively close to each other in the nucleic acid to be identified. The probes must be nonoverlapping to one another.
The probes can be prepared synthetically, semisynthetically, 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 radioac lively labeled compounds may be used as the labels. The label substance may also be fluores cent, luminescent, light-emitting ,enzymatically or immunologically demonstrable, etc. Labels based on biotin and avidin or streptavidin, lanthanide chelates, ferritin and heme compounds, and immunologically demonstrable heptenes such as acetoxyacetylfluonene 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. All currently known label substances suitable for nucleic acid hybridization, or those developed in the future, can be freely applied to the method.
To the other probe, the so-called capturing probe a component having affinity for another component is attached. Biotin-avidin or streptavidin, heavy-metal derivatives-thio groups, vari ous homopolynucleotides, such as poly dG-poly dC, poly dA-poly dT, and poly dA-poly U are suitable affinity pairs. But also 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 is carried out the specimen is treated so 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 solutions. The hybridization takes place within the temperature range 0-80"C, but it is advantageous to use a temperature of 65"C. If the hybridization solution contains formamide (40-55%), a temperature of 37"C cn be used. An hour is sufficient as 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 surfaces, 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, celiulose, 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 surface. It is a prerequisite for the material selected that it is possible to affix to it a component having affinity for the component atached to the capturing probe.
If the specimen contains 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 fractionation.
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 l251-labeled recombinant phage mKTH 1206, which contains a Bgl Il-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 Mikroorganismen under the number DSM 2827. Its specific activity is 7X107 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 1202, which has been deposited in the culture collection Deutsche Sammlung von Mikroorganismen under the number DSM 2824, and it comprises a BamHl D fragment of the adenovirus (map position 29-42%) cloned into the plasmid pBR322. The recombinant plasmid pKTH 1202 (DSM 2824) was fragmented by using the restriction enzyme Hae Ill, and a poly A tail was linked to the 3' ends of the fragments by using terminal transferase enzyme. The length of the tail was measured by 3H-A incorporation.
The length was on average about 70 A-residues. Before being used the capturing probe was denatured by boiling.
The sample used consisted of adenovirus-infected A-549 cells. The cells were incubated for 21 hours after the infection. The cells were collected and lysed by using a 1% sodium dodecyl sulfate solution. The solution contained about 106 cells/ml. Its viscosity was lowered by sonication. Identically treated non-infected A-549 cells were used as controls. Before hybridization the specimen was boiled for 5 minutes in 0.02 M NaOH, cooled to O"C, and neutralized with acetic acid.
For the test, 500,000 cpm detector probe, 50 ng capturing probe DNA and 10 ,al specimen were combined in a test tube. The volume was adjusted to solid, 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 dodecyl sulfate. The mixture was incubated for 1 hour at 65"C.
After the hybridization the solution was cooled to +20"C and run slowly through a chromatography column having 1 ml oligo dT cellulose. The solution 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 sulfate. The attached DNA was finally detached by using 1 ml 0.02 M NaOH. This solution was recovered and its radioactivity was determined.
Result: 1251 activity (cpm) Target nucleic acid: infected cells Control cells 5230 325 Example 2 Identification of the DNA of Chlamydia trachomatis by the aid of biotin-streptavidin The detector probe used was l251-labeled recombinant phage mKTH 1245, which contains two BamHI-Sal IDNA 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 Microbiology Letters 23 pp. 83-89, 1984.
The capturing probe used was the recombinant plasmid pKTH 1250. This plasmid consists of a 2.9 kb Sall-Clal 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 nicktranslation method (Rigby et al. J.Mol.Biol. 113, pp. 237-251, 1977) and the biotin-1 1-UTP as the substrate (Bethesda Research Laboratories). The capturing probe DNA was boiled in a buffer comprising 10 mM Tris-CI 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 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 20C1l, and the buffer solution was the same as in Example 1. The control DNA was calf thymus PNA.
The mixture was incubated for 60 min at 650C. Thereafter, 500,ul of a buffer solution having the composition 0.1 M Tris-CI pH 7.5, 0.1 M NaCI, 2 mM MgCI2, 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.
1251 activity (cpm) Result: Target nucleic acid: 10 ng pkTH 1220 10 ng control DNA 1350 115 Example 3 Identification of the plasmid pBR322 DNA by the aid of an antigen-antibody pair The detector probe used is a derivative of the plasmid pBR322 (commercially available from several sources) from which the Pstl-Sall (3613-651) fragment has been removed. The plasmid was labeled with photobiotin using a known method (Forster et al. Nucleic Acids Res. 13, pp.
745-761, 1985) and commercial reagents (Bresa, Adelaide, Australia).
The capturing probe used was DNA from a recombinant phage M13mp11 into which the pBR322 Pstl-Sall 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 5x105 lysed E.coli cells (both with and without pBR322) were combined with 100 ng each of capturing DNA and detector probe in a 50/xl 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,us by the addition of 0.02 M sodium phosphate (pH 7.6), whereafter the solution was transferred to the antibody-coated microtiter well. This was followed by incubation for 2 h at 37"C. The well was then washed with a solution containing 0. 15 M sodium chloride, 0.02 M sodium phosphate pH 7.6 and 0.05% triton X-100. The presence of detector probe was visualized by addition of streptavidin (Bethesda Research Laboratories BRL), washing, addition of biotinylated alkaline phosphatase (BRL), and washing as described by Leary et al. Proc. Natl.
Acad. Sci. USA 80, pp. 4045-4049, 1983. Finally 250p1 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: A4tOnm Target nucleic acid: cells with pBR 322 Cells without pBR322 > 2 0.15

Claims (1)

1. A method for the identification of nucleic acids, wherein at least two probes which are in the same solution phase are used in a hybridization method, the two probes being at least a detector probe labeled with a detectable label and a capturing probe having one member of an affinity pair attached, the capturing probe: target nucleic acid: detector probe hybrid formed in the hybridization reaction being isolated by means of the other member of the affinity pair.
2. A method of probing nucleic acid comprising the steps of: i. hybridizing the nucleic acid to at least two probes contained in a solution phase, the probes being a detector probe labeled with a detectable label and a capturing probe having attached to it a moiety which is one member of an affinity pair, and ii. isolating the capturing probe: nucleic acid: detector probe hybrid thereby formed, by means of the other member of the affinity pair.
3. A method according to claim 1 or claim 2, wherein the affinity pair is biotin-streptavidin or biotin-avidin.
4. A method according to any preceding claim, wherein the affinity pair is a homopolynucleotide pair.
5. A method according to any of claims 1, 2 or 4, characterized in that the affinity pair is poly dC-dG.
6. A method according to any of claims 1, 2 or 4, wherein the affinity pair if poly dA-poly dT.
7. A method according to any of claims 1, 2 or 4, wherein the affinity pair is poly dA-poly U.
8. A method according to claim 1 or claim 2, wherein the affinity pair is a heavy-metal derivative-a thio group.
9. A method according to claim 1 or claim 2, wherein the affinity pair is an antigen-an antibody.
10. A method according to any preceding claim wherein the hybridizing step is completed in about 1 hour.
11. A method according to any preceding claim wherein the probes are nucleic acid fragments which are sufficiently homologous to the target nucleic acid to be probed that they will hybridize thereto.
12. A method according to any preceding claim wherein the two probes do not overlap with each other when hybridized to the target nucleic acid.
13. A method according to any preceding claim wherein at least one of the probes is bound to a vector.
14. A method according to any preceding claim wherein the detectable label is radioactive in nature.
15. A method according to any preceding claim wherein the nucleic acid is in single stranded form.
16. A method substantially as described herein with reference to any of the examples.
CLAIMS Amendments to the claims have been filed, and have the following effect: Claims 1 above have been deleted or textually amended.
New or textually amended claims have been filed as follows:
1. A method for the identification of nucleic acids, wherein at least two probes which are in the same solution phase are used in a hybridization method, including a detector probe labeled with a detectable label and a capturing probe having one member of an affinity pair attached, the capturing probe:target nucleic acid:detector probe hybrid formed in any hybridization reaction being isolated by means of the other member of the affinity pair.
GB08531414A 1985-01-02 1985-12-20 Identification of nucleic acids Expired GB2169403B (en)

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FI850004A FI72146C (en) 1985-01-02 1985-01-02 Procedure for Identifying Nucleic Acids.

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CA (1) CA1271705A (en)
CH (1) CH666696A5 (en)
DE (1) DE3546312A1 (en)
DK (1) DK164932C (en)
FI (1) FI72146C (en)
FR (1) FR2575493B1 (en)
GB (1) GB2169403B (en)
HU (1) HU196453B (en)
IE (1) IE58290B1 (en)
IL (1) IL77489A (en)
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LU (1) LU86238A1 (en)
NL (1) NL189427C (en)
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NL8503597A (en) 1986-08-01
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LU86238A1 (en) 1986-04-14
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FI850004L (en) 1986-07-03
GB2169403B (en) 1988-06-08
AU561382B2 (en) 1987-05-07
JPS61185200A (en) 1986-08-18
DK164932B (en) 1992-09-07
CA1271705A (en) 1990-07-17
NL189427C (en) 1993-04-01
JPH0669400B2 (en) 1994-09-07
AU5174885A (en) 1986-07-17
NO166743B (en) 1991-05-21
FR2575493A1 (en) 1986-07-04
AT397514B (en) 1994-04-25
IT1201514B (en) 1989-02-02
ATA376785A (en) 1993-09-15
BE903937A (en) 1986-04-16
DK164932C (en) 1993-01-25
ZA859895B (en) 1986-08-27
NL189427B (en) 1992-11-02
GB8531414D0 (en) 1986-02-05
DK386A (en) 1986-07-03
SE8600011L (en) 1986-07-03
NO166743C (en) 1991-08-28
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IL77489A (en) 1991-01-31
RO94651A (en) 1988-06-30
SE463212B (en) 1990-10-22
HUT40166A (en) 1986-11-28
FR2575493B1 (en) 1990-01-26
RO94651B (en) 1988-07-01
CH666696A5 (en) 1988-08-15
HU196453B (en) 1988-11-28
IT8523408A0 (en) 1985-12-30
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IE853333L (en) 1986-07-02
FI72146B (en) 1986-12-31
IE58290B1 (en) 1993-08-25

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