WO1996027680A1 - Detection d'acides nucleiques specifique aux sequences - Google Patents

Detection d'acides nucleiques specifique aux sequences Download PDF

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Publication number
WO1996027680A1
WO1996027680A1 PCT/EP1996/000893 EP9600893W WO9627680A1 WO 1996027680 A1 WO1996027680 A1 WO 1996027680A1 EP 9600893 W EP9600893 W EP 9600893W WO 9627680 A1 WO9627680 A1 WO 9627680A1
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WIPO (PCT)
Prior art keywords
nucleic acid
sequence
detected
nucleic acids
information
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PCT/EP1996/000893
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German (de)
English (en)
Inventor
Jörg KLEIBER
Henrik ØRUM
Ane-Ullerup Lester
Albert Geiger
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Boehringer Mannheim Gmbh
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Priority claimed from DE19548590A external-priority patent/DE19548590A1/de
Application filed by Boehringer Mannheim Gmbh filed Critical Boehringer Mannheim Gmbh
Priority to AU50029/96A priority Critical patent/AU5002996A/en
Priority to US08/894,808 priority patent/US6475721B2/en
Priority to EP96906732A priority patent/EP0813610A1/fr
Priority to JP08526599A priority patent/JP2000513921A/ja
Publication of WO1996027680A1 publication Critical patent/WO1996027680A1/fr

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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
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • 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
    • 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
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

Definitions

  • the invention relates to a solid support, to the surface of which two or more nucleic acid analogs of different base sequences are bound at predetermined locations, and to methods for the detection of nucleic acids using such a support.
  • nucleic acids can then be used to infer the presence of an infectious agent or the genetic condition of an organism.
  • Detections based on the presence of special nucleotide sequences have been made easier in particular by the fact that methods for amplifying nucleic acids, which are only present in small numbers, are now available.
  • the specific detection of nucleotide sequences still poses a great challenge to reagents and methods of carrying out analyzes based on the detection from Nuklein- acid sequences are based.
  • the nucleotide sequences to be detected are not known as such, but should only be determined by the nucleic acid detection.
  • EP-B-0-237 362 describes a method for the detection of nucleotide sequences of the HLA gene, with which a clinically relevant point mutation can be found.
  • an oligonucleotide which is bound to a membrane and which has a nucleotide sequence which is exactly complementary to one of the two nucleic acids to be distinguished is brought into contact with the sample. If certain conditions are met, only the exactly complementary nucleic acid binds to the solid-phase bound oligonucleotide and can be detected.
  • US Pat. No. 5,202,231 describes a method in which the sequence of a nucleic acid can theoretically be determined by bringing oligonucleotides with a predetermined and known sequence into contact with the sample of the unknown nucleic acid under hybridization conditions. All possible permutations of the nucleotide sequence must be immobilized at known locations on a solid phase. By determining at which locations the nucleic acid hybridizes with the sequence to be determined, it can theoretically be determined which sequences are present in the nucleic acid.
  • the main problem of the prior art is that the melting temperatures of the selected sequence-specific oligonucleotides differ with the nucleic acid to be sequenced or detected. This has to be optimized by elaborate selection of the length of the oligonucleotides, their base composition and optimization of the position of the mismatches within the oligonucleotide and the salt concentration of the hybridization mixture. In many cases, it is still practically impossible to be closely related Differentiate sequences from each other simultaneously.
  • the hybridization temperature is also critical. Even variations of 1 to 2 ° C can lead to modifications in the intensity or to false negative results. Faulty analysis results are very serious, especially in the diagnosis based on the presence of point mutations.
  • the object of the present invention was therefore to provide an alternative method for the sequence-specific detection of nucleic acids and materials suitable therefor.
  • the invention is achieved by a solid support, on the surface of which two or more nucleic acid analogs of different base sequences are bound at predetermined, different locations.
  • the invention also relates to a method for the sequence-specific detection of a nucleic acid using this solid support.
  • a solid support is understood to mean an object which has a surface which is so extensive that individual areas can be distinguished on it. This surface is preferably flat and larger than 5 mm 2 , preferably between 10 mm 2 and approximately 100 cm 2 .
  • the material of the carrier is not liquid and not gaseous and preferably does not dissolve or does not completely dissolve in sample liquids or reaction mixtures which are used for the immobilization of nucleic acids on the surface. Examples of such materials include glass, plastics (e.g. polystyrene, polyamide, polyethylene, polypropylene), gold or the like. However, the material does not necessarily have to be completely solid itself, but can be made firm, for example, by attachment to support materials.
  • the external shape of the solid support depends essentially on the manner in which the presence of nucleic acids on this solid support is to be detected. It has proven useful, for example, to choose an essentially planar shape, e.g. B. a tile.
  • Particularly suitable solid supports are, for. B. polystyrene plates with a thickness of about 1 to 5 mm, an area of about 1 to 5 cm 2 . Membranes made of polyamide with a size of 4 x 2.5 cm 2 have proven to be particularly suitable.
  • Two or more nucleic acid analogs of different base sequences are bound at different points on the surface of this support. These locations or areas preferably do not overlap together. They are preferably separated from one another by surface areas to which no nucleic acid analogs are bound. The locations at which the nucleic acid analogs are bound are referred to below as binding regions.
  • the binding regions can have different shapes, which are also essentially determined by the type of preparation of the solid support or by the method by which the nucleic acid analogs are bound in the binding regions.
  • the minimum size of the binding regions is essentially determined by the device with which the event of the binding of a nucleic acid to nucleic acid analogues of a region is detected. Devices already exist that can detect binding in areas of approximately 1 ⁇ m. An upper limit on the size of the binding areas results from economic reasons and the desire to be able to handle the solid support in a sensible manner.
  • the size of the binding areas results in particular from the methods by which the nucleic acid analogs are applied to the surface. Such procedures will be described later.
  • binding areas on the solid support depends on the intended use of the solid support. In the simplest case, only two binding areas are required for the detection of a specific point mutation.
  • One binding region contains nucleic acid analogs which contain a base at the position at which the point mutation is to be detected, which base is complementary to the base in the position of the normal sequence, while the other binding region contains a nucleic acid analogue which corresponds to the base sequence the position contains a base that is complementary to the base of the mutated sequence.
  • a solid support to the surface of which two nucleic acid analogs that are completely different from one another in the sequence are bound, can simultaneously detect two nucleic acids or nucleic acid sequences that are not very closely related to one another.
  • Non-naturally occurring molecules that can recognize nucleic acids via base pairings are understood as nucleic acids analog. They therefore contain a specific base sequence that is completely complementary to a base sequence of a nucleic acid to be detected.
  • the base sequence is therefore preferably composed essentially of the naturally occurring nucleobases. If the specificity of the base pairing is not lost, modifications to the nucleobases are also permitted.
  • Complementary to a nucleic acid are in particular nucleic acid analogs which have a base sequence which, in the state bound to the nucleic acid, forms water bridges to a base sequence of the nucleic acid according to the principle of base pairing. This sequence is preferably at least 8 bases long and particularly preferably between 8 and 25 bases long.
  • nucleic acid analogs are further defined by the fact that they differ structurally from nucleic acids, at least in the backbone.
  • a basic structure in nucleic acids or in a nucleic acid analog is understood to be a structure of essentially identical units, each of which contains a base bound. In the naturally occurring nucleic acids, the basic structure is a sugar phosphate basic structure. In nucleic acid analogs, this backbone is structurally modified, for example by completely or partially replacing the sugar or phosphate portion with other chemical units, e.g. B. non-cyclic components. In the basic structure, essentially identical units can also alternate.
  • nucleic acid analogs Some properties of nucleic acid analogs are given below, which are intended to facilitate the selection of nucleic acid analogs suitable for the present invention. It is an advantageous property of nucleic acid analogs if they have a higher affinity for sequence-complementary nucleic acids than an oligonucleotide with an identical base sequence. Furthermore, nucleic acid analogs are preferred which carry fewer charges than a corresponding oligonucleotide of the same length or which are able to compensate charges with opposite charges. Essentially uncharged nucleic acid analogs are particularly preferred. Particularly preferred nucleic acid analogs are those whose affinity for complementary nucleic acids essentially does not depend on the salt content of the hybridization mixture.
  • nucleic acid analogs are the compounds described in WO 92/20702 and WO 92/20703 (peptides nucleic acid, PNA, e.g. Nature 365, 566-568 (1993) and Nucl. Acids Res. 21, 5332-5 (1993)).
  • PNA peptides nucleic acid
  • PNA peptides nucleic acid
  • PNA nucleic acid
  • Preferred nucleic acid analogs are compounds which have a polyamide backbone which contains a multiplicity of bases bonded along the backbone, each base being bound to a nitrogen atom of the backbone.
  • nucleic acid analogs are also said to fall into compounds as described in EP-A-0 672 677.
  • nucleic acid analogs are described in Recueil 91, 1069-1080 (1971), Methods in Molecular Biology 29, 355-389 (1993), Tetrahedron 31, 73-75 (1975), J. Org. Chem. 52, 4202-4206 ( 1987), Nucl. Acids Res. 17, 6129-6141 (1989), Unusual Properties of New Polymers (Springer Verlag 1983), 1-16, Specialty polymers (Springer Verlag 1981), 1-51, WO 92/20823, WO 94/06815, WO 86/05518 and WO 86/05519. Further nucleic acid analogs are described in Proc. Natl. Acad. Be. USA 91: 7864-7868 (1994), Proc. Natl.
  • the nucleic acid analogs mentioned have a length of 8 to 30 bases, a length of 10 to 25 bases is particularly advantageous.
  • the nucleic acid analogs mentioned are bound directly or indirectly to the surface of the solid support. The type of binding essentially depends on which reactive groups on the solid support are available for binding and which reactive groups on the nucleic acid analog are available for binding without inhibiting the ability of the nucleic acid analog to bind to a complementary nucleic acid, and whether the nucleic acid analogs are to be simultaneously bound to different sites or to be built up at these sites.
  • Reactive groups on the surface of a solid support are usually selected from the group -OH, -NH 2 and SH.
  • Reactive groups of nucleic acid analogs are preferably selected from the group -OH, -NH 2 , -SH, -COOH, -SO 3 H and -PO 3 H 2 .
  • the reactive groups of the surface and the nucleic acid analog are particularly preferably covalently bonded to one another, in particular by means of a linker of more than 15 atoms and less than 200 atoms in length.
  • a linker is understood to mean a part of a molecule which essentially has the function of removing the nucleic acid analogs in a sterically accessible manner on the surface of the solid support.
  • the linker is usually selected so that it contains, in addition to carbon atoms (eg in alkylene units), a plurality of hetero atoms (eg -O- or -NH- or -NR-) which facilitate solvation.
  • the linker preferably contains one or more ethyleneoxy units and / or peptide groups.
  • the linker particularly preferably contains one or more units as described in DE-A 3924705. Particular preference is given to the units described by way of example, hereinafter referred to as ado (8-amino-3,6-dioxa-octanoic acid) be designated.
  • ado (8-amino-3,6-dioxa-octanoic acid) be designated.
  • the nucleic acid analog that is bound at one site can also be a mixture of two or more analogs of different but known sequences. This can reduce the number of digits required for a multiple determination.
  • the surface of the carrier is preferably not charged and is preferably hydrophilic. It has been shown as a result of the invention that the use of essentially uncharged surfaces is advantageous for the detection of nucleic acids.
  • a solid support loaded with nucleic acid analogs at different locations in the sense of the invention can be produced in different ways.
  • suitable amounts of solutions, each containing different nucleic acid analogs are applied to the surface of the solid support at different points, e.g. B. pipetted.
  • the amounts of liquid added should not mix here. This can be achieved, for example, by the fact that the feed points are far apart or the spreading of the liquid is stopped by a hydrophobic barrier between the different points.
  • Either the nucleic acid analogs or the surface of the solid support are preferably activated for the reaction.
  • Such activation can be carried out, for example, in that one of the abovementioned groups, by generating a reactive species, in the case of a carboxyl group, for example an activated ester (for example an N-hydroxysuccinimide ester), which rapidly binds an ester to a hydroxyl group without further activation is activated.
  • Suitable activated polyamide membranes carry, for example, triazm groups which can react with amino groups of nucleic acid analogs to form a covalent bond.
  • the activation can also take place via bifunctional reagents, squaric acid derivatives according to WO 95/15983 or by means of glutaraldehyde (GB 2197720).
  • the binding of the analogs can also be achieved by coating the support surface with nucleotide sequences that are complementary to part of the sequence the nucleotide analogs.
  • the different nucleic acid analogs can be bound to the binding regions simultaneously or in succession.
  • nucleic acid analogs successively from monomer units at the different locations on the surface.
  • the technology described in WO 92/10092 or WO 90/15070 can be used for this purpose.
  • Corresponding monomers are z. B. described in WO 92/20702.
  • the invention also relates to a method for the sequence-specific detection of a nucleic acid with the aid of the solid support according to the invention.
  • Detectable nucleic acids are natural or artificial nucleic acids. Nucleic acids are accordingly also understood to mean nucleic acid analogs. In particular, however, the nucleic acid to be detected is RNA or DNA, which is suitable for a nucleic acid-containing organism, e.g. B. a virus, a bacterium, a multi-cell, a plasmid or a genetic state, e.g. B. a predisposition or predisposition to a certain disease or spontaneous mutation in a gene are characteristic. These are essentially RNA and DNA, both genomic and derived from them. An important class of nucleic acids in the sense of the invention are the results of a nucleic acid amplification.
  • the nucleic acids can either be in their raw form or in a purified or processed form.
  • a purification can be present, for example, in that the nucleic acids are separated from cell components in a prepared step, for example by an affinity separation step.
  • the nucleic acids can be enzymatically extended, specifically amplified or transcribed.
  • the carrier according to the invention has a nucleic acid analog bound to one site which has a base sequence which is complementary to a base sequence of the nucleic acid to be detected.
  • This base sequence is chosen so that it allows a statement to be made about the specific presence of the nucleic acid. As a rule, this means that as little as possible, but preferably no further nucleic acid of the same total sequence is present in the mixture.
  • it must be established that it is also possible with the aid of the carrier according to the invention to specifically detect groups of nucleic acids.
  • the base sequence of the nucleic acid analog can be chosen so that it lies in a conserved area, but this sequence only occurs in members of this taxonomic group.
  • nucleic acid analog is preferably bound at a different location on the surface, which has a base sequence that is not complementary to the same base sequence. It can be a nucleic acid analogue whose base sequence can be shorter or longer or which differs in its base sequence from the first nucleic acid analogue in one or more bases.
  • the difference in the base sequence depends on the task to be solved. The differences may include e.g. B. point mutations, minor deletions and insertions. With some inherited diseases, e.g. B. cystic fibrosis, the sequences of the nucleic acid analogs differ in individual positions (point mutations) and several positions (deletions, see at ⁇ 508).
  • the mutation to be detected is preferably positioned near the center of the base sequence of the analog.
  • sequences of the analogs can also be chosen such that their hybridization positions differ by one base each (overlapping) while the length remains the same.
  • the sequences can also be chosen so that the hybridization areas are adjacent to the nucleic acid to be detected.
  • nucleic acids analogous the sequence of which is complementary to sequences on these nucleic acids.
  • two nucleic acid analogs are bound at different locations on the surface of the solid support, the base sequence of which differ exactly in position which also differentiate the alleles.
  • a nucleic acid analogue complementary to a certain sequence of the one alley and the other nucleic acid analogue complementary to the sequence of the other alien are chosen.
  • the length and hybridization site of the nucleic acid analogs are the same.
  • cystic fibrosis for example, detection of all alleles is usually carried out.
  • the wild-type contains two healthy alleles, heterozygotes contain a mutated and a wild-type sequence and homozygous mutants contain two mutant nucleic acids. In this case, it should not only be determined whether there are mutants, but whether it is a heterozygous or homozygous case. According to the present invention, it is possible to quantitatively detect both alleles simultaneously and thus differentiate between the three cases mentioned.
  • mutated cells / particles are often present in the background of non-mutated / normal cells.
  • the selective detection is not certain or not possible with methods of the prior art.
  • the analysis of ras mutations from DNA from stool requires the reliable detection of a mutated sequence in the presence of approximately 100 normal sequences (Science 256, 102-105, (1992)).
  • some of these mutants are less than 2% of all HIV sequences. It is therefore particularly possible with the method according to the invention to investigate mixtures of nucleic acids which are very similar to one another, even if one of the nucleic acids is in a very large excess compared to the nucleic acid to be determined.
  • the lengths of the bound nucleic acid analogs are preferably the same.
  • the selection of a length of between 10 and 100, preferably 10 and 50, bases has proven to be expedient. Particularly good results can be achieved if nucleic acid analogs with a length of between 10 and 25 bases are used.
  • the nucleic acid-containing sample is brought into contact with the sites on the surface of the support which have bound the nucleic acid analogs. This can be done, for example, by introducing the solid support into the sample liquid or pouring the sample liquid onto the solid support in one or more portions.
  • the nucleic acids of the sample liquid can be in a denatured (single-stranded) state before being brought into contact with the support.
  • a great advantage of the invention is, however, that, for. B. by using PNAs, denaturation before contacting can be avoided.
  • the PNAs displace a strand of possibly double-stranded nucleic acid to be determined. It is only essential that the contacting takes place under conditions in which the nucleic acid to be detected specifically binds to the relevant location on the surface via the nucleic acid analogs which are complementary to a sequence of the nucleic acid to be detected. Although these conditions can be different for different types of nucleic acid analogs, they can be obtained in a simple manner by testing for given nucleic acid analogs. As a rule, these conditions will be based on the conditions known for carriers loaded with oligonucleotides.
  • nucleic acid analogs according to WO 92/20702 are used, however, conditions can be selected which are significantly different from the hybridization conditions of corresponding oligonucleotides.
  • the presence of less than 100 mM, particularly preferably less than 50 mM and very particularly preferably less than 10 mM salts should be emphasized. Under these conditions, sufficient differentiation between sequence-like nucleic acids could not be achieved with oligonucleotides of the same sequence.
  • the sample is kept in contact with the surface for as long as is necessary for sufficient binding of the nucleic acid to the relevant point on the surface. This period is usually in the range of a few minutes.
  • nucleic acid It is then determined whether and at what point a nucleic acid has bound to the surface. This is regarded as a sign of the presence of a nucleic acid which is an analogue of the nucleic acid bound at this point contains complementary base sequence.
  • the determination of the binding that has taken place can be done in different ways.
  • the nucleic acid to be detected can be bound to the nucleic acid analog in an advantageous manner by detecting a mark made in the nucleic acid to be detected in a step prior to contacting the surface.
  • This can be, for example, a detectable group, e.g. B. act a fluorescent residue.
  • This determination can be made optically using a microscope or in a measuring cell provided for this purpose. While the location of the binding that took place is an indication of the presence of a nucleic acid with a certain sequence, the amount of label at a predetermined location can be used as a sign of the amount of the presence of the nucleic acid to be detected.
  • the nucleic acids to be detected are particularly preferably results of a nucleic acid amplification reaction such as the polymerase chain reaction according to EP-B-0 202 362 or NASBA according to EP-A-0 329 822. It is essential for the amplification that the nucleic acid sequence, the is intended for binding to the nucleic acid analogs, is amplified by the amplification process. The more true to the sequence the amplification takes place, ie the fewer errors are built into the sequence during the amplification, the more suitable the amplification method is.
  • the polymerase chain reaction has proven to be particularly suitable.
  • the primers for the amplification are chosen such that the nucleic acid sequence to be detected lies in the area between the primer hybridization sites.
  • a direct detection of the binding that has taken place without incorporation of a label is possible, for example, by using an intercalating agent.
  • intercalating agents have the property of being selectively incorporated into double-stranded base-containing compounds, including the complex of the nucleic acid analogs and the sequence-specifically bound nucleic acid.
  • the intercalating agents e.g. B. a fluorescence
  • a particularly suitable agent is ethidium bromide.
  • the surface is coated with a solution of a detectably labeled antibody against the complex of nucleic acid analogs. and the nucleic acid to be detected.
  • a detectably labeled antibody against the complex of nucleic acid analogs. and the nucleic acid to be detected are described, for example, in WO 95/17430.
  • the detection of the hybrid depends on the type of marking. It can be, for example, a scanner, a CCD camera or a microscope.
  • the solid support is used to detect known mutations and polymorphisms.
  • the number of mutations and polymorphisms to be determined is one Measure of the required number of different nucleic acid analogs or sites on the surface.
  • the sequence of the nucleic acid analogs is specially matched to the sequence of the nucleic acids around the mutations and polymorphisms.
  • the sequences are preferably chosen so that the base, in which sequence-like nucleic acid analogs differ, lies in or near the center of the sequence.
  • Oncology detection of mutations in tumor suppressor and oncogenes and determination of the ratio of mutated to normal cells.
  • hereditary diseases cystic fibrosis, sickle cell anemia, etc.
  • a sequence of short nucleic acid fragments can be determined using the so-called sequencing by hybridization method.
  • sequencing by hybridization method for this purpose, as many different nucleic acid analogs are immobilized as there are per mutations from the selected length of the sequence.
  • 4 N sites are required if N is the number of bases in each nucleic acid analog.
  • N is preferably between 5 and 12.
  • Correspondingly fewer sites are required for the sequencing of very short DNA fragments.
  • the method for sequencing unknown nucleic acids by sequencing by hybridization is described in WO 92/10588.
  • An advantage of the present invention is that the specificity of the hybridization is largely independent of the conditions in the sample. This facilitates the simultaneous binding of nucleic acids to different areas of the surface.
  • nucleic acid analogs such as PNA
  • PNA nucleic acid analogs
  • the carriers according to the invention are even suitable for determining nucleic acids several times in succession.
  • the carrier is subjected to a heat treatment in contact with a liquid.
  • the temperature chosen is one at which the binding of the nucleic acid analog to the optionally bound nucleic acid is released.
  • the carrier is then available for a new determination.
  • FIG. 1b shows the sequences of the DNA molecules which are homologous to the PNA sequences from FIG. la and were used for the DNA / ODN hybridization experiments.
  • ODN complementary oligonucleotides
  • FIG. 2 shows the hybridization results from Example 4, which are intended to illustrate the selectivity of the method.
  • the conditions were: 200 nl spot volume (100; 10; 1; 0, 1 ⁇ M PNA, one concentration per column), incubation at 45 ° C.
  • FIG. 3 shows the hybridization results from Example 6, which demonstrates the detection of PCR amplicons by immobilized PNA probes.
  • the conditions were: 200 nl spot volume (100; 10; 1; 0.1 ⁇ M PNA, one concentration per column) incubation at 45 ° C. Also mean:
  • ODN la (lpMol, InM), line 1 (PNA1), line 2 (PNA2) line 3 (PNA3)
  • FIG. 4 shows the results of the qualitative and quantitative analysis of analyte mixtures by PNA arrays.
  • the conditions were: 200 nl spot volume (100 ⁇ M PNA, one of the PNA per line, one analyte mixture per column).
  • FIG. 5 shows the influence of linker length on hybridization.
  • the conditions were: 1 ⁇ l spot volume (100; 40; 20; 10; 5; 1 ⁇ M PNA, one concentration per column, (Ado) 3 - PNA in row 1, (Ado) ⁇ -PNA in row 2, (Ado) o-PNA in row 3).
  • 1 ⁇ l spot volume 100; 40; 20; 10; 5; 1 ⁇ M PNA, one concentration per column, (Ado) 3 - PNA in row 1, (Ado) ⁇ -PNA in row 2, (Ado) o-PNA in row 3).
  • Ado 1 ⁇ l spot volume
  • FIG. 6a-6c demonstrate the possibility of using PNA-derivatized membranes several times after regeneration.
  • the conditions were: 1 ⁇ l spot volume (100; 40; 20; 10; 5, 1 ⁇ M PNA, one concentration per column).
  • 1 ⁇ M PNA one concentration per column.
  • FIG. 6 mean:
  • Membrane 2 regeneration with 0. IM sodium hydroxide solution, RT In, 2x 10min. bidest.
  • Water RT membrane 3 regeneration with IM sodium hydroxide solution, RT lh, 2x 10min. bidest.
  • Water RT membrane 4 regeneration with dist. Water, 70 ° C lh, 2x 10min. bidest.
  • Water RT membrane 5 regeneration with 0. IM sodium hydroxide solution, 70 ° C lh, 2x 10min. bidest. Water RT
  • nucleic acid analogs used were prepared in accordance with WO 92/20702. Unless otherwise specified, chemicals and reagents were from Boehnnger Mannheim GmbH.
  • the membrane is derivatized using 100 ⁇ M, 10 ⁇ M 1 ⁇ M and 0.1 ⁇ M PNA solutions as described in Example 1. It is then prehybridized in a 50 ml hybridization vessel with 10 ml hybridization buffer (10 mM sodium phosphate pH 7.2, 0.1% SDS (sodium dodecyl sulfate)) in the hybridization oven at 45 ° C. After 30 min. 10 ⁇ l of a solution which contains DIG-labeled oligonucleotide in a concentration of 1 ⁇ M are added, and the mixture is stirred for a further 60 min. hybridizes. Then 2 x 10 min.
  • 10 ml hybridization buffer 10 mM sodium phosphate pH 7.2, 0.1% SDS (sodium dodecyl sulfate)
  • the membrane is derivatized using 100 ⁇ M, 10 ⁇ M and 1 ⁇ M PNA solution as described in example 1.
  • the membrane is prehybridized in a 50 ml screw vessel with 10 ml hybridization buffer (cf. Example 2) in the oven at 45 ° C. After 30 min. 10 ⁇ l of a solution which contains a fluorescence-labeled oligonucleotide in a concentration of 1 ⁇ M are added, and there are a further 60 min. hybridizes.
  • the membrane is then 2 x 10 min. washed with 25 ml wash buffer (see Example 2) at 45 ° C. The fluorescence intensities are measured after the membrane has dried.
  • Three membrane strips are each with three (AdoVPNA molecules that differ in their base sequence at one or two positions (see FIG. La, SEQ.ID.NOS. 1, 2, 3)) using PNA solutions in the Concentration range between 100 ⁇ M and 0.1 ⁇ M derivatized analogously to example 1.
  • the membrane strips are prehybridized in 50 ml screw-top containers with 10 ml hybridization buffer for 30 min. Then one of the three DIG-labeled oligonucleotides (FIG. 1b, SEQ.ID.NOS 4, 5, 6) After 60 minutes of hybridization, 2 ⁇ 10 minutes are washed with 25 ml of washing buffer each, and the hybridization events are demonstrated as described in Example 2.
  • Hybrid PNA / ODN
  • Hybrid S / N signal (hybrid) / signal (match)
  • Membrane strips are derivatized with three (AdoVPNA molecules, which differ in their base sequence (FIG. La, SEQ.ID.NOS. 1, 2, 3), in a concentration of 100 ⁇ M analogously to Example 1. They are then in 20 ml Hybridization tubes with 10 ml hybridization buffer (cf. Example 2) prehybridized at 45 ° C. The buffer is changed after 30 min .. In experiments 1 to 7, the added buffer differs in the analyte concentrations of the DIG-labeled components oligonucleotide 1, 2 and 3, SEQ.ID.NOS. 4, 5, 6.
  • Example 2 After 60 minutes of hybridization at 45 ° C., 2 ⁇ 10 minutes are washed with 10 ml of washing buffer and the detection of a hybridization event is carried out as in Example 2. The luminescence signal is also monitored recorded by a luminescence imager and then evaluated (FIG. 4).
  • the signal intensities found enable both a qualitative and semi-quantitative statement about the composition of the analyte mixture. Absolute quantitative statements are possible after calibration of the signal intensities.
  • a double-stranded DNA fragment is ligated into a pUC19 plasmid, the sequence of which is complementary to the PNA probe PNA1.
  • the plasmid is transformed into E. coli, cloned and then sequenced.
  • a section of the plasmid sequence is amplified and DIG- marked during the amplification reaction. The amplification is carried out in a total volume of 50 ⁇ l.
  • the amplification mixture consists of 1 ⁇ l plasmid (1 ng / ⁇ l), 1 ⁇ l primer Fl (10 ⁇ M), 1 ⁇ l DIG primer R1 (10 ⁇ M), 5 ⁇ l 10 ⁇ PCR buffer (100 mM Tris / HCl, 15 mM MgCl 2, 500 mM KC1, pH 8.3), 2 ⁇ l dNTP solution (10 mM dATP, 10 mM dCTP, 10 mM dGTP, 10 mM dTTP in distilled water pH 7.0), 0.5 ⁇ l Taq polymerase (5 unit / ⁇ l) and 38.5 ⁇ l water .
  • Primer Fl 5'-GTA AAA CGA CGG CCA GT-3 '(SEQ.ID.NO. 12)
  • Each reaction batch is 3 min. heated to 96 ° C and then 30 rounds of a 3-step PCR cycle are carried out (45 sec. 96 ° C, 30 sec. 48 ° C, 1 min. 72 ° C). In the last cycle, the elongation step is extended by 5 minutes at 72 ° C.
  • the membranes are each with three (AdoVPNA sequences, which differ in their base sequence at one or two positions (see FIG. La, SEQ.ID.NOS. 1, 2, 3), using PNA solutions in Concentration range between 100 ⁇ M and 0.1 ⁇ M derivatized analogously to Example 1.
  • the membrane is pretreated in a 20 ml hybridization vessel with 5 ml hybridization buffer at 45 ° C.
  • the buffer is changed and the analyte solution is added of the analyte solution, the amplification mixture is diluted directly (ds Amplicon) or after 5 min heat denaturation (ss Amplicon) in 1 ml hybridization buffer, after 1 h, 2 h 30 min or 4 h hybridization at 45 ° C, 2 x 10 min. washed with 5 ml of washing buffer each Hybridization events are detected as described in Example 2 (FIG. 3).
  • FIG. 3 9 fields can be seen.
  • the rows differ in the length of the incubation period (4 h, 2 l ⁇ h and 1 h).
  • the columns differ in the type of nucleic acid to be detected.
  • In each of the 3 fields of column I there are 3 rows of spots on top of each other.
  • the rows differ in the sequence of the PNAs, while the columns of each field differ in concentration. Both the specificity and the quantifiability in the case of oligonucleotides as the detecting nucleic acid are shown in column I.
  • the influence of the incubation time can be seen in the figure. It is clear that an excellent sequence discrimination for ODN la and the amplificates is achieved even with one hour's hybridization.
  • Membrane strips are each with three (Ado) 6 -PNA sequences (SEQ.ID.NOS. 1, 2, 3) and three DNA molecules (SEQ.ID.NOS. 8, 10, 11), which differ in their base sequence differentiate at one or two positions (see FIG. la and lb), derivatized using 50 ⁇ M solutions analogous to Example 1.
  • the spot volume is 400 nl instead of 200 nl.
  • the membrane strips are placed in 20 ml hybridization vessels with either 5 ml of low salt buffer (see example 2) or high salt buffer (6 x SSC: 0.9 M NaCl, 90 mM sodium citrate, 0.1% SDS, pH 7.0) at 37 ° C or alternatively 45 ° C 30 min. pre-hybridized.
  • Both the DNA and the PNA probes are able to distinguish completely complementary single-stranded target sequences from single and double mismatched sequences.
  • ODN1 PNA 45'C low salt 100.0% 1.2% 1.5% DNA 37 * C, high salt 100.0% 1.2% 6.4%
  • Membrane strips are made with PNA molecules (see FIG. La, SEQ.ID.NOS. 7, 1, 9), which differ in the length of the linker (Ado 3 , Ado 6 and Ado 9 ), using PNA - Derivatized solutions in the concentration range between 100 uM and 1 uM analogous to Example 1.
  • the spot volume is 1 ⁇ l instead of 200 nl.
  • the membrane strips are in hybridization vessels with 10 ml hybridization buffer (5, 10 or 25 mM sodium phosphate, 0.1% SDS, pH 7.0) for 30 min. Prehybridized at 35 ° C. Then 10 pmol 3 P-labeled oligonucleotide (see FIG.
  • a membrane is derivatized with (Ado) 6 -PNA molecules (see FIG. La: PNA lb, SEQ.ID.NO. 1) using PNA solutions in the concentration range between 100 ⁇ M and 1 ⁇ M analogously to Example 1.
  • the PNA is applied in five identical concentration series.
  • the spot volume is 1 ⁇ l.
  • the membrane is in a hybridization vessel with 10 ml hybridization buffer (10 mM sodium phosphate, 0.1% SDS, pH 7.0) for 30 min. Prehybridized at 35 ° C. Then 10 pmol 3 P-labeled oligonucleotide (see FIG. 1c: ODN 1b, SEQ.ID.NO. 4) are added and 60 min. hybridized at 50 ° C.
  • the membrane is 2 x 10 min. washed with 50 ml washing buffer (5 mM sodium phosphate, 0.1% SDS, pH 7.0) at 50 ° C. Hybridization events are detected by autoradiography. (FIG. 6a). After autoradiography, the membrane is cut into five identical strips. These membrane strips are treated differently in the course of the further experiment.
  • Membrane 1 is not incubated and serves as a control membrane, membrane 2 becomes 60 min. incubated at room temperature with 50 ml of 0.1 M sodium hydroxide solution, membrane 3 for the same period with 50 ml of 1 M sodium hydroxide solution, membrane 4 becomes 60 min. at 70 ° C with 50 ml dist. Incubated water and membrane 5 is 60 min.
  • FIG. 6b shows very different results are obtained through the different treatment methods.
  • Treatment with bidest water at 70 ° C (membrane 4) causes the membrane to regenerate almost completely.
  • the success of the regeneration is worse if conditions are used that i.a. are common for denaturing nucleic acids.
  • Incubation of membrane 3 with 1 M sodium hydroxide solution at room temperature thus has hardly any regeneration effect.
  • Lowering the sodium hydroxide concentration from 1 M to 0.1 M increases the degree of regeneration both at room temperature (membrane 2) and 70 ° C (membrane 5).
  • none of these conditions leads to an approximately good degree of regeneration, as is the case with bidest. Water is the case (membrane 4).
  • the example shows that these conditions are important parameters for the efficient denaturation of membrane-bound PNA / DNA double strands.

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Abstract

L'invention concerne un procédé permettant de détecter des mutations ponctuelles et des polymorphismes dans des acides nucléiques, et de séquencer des acides nucléiques inconnus au moyen d'un procédé simple utilisant des séries. Selon ce procédé, on utilise des analogues d'acides nucléiques sous forme de discrimateurs de séquence, ce qui facilite la technique opératoire même en cas de problèmes complexes.
PCT/EP1996/000893 1995-03-04 1996-03-04 Detection d'acides nucleiques specifique aux sequences WO1996027680A1 (fr)

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AU50029/96A AU5002996A (en) 1995-03-04 1996-03-04 Sequence-specific detection of nucleic acids
US08/894,808 US6475721B2 (en) 1995-03-04 1996-03-04 Sequence specific detection of nucleic acids using a solid carrier bound with nucleic acid analog probes
EP96906732A EP0813610A1 (fr) 1995-03-04 1996-03-04 Detection d'acides nucleiques specifique aux sequences
JP08526599A JP2000513921A (ja) 1995-03-04 1996-03-04 核酸の配列特異的検出

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EP95118843.2 1995-11-30
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DE19548590A DE19548590A1 (de) 1995-12-23 1995-12-23 Sequenzspezifischer Nachweis von Nukleinsäuren
DE19548590.4 1995-12-23

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EP0742287A2 (fr) * 1995-05-10 1996-11-13 McGall, Glenn H. Sondes d'acide nucleique modifiés
WO1999005320A1 (fr) * 1997-07-22 1999-02-04 Rapigene, Inc. Fonctionnalites multiples a l'interieur d'un element matriciel et utilisations en decoulant
WO2001001144A2 (fr) * 1999-06-30 2001-01-04 Iris Bio Technologies Hybridation d'adn cible avec des analogues d'acide nucleique immobilises
WO2001046461A2 (fr) * 1999-12-22 2001-06-28 Biochip Technologies Gmbh Acides nucleiques modifies et leur utilisation
WO2001071039A2 (fr) * 2000-03-20 2001-09-27 Incyte Genomics, Inc. Sequences polynucleotidiques combinees en tant que resultats d'essais discrets
US6306588B1 (en) 1997-02-07 2001-10-23 Invitrogen Corporation Polymerases for analyzing or typing polymorphic nucleic acid fragments and uses thereof
US6365349B1 (en) 1997-07-22 2002-04-02 Qiagen Genomics, Inc. Apparatus and methods for arraying solution onto a solid support
US6458530B1 (en) 1996-04-04 2002-10-01 Affymetrix Inc. Selecting tag nucleic acids
US6528256B1 (en) 1996-08-30 2003-03-04 Invitrogen Corporation Methods for identification and isolation of specific nucleotide sequences in cDNA and genomic DNA
WO2003038122A2 (fr) * 2001-10-26 2003-05-08 Febit Ag Sondes asymetriques
US7108971B2 (en) 1999-05-14 2006-09-19 Iris Biotechnologies, Inc. Reversible binding of molecules to metal substrates through affinity interactions
US7375198B2 (en) 1993-10-26 2008-05-20 Affymetrix, Inc. Modified nucleic acid probes
US8017758B2 (en) * 2002-03-21 2011-09-13 Boston Probes, Inc. PNA oligomers, oligomer sets, methods and kits pertaining to the detection of Bacillus anthracis

Families Citing this family (1)

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JP4524392B2 (ja) * 2006-04-25 2010-08-18 国立大学法人 千葉大学 プローブポリヌクレオチド固定化担体の再生方法

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WO1989011545A1 (fr) * 1988-05-17 1989-11-30 Institute For Animal Health Limited Detection de sensibilite a l'encephalopathie spongiforme bovine
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WO1993025706A1 (fr) * 1992-06-05 1993-12-23 Buchardt, Dorthe Utilisations d'analogues d'acide nucleique pour inhiber les procedures d'amplification de l'acide nucleique
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Cited By (21)

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Publication number Priority date Publication date Assignee Title
US7375198B2 (en) 1993-10-26 2008-05-20 Affymetrix, Inc. Modified nucleic acid probes
US7794943B2 (en) 1993-10-26 2010-09-14 Affymetrix, Inc. Modified nucleic acid probes
EP0742287A2 (fr) * 1995-05-10 1996-11-13 McGall, Glenn H. Sondes d'acide nucleique modifiés
EP0742287A3 (fr) * 1995-05-10 1997-12-29 McGall, Glenn H. Sondes d'acide nucleique modifiés
US6458530B1 (en) 1996-04-04 2002-10-01 Affymetrix Inc. Selecting tag nucleic acids
US6528256B1 (en) 1996-08-30 2003-03-04 Invitrogen Corporation Methods for identification and isolation of specific nucleotide sequences in cDNA and genomic DNA
US7501237B2 (en) 1997-02-07 2009-03-10 Life Technologies Corporation Polymerases for analyzing or typing polymorphic nucleic acid fragments and uses thereof
US6306588B1 (en) 1997-02-07 2001-10-23 Invitrogen Corporation Polymerases for analyzing or typing polymorphic nucleic acid fragments and uses thereof
US6365349B1 (en) 1997-07-22 2002-04-02 Qiagen Genomics, Inc. Apparatus and methods for arraying solution onto a solid support
AU742599B2 (en) * 1997-07-22 2002-01-10 Qiagen Genomics, Inc. Multiple functionalities within an array element and uses thereof
WO1999005320A1 (fr) * 1997-07-22 1999-02-04 Rapigene, Inc. Fonctionnalites multiples a l'interieur d'un element matriciel et utilisations en decoulant
US7108971B2 (en) 1999-05-14 2006-09-19 Iris Biotechnologies, Inc. Reversible binding of molecules to metal substrates through affinity interactions
WO2001001144A3 (fr) * 1999-06-30 2001-05-03 Iris Bio Technologies Hybridation d'adn cible avec des analogues d'acide nucleique immobilises
WO2001001144A2 (fr) * 1999-06-30 2001-01-04 Iris Bio Technologies Hybridation d'adn cible avec des analogues d'acide nucleique immobilises
WO2001046461A3 (fr) * 1999-12-22 2002-05-23 Biochip Technologies Gmbh Acides nucleiques modifies et leur utilisation
WO2001046461A2 (fr) * 1999-12-22 2001-06-28 Biochip Technologies Gmbh Acides nucleiques modifies et leur utilisation
WO2001071039A3 (fr) * 2000-03-20 2002-10-10 Incyte Genomics Inc Sequences polynucleotidiques combinees en tant que resultats d'essais discrets
WO2001071039A2 (fr) * 2000-03-20 2001-09-27 Incyte Genomics, Inc. Sequences polynucleotidiques combinees en tant que resultats d'essais discrets
WO2003038122A3 (fr) * 2001-10-26 2004-03-25 Febit Ag Sondes asymetriques
WO2003038122A2 (fr) * 2001-10-26 2003-05-08 Febit Ag Sondes asymetriques
US8017758B2 (en) * 2002-03-21 2011-09-13 Boston Probes, Inc. PNA oligomers, oligomer sets, methods and kits pertaining to the detection of Bacillus anthracis

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JP2000513921A (ja) 2000-10-24
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AU5002996A (en) 1996-09-23
CA2214430A1 (fr) 1996-09-12

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