CA2214430A1 - Sequence-specific detection of nucleic acids - Google Patents
Sequence-specific detection of nucleic acids Download PDFInfo
- Publication number
- CA2214430A1 CA2214430A1 CA002214430A CA2214430A CA2214430A1 CA 2214430 A1 CA2214430 A1 CA 2214430A1 CA 002214430 A CA002214430 A CA 002214430A CA 2214430 A CA2214430 A CA 2214430A CA 2214430 A1 CA2214430 A1 CA 2214430A1
- Authority
- CA
- Canada
- Prior art keywords
- nucleic acid
- sequence
- detected
- site
- nucleic acids
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The invention concerns a method of detecting point mutations and polymorphisms in nucleic acids and of sequencing unknown nucleic acids by means of a simple method using arrays. According to the method, nucleic acid analogues are used as sequence discriminators. This method facilitates the operating method, even in complex cases.
Description
Sequence-Specific Detection of Nucleic Acids Subject matter of the invention is a solid carrier having two or more nucleic acid analogs with di~erenl base sequences bound to predetermined sites on its surface. The invention also addresses a method for the detection of nucleic acids using a carrier of this nature.
Sample analysis has undergone rapid development in recent dec~des While analytes were initially detected primarily by means of their reaction with conventional chemical re~gent~, and later on with enzymes, tests that utilize the immlln~logical characteri~tics of the analyte have become the standard recently, especially in m~lic~ p;nostics. This is especially true in the field of infectious diseases. However, immllnf~logical procedures can basically only detect analy-tes with which imm-lnologically active compounds such as antigens or antibodies play a role. These procedures have resulted in promising poten~ial applications for many infections caused by viruses or bacteria. Genetic ~ e~eS or predispositions that are not expressed as a change in protein patterns--or only to an in~llffici~nt extent--are either diffi'-,lllt or impossible to detect using immllnological procedures, however. Nucleic acids have therefore ~ecell~ly become the object of detectinn in many cases. The presence of certain nucleic acids can infer the presence of an infectious agent or the genetic condition of an organism. Detection procedures based on the presence of special nucleotide sequences in particular were f~ilit~ted recently when methods for the amplification of nucleic acids that are present in small numbers became available. Due to the large quantity of sequence information and the fact that two nucleotide sequences with completely di~erellL functions often differ by just one base unit, the specific detection of nucleotide sequences still poses a considerable ch~ nP~e for reagents and analytical methods that are based on the detection of nucleic acid sequences. In addition, the nucleotide sequences are often not even known, but rather are det~rmined for the first time in the nucleic acid detectinn method itself.
A method for the detection of nucleotide sequences of the E~A gene is described in EP-B-0 237 362 with which a clinically relevant point mutation can be detected In this 4300ENGL.DOC
method, an oligonucleotide that is bound to a membrane and has a nucleotide sequence that is exactly complçm~nt~ry to one of the two nucleic acids to be di~er~ ted is brought in contact with the sample. While certain conditions are ..~ e~l~ only that nucleic acid that is exactly complementary binds to the oligoml~.lçoti(le that is bound to the solid phase, and can be detected.
A method is described in Proc. Natl. Acad. Sci. USA 86, 6230-6234 (1989) in which a large number of oligonucleotides that are bound to di~relenl, predeterminecl sites of a nylon membrane by means of poly-dT are used for the simlllt~neous detection of all known allelic variants of an amplified region of a nucleic acid.
A method is described in US-A 5,202,231 in which the sequence of a nucleic acid can be det~rrnined theoretically by bringing oligonucleotides having a predetermined, known sequence in contact with a sample of the unknown nucleic acids under hybridization conditions. This requires that all possible permutations of the nucleotide sequence be immobilized on known sites of a solid phase. By determining the sites to which the nucleic acids cont~ining the sequence to be deterrnined hybridize, it can theoretically be determined which sequences are present in the nucleic acid.
Prior art in the field of the analysis of genetic polymorphisms using "oligonucleotide arrays"
is described in Nucleic Acids Research 22, 5456-5465 (1994) and Clin. Chem. 41/5, 700-6 (1995).
The main problem with the prior art is the fact that the melting temperatures of the selected sequence-specific oligonucleotides cnnt~inin~ the nucleic acids to be sequenced or detected are di~ele"~. To remedy this sit-l~tiQn~ one has to perform the complex method of selecting the length ofthe oligonucleotide and its base composition, and opl;...;,;i-g the position of the mi.cm~trhes within the oligonucleotide as well as the salt concentration of the hykri~i7~tion complex. In many cases, however, it is practically impossible to .~imlllt~n~.ously tli.~tin~ h closely related sequences from each other. The hybridization temperature is another critical parameter. Variations of as little as 1 to 2 ~C can change the 430WiNGLDOC
intensity or produce false-negative results. Incorrect analytical results based on the presence of point mutations have serious impliç~tiQn~ for diagnosis.
The object of this invention was, thereIore, to provide an ~ltern~tive method for the sequence-specific detection of nucleic acids and to provide suitable m~teri~l~ for this method.
This object was accomplished by providing a solid carrier having two or more nucleic acid analogs with di~ele-l~ base sequences bound to predetermined sites on its surface. Another object of the invention is a method for the sequence-specific detection of a nucleic acid using this solid carrier.
A"solid carrier" as described by this invention refers to an object that has a surface that is so broad that specific areas can be di~tin~lish~d upon it. This surface is preferably flat and larger than 5 mmZ, and is preferably between 10 mm2 and approx. 100 cm2. The carrier material is not liquid or gaseous, and preferably dissolves either not at all or incompletely in the sample fluids or reaction preparations that are used to immobilize nucleic acids to the surface. Examples of such materials are glass, plastics (e.g. poly~Lyl~i, e, polyamide, polyethylene, polypropylene), gold, etc. The m~teri~l does not n~cess~rily have to be completely solid itself, but rather can be made solid by the ~tt~hmPnt of supporting m~t~ri~
The external shape of the solid carrier basically depends on the method used to detect the presence of nucleic acids on this solid carrier. It has proven to be applopliate, for in~t~nc.
to select a basically planar form, e.g. a chip.
Solid carriers that are especially suitable are, therefore, polystyrene chips that are from 1 to 5 mm thick and have a surface area of from 1 to 5 cm2, for in~t~nce Polyamide membranes that are 4 x 2.5 cm2 in size have proven to be especially well-suited for use with this invention. Two or more nucleic acid analogs having different base sequences are bound to different sites of the surface of this carrier. These sites or regions preferably do not overlap with each other. They are preferably separated from each other by regions on the surface to 43~EN~.DOC
which no nucleic acid analogs are bound. The sites to which the nucleic acid analogs are bound are referred to as "binding regions" below. The binding regions can have di~renl shapes. These shapes are basically det~rmin~d by the method of m~mlf~ctllring the solid carrier or by the method used to det~ormine the nucleic acid analogs in the binding regions.
The minimllm size of the binding regions is basically detprmined by the instrument with which the event--the binding of a nucleic acid to nucleic acid analogs of a region--is detecte~l Instruments are already available that can detect binding to regions that are approx. 1 mm in size. The upper limit of the size of the binding regions is determined by cost effectiveness and h~ntlling considerations.
The size of the binding regions is also basically determined by the methods used to apply the nucleic acid analogs to the surface. Such methods will be described later.
The number of binding regions on the solid carrier depends on the intended use of the solid carrier. In the simplest case, just two binding regions are needed to detect a certain point mutation. In this case, a binding region contains nucleic acid analogs that have a base in the position at which the point mutation is to be detected This base is compl~m~ont~ry to the base in the position of the normal sequence. The other binding region, on the other hand, contains a nucleic acid analog that has a base in the corresponding position that is complement~ry to the base ofthe ml-t:~ted sequence. In another case, two nucleic acids or nucleic acid sequences that are only slightly related to each other can be detected ~iml~lt~neously using a solid carrier that has two completely di~e~ nucleic acid analogs bound to its surface.
"Nucleic acid analogs" refer to non-naturally occurring molecules that can detect nucleic acids by means of base pairings. They therefore contain a specific base sequence that is completely complem~nt~ry to the base sequence of a nucleic acid to be detecte~1 The base sequence is therefore preferably composed of two naturally occllrring nucleobases. As long as the specificity of the base pairing is not lost, modifications to the nucleobases are also allowed, however.
430(ENGL~DOC
Those nucleic acid analogs are considered compl~ment~ry to a nucleic acid that have a base sequence that forms hydrogen bridges with a base sequence of the nucleic acid per the principle of base pairing when it is bound to the nucleic acid. This sequence is preferably at least 8 bases long and, more preferably, between 8 and 25 bases long.
The nucleic acid analogs are further defined by the fact that they are structurally di~ert;l,~
from nucleic acids, at least in terrns of the backbone. The "backbone" in nucleic acids or a nucleic acid analog refers to a structure that is basically composed of i~l~ntic~l units that each contain a base. In naturally occ~ ng nucleic acids, the backbone is a sugar phosphate backbone. This backbone is structurally modified in nucleic acid analogs, e.g. in that the sugar or phosphate portion is completely or partially replaced with other çhPmiczil units such as non-cyclic components. Basically, icl~ntic~l units can also replace each other in the backbone.
A few characteristics of nucleic acid analogs are described below to f~rilit~te the selection of nucleic acid analogs that are suitable for use with this invention. It is advantageous for nucleic acid analogs to have a higher affinity to sequence-complementary nucleic acids than an oligonucleotide with an idçn~ic~l base sequence. In addition, those nucleic acid analogs are plere-led that carry fewer charges than a corresponding oligonucleotide ofthe same length, or that can compensate charges with the opposite charges. Basically, uncharged nucleic acid analogs are especially prefelled. Especially p-efelled nucleic acid analogs are those whose affinity to complçmPnt~ry nucleic acids basically does not depend on the salt content of the hybridization complex.
The nucleic acid analogs that are especially suitable are the nucleic acid analogs described in 2 and WO 92/20703 ~Peptide Nucleic Acid, PNA, e.g. Nature 365, 566-568(1993) and Nucl. Acids Res. 21, 533~-5 (1993)). These patent applications are referred to for the description of the structure of the nucleic acid analogs. Preferred nucleic acid analogs are compounds that have a polyamide backbone that contains a number of bases bound along the backbone, with each base bound to a nitrogen atom of the backbone.
Nucleic acid analogs should also include compounds, however, like those described in EP-A-0 672 677. Additional nucleic acid analogs are described in Recueil 91, 1069-1080 ~3~ no~
(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.
Additional nucleic acid analogs are described in Proc. Natl. Acad. Sci. USA 91, 7864-7868 (1994), Proc. Nat. Acad. Sci. USA 92, 6097-6101 (1995) and J. Am. Chem. Soc. 117, 6140-6141 (1995). The nucleic acid analogs described are from 8 to 30 bases long, while a length offrom 10 to 25 bases is especially advantageous. The nucleic acid analogs named are bound directly or indirectly to the surface of the solid carrier. The type of binding basically depends on which reactive groups are available for binding on the solid carrier, and which reactive groups are available for binding to the nucleic acid analog without restricting the ability of the nucleic acid analogs to bind to a complçm~nt~ry nucleic acid. The type of binding also depends on whether the intent is to ~iml~lt~neously bind the nucleic acid analogs to different sites, or to build upon them. It can also be applupliate to cover the surface of the solid carrier with a layer of a material that has a greater ability to bind, or to activate the surface by means of a çhemic~l reaction. Reactive groups on the surface of a solid carrier are usually selected from the group -OH, -NH2 and SH. Reactive groups of nucleic acid analogs are preferably selected from the group -~X N H2,-S X -COO X -SO3H and -PO3H2.
In an especially preferred embodiment, the reactive groups of the surface and the nucleic acid analog are covalently bound to each other, especially by means of a linker that is more than 15 atoms and less than 200 atoms long. A "linker" refers to a portion of a molecule that basically has the function of removing the nucleic acid analogs that are sterically available on the surface of the solid carrier. A linker is usually selected that has hydrogen atoms (e.g. in alkylene units) and numerous heteroatoms (e.g. -O- or -NH- or -NR-) that f~.ilit~te solvation. The linker preferably contains one or more ethylene oxy units and/or peptide groups. In an especially plerell~d embodiment, the linker contains one or more units as described in DE-A 3924705. Especially plerel-ed are the units described as an example which is referred to as Ado (8-amino-3,6-dioxa-octanic acid), below. A slight dependence of the binding of nucleic acids to the PNA surface can be reduced by using longer linkers between PNA and the solid carrier.
4300~NGL.DOC
The nucleic acid analog that is bound to a site can also be a mixture of two or more analogs having difrelen~ but known sequences. This can reduce the number of sites required for a multiple dete, ,,,i~ ion The surface of the carrier is preferably not charged, and is preferably hydrophilic. The invention demonstrated that the use of basically uncharged s~ ce~e is an advantage when cletecting nucleic acids.
A solid carrier loaded with nucleic acid analogs at di~elenL sites as provided by this invention can be m~mlf~ctllred in different ways. In one embodiment, suitable qu~ntities of solutions that each contain dirre~ nucleic acid analogs are applied to di~le-ll sites on the surface of the solid carrier, e.g. via pipette. The liquid sarnples should not mix with each other on the surface of the solid carrier. This can be accomplished, for in.~t~nr.e, by locating the application sites far apart from each other or by using a hydrophobic barrier to stop the exr~n.eiQn of the liquid between the various sites. Either the nucleic acid analogs or the surface of the solid carrier is preferably activated for the reaction. This activation can be achieved, for in.et~nre, in that one of the groups described above is activated by the creation of a reactive species. In the case of a carboxyl group, this would be an activated ester (e.g. a N-hydlo~y srlcrinimide ester) that quicldy enters into an ester bond with a hylllo~yl group without further activation. Suitably activated polyamide membranes carry triazine groups, for inst~nce~ that can react with amino groups of nucleic acid analogs with the forrnation of a covalent bond. The activation can also take place by means of bifunctional re~g~nte, squaric acid derivatives of WO 95/15983 or glutaraldehyde (GB 2197720).
The binding of the analogs can also be realized by coating the carrier surface with nucleotide sequences that are complementary to a part of the sequence of the nucleotide analogs. The binding of the di~el ell~ nucleic acid analogs to the binding regions can take place simlllt~neously or sequentially.
Af[er a s--ffiçient amount of time has passed for the binding to take place, it is advantageous to wash away any nucleic acid analogs ~hat have not bound or are bound in~l-fficiently, 430(1~NGL.DOC
along with any binding reagents that were used. This is performed preferably under conditions in which non-bound nucleic acid analogs cannot bind with nucleic acid analogs that are bound to other regions.
R~ie.~lly, it is also possible to build upon the nucleic acid analogs on the different sites of the surface by means of monomeric units. The technolog,v described in WO 92/10092 or WO 90/15070 can be used for this purpose. Appropriate monomers are described in WO 92/20702, for instance.
Another subject matter of this invention is a method for the sequence-specific detection of a nucleic acid using the solid carrier provided by this invention.
Detectable nucleic acids are natural or artificial nucleic acids. "Nucleic acids" therefore also refer to nucleic acid analogs. The nucleic acid to be detected, however, is the RNA or DNA in particular that is characteristic for an organism cont~inin~ nucleic acids, e.g. a virus, a b~ct~rillm, a multicellular organism, a plasmid or a genetic condition such as a predisposition or a disposition for a certain disease or a spontaneous genetic mutation. The RNA and DNA in this case is basically of genomic origin or an origin derived therefrom. An important class of nucleic acids in the context of this invention are the results of a nucleic acid amplification. These results are also referred to as "~mplific~tes" or "amplicons" below.
The nucleic acids can be present in either their raw form or in a purified or processed form.
A purification can also take place by separating the nucleic acids from cell components in a preparatory step, e.g. an affinity separation step. The nucleic acids can also be enzym~ti~ ~lly ~Yt~nr1erl, specifically amplified or transcribed.
For the sequence-specific detectic~n of nucleic acids, the carrier provided by this invention has a nucleic acid analog bound to a site that has a base sequence that is complement~ry to a base sequence of the nucleic acid to be detected This base sequence is selected intentionally so that it can specifically reveal the presence of the nucleic acid. In the normal case this means that the mixture contains as few as possible--and preferably no--additional nucleic acids having the same total sequence. It must be mentioned, however, that the carrier provided by this invention can also be used to specifically detect groups of 4300ENGL.DOC
nucleic acids. It can be a task of the method, for in~t~nf ~ to detect any member of a certain taxonomic group, e.g. a family of bacteria, by means of its nucleic acids. In this case, the base sequence of the nucleic acid analog can be intentionally selected so that it lies in a conserved region but only occurs in members of this taxonomic group.
An additional nucleic acid analog that has a base sequence that is not compl~Pmpnt~ry to the same base sequence is preferably bound to a di~Te~ L site of the surface. It can be a nucleic acid analog, the base sequence of which can be shorter or longer, or which can differ from the first nucleic acid analog by one or more bases. The difference in the base sequence depends on the task to be solved. The differences can include point mutations, or smaller dçlçtiom and insertions, for instance. In many genetic tii~e~es, such as cystic fibrosis, the sequences of the nucleic acid analogs differ in terms of individual positions (point mllt~tion~) and numerous positions (deletions, for example at ~ 508).
The mutation to be detected is preferably positioned close to the middle of the base sequence of the analog.
The sequences of the analog can also be intpntic~n~lly selected so that their hybridization positions differ by one base each, even though the lengths are idPntic~l (overlap). The sequences can also be intentionally selected so that the hybridization regions are ~ cent to each other on the nucleic acid to be cletecte~1 Numerous mutations can also be detected in the same or di~elent nucleic acids by sf-lecting nucleic acid analogs with sequences that are complpm~nt~ry to the sequence on these nucleic acids.
In an especially easy case in which the purpose is to determine which of two alleles is present in a complex, two nucleic acid analogs are bound to di~erellL sites of the surface of the solid carrier, the base sequences of which differ in exactly that position where the alleles also differ. A nucleic acid analog is therefore selected that is complçm~nt~ry to a certain sequence of one allele, while the other nucleic acid analog is comrlem~nt~ry to the sequence 43001~NGL.DOC
.
of the other allele. The length and hybridization sites of the nucleic acid analogs are identical.
All alleles are usually detected for cystic fibrosis, for in.~t~nce The wild-type contains two healthy alleles. Heterozygotes contain one m-lt~ted and one wild-type sequence, and homozygotic mllt~nt~ contain two mutant nucleic acids. In this case, the intent is not just to determine if mllt~nts are present, but to determine if it is a heterozygotic or homozygotic case. In accordance with this invention it is possible to sim~llt~nçously q~ ely detect both alleles and thereby differentiate between the three cases described.
In many cases, especially in oncology and in the determination of infectious parameters, mllt~ted cells/particles are usually located in the background of non-m~lt~te~l/normal cells.
In these cases, selective detecti~n can not be performed reliably or at all using methods provided by the state of the art. The analysis of ras mutations from DNA from stool samples, for instance, requires that a m--t~ted sequence be reliably detected in the presence of approx. 100 normal sequences (Science 256, 102-105 (1992)). In the field of infectious diseases, it would be desirable to det~rmirle different HIV populations in one infected patient. The quantity of many mnt~nt~: of these HlV pop--l~ti~-n.~ is less than 2% compared with all HrV sequences, however. The method provided by this invention therefore makes it especially quite possible to investigate mixtures of nucleic acids that are very similar to each other, even if one of the nucleic acids is present in a much greater quantity than the nucleic acid to be determined.
The lengths of the bound nucleic acid analogs are preferably identical. An applop,iate length has proven to be between 10 and 100, and preferably between 10 and 50 bases. Especially good results are obtained with nucleic acid analogs that are between 10 and 25 bases long.
To perform the method provided by this invention, the sample c~ nucleic acid is brought in contact with the sites on the surface of the carrier that have bound the nucleic acid analogs. This can be performed, for in~t~nce7 by bringing the solid carrier into the sample fluid or pouring the sample fluid onto the solid carrier in one or more portions. The nucleic acids in the sample fluid can be denatured (single-strand) before they are brought in 430W~NGLDOC
contact with the carrier. A major advantage of the invention, however, is the fact that a prelimin~ry denaturation step can be çiimin~te~1, e.g. by using PNAs. The PNAs force a strand out of the double-stranded nucleic acid to be determinp~rl The only important requirement is that the sample be brought in contact with the solid carrier under con~litione in which the nucleic acid to be ~letected binds spe~ifie~lly to the appropriate site on the surface by means of the nucleic acid analog which is compl~mPnt~ry to one sequence of the nucleic acid to be determined. These conditions can be ~ for ~li~elel~ types of nucleic acid analogs, of course, but they are easily determined for given nucleic acid analogs by pelr~ ning tests. In the normal case, these con-litions are based on the conditions that are known for carriers loaded with oligonucleotides. If nucleic acid analogs are used as described in WO 92/20702, however, con~ ione can be selected that are much di~ele-.l from the hybricli~tion conditions for the corresponding oligonucleotides. It has proven to be appropriate, however, to use much less salt than when the corresponding oli~oml~l~otides are used. For instance, the presence of less than 100 mM and, more preferably, less than 50 mM, and most preferably, less than 10 rnM salt is recommPn~ed Under these conditions, it would not be possible to s~lffi~iently di~lel-liate between nucleic acids having similar sequences using oligonucleotides having the same sequence.
The sample is kept in contact with the surface as long as necessary to achieve a s .ffi~.ient binding of the nucleic acids to the appropriate site on the surface. This period is usually a few mimltes In the next step, it is determined whether the nucleic acid has bound to the surface and, if so, to which site. This is considered an in~1ic~tion of the presence of a nucleic acid that contains a base sequence that is compl~omPnt~ry to the nucleic acid analog bound to this site.
The binding can be determined using various methods.
Instruments are already available with which changes on specific sites of surfaces can be determined directly. For methods that use these types of instruments it is not even necesc~ry to remove the sample cont~ining the nucleic acid from the surface after it has been applied.
Normally, however, it is preferable to remove the fiuid from the surface and use a wash solution to remove any . ~ .g reagent that is still adhered to the surface. This step .
43f~.~r.T. n~c provides the advantage of also washing away sample components that can interfere with the determin~tion of the binding In a prerel~ ed embodiment, the binding of the nucleic acid to be detected with the nucleic acid analog is determined by means of a label that is inserted in the nucleic acid to be determined in a step that is performed before the sample is brought in contact with the sl-Tf~ee The label can be a detect~ble group such as a fluorescent group, for in~t~nce. This determination can be performed optically using a microscope or in a measuring cell provided for this purpose. While the site at which the binding took place is an indication of the presence of a nucleic acid having a certain sequence, the quantity of label at a predetermined site can be used as an indication of the quantity of the nucleic acid to be determined.
In an especially p,erel.~d embodiment, the nucleic acids to be detected are the products of a nucleic acid amplification method such as the polymerase chain reaction as described in EP-B-0 202 362, or NASBA as described in EP-A-0 329 822. It is important that the nucleic acid sequence to bind with the nucleic acid analog be amplified by the amplification method. The better the amplification method m~int~ine the original sequence--that is~ the fewer errors that are incorporated into the sequence during amplification--the more suitable the amplification method. The polymerase chain reaction has proven to be especially suitable. The amplification primers are selected specifically so that the nucleic acid sequence to be detected lies in the region between the hybridization sites.
It has also proven advantageous to insert the label required for the detectinn reaction into the amplificate during amplific~tion This can be performed, for in~t~nr~ by using labelled primers or labelled monon-lcleoside triphosphates.
The binding that took place can be detected directly without inserting a label, for in~t~nce, by using an interc~l~ting agent. These agents have the characteristic of depositing selectively on double-stranded compounds that contain bases, inr,~ lin~ the complex of the nucleic acid analog and the nucleic acid bound in sequence-specific fashion. The presence of the 43(~.~r.T T7~C
CA 022l4430 l997-09-02 complexes can be detected using specific characteristics of intercalating agents, e.g.
fluorescence. Fthi~ m bromide is an especially suitable agent.
Another method for detectin~ hybrids without inserting a label is based on surface plasmon resonance, as described in EP-A-0 618 441, for in~t~n~e.
According to another possible method ~or determinin~ the bin(lin~;~ the surface is brought in contact with a solution of an antibody labelled for detection. This antibody is directed against the complex con~isting of the nucleic acid analog and the nucleic acid to be determined. Antibodies of this nature are described in WO 95/17430, for in~t~nce The detection of the hybrids depends on the type of labelling used. The hybrids can be detected with a scanner, a CCD camera or a microscope, for in~t~nce This invention provides numerous advantages. In particular, it allows detecting sequence differences in nucleic acid regions located within secon-l~ry structures. With this invention it is also possible to increase the sensitivity of detection> because it can use a greater absolute quantity of bound nucleic acid to be determined than traditional methods. It is also possible, in particular, to increase the signal-to-noise ratio compared with methods that use oli~on~lcleotides. In the first attempt, a signal-to-noise ratio of less than 1: 1000 was obtained.
The invention can be used in at least two fields. In the first case, the solid carrier is used to detect known mutations and polymorphisms. In this case, the number of mllt~tion.~ and polymorphisms to be determined is an indicator of the number of ~lirre~ nucleic acid analogs or sites required on the surface. The sequence of the nucleic acid analogs is specially coordinated with the sequence of the nucleic acids around the mutations and polyrnorphisms. Preferably, the sequences are selected in such a way that the base by which nucleic acid analogs having similar sequences differ is located in or near the middle of the sequence.
The carriers provided by this invention can be used in the following fields:
430~NGI~DOC
I~Lfectious (li~e~ses, the simlllt~neous det~ n of different analytes/parameters, and in investig~ti~ns of the condition of a gene in a b~ct~rillm or virus, e.g. for multi-drug rP~ict~nr.e studies.
Oncology (detection of mutations in tumor suppressor genes and oncogenes, and in the det~rrnin~tion of the relationship between mllt~ted and normal cells).
Investigation of inherited ~i~e~es (cystic fibrosis, sickle cell anemia, etc.).
Tissue and bone marrow typing (MHC complex) (see Clin. Chem. 41/4, 553-5 (1995)).
In a second potential application, a sequence of short nucleic acid fr~mentc can be determined using the méthod called "sequencing by hybridization". In this method, the same number of different nucleic acid analogs are immobilized as there are pelllluLalions of the s~lected length of the sequence. To achieve a sufficient level of sensitivity, 4N sites are required, with N equal to the number of bases in each nucleic acid analog. Preferably, N is between 5 and 12. Correspondingly fewer sites are required to sequence very short DNA
fr~gment~ The method for sequencing unknown nucleic acids using the "sequencing by hybri~li7~tion" method is described in WO 92/10588.
An advantage of this invention is the ~act that the specificity of the hybridization is largely independent of the contlitic~n~ in the sample. This f~cilit~t~ ~imlllt~neous binding of nucleic acids to different regions on the surface.
Surprisingly, it was shown that nucleic acid analogs such as PNA have an Pxc~ nt ability to discriminate between sequences on the surface. This disc~ n was better than was to be expected from the melting temperatures of analogous, dissolved compounds.
Surprisingly, the carriers provided by this invention are even suitable for use in numerous, consecutive det~rmin~tions of nucleic acids. After a determination is performed, the carrier that is in contact with a fluid undergoes heat tre~tm~nt A temperature is selected at which 43001~NGLDOC
the bond between the nucleic acid analog and the nucleic acid is dissolved. The carrier is then available to perform another dete~ ;Qn With the method provided by this invention, it is possible for the first time to determine relative q~l~nti~ies of very similar nucleic acids located next to each other in a sample in a concentration range of at least two logs. It has been possible to q~l~ntit~tively dele~ .e ~ using seq l~neing methods, for instance. The m~imllm level of discrimin~tion available with this method was l: l0, however.
With the method provided by this invention it is also possible to bind double-stranded DNA
to the immobilized nucleic acid analogs of the solid carrier without a denaturation step.
Comparison studies have shown that this is not possible with immobilized DNA. It has also been shown that mi~m~tches that are not located in the center of the hybrid can also be ~listin~ hed with a high degree of selectivity.
The sequences of the PNA molecules are shown in Fig. la. They are used as examples to explain the method. The PNAs were prepared as described in WO 92/20702.
Fig. lb shows the sequences of the DNA molecules that are homologous to the PNA
sequences from Fig. la and that were used for the DN~/ODN hybridization experiments.
Fig. 1c shows the sequences of the complementary oligom~cleotides (ODN) used that were labelled with digoxigenin on the S'-phosphate end using the S'-DIG End Labelling Kit ~13Oehringer l~nnheim) and that were phosphorylated with polynucleotide kinase and 32p_ g-ATP 5'.
Fig. ld shows the feasible combinations of oligonucleotide (ODN) and PNA for forming hybrids. Td~.ntic~l col~hil1~tions apply for hybri~li7~ti~ ns between DNA probes (DNA, see Pig. lb) and oligonucleotides (ODN, see Fig. lc).
Fig. 2 shows the hybridization results from Exarnple 4 to illustrate the selectivity of the method. The conditions were: 200 nl spot volume (l00; l0; l; 0. l mM PNA, one concentration per cleavage), incubation at 45 ~C.
4300E~NGLDOC
CA 022l4430 l997-09-02 Fig. 3 shows the hybritli7~tic)n results from Exarnple 6. They verify that PCR amplicons are detected by immobilized PNA probes. The con~litionc were: 200 nl spot volume (100; 10; 1;
0.1 mM PNA, one concentration per cleavage), incubation at 45 ~C. The labels in Fig. 3 mean:
Control experiment, ODN la (lpMol, lnM), line I (PNA 1), line 2 (PNA 2) line 3 ~PNA 3) II ss Amplificate (118 bp, one-fold DIG-labelled) 5 min heat denaturation at 94 ~C
(50 ml PCR preparation diluted in 1 ml hybridization buffer) Line 1 (PNA 1), line 2 (PNA 2), line 3 CPNA 3) m ds Amplificate (118 bp, one-fold DIG-labelled) (50 ml PCR preparation diluted directly in 1 ml hybridization buffer) Line 1 OENA 1), line 2 ~PNA 2), line 3 (PNA 3) Fig. 4 shows the results of the qualitative and q ~~ntit~tive analysis of analyte mixtures by means of PNA arrays. The conditions were: 200 nl spot volume (100 rnM PNA, one PNA
per line, one analyte mixture per cleavage).
Fig. 5 shows the effect of linker length on the hybridization. The con~itit~ns were:
1 rnl spot volume (100; 40; 20; 10; 5; 1 mM PNA, one concentration per cleavage, (Ado)3-PNA in row 1, (Ado)6-PNA in row 2, (Ado)g-PNA in row 3). The labels in Fig. 5 mean:
A. Prehybridization / hybridization in S mM sodium phosphate, 0.1% SDS, pH 7.0 B. Prehybridization / hybridization in 10 mM sodium phosphate, 0.1% SDS, pH 7.0 C. Prehybridization / hybridi7~ltion in 25 mM sodium phosphate, 0.1% SDS, pH 7.0 Figures 6a - 6c d~mon~rate that PNA-derivatized membranes can be used many times aflcer regeneration. The con~litic)ns were: 1 ml spot volume (100; 40; 20; 10; 5; 1 mM PNA, one concentration per cleavage).
43~
CA 022l4430 l997-09-02 The labels in Fig. 6 mean:
6a: Signal intçn~i~ies after the first hybritli7~ti~n 6b: Signal intçn.~ities after the regeneration procedure Membrane 1: No regeneration (controls) ~embrane 2: Regeneration with 0.1 M sodium hydroxide solution, RT 1 h, 2 x 10 min hicli~tilled water RT
~embrane 3: Regeneration with 1 M sodium hydroxide solution, RT, 1 h, 2 x 10 min bidistilled water RT
~embrane 4: Regeneration with ~ tilled water, 70 ~C 1 h, 2 x 10 min bidistilled water RT
~embrane 5: Regeneration with 0.1 M sodium hydroxide solution, 70 ~C 1 h, 2 x 10 min bidistilled water RT
6c: Signal intçn~ities after rehybrirli7~tion ~his invention is explained in fu~ther detail using the following examples:
4300ENGL.DOC
Examples General:
The nucleic acid analogs used were m~mlfact lred as described in WO 92/20702. Unless indicated otherwise, chçmie~l~ and reagents were products of Boehringer ~annhP.im GmbH.
Example l:
Covalent Derivatization of Nylon Membranes 200 nl of a solution that contains PNA in the desired concentration in 0.5 M sodium carbonate pH 9.0 are applied to an Tmnlllnodyne ABC membrane (Pall) with a pipette. After the spots are dry, the membrane is washed with 0. l M sodium hydroxide solution to deactivate any reactive functional surface groups that may still be present. The membrane is washed a second time with water and then dried.
Example 2:
Detection of a Hybri~li7ation Event Using L ~minesc~nce The membrane is derivatized as described in Example l using lO0 IlM, lO mM, 1 IlM and 0.1 mM PNA solutions. It is then prehybridized in a 50 ml hybridization vessel with lO ml hybri(li7~tion buffer (lO mM sodium phosphate, pH 7.2, 0.1% SDS (sodium dodecylsulfate)) in a hybridization oven at 45 ~C. After 30 mimltç~, lO ml of a solution that contains the DIG-labelled oligonucleotide in a 1 mM concentration is added and the complex is hybridized for another 60 min~ltes It is then washed for 2 x lO mimltes with 25 ml wash buffer each time (5 mM sodium phosphate pH 7.2, 0.05% SDS) at 45 ~C. Thedetection reaction is performed according to the protocol for digoxigenin detection (DIG
Detection Kit, Boehringer l~annh~im GmbH, BRD). The anti-DIG-AP conjugate is used in 430t~NGLDOC
-CA 022l4430 l997-09-02 a 1:10000 dilution. CDP-StarTM is used in a 1:10000 dilution as the substrate for the alkaline phosphatase.
Example 3:
Detection of a Hybri~1i7~tion Event Using Fluorescence The membrane is derivatized as described in Example 1 using 100 ,uM, 10 ,uM and 1 ~M
PNA solution. The membrane is prehybridized in a 50 ml screw-top container with 10 ml hybridization buffer (see Example 2) in the oven at 45 ~C. A~er 30 min-ltç~, 10 m1 of a solution that contains a fluorescent-labelled oligonucleotide in a concentration of 1 IlM is added, and the preparation is hybridized for another 60 mimltes The membrane is then washed for 2 x 10 mimltes with 25 ml wash buffer each time (see Example 2) at 45 ~C. The membrane is dried, then the intensity of the fluorescence is measured.
Example 4:
Selectivity of the Method Three membrane strips are derivatized with three (Ado)6-PNA molecules each that differ according to one or two positions of their base sequence (see Fig. la, SEQ.ID.NOS. 1, 2, 3), using PNA solutions in a concentration range of between 100 mM and 0.1 mM asdescribed in Example 1. The membrane strips are prehybridized with 10 ml hybridi7~tinn buffer for 30 min~ltes in 50 ml screw-top cont~in~rs In the next step, one of the three DIG-labelled oligonucleotides (Fig. lb, SEQ.ID.NOS. 4, 5, 6) is added. After hybridizing for 60 mimlte~, the membranes are washed for 2 x 10 mimltes with 25 ml wash buffer each time.
The hybridization events are detecte~l as described in Example 2.
All possible double-stranded hybrids between the PNA molecules involved and the oligonucleotides are shown in Fig. ld. Figure 2 illustrates that, in almost every case, the only oligonucleotide detected is the one that is exactly complçm~nt~ry to the immobilized nucleic acid analog (PNA 1, PNA 2, PNA 3). The signal-to-noise ratios (S/N) can also be 4300ENGL.DOC
estim~ted from the figure. They were evaluated q~l~ntit~tively, and the results are presented in Table 1.
Table 1 Hybrid (PNA/ODN) S/NSignal (Hybrid)/Signal ~Match) 1/1 655.2 100.0%
2/1 20.7 3.2%
3/1 10.3 1.6%
1/2 23 . 1 2.6%
2/2 871.8 100.0%
3/2 6.4 0.7%
1/3 109.4 22.7%
2/3 12.3 2.5%
3/3 481.1 100.0%
Example 5:
Q~l~ntific~tiQn Membrane strips are derivatized with three (Ado)6-PNA molecules with different base sequences (Fig. la, SEQ.ID.NOS. 1, 2, 3) in a concentration of 100 mM as described in Fx~mrle 1. They are then prehybridized in 20 ml hybridization vessels with 10 mlhybridization buffer (see Example 2) at 45 ~C. The buffer is replaced after 30 mimltes In eXpPrim~nt~ 1 through 7, the buffer to be added differs according to the analyteconcentrations of the DIG-labelled components - oligonucleotide 1, 2 and 3, SEQ.ID.NOS.
Sample analysis has undergone rapid development in recent dec~des While analytes were initially detected primarily by means of their reaction with conventional chemical re~gent~, and later on with enzymes, tests that utilize the immlln~logical characteri~tics of the analyte have become the standard recently, especially in m~lic~ p;nostics. This is especially true in the field of infectious diseases. However, immllnf~logical procedures can basically only detect analy-tes with which imm-lnologically active compounds such as antigens or antibodies play a role. These procedures have resulted in promising poten~ial applications for many infections caused by viruses or bacteria. Genetic ~ e~eS or predispositions that are not expressed as a change in protein patterns--or only to an in~llffici~nt extent--are either diffi'-,lllt or impossible to detect using immllnological procedures, however. Nucleic acids have therefore ~ecell~ly become the object of detectinn in many cases. The presence of certain nucleic acids can infer the presence of an infectious agent or the genetic condition of an organism. Detection procedures based on the presence of special nucleotide sequences in particular were f~ilit~ted recently when methods for the amplification of nucleic acids that are present in small numbers became available. Due to the large quantity of sequence information and the fact that two nucleotide sequences with completely di~erellL functions often differ by just one base unit, the specific detection of nucleotide sequences still poses a considerable ch~ nP~e for reagents and analytical methods that are based on the detection of nucleic acid sequences. In addition, the nucleotide sequences are often not even known, but rather are det~rmined for the first time in the nucleic acid detectinn method itself.
A method for the detection of nucleotide sequences of the E~A gene is described in EP-B-0 237 362 with which a clinically relevant point mutation can be detected In this 4300ENGL.DOC
method, an oligonucleotide that is bound to a membrane and has a nucleotide sequence that is exactly complçm~nt~ry to one of the two nucleic acids to be di~er~ ted is brought in contact with the sample. While certain conditions are ..~ e~l~ only that nucleic acid that is exactly complementary binds to the oligoml~.lçoti(le that is bound to the solid phase, and can be detected.
A method is described in Proc. Natl. Acad. Sci. USA 86, 6230-6234 (1989) in which a large number of oligonucleotides that are bound to di~relenl, predeterminecl sites of a nylon membrane by means of poly-dT are used for the simlllt~neous detection of all known allelic variants of an amplified region of a nucleic acid.
A method is described in US-A 5,202,231 in which the sequence of a nucleic acid can be det~rrnined theoretically by bringing oligonucleotides having a predetermined, known sequence in contact with a sample of the unknown nucleic acids under hybridization conditions. This requires that all possible permutations of the nucleotide sequence be immobilized on known sites of a solid phase. By determining the sites to which the nucleic acids cont~ining the sequence to be deterrnined hybridize, it can theoretically be determined which sequences are present in the nucleic acid.
Prior art in the field of the analysis of genetic polymorphisms using "oligonucleotide arrays"
is described in Nucleic Acids Research 22, 5456-5465 (1994) and Clin. Chem. 41/5, 700-6 (1995).
The main problem with the prior art is the fact that the melting temperatures of the selected sequence-specific oligonucleotides cnnt~inin~ the nucleic acids to be sequenced or detected are di~ele"~. To remedy this sit-l~tiQn~ one has to perform the complex method of selecting the length ofthe oligonucleotide and its base composition, and opl;...;,;i-g the position of the mi.cm~trhes within the oligonucleotide as well as the salt concentration of the hykri~i7~tion complex. In many cases, however, it is practically impossible to .~imlllt~n~.ously tli.~tin~ h closely related sequences from each other. The hybridization temperature is another critical parameter. Variations of as little as 1 to 2 ~C can change the 430WiNGLDOC
intensity or produce false-negative results. Incorrect analytical results based on the presence of point mutations have serious impliç~tiQn~ for diagnosis.
The object of this invention was, thereIore, to provide an ~ltern~tive method for the sequence-specific detection of nucleic acids and to provide suitable m~teri~l~ for this method.
This object was accomplished by providing a solid carrier having two or more nucleic acid analogs with di~ele-l~ base sequences bound to predetermined sites on its surface. Another object of the invention is a method for the sequence-specific detection of a nucleic acid using this solid carrier.
A"solid carrier" as described by this invention refers to an object that has a surface that is so broad that specific areas can be di~tin~lish~d upon it. This surface is preferably flat and larger than 5 mmZ, and is preferably between 10 mm2 and approx. 100 cm2. The carrier material is not liquid or gaseous, and preferably dissolves either not at all or incompletely in the sample fluids or reaction preparations that are used to immobilize nucleic acids to the surface. Examples of such materials are glass, plastics (e.g. poly~Lyl~i, e, polyamide, polyethylene, polypropylene), gold, etc. The m~teri~l does not n~cess~rily have to be completely solid itself, but rather can be made solid by the ~tt~hmPnt of supporting m~t~ri~
The external shape of the solid carrier basically depends on the method used to detect the presence of nucleic acids on this solid carrier. It has proven to be applopliate, for in~t~nc.
to select a basically planar form, e.g. a chip.
Solid carriers that are especially suitable are, therefore, polystyrene chips that are from 1 to 5 mm thick and have a surface area of from 1 to 5 cm2, for in~t~nce Polyamide membranes that are 4 x 2.5 cm2 in size have proven to be especially well-suited for use with this invention. Two or more nucleic acid analogs having different base sequences are bound to different sites of the surface of this carrier. These sites or regions preferably do not overlap with each other. They are preferably separated from each other by regions on the surface to 43~EN~.DOC
which no nucleic acid analogs are bound. The sites to which the nucleic acid analogs are bound are referred to as "binding regions" below. The binding regions can have di~renl shapes. These shapes are basically det~rmin~d by the method of m~mlf~ctllring the solid carrier or by the method used to det~ormine the nucleic acid analogs in the binding regions.
The minimllm size of the binding regions is basically detprmined by the instrument with which the event--the binding of a nucleic acid to nucleic acid analogs of a region--is detecte~l Instruments are already available that can detect binding to regions that are approx. 1 mm in size. The upper limit of the size of the binding regions is determined by cost effectiveness and h~ntlling considerations.
The size of the binding regions is also basically determined by the methods used to apply the nucleic acid analogs to the surface. Such methods will be described later.
The number of binding regions on the solid carrier depends on the intended use of the solid carrier. In the simplest case, just two binding regions are needed to detect a certain point mutation. In this case, a binding region contains nucleic acid analogs that have a base in the position at which the point mutation is to be detected This base is compl~m~ont~ry to the base in the position of the normal sequence. The other binding region, on the other hand, contains a nucleic acid analog that has a base in the corresponding position that is complement~ry to the base ofthe ml-t:~ted sequence. In another case, two nucleic acids or nucleic acid sequences that are only slightly related to each other can be detected ~iml~lt~neously using a solid carrier that has two completely di~e~ nucleic acid analogs bound to its surface.
"Nucleic acid analogs" refer to non-naturally occurring molecules that can detect nucleic acids by means of base pairings. They therefore contain a specific base sequence that is completely complem~nt~ry to the base sequence of a nucleic acid to be detecte~1 The base sequence is therefore preferably composed of two naturally occllrring nucleobases. As long as the specificity of the base pairing is not lost, modifications to the nucleobases are also allowed, however.
430(ENGL~DOC
Those nucleic acid analogs are considered compl~ment~ry to a nucleic acid that have a base sequence that forms hydrogen bridges with a base sequence of the nucleic acid per the principle of base pairing when it is bound to the nucleic acid. This sequence is preferably at least 8 bases long and, more preferably, between 8 and 25 bases long.
The nucleic acid analogs are further defined by the fact that they are structurally di~ert;l,~
from nucleic acids, at least in terrns of the backbone. The "backbone" in nucleic acids or a nucleic acid analog refers to a structure that is basically composed of i~l~ntic~l units that each contain a base. In naturally occ~ ng nucleic acids, the backbone is a sugar phosphate backbone. This backbone is structurally modified in nucleic acid analogs, e.g. in that the sugar or phosphate portion is completely or partially replaced with other çhPmiczil units such as non-cyclic components. Basically, icl~ntic~l units can also replace each other in the backbone.
A few characteristics of nucleic acid analogs are described below to f~rilit~te the selection of nucleic acid analogs that are suitable for use with this invention. It is advantageous for nucleic acid analogs to have a higher affinity to sequence-complementary nucleic acids than an oligonucleotide with an idçn~ic~l base sequence. In addition, those nucleic acid analogs are plere-led that carry fewer charges than a corresponding oligonucleotide ofthe same length, or that can compensate charges with the opposite charges. Basically, uncharged nucleic acid analogs are especially prefelled. Especially p-efelled nucleic acid analogs are those whose affinity to complçmPnt~ry nucleic acids basically does not depend on the salt content of the hybridization complex.
The nucleic acid analogs that are especially suitable are the nucleic acid analogs described in 2 and WO 92/20703 ~Peptide Nucleic Acid, PNA, e.g. Nature 365, 566-568(1993) and Nucl. Acids Res. 21, 533~-5 (1993)). These patent applications are referred to for the description of the structure of the nucleic acid analogs. Preferred nucleic acid analogs are compounds that have a polyamide backbone that contains a number of bases bound along the backbone, with each base bound to a nitrogen atom of the backbone.
Nucleic acid analogs should also include compounds, however, like those described in EP-A-0 672 677. Additional nucleic acid analogs are described in Recueil 91, 1069-1080 ~3~ no~
(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.
Additional nucleic acid analogs are described in Proc. Natl. Acad. Sci. USA 91, 7864-7868 (1994), Proc. Nat. Acad. Sci. USA 92, 6097-6101 (1995) and J. Am. Chem. Soc. 117, 6140-6141 (1995). The nucleic acid analogs described are from 8 to 30 bases long, while a length offrom 10 to 25 bases is especially advantageous. The nucleic acid analogs named are bound directly or indirectly to the surface of the solid carrier. The type of binding basically depends on which reactive groups are available for binding on the solid carrier, and which reactive groups are available for binding to the nucleic acid analog without restricting the ability of the nucleic acid analogs to bind to a complçm~nt~ry nucleic acid. The type of binding also depends on whether the intent is to ~iml~lt~neously bind the nucleic acid analogs to different sites, or to build upon them. It can also be applupliate to cover the surface of the solid carrier with a layer of a material that has a greater ability to bind, or to activate the surface by means of a çhemic~l reaction. Reactive groups on the surface of a solid carrier are usually selected from the group -OH, -NH2 and SH. Reactive groups of nucleic acid analogs are preferably selected from the group -~X N H2,-S X -COO X -SO3H and -PO3H2.
In an especially preferred embodiment, the reactive groups of the surface and the nucleic acid analog are covalently bound to each other, especially by means of a linker that is more than 15 atoms and less than 200 atoms long. A "linker" refers to a portion of a molecule that basically has the function of removing the nucleic acid analogs that are sterically available on the surface of the solid carrier. A linker is usually selected that has hydrogen atoms (e.g. in alkylene units) and numerous heteroatoms (e.g. -O- or -NH- or -NR-) that f~.ilit~te solvation. The linker preferably contains one or more ethylene oxy units and/or peptide groups. In an especially plerell~d embodiment, the linker contains one or more units as described in DE-A 3924705. Especially plerel-ed are the units described as an example which is referred to as Ado (8-amino-3,6-dioxa-octanic acid), below. A slight dependence of the binding of nucleic acids to the PNA surface can be reduced by using longer linkers between PNA and the solid carrier.
4300~NGL.DOC
The nucleic acid analog that is bound to a site can also be a mixture of two or more analogs having difrelen~ but known sequences. This can reduce the number of sites required for a multiple dete, ,,,i~ ion The surface of the carrier is preferably not charged, and is preferably hydrophilic. The invention demonstrated that the use of basically uncharged s~ ce~e is an advantage when cletecting nucleic acids.
A solid carrier loaded with nucleic acid analogs at di~elenL sites as provided by this invention can be m~mlf~ctllred in different ways. In one embodiment, suitable qu~ntities of solutions that each contain dirre~ nucleic acid analogs are applied to di~le-ll sites on the surface of the solid carrier, e.g. via pipette. The liquid sarnples should not mix with each other on the surface of the solid carrier. This can be accomplished, for in.~t~nr.e, by locating the application sites far apart from each other or by using a hydrophobic barrier to stop the exr~n.eiQn of the liquid between the various sites. Either the nucleic acid analogs or the surface of the solid carrier is preferably activated for the reaction. This activation can be achieved, for in.et~nre, in that one of the groups described above is activated by the creation of a reactive species. In the case of a carboxyl group, this would be an activated ester (e.g. a N-hydlo~y srlcrinimide ester) that quicldy enters into an ester bond with a hylllo~yl group without further activation. Suitably activated polyamide membranes carry triazine groups, for inst~nce~ that can react with amino groups of nucleic acid analogs with the forrnation of a covalent bond. The activation can also take place by means of bifunctional re~g~nte, squaric acid derivatives of WO 95/15983 or glutaraldehyde (GB 2197720).
The binding of the analogs can also be realized by coating the carrier surface with nucleotide sequences that are complementary to a part of the sequence of the nucleotide analogs. The binding of the di~el ell~ nucleic acid analogs to the binding regions can take place simlllt~neously or sequentially.
Af[er a s--ffiçient amount of time has passed for the binding to take place, it is advantageous to wash away any nucleic acid analogs ~hat have not bound or are bound in~l-fficiently, 430(1~NGL.DOC
along with any binding reagents that were used. This is performed preferably under conditions in which non-bound nucleic acid analogs cannot bind with nucleic acid analogs that are bound to other regions.
R~ie.~lly, it is also possible to build upon the nucleic acid analogs on the different sites of the surface by means of monomeric units. The technolog,v described in WO 92/10092 or WO 90/15070 can be used for this purpose. Appropriate monomers are described in WO 92/20702, for instance.
Another subject matter of this invention is a method for the sequence-specific detection of a nucleic acid using the solid carrier provided by this invention.
Detectable nucleic acids are natural or artificial nucleic acids. "Nucleic acids" therefore also refer to nucleic acid analogs. The nucleic acid to be detected, however, is the RNA or DNA in particular that is characteristic for an organism cont~inin~ nucleic acids, e.g. a virus, a b~ct~rillm, a multicellular organism, a plasmid or a genetic condition such as a predisposition or a disposition for a certain disease or a spontaneous genetic mutation. The RNA and DNA in this case is basically of genomic origin or an origin derived therefrom. An important class of nucleic acids in the context of this invention are the results of a nucleic acid amplification. These results are also referred to as "~mplific~tes" or "amplicons" below.
The nucleic acids can be present in either their raw form or in a purified or processed form.
A purification can also take place by separating the nucleic acids from cell components in a preparatory step, e.g. an affinity separation step. The nucleic acids can also be enzym~ti~ ~lly ~Yt~nr1erl, specifically amplified or transcribed.
For the sequence-specific detectic~n of nucleic acids, the carrier provided by this invention has a nucleic acid analog bound to a site that has a base sequence that is complement~ry to a base sequence of the nucleic acid to be detected This base sequence is selected intentionally so that it can specifically reveal the presence of the nucleic acid. In the normal case this means that the mixture contains as few as possible--and preferably no--additional nucleic acids having the same total sequence. It must be mentioned, however, that the carrier provided by this invention can also be used to specifically detect groups of 4300ENGL.DOC
nucleic acids. It can be a task of the method, for in~t~nf ~ to detect any member of a certain taxonomic group, e.g. a family of bacteria, by means of its nucleic acids. In this case, the base sequence of the nucleic acid analog can be intentionally selected so that it lies in a conserved region but only occurs in members of this taxonomic group.
An additional nucleic acid analog that has a base sequence that is not compl~Pmpnt~ry to the same base sequence is preferably bound to a di~Te~ L site of the surface. It can be a nucleic acid analog, the base sequence of which can be shorter or longer, or which can differ from the first nucleic acid analog by one or more bases. The difference in the base sequence depends on the task to be solved. The differences can include point mutations, or smaller dçlçtiom and insertions, for instance. In many genetic tii~e~es, such as cystic fibrosis, the sequences of the nucleic acid analogs differ in terms of individual positions (point mllt~tion~) and numerous positions (deletions, for example at ~ 508).
The mutation to be detected is preferably positioned close to the middle of the base sequence of the analog.
The sequences of the analog can also be intpntic~n~lly selected so that their hybridization positions differ by one base each, even though the lengths are idPntic~l (overlap). The sequences can also be intentionally selected so that the hybridization regions are ~ cent to each other on the nucleic acid to be cletecte~1 Numerous mutations can also be detected in the same or di~elent nucleic acids by sf-lecting nucleic acid analogs with sequences that are complpm~nt~ry to the sequence on these nucleic acids.
In an especially easy case in which the purpose is to determine which of two alleles is present in a complex, two nucleic acid analogs are bound to di~erellL sites of the surface of the solid carrier, the base sequences of which differ in exactly that position where the alleles also differ. A nucleic acid analog is therefore selected that is complçm~nt~ry to a certain sequence of one allele, while the other nucleic acid analog is comrlem~nt~ry to the sequence 43001~NGL.DOC
.
of the other allele. The length and hybridization sites of the nucleic acid analogs are identical.
All alleles are usually detected for cystic fibrosis, for in.~t~nce The wild-type contains two healthy alleles. Heterozygotes contain one m-lt~ted and one wild-type sequence, and homozygotic mllt~nt~ contain two mutant nucleic acids. In this case, the intent is not just to determine if mllt~nts are present, but to determine if it is a heterozygotic or homozygotic case. In accordance with this invention it is possible to sim~llt~nçously q~ ely detect both alleles and thereby differentiate between the three cases described.
In many cases, especially in oncology and in the determination of infectious parameters, mllt~ted cells/particles are usually located in the background of non-m~lt~te~l/normal cells.
In these cases, selective detecti~n can not be performed reliably or at all using methods provided by the state of the art. The analysis of ras mutations from DNA from stool samples, for instance, requires that a m--t~ted sequence be reliably detected in the presence of approx. 100 normal sequences (Science 256, 102-105 (1992)). In the field of infectious diseases, it would be desirable to det~rmirle different HIV populations in one infected patient. The quantity of many mnt~nt~: of these HlV pop--l~ti~-n.~ is less than 2% compared with all HrV sequences, however. The method provided by this invention therefore makes it especially quite possible to investigate mixtures of nucleic acids that are very similar to each other, even if one of the nucleic acids is present in a much greater quantity than the nucleic acid to be determined.
The lengths of the bound nucleic acid analogs are preferably identical. An applop,iate length has proven to be between 10 and 100, and preferably between 10 and 50 bases. Especially good results are obtained with nucleic acid analogs that are between 10 and 25 bases long.
To perform the method provided by this invention, the sample c~ nucleic acid is brought in contact with the sites on the surface of the carrier that have bound the nucleic acid analogs. This can be performed, for in~t~nce7 by bringing the solid carrier into the sample fluid or pouring the sample fluid onto the solid carrier in one or more portions. The nucleic acids in the sample fluid can be denatured (single-strand) before they are brought in 430W~NGLDOC
contact with the carrier. A major advantage of the invention, however, is the fact that a prelimin~ry denaturation step can be çiimin~te~1, e.g. by using PNAs. The PNAs force a strand out of the double-stranded nucleic acid to be determinp~rl The only important requirement is that the sample be brought in contact with the solid carrier under con~litione in which the nucleic acid to be ~letected binds spe~ifie~lly to the appropriate site on the surface by means of the nucleic acid analog which is compl~mPnt~ry to one sequence of the nucleic acid to be determined. These conditions can be ~ for ~li~elel~ types of nucleic acid analogs, of course, but they are easily determined for given nucleic acid analogs by pelr~ ning tests. In the normal case, these con-litions are based on the conditions that are known for carriers loaded with oligonucleotides. If nucleic acid analogs are used as described in WO 92/20702, however, con~ ione can be selected that are much di~ele-.l from the hybricli~tion conditions for the corresponding oligonucleotides. It has proven to be appropriate, however, to use much less salt than when the corresponding oli~oml~l~otides are used. For instance, the presence of less than 100 mM and, more preferably, less than 50 mM, and most preferably, less than 10 rnM salt is recommPn~ed Under these conditions, it would not be possible to s~lffi~iently di~lel-liate between nucleic acids having similar sequences using oligonucleotides having the same sequence.
The sample is kept in contact with the surface as long as necessary to achieve a s .ffi~.ient binding of the nucleic acids to the appropriate site on the surface. This period is usually a few mimltes In the next step, it is determined whether the nucleic acid has bound to the surface and, if so, to which site. This is considered an in~1ic~tion of the presence of a nucleic acid that contains a base sequence that is compl~omPnt~ry to the nucleic acid analog bound to this site.
The binding can be determined using various methods.
Instruments are already available with which changes on specific sites of surfaces can be determined directly. For methods that use these types of instruments it is not even necesc~ry to remove the sample cont~ining the nucleic acid from the surface after it has been applied.
Normally, however, it is preferable to remove the fiuid from the surface and use a wash solution to remove any . ~ .g reagent that is still adhered to the surface. This step .
43f~.~r.T. n~c provides the advantage of also washing away sample components that can interfere with the determin~tion of the binding In a prerel~ ed embodiment, the binding of the nucleic acid to be detected with the nucleic acid analog is determined by means of a label that is inserted in the nucleic acid to be determined in a step that is performed before the sample is brought in contact with the sl-Tf~ee The label can be a detect~ble group such as a fluorescent group, for in~t~nce. This determination can be performed optically using a microscope or in a measuring cell provided for this purpose. While the site at which the binding took place is an indication of the presence of a nucleic acid having a certain sequence, the quantity of label at a predetermined site can be used as an indication of the quantity of the nucleic acid to be determined.
In an especially p,erel.~d embodiment, the nucleic acids to be detected are the products of a nucleic acid amplification method such as the polymerase chain reaction as described in EP-B-0 202 362, or NASBA as described in EP-A-0 329 822. It is important that the nucleic acid sequence to bind with the nucleic acid analog be amplified by the amplification method. The better the amplification method m~int~ine the original sequence--that is~ the fewer errors that are incorporated into the sequence during amplification--the more suitable the amplification method. The polymerase chain reaction has proven to be especially suitable. The amplification primers are selected specifically so that the nucleic acid sequence to be detected lies in the region between the hybridization sites.
It has also proven advantageous to insert the label required for the detectinn reaction into the amplificate during amplific~tion This can be performed, for in~t~nr~ by using labelled primers or labelled monon-lcleoside triphosphates.
The binding that took place can be detected directly without inserting a label, for in~t~nce, by using an interc~l~ting agent. These agents have the characteristic of depositing selectively on double-stranded compounds that contain bases, inr,~ lin~ the complex of the nucleic acid analog and the nucleic acid bound in sequence-specific fashion. The presence of the 43(~.~r.T T7~C
CA 022l4430 l997-09-02 complexes can be detected using specific characteristics of intercalating agents, e.g.
fluorescence. Fthi~ m bromide is an especially suitable agent.
Another method for detectin~ hybrids without inserting a label is based on surface plasmon resonance, as described in EP-A-0 618 441, for in~t~n~e.
According to another possible method ~or determinin~ the bin(lin~;~ the surface is brought in contact with a solution of an antibody labelled for detection. This antibody is directed against the complex con~isting of the nucleic acid analog and the nucleic acid to be determined. Antibodies of this nature are described in WO 95/17430, for in~t~nce The detection of the hybrids depends on the type of labelling used. The hybrids can be detected with a scanner, a CCD camera or a microscope, for in~t~nce This invention provides numerous advantages. In particular, it allows detecting sequence differences in nucleic acid regions located within secon-l~ry structures. With this invention it is also possible to increase the sensitivity of detection> because it can use a greater absolute quantity of bound nucleic acid to be determined than traditional methods. It is also possible, in particular, to increase the signal-to-noise ratio compared with methods that use oli~on~lcleotides. In the first attempt, a signal-to-noise ratio of less than 1: 1000 was obtained.
The invention can be used in at least two fields. In the first case, the solid carrier is used to detect known mutations and polymorphisms. In this case, the number of mllt~tion.~ and polymorphisms to be determined is an indicator of the number of ~lirre~ nucleic acid analogs or sites required on the surface. The sequence of the nucleic acid analogs is specially coordinated with the sequence of the nucleic acids around the mutations and polyrnorphisms. Preferably, the sequences are selected in such a way that the base by which nucleic acid analogs having similar sequences differ is located in or near the middle of the sequence.
The carriers provided by this invention can be used in the following fields:
430~NGI~DOC
I~Lfectious (li~e~ses, the simlllt~neous det~ n of different analytes/parameters, and in investig~ti~ns of the condition of a gene in a b~ct~rillm or virus, e.g. for multi-drug rP~ict~nr.e studies.
Oncology (detection of mutations in tumor suppressor genes and oncogenes, and in the det~rrnin~tion of the relationship between mllt~ted and normal cells).
Investigation of inherited ~i~e~es (cystic fibrosis, sickle cell anemia, etc.).
Tissue and bone marrow typing (MHC complex) (see Clin. Chem. 41/4, 553-5 (1995)).
In a second potential application, a sequence of short nucleic acid fr~mentc can be determined using the méthod called "sequencing by hybridization". In this method, the same number of different nucleic acid analogs are immobilized as there are pelllluLalions of the s~lected length of the sequence. To achieve a sufficient level of sensitivity, 4N sites are required, with N equal to the number of bases in each nucleic acid analog. Preferably, N is between 5 and 12. Correspondingly fewer sites are required to sequence very short DNA
fr~gment~ The method for sequencing unknown nucleic acids using the "sequencing by hybri~li7~tion" method is described in WO 92/10588.
An advantage of this invention is the ~act that the specificity of the hybridization is largely independent of the contlitic~n~ in the sample. This f~cilit~t~ ~imlllt~neous binding of nucleic acids to different regions on the surface.
Surprisingly, it was shown that nucleic acid analogs such as PNA have an Pxc~ nt ability to discriminate between sequences on the surface. This disc~ n was better than was to be expected from the melting temperatures of analogous, dissolved compounds.
Surprisingly, the carriers provided by this invention are even suitable for use in numerous, consecutive det~rmin~tions of nucleic acids. After a determination is performed, the carrier that is in contact with a fluid undergoes heat tre~tm~nt A temperature is selected at which 43001~NGLDOC
the bond between the nucleic acid analog and the nucleic acid is dissolved. The carrier is then available to perform another dete~ ;Qn With the method provided by this invention, it is possible for the first time to determine relative q~l~nti~ies of very similar nucleic acids located next to each other in a sample in a concentration range of at least two logs. It has been possible to q~l~ntit~tively dele~ .e ~ using seq l~neing methods, for instance. The m~imllm level of discrimin~tion available with this method was l: l0, however.
With the method provided by this invention it is also possible to bind double-stranded DNA
to the immobilized nucleic acid analogs of the solid carrier without a denaturation step.
Comparison studies have shown that this is not possible with immobilized DNA. It has also been shown that mi~m~tches that are not located in the center of the hybrid can also be ~listin~ hed with a high degree of selectivity.
The sequences of the PNA molecules are shown in Fig. la. They are used as examples to explain the method. The PNAs were prepared as described in WO 92/20702.
Fig. lb shows the sequences of the DNA molecules that are homologous to the PNA
sequences from Fig. la and that were used for the DN~/ODN hybridization experiments.
Fig. 1c shows the sequences of the complementary oligom~cleotides (ODN) used that were labelled with digoxigenin on the S'-phosphate end using the S'-DIG End Labelling Kit ~13Oehringer l~nnheim) and that were phosphorylated with polynucleotide kinase and 32p_ g-ATP 5'.
Fig. ld shows the feasible combinations of oligonucleotide (ODN) and PNA for forming hybrids. Td~.ntic~l col~hil1~tions apply for hybri~li7~ti~ ns between DNA probes (DNA, see Pig. lb) and oligonucleotides (ODN, see Fig. lc).
Fig. 2 shows the hybridization results from Exarnple 4 to illustrate the selectivity of the method. The conditions were: 200 nl spot volume (l00; l0; l; 0. l mM PNA, one concentration per cleavage), incubation at 45 ~C.
4300E~NGLDOC
CA 022l4430 l997-09-02 Fig. 3 shows the hybritli7~tic)n results from Exarnple 6. They verify that PCR amplicons are detected by immobilized PNA probes. The con~litionc were: 200 nl spot volume (100; 10; 1;
0.1 mM PNA, one concentration per cleavage), incubation at 45 ~C. The labels in Fig. 3 mean:
Control experiment, ODN la (lpMol, lnM), line I (PNA 1), line 2 (PNA 2) line 3 ~PNA 3) II ss Amplificate (118 bp, one-fold DIG-labelled) 5 min heat denaturation at 94 ~C
(50 ml PCR preparation diluted in 1 ml hybridization buffer) Line 1 (PNA 1), line 2 (PNA 2), line 3 CPNA 3) m ds Amplificate (118 bp, one-fold DIG-labelled) (50 ml PCR preparation diluted directly in 1 ml hybridization buffer) Line 1 OENA 1), line 2 ~PNA 2), line 3 (PNA 3) Fig. 4 shows the results of the qualitative and q ~~ntit~tive analysis of analyte mixtures by means of PNA arrays. The conditions were: 200 nl spot volume (100 rnM PNA, one PNA
per line, one analyte mixture per cleavage).
Fig. 5 shows the effect of linker length on the hybridization. The con~itit~ns were:
1 rnl spot volume (100; 40; 20; 10; 5; 1 mM PNA, one concentration per cleavage, (Ado)3-PNA in row 1, (Ado)6-PNA in row 2, (Ado)g-PNA in row 3). The labels in Fig. 5 mean:
A. Prehybridization / hybridization in S mM sodium phosphate, 0.1% SDS, pH 7.0 B. Prehybridization / hybridization in 10 mM sodium phosphate, 0.1% SDS, pH 7.0 C. Prehybridization / hybridi7~ltion in 25 mM sodium phosphate, 0.1% SDS, pH 7.0 Figures 6a - 6c d~mon~rate that PNA-derivatized membranes can be used many times aflcer regeneration. The con~litic)ns were: 1 ml spot volume (100; 40; 20; 10; 5; 1 mM PNA, one concentration per cleavage).
43~
CA 022l4430 l997-09-02 The labels in Fig. 6 mean:
6a: Signal intçn~i~ies after the first hybritli7~ti~n 6b: Signal intçn.~ities after the regeneration procedure Membrane 1: No regeneration (controls) ~embrane 2: Regeneration with 0.1 M sodium hydroxide solution, RT 1 h, 2 x 10 min hicli~tilled water RT
~embrane 3: Regeneration with 1 M sodium hydroxide solution, RT, 1 h, 2 x 10 min bidistilled water RT
~embrane 4: Regeneration with ~ tilled water, 70 ~C 1 h, 2 x 10 min bidistilled water RT
~embrane 5: Regeneration with 0.1 M sodium hydroxide solution, 70 ~C 1 h, 2 x 10 min bidistilled water RT
6c: Signal intçn~ities after rehybrirli7~tion ~his invention is explained in fu~ther detail using the following examples:
4300ENGL.DOC
Examples General:
The nucleic acid analogs used were m~mlfact lred as described in WO 92/20702. Unless indicated otherwise, chçmie~l~ and reagents were products of Boehringer ~annhP.im GmbH.
Example l:
Covalent Derivatization of Nylon Membranes 200 nl of a solution that contains PNA in the desired concentration in 0.5 M sodium carbonate pH 9.0 are applied to an Tmnlllnodyne ABC membrane (Pall) with a pipette. After the spots are dry, the membrane is washed with 0. l M sodium hydroxide solution to deactivate any reactive functional surface groups that may still be present. The membrane is washed a second time with water and then dried.
Example 2:
Detection of a Hybri~li7ation Event Using L ~minesc~nce The membrane is derivatized as described in Example l using lO0 IlM, lO mM, 1 IlM and 0.1 mM PNA solutions. It is then prehybridized in a 50 ml hybridization vessel with lO ml hybri(li7~tion buffer (lO mM sodium phosphate, pH 7.2, 0.1% SDS (sodium dodecylsulfate)) in a hybridization oven at 45 ~C. After 30 mimltç~, lO ml of a solution that contains the DIG-labelled oligonucleotide in a 1 mM concentration is added and the complex is hybridized for another 60 min~ltes It is then washed for 2 x lO mimltes with 25 ml wash buffer each time (5 mM sodium phosphate pH 7.2, 0.05% SDS) at 45 ~C. Thedetection reaction is performed according to the protocol for digoxigenin detection (DIG
Detection Kit, Boehringer l~annh~im GmbH, BRD). The anti-DIG-AP conjugate is used in 430t~NGLDOC
-CA 022l4430 l997-09-02 a 1:10000 dilution. CDP-StarTM is used in a 1:10000 dilution as the substrate for the alkaline phosphatase.
Example 3:
Detection of a Hybri~1i7~tion Event Using Fluorescence The membrane is derivatized as described in Example 1 using 100 ,uM, 10 ,uM and 1 ~M
PNA solution. The membrane is prehybridized in a 50 ml screw-top container with 10 ml hybridization buffer (see Example 2) in the oven at 45 ~C. A~er 30 min-ltç~, 10 m1 of a solution that contains a fluorescent-labelled oligonucleotide in a concentration of 1 IlM is added, and the preparation is hybridized for another 60 mimltes The membrane is then washed for 2 x 10 mimltes with 25 ml wash buffer each time (see Example 2) at 45 ~C. The membrane is dried, then the intensity of the fluorescence is measured.
Example 4:
Selectivity of the Method Three membrane strips are derivatized with three (Ado)6-PNA molecules each that differ according to one or two positions of their base sequence (see Fig. la, SEQ.ID.NOS. 1, 2, 3), using PNA solutions in a concentration range of between 100 mM and 0.1 mM asdescribed in Example 1. The membrane strips are prehybridized with 10 ml hybridi7~tinn buffer for 30 min~ltes in 50 ml screw-top cont~in~rs In the next step, one of the three DIG-labelled oligonucleotides (Fig. lb, SEQ.ID.NOS. 4, 5, 6) is added. After hybridizing for 60 mimlte~, the membranes are washed for 2 x 10 mimltes with 25 ml wash buffer each time.
The hybridization events are detecte~l as described in Example 2.
All possible double-stranded hybrids between the PNA molecules involved and the oligonucleotides are shown in Fig. ld. Figure 2 illustrates that, in almost every case, the only oligonucleotide detected is the one that is exactly complçm~nt~ry to the immobilized nucleic acid analog (PNA 1, PNA 2, PNA 3). The signal-to-noise ratios (S/N) can also be 4300ENGL.DOC
estim~ted from the figure. They were evaluated q~l~ntit~tively, and the results are presented in Table 1.
Table 1 Hybrid (PNA/ODN) S/NSignal (Hybrid)/Signal ~Match) 1/1 655.2 100.0%
2/1 20.7 3.2%
3/1 10.3 1.6%
1/2 23 . 1 2.6%
2/2 871.8 100.0%
3/2 6.4 0.7%
1/3 109.4 22.7%
2/3 12.3 2.5%
3/3 481.1 100.0%
Example 5:
Q~l~ntific~tiQn Membrane strips are derivatized with three (Ado)6-PNA molecules with different base sequences (Fig. la, SEQ.ID.NOS. 1, 2, 3) in a concentration of 100 mM as described in Fx~mrle 1. They are then prehybridized in 20 ml hybridization vessels with 10 mlhybridization buffer (see Example 2) at 45 ~C. The buffer is replaced after 30 mimltes In eXpPrim~nt~ 1 through 7, the buffer to be added differs according to the analyteconcentrations of the DIG-labelled components - oligonucleotide 1, 2 and 3, SEQ.ID.NOS.
4, 5, 6. The strips are hybridized for 60 mimltes at 45 ~C and then washed for 2 x 10 mimltes with 10 ml wash buffer. The detection is peTformed using the procedure described in F.x~mrle 2. The Illminescçnce signal is recorded with a l~lminçsc~nce imager and then evaluated (Fig. 4).
~3c~.~rTT no~
The signal intçn~ities found can be used to reach a qualitative or semi-q l~ntit~tive finding regarding the composition of the analyte complex. Absolutely qu~ntit~tive finflin~ can be reached after the signal in~Pn~iti~e are calibrated.
Example 6:
Detec~ion of PCRAmplicons A. Obtaining a Suitable Analyte (Amplificate) A double-stranded DNA fragment is ligated in a pUCl9 plasrnid, the sequence of which is complem~nt~ry to the PNA probe PNA 1. The plasmid is transformed in E. coli, cloned, and then sequenced. For the subsequent hybridization experiments, a section of the plasmid sequence is amplified and DIG-labelled during the amplification reaction. The amplification is performed in a total volume of 50 Ill. The amplification complex consists of 1 ,ul plasmid (1 ng/~ l primer Fl (10 ,uM~, 1 ,ul DIG primer Rl (10 ll~, 5 ,ul 10 x PCR buffer (100 mM Tris/HCl, 15 mM MgCl2, 500 mM KCI, 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 Taqpolymerase (5 units/~ll) and 38.5 ml water.
Primer Fl: 5'-GTA AAA CGA CGG CCA GT-3' (SEQ.ID.NO. 12) Primer R1: 5'-DIG-AAC AGC TAT GAC CAT GA-3' (SEQ.ID.NO. 13) Each reaction mixture is warmed to 96 ~C for 3 mimltes In the next step, 30 rounds of a 3-level PCR cycle are performed (45 sec. 96 ~C, 30 sec, 48 ~C, 1 min 72 ~C). In the last cycle, the elongation step is increased by 5 mimltes at 72 ~C.
B. Hybridization Reaction The membranes are derivatized with three (Ado)6-PNA sequences each that differ according to ome or two positions in their base sequence (see Fig. la, SEQ.ID.NOS. 1, 2, 3) using ~13~N~'TT T~9C
PNA solutions in a concentration range between 100 ,uM and 0.1 ,uM as described in Example 1. The membrane is p~ ealed in a 20 ml hybridization vessel with 5 ml hybridization buffer at 45 ~C. The buffer is replaced after 30 rnim-tes and the analyte solution is added. To make the analyte solution, the amplification complex is diluted directly (ds a-m--plicon) and, after 5 mimltcs of heat denaturation (ss arnplicon), in 1 m1 hybridi7~tion buffer. Ai~cer hybridization for 1 h, 2h 30 min and 4 h at 45 ~C, the membranes are washed for 2 x 10 minlltes with 5 ml wash buffer each time. Hybri~1i7~tion events are c~etected as described in Exarnple 2 ~Fig. 3).
Nine fields are shown in Figure 3. The difference between each row is the incubation period (4 h, 2-1/2 h, and 1 h). The difference between each column is the type of nucleic acid to be detected Three overlapping rows of spots are applied to each of the 3 fields of column I.
The difference between the rows is the sequence of the PNAs, while the difference between the columns of each field is the concentration. The specificity and the ability to be qll~ntified are in~lic~ted in column I for the case in which oligonucleotides are used as the detecting nucleic acid.
The figure illustrates the infl~nce of inc~b~tiQn time. It is clear that an excellent sequence di~ rimin~tion for ODN l a and the amplificates is obtained after hybridization for just one hour. The difference between columns II and III in Fig. 3 is that an amplificate that was previously made single-stranded is used in one case as the nucleic acid to be detected In column III, an amplificate that was not previously made single-stranded is used as the nucleic acid to be detected The signals in~lic~te clearly that it is not necessary to denature double-stranded nucleic acids before applying them to the solid carrier. This decreases the number of working steps (heating step, single strand separation, wash step) and, therefore, reduces the danger of cont~min~iQn. PNA probes in combination with low-salt conditions therefore offer clear advantages over DNA probes.
430aENG~DOC
Example 7 Comparison of PNA / DNA Hyhri~li7~tion Membrane strips are del;v~lized with three (Ado)6-PNA sequences each (SEQ.ID.NOS. 1, 2, 3) and three DNA molecules each (SEQ.ID.NOS. 8, 10, 11) that differ according to one or two positions in their base sequence (see Fig. la and lb) using 50 mM solutions as described in Fx~mple 1. Unlike Example 1, the spot volume is 400 nl instead of 200 nl. The membrane strips are prehybridized in 20 ml hybricli7~ti~n vessels with either 5 ml 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 either 37 ~C or 45 ~C for 30 min~-tes In the next step, one ofthe three DIG-labelled oligomlrleotides ~Fig. lc, SEQ.ID.NOS. 4, 5, 6) is added. After a hyhri~li7~tion step of 60 min~lte~, the strips are washed for 2 x 10 min-ltes with 5 ml wash buffer each time at 37 ~C or 45 ~C. The wash buffer from Example 2 is used for the low-salt experiments. For the high-salt experiments, a 1 x SSC buffer with 0.02% SDS, pH 7.0 is used. The hybridization results are detected as described in Example 2. The eva~ ti~n is performed q~ntit~tively and is illustrated in Table 2.
Both the DNA and PNA probes are able to completely disc~ a~e between compl~mlont~ry, single-stranded target sequences of single and double mi~m~tchedsequences. PNA probes demonstrate clear advantages over the DNA probes for certain types of mi~m~trhe~, especially when they are not located in the middle of the sequence, but rather shifted to the end. This becomes especially clear in the r~mrle of a decentral G/T
mi~m~trh (probe 1 / ODN 3), which is tolerated by the DNA probe much more strongly than by the PNA probe having the identical sequence.
430~ENGLDOC
Table 2 ~ ,~," s: ~s ~33~ 3~,S, ~,3 ~ 7- ~ P~r ~s ODN 1 PNA45 ~C, low salt 100.0% 1.2% .5~
DNA 37 ~C, high 100.0% 1.2% 6.4%
salt ODN 2 PNA 45 ~C, low salt 1.1% 100.0% 2.9%
DNA 37 ~C, higlî < 2%* 100.0% < 2%*
salt ODN 3 PNA 45 ~C, low salt 26.0% 1.5% 100.0%
DNA 37 ~C, high 66.5% < 2%* 100.0%
salt * A more exact value cannot be detennined because the spot intensity is lower than the standard deviation of the background signal.
~13nn~1~Tr.T T70C
Example 8 TnflllP.n- e of the Length of the Linker Between the Membrane and PNA Probes Membrane strips are derivatized with PNA molecules (see Fig. la, SEQ.ID.NOS. 7, 1, 9) that differ according to the length of the linker (Ado3, Ado6 or Ado9) using PNA solutions in the concentration range between 100 ~lM and 1 ~lM as described in Example 1. Unlike Example 1, the spot volume is 1 111 instead of 200 nl. The membrane strips are pl~hyl~lidized in hybril1i7~tion cont~iners with 10 ml hybricii7~tion buffer (5, 10 or 25 rnM
sodium phosphate, 0.1% SDS, pH 7.0) for 30 min~ltes at 35 ~C. In the next step, 10 pMol 32P-labelled oligonucleotide (see Fig. lc: ODN lb, SEQ.ID.NO. 4) is added and the preparation is hybridized for 60 min~ltes at 50 ~C. The membranes are washed for 2 x 10 minlltes with 50 ml wash buffer (5 mM[ sodium phosphate, 0.1% SDS, pH 7.0) at 50 ~C.
The hybridi7~tion events are detected using autoradiography (Fig. 5). The figure shows that a longer linker greatly improves the hybridization.
Example 9 Reuse of PNA Membranes A membrane is derivatized with (Ado)6-PNA molecules (see Fig. 1 a: PNA lb, SEQ.ID.NO.
1) using PNA solutions in a concentration range between 100 ~lM and 1 ,uM as described in Example 1. The PNA is applied in five identical concentration sequences. The spot volume is 1 ,ul, as in Example 8. The membrane is plehyl .idized in a hybridi:zation vessel with 10 m1 hybridization buffer (10 mM sodium phosphate, 0.1% SDS, pH 7.0) for 30 mimltes at 35 ~C. In the next step, 10 pMol 32P-labelled oligonucleotide (see Fig. lc: ODN lb,SEQ.ID.NO. 4) is added and the prepalalion is hybridized for 60 minllt~i at 50 ~C. The membrane is washed for 2 x 10 mimltes with 50 ml wash buffer (5 mM sodium phosphate, 0.1% SDS, pH 7.0) at 50 ~C. The hybri~i7~tic)n events are detected using autoradiography (Fig. 6a). AP~er the autoradiography is performed, the membrane is cut into five iclenti strips. These membrane strips are each treated di~el elllly in the rest of the experiment.
43WENGL.DOC
Membrane 1 is not inr~lb~ted and serves as the control membrane. Membrane 2 is incub~ted for 60 min-ltcs at room temperature with 50 ml 0.1 M sodium hydroxide solution.
Membrane 3 is inc~lbated for 60 mimltes as well, with 50 ml 1 M sodium hydroxidesolution. Membrane 4 is inc~lb~ted for 60 minll~eS at 70 ~C with 50 ml distilled water.
Membrane 5 is inr,~lb~ted for 60 min~tes at 70 ~C with 50 ml 0.1 N sodium hydroxide solution. All membranes are then washed with tli.~tilled water for 2 x 10 minlltes After this procedure is ct)mrlete~l, autoradiography is pP.rfoTmed once more (Fig. 6b). These membrane strips are then used a second time in a hybridization reaction as described, and the hybridization events are detected using autoradiography (Fig. 6c).
As shown in Fig. 6b, the difre~ tre~tment methods yield very di~le..~ results. Tre~tmPnt with bidistilled water at 70 ~C (membrane 4) causes the membrane to regenerate almost completely. An unexpected discovery was the fact that the success of the regeneration is poorer if conditions are used that are common for the denaturation of nucleic acids. As such, the incub~tion of membrane 3 with 1 M sodium hydroxide solution at room temperature yields virtually no regeneration effect. Decreasing the concentration of sodium hydroxide solution from 1 M to 0.1 ~ increases the degree of regeneration at room temperature (membrane 2) and at 70 ~C (membrane 5). None of these conditions, however, results in an even slightly good degree of regeneration, as is the case with bi~ tillecl water (membrane 4). The example shows that these conditions are important parameters for the ~ffici~nt denaturation of membrane-bound PNA/DNA double-strands.
Regardless of the regeneration method, all membranes can be reused for hybridization (Fig. 6c), without considerably worsening the signal-to-noise ratio. Up to 6 rehybridizations could be performed without a noticeable effect on the PNA membranes' ability to regenerate or rehybridize.
43001~NGL.DOC
SEQUENCE LIST~G
(1) GENERAL ~FORMATION:
(i) APPLICANT:
(A) NAME: Boehringer ~nnh~im GrnbH
(B) STREET: S~n~lhoferstr~ 116 (C) CITY: M~nnh~im (E) COUNTRY: DE
(F) POSTAL CODE (Zn?): 68298 (G) TEI~PHONE: 0621 759 4348 (EI) TELEFAX: 0621 759 4457 (ii) TITLE OF ~IVENTION: Sequence-Specific Detection of Nucleic Acids (iii) NUMBER OF SEQUENCES: 13 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC comp~til~le (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO) (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) sTRANDFnNEss: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(ix) FEATURE:
(A) NAMEfKEY: Modified-site (B) LOCATION:15 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l-adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:14 43001~NGLDOC
, (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l-~hyll~illyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) ~EATURE:
(A) NAME/KEY: Modified-site ~3) LOCATION:13 (D) OTHERINFORMATION:/product= "OTHER"
/note= "Xaa is N-((1 -cytosyl)acetyl)-N-(2-arninoethyl)-beta-alanine"
(ix) F~ATllRE:
(A) NA~JKEY: Modified-site (13) LOCATION:11..12 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l-adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:10 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((1-cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (13) LOCATION:9 ~D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-(( 1 -adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATI~RE:
(A) NAME/KEY: Modified-site ~3) LOCATION:8 ~D) OTEIER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((1 -cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAMEIKEY: Modified-site (B) LOCATION:7 (D) OTHERINFORMATION:/product= "OTHER"
/note= "Xaa is N-((1 -thyminyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site 4300E~GL.DOC
(B) LOCATION:6 ~D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l -guaninyl)acetyl)-N-(z-~minoettlyl)-beta-alanine (ix) ~EATI~
(A) NAMEtKEY: Modified-site Q3) LOCATION:5 (D) OTE~R INFORMATION:/product= "OTHER"
/note= "Xaa is N-((1-cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATI~E:
(A) NAMElKEY: Modified-site (13) LOCATION:4 (D) OTHER lNFORMATION:/product= "OT~R"
/note= "Xaa is N-((l-adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:3 (D) OTHER INFORMATION:/product= "OTE~ER"
/note= "Xaa is N-((l-Lhy~ yl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:2 ~D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l -guaninyl)acetyl)-N-(~-aminoethyl)-beta-alanine"
(ix) ~EATURE:
(A) NAMEIKEY: Modified-site ~B) LOCATION: 1 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((1 -thyminyl)acetyl)-N-(2-amino-N'-(hexa(8-amino-3 ,6-dioxa-oc tano-l-yl)-ethyl)-beta-alanine"
.
(lX) FEATllRE:
(A) NAMEIKEY: Modified-site Q3) LOCATION:16 ~D) Ol~RINFORMATION:/product= "Ol~R"
/note= "Amide"
.~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly (2) INFORMATION FOR SEQ ID NO: 2:
Cl) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECUIE TYPE: peptide (iii) HYPOTHETICAL: NO
(ix) FEATURE:
(A) NAME/KEY: Modified-site Q3) LOCATION:15 (D) OTHER~FORMATION:/product= "OTHER"
/note= "Xaa is N~ adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
~ (A) NAME/KEY: Modified-site ~13) LOCATION: 14 (D) OTHER ~FORMATION:/product= "OTHER"
/note= "Xaa is N-((1-thy~ yl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (13) LOCATION: 13 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((1 -cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:11..12 ~D) OTHERINFORMATION:/product= "OTHER"
/note= "Xaa is N-((1 -adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:10 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l-cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) ~EATURE:
(A) NA~/K~Y: Modified-site (B) LOCATION:9 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l -adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (13) LOCATION:8 (D) OTEIER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l -guaninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:7 (D) O l~R ~IFORMATION:/product= "OT~R"
/note= "Xaa is N-((l -thyminyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:6 (:D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l -guaninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) ~EATURE:
(A) N~¢/KEY: Modified-site (B) LOCATION:5 (D) Ol~RINFORMATION:/product= "OTHER"
/note= "Xaa is N-((l -cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAMEIKEY: Modified-site (B) LOCATION:4 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-~l-adeninyl)acetyl)-N-(2-arninoethyl)-beta-alanine"
430~1ENGLDOC
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:3 Q~) OTHER INFO~MATION:/product= "OTHER"
/note= "Xaa is N-((l-ll~y~ lyl)acetyl)-N-(2-~min~ethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site ~B) LOCATION:2 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((1 -guaninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION: 1 (D) OTHER INFORMATION:/producP "OT~R"
/note= "Xaa is N-((l -thyminyl)acetyl)-N-(2-amino-N'-(hexa(8-amino-3 ,6-dioxa-oc tano- 1 -yl)-ethyl)-beta-alanine"
(ix) FEATllRE:
(A) NAME/KEY: Modified-site ~B) LOCATION: 16 - (D) OTHER INFORMATION:/product= "OTHER"
/note= "Amide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly (2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
430()ENGL.DOC
x) FEATI~
(A) NAME/KEY: Modified-site ~B) LOCATION:15 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l-adeninyl)acetyl)-N-(2-~minoethyl)-beta-alanine"
~lX) ~ATInRE:
(A) NAME/KEY: Modified-site ~13) LOCATION: 14 ~D) OTE~R INFORMATION:tproduct= "OTHER"
/note= "Xaa is N-((1-LLy~ yl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (13) LOCATION:13 ~D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((1-cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
~ix) ~EATURE:
(A) NA~/KEY: Modified-site ~B) LOCATION: 11 . .12 ~D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l-adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
~ix) ~EATURE:
(A) NAME/KEY: Modified-site (13) LOCATION:10 (D) Ol~RINFORMATION:/product= "OTHER"
/note= "Xaa is N-((1-cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (13) LOCATION:9 (D) Ol~R INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l-adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
~ix) FEATI~E:
(A) NAME/KEY: Modified-site (13) LOCATION:8 (D) Ol~R~FORMATION:/product= "OTHER"
/note= "Xaa is 43~;Nt'.T. T'W
N-((l-cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/~Y: Modified-site (B) LOCATION:7 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l-~hymll~yl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site ~13) LOCATION:6 (D) OTHER INl~ORMATION:/product= "OTHER"
/note= "Xaa is N-((1-adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAMEIKEY: Modified-site (13) LOCATION:5 ~D) OTHERINFORMATION:/product= "OTHER"
/note= "Xaa is N-((1-cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:4 (D) OTHER I~lFORMATION:/product= "OTHER"
/note= "Xaa is N-((l-adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATllRE:
(A) NAME/KEY: Modified-site (13) LOCATION:3 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((1 -~Lylllillyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAMElKEY: Modified-site (B) LOCATION:2 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l -guaninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION: 1 ~D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l-thyminyl)acetyl)-N-(2-amino-N'-(hexa(8-amino-3,6-dioxa-oc - tano-l-yl)-ethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site ~13) LOCATION:16 ~D) OTHER INFORMATION:/product= "OTHER"
/note= "Amid"
.
(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly (2) SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 Base pairs (B) TYPE: Nucleotide (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid (A) DESCRIPTION: /desc = "oligodesoxyribonucleotide"
(iii) HYPOTHETICAL: NO
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION:15 (D) OTHER INFORMATION:/note= "labelled at the 5'-phosphate with digoxigenin via aminolinker (Boehringer ~nnh~im GmbX BRD) or 32-P"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
(2) lNFORMATION FOR SEO ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 Base pairs 4300ENGL.DOC-(B) TYPE: Nucleotide (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid (A) DESCRIPTION: /desc = "oligodesoxyribonucleotide"
(iii) HYPOTHETICAL: NO
(ix) FEATURE:~
(A) NAME/KEY: rnisc_feature (B) LOCATION:15 (D) OTHER INFORMATION:/note= "labelled at the 5'-phosphate with digoxigenin via aminolinker (Boehringer ~nnheim GmbX BRD)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 Base pairs ~13) TYPE: Nucleotide (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid (A) DESCRIPTION: /desc = "oligodesoxyribonucleotide"
(iii) HYPOTHETICAL: NO
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION:15 (D) OTHER INFORMATION:/note= "labelled at the 5'-phosphate with oxigf~nin via arninolinker (Boehringer ~nnhPim GmbX BRD)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
(2) INFORMATION FOR SEQ ID NO: 7:
430(ENGI~DOC
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids ~13) TYPE: amino acid (C) STRANDEDNESS: Single ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(ix) ~EATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:15 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((1 -adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:14 ~D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l -lhyll~illyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION: 13 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l-cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site ~3) LOCATION: 11..12 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l -adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site Q3) LOCATION: 10 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((1-cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site 4~ t'.T T10~
(B) LOCATION:9 ~D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l-adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (13) LOCATION:8 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l-cytosyl)acetyl)-N-(2 -aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:7 ~) OTHER INFORMATIO~:/product= "OTHER"
/note= "Xaa is N-((l -thyrninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (13) LOCATION:6 ~D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l -guaninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(IX) FEAllJlRE:
(A) NAME/KEY: Modified-site (B) LOCATION:5 (D) OTHERlNFORMATION:/product= "OTHER"
/note= "Xaa is N-((l -cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:4 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-(( 1 -adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site ~13) LOCATION:3 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l-~llyll~ yl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
430QI~NGLDOC
(A) NAME/KEY: Modified-site (B) LOCATION:2 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((1 -guaninyl)acetyl)-N-(2-aminoethyl)-beta-alar~ine"
(ix) FEATURE:
(A) NAME/K~Y: Modified-site (B) LOCATION: 1 (D) OTHER INFORMATION:/product= "OT~R"
/note= "Xaa is N-~l-~ymillyl)acetyl)-N-(2-amino-N'-(tri(8-amino-3 ,6-dioxa-oct ano-1-yl)-ethyl)-beta-alanine"
(ix) FEATURE:
(A) NAMEIKEY: Modified-site (B) LOCATION: 16 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Amide"
(xi) SEQUENCE DESCR~PTION: SEQ ID NO: 7:
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly (2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 Base pairs (13) TYPE: Nucleotide (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid (A) DESCRIPTION: /desc = "oligodesoxyribonucleotide"
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
1111111111 llllllGTACGTCACAACTA 30 (2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
430al3NGL.DOG
(A) LENGTH: 16 Amino acids (}3) TYPE: amino acid (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOT~TICAL: NO
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:15 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l -adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION: 14 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l-~hylnillyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION: 13 ~D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-(( 1 -cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NA~3/~Y: Modified-site ~13) LOCATION: 11 . .12 (D) OTHER ~FORMATION:/product= "OTHER"
/note= "Xaa is N-((l -adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME~KEY: Modified-site ~B) LOCATION: 10 (D) OTHER INFO:~MATION:/product--"OTHER"
/note= "Xaa is N-((1 -cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) ~EATURE:
(A) NAMEIKEY: Modified-site (B) LOCATION:9 430~XENGI,DOC
-(I)) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l-adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATVRE:
(A) NAME/KEY: Modified-site (B) LOCATION:8 (D) OTHER INFORMATION:/producP "OTHER"
/note= "Xaa is N-((l-cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) ~EATURE:
(A) NAME~KEY: Modified-site (E3) LOCATION:7 ~D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-(( 1 -~11yl~lillyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site ~13) LOCATION:6 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((1-guaninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEAT~RE:
(A) NAMEIKEY: Modified-site (13) LOCATION:S
~D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l -cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/~EY: Modified-site (13) LOCATION:4 (D) OTHER ~IFORMATION:/product= "OTEIER"
/note= "Xaa is N-(( l -adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) ~EATURE:
(A) NAME/KEY: Modified-site (13) LOCATION:3 ~D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-~l -lhymi~yl)acetyl)-N-(2-aminoethyl)-beta-alanine~
(ix) FEATVRE:
(A) NAME~KEY: Modified-site 430~.Nr.r. noC
(B) LOCATION:2 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((1 -guaninyl)acetyl)-N-(2-arninoethyl)-beta-alanine"
(~x) FEATURE:
(A) NAME~KEY: Modified-site ~B) LOCATION: 1 ~1)) OTHER INFORMATION:/producP "OTHER"
/note= "Xaa is N-((l-thyminyl)acetyl)-N-(2-amino-Nl-(nona(8-amino-3,6-dioxa-oc tano-1-yl)-ethyl)-beta-alanine"
(ix) FEATI~RE:
(A) NAME/KEY: Modified-site (B) LOCATION:16 (D) OTHERINFORMATION:/product= "OTHER"
/note= "Arnide"
(x~) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly (2) rNFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 Base pairs (B) TYPE: Nucleotide (C) STRANDEDNESS: Single (D) TOPOLOGY: linear ~ (ii) MOLECULE TYPE: Other nucleic acid (A) DESCRIPTION: /desc = "oligodesoxyribonucleotide"
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
1111111111 llllllGTACGTGACAACTA 30 (2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 Base pairs 4300ENGL.DOC
(B) TYPE: Nucleotide (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid (A) DESCRIPTION: /desc = "oligodesoxyribonucleotide"
(iii) HYPOTHETICAL: NO
(xi) S}~QUENCE DESCRIPTION: SEQ ID NO: 11:
l l l l l l l l l L l l l l l lGTAC ATCACAACTA 30 (2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CH.9R~CTERISTICS:
(A) LENGTH: 17 Base pairs ~13) TYPE: Nucleotide (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid (A) DESCRIPTION: /desc= "Oligodesoxyribonucleotide"
(iii) HYPOl~TICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 Base pairs (B) TYPE: Nucleotide (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid (A) DESCRIPTION: /desc = "Oligodesoxyribonucleotide"
(iii? HYPOTHETICAL: NO
(ix) FEATURE:
(A) NAME/KEY: misc_feature 43001~NGL.DOC
-~B) LOCATION: 1 (D) OTHER INFORMATION:/note= "A at the 5'-terminus is bound via aminomodifier ~Boehringer l!~nnh~im GmbH) to digoxigenin"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
43Q~.Nr.r. r~oc
~3c~.~rTT no~
The signal intçn~ities found can be used to reach a qualitative or semi-q l~ntit~tive finding regarding the composition of the analyte complex. Absolutely qu~ntit~tive finflin~ can be reached after the signal in~Pn~iti~e are calibrated.
Example 6:
Detec~ion of PCRAmplicons A. Obtaining a Suitable Analyte (Amplificate) A double-stranded DNA fragment is ligated in a pUCl9 plasrnid, the sequence of which is complem~nt~ry to the PNA probe PNA 1. The plasmid is transformed in E. coli, cloned, and then sequenced. For the subsequent hybridization experiments, a section of the plasmid sequence is amplified and DIG-labelled during the amplification reaction. The amplification is performed in a total volume of 50 Ill. The amplification complex consists of 1 ,ul plasmid (1 ng/~ l primer Fl (10 ,uM~, 1 ,ul DIG primer Rl (10 ll~, 5 ,ul 10 x PCR buffer (100 mM Tris/HCl, 15 mM MgCl2, 500 mM KCI, 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 Taqpolymerase (5 units/~ll) and 38.5 ml water.
Primer Fl: 5'-GTA AAA CGA CGG CCA GT-3' (SEQ.ID.NO. 12) Primer R1: 5'-DIG-AAC AGC TAT GAC CAT GA-3' (SEQ.ID.NO. 13) Each reaction mixture is warmed to 96 ~C for 3 mimltes In the next step, 30 rounds of a 3-level PCR cycle are performed (45 sec. 96 ~C, 30 sec, 48 ~C, 1 min 72 ~C). In the last cycle, the elongation step is increased by 5 mimltes at 72 ~C.
B. Hybridization Reaction The membranes are derivatized with three (Ado)6-PNA sequences each that differ according to ome or two positions in their base sequence (see Fig. la, SEQ.ID.NOS. 1, 2, 3) using ~13~N~'TT T~9C
PNA solutions in a concentration range between 100 ,uM and 0.1 ,uM as described in Example 1. The membrane is p~ ealed in a 20 ml hybridization vessel with 5 ml hybridization buffer at 45 ~C. The buffer is replaced after 30 rnim-tes and the analyte solution is added. To make the analyte solution, the amplification complex is diluted directly (ds a-m--plicon) and, after 5 mimltcs of heat denaturation (ss arnplicon), in 1 m1 hybridi7~tion buffer. Ai~cer hybridization for 1 h, 2h 30 min and 4 h at 45 ~C, the membranes are washed for 2 x 10 minlltes with 5 ml wash buffer each time. Hybri~1i7~tion events are c~etected as described in Exarnple 2 ~Fig. 3).
Nine fields are shown in Figure 3. The difference between each row is the incubation period (4 h, 2-1/2 h, and 1 h). The difference between each column is the type of nucleic acid to be detected Three overlapping rows of spots are applied to each of the 3 fields of column I.
The difference between the rows is the sequence of the PNAs, while the difference between the columns of each field is the concentration. The specificity and the ability to be qll~ntified are in~lic~ted in column I for the case in which oligonucleotides are used as the detecting nucleic acid.
The figure illustrates the infl~nce of inc~b~tiQn time. It is clear that an excellent sequence di~ rimin~tion for ODN l a and the amplificates is obtained after hybridization for just one hour. The difference between columns II and III in Fig. 3 is that an amplificate that was previously made single-stranded is used in one case as the nucleic acid to be detected In column III, an amplificate that was not previously made single-stranded is used as the nucleic acid to be detected The signals in~lic~te clearly that it is not necessary to denature double-stranded nucleic acids before applying them to the solid carrier. This decreases the number of working steps (heating step, single strand separation, wash step) and, therefore, reduces the danger of cont~min~iQn. PNA probes in combination with low-salt conditions therefore offer clear advantages over DNA probes.
430aENG~DOC
Example 7 Comparison of PNA / DNA Hyhri~li7~tion Membrane strips are del;v~lized with three (Ado)6-PNA sequences each (SEQ.ID.NOS. 1, 2, 3) and three DNA molecules each (SEQ.ID.NOS. 8, 10, 11) that differ according to one or two positions in their base sequence (see Fig. la and lb) using 50 mM solutions as described in Fx~mple 1. Unlike Example 1, the spot volume is 400 nl instead of 200 nl. The membrane strips are prehybridized in 20 ml hybricli7~ti~n vessels with either 5 ml 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 either 37 ~C or 45 ~C for 30 min~-tes In the next step, one ofthe three DIG-labelled oligomlrleotides ~Fig. lc, SEQ.ID.NOS. 4, 5, 6) is added. After a hyhri~li7~tion step of 60 min~lte~, the strips are washed for 2 x 10 min-ltes with 5 ml wash buffer each time at 37 ~C or 45 ~C. The wash buffer from Example 2 is used for the low-salt experiments. For the high-salt experiments, a 1 x SSC buffer with 0.02% SDS, pH 7.0 is used. The hybridization results are detected as described in Example 2. The eva~ ti~n is performed q~ntit~tively and is illustrated in Table 2.
Both the DNA and PNA probes are able to completely disc~ a~e between compl~mlont~ry, single-stranded target sequences of single and double mi~m~tchedsequences. PNA probes demonstrate clear advantages over the DNA probes for certain types of mi~m~trhe~, especially when they are not located in the middle of the sequence, but rather shifted to the end. This becomes especially clear in the r~mrle of a decentral G/T
mi~m~trh (probe 1 / ODN 3), which is tolerated by the DNA probe much more strongly than by the PNA probe having the identical sequence.
430~ENGLDOC
Table 2 ~ ,~," s: ~s ~33~ 3~,S, ~,3 ~ 7- ~ P~r ~s ODN 1 PNA45 ~C, low salt 100.0% 1.2% .5~
DNA 37 ~C, high 100.0% 1.2% 6.4%
salt ODN 2 PNA 45 ~C, low salt 1.1% 100.0% 2.9%
DNA 37 ~C, higlî < 2%* 100.0% < 2%*
salt ODN 3 PNA 45 ~C, low salt 26.0% 1.5% 100.0%
DNA 37 ~C, high 66.5% < 2%* 100.0%
salt * A more exact value cannot be detennined because the spot intensity is lower than the standard deviation of the background signal.
~13nn~1~Tr.T T70C
Example 8 TnflllP.n- e of the Length of the Linker Between the Membrane and PNA Probes Membrane strips are derivatized with PNA molecules (see Fig. la, SEQ.ID.NOS. 7, 1, 9) that differ according to the length of the linker (Ado3, Ado6 or Ado9) using PNA solutions in the concentration range between 100 ~lM and 1 ~lM as described in Example 1. Unlike Example 1, the spot volume is 1 111 instead of 200 nl. The membrane strips are pl~hyl~lidized in hybril1i7~tion cont~iners with 10 ml hybricii7~tion buffer (5, 10 or 25 rnM
sodium phosphate, 0.1% SDS, pH 7.0) for 30 min~ltes at 35 ~C. In the next step, 10 pMol 32P-labelled oligonucleotide (see Fig. lc: ODN lb, SEQ.ID.NO. 4) is added and the preparation is hybridized for 60 min~ltes at 50 ~C. The membranes are washed for 2 x 10 minlltes with 50 ml wash buffer (5 mM[ sodium phosphate, 0.1% SDS, pH 7.0) at 50 ~C.
The hybridi7~tion events are detected using autoradiography (Fig. 5). The figure shows that a longer linker greatly improves the hybridization.
Example 9 Reuse of PNA Membranes A membrane is derivatized with (Ado)6-PNA molecules (see Fig. 1 a: PNA lb, SEQ.ID.NO.
1) using PNA solutions in a concentration range between 100 ~lM and 1 ,uM as described in Example 1. The PNA is applied in five identical concentration sequences. The spot volume is 1 ,ul, as in Example 8. The membrane is plehyl .idized in a hybridi:zation vessel with 10 m1 hybridization buffer (10 mM sodium phosphate, 0.1% SDS, pH 7.0) for 30 mimltes at 35 ~C. In the next step, 10 pMol 32P-labelled oligonucleotide (see Fig. lc: ODN lb,SEQ.ID.NO. 4) is added and the prepalalion is hybridized for 60 minllt~i at 50 ~C. The membrane is washed for 2 x 10 mimltes with 50 ml wash buffer (5 mM sodium phosphate, 0.1% SDS, pH 7.0) at 50 ~C. The hybri~i7~tic)n events are detected using autoradiography (Fig. 6a). AP~er the autoradiography is performed, the membrane is cut into five iclenti strips. These membrane strips are each treated di~el elllly in the rest of the experiment.
43WENGL.DOC
Membrane 1 is not inr~lb~ted and serves as the control membrane. Membrane 2 is incub~ted for 60 min-ltcs at room temperature with 50 ml 0.1 M sodium hydroxide solution.
Membrane 3 is inc~lbated for 60 mimltes as well, with 50 ml 1 M sodium hydroxidesolution. Membrane 4 is inc~lb~ted for 60 minll~eS at 70 ~C with 50 ml distilled water.
Membrane 5 is inr,~lb~ted for 60 min~tes at 70 ~C with 50 ml 0.1 N sodium hydroxide solution. All membranes are then washed with tli.~tilled water for 2 x 10 minlltes After this procedure is ct)mrlete~l, autoradiography is pP.rfoTmed once more (Fig. 6b). These membrane strips are then used a second time in a hybridization reaction as described, and the hybridization events are detected using autoradiography (Fig. 6c).
As shown in Fig. 6b, the difre~ tre~tment methods yield very di~le..~ results. Tre~tmPnt with bidistilled water at 70 ~C (membrane 4) causes the membrane to regenerate almost completely. An unexpected discovery was the fact that the success of the regeneration is poorer if conditions are used that are common for the denaturation of nucleic acids. As such, the incub~tion of membrane 3 with 1 M sodium hydroxide solution at room temperature yields virtually no regeneration effect. Decreasing the concentration of sodium hydroxide solution from 1 M to 0.1 ~ increases the degree of regeneration at room temperature (membrane 2) and at 70 ~C (membrane 5). None of these conditions, however, results in an even slightly good degree of regeneration, as is the case with bi~ tillecl water (membrane 4). The example shows that these conditions are important parameters for the ~ffici~nt denaturation of membrane-bound PNA/DNA double-strands.
Regardless of the regeneration method, all membranes can be reused for hybridization (Fig. 6c), without considerably worsening the signal-to-noise ratio. Up to 6 rehybridizations could be performed without a noticeable effect on the PNA membranes' ability to regenerate or rehybridize.
43001~NGL.DOC
SEQUENCE LIST~G
(1) GENERAL ~FORMATION:
(i) APPLICANT:
(A) NAME: Boehringer ~nnh~im GrnbH
(B) STREET: S~n~lhoferstr~ 116 (C) CITY: M~nnh~im (E) COUNTRY: DE
(F) POSTAL CODE (Zn?): 68298 (G) TEI~PHONE: 0621 759 4348 (EI) TELEFAX: 0621 759 4457 (ii) TITLE OF ~IVENTION: Sequence-Specific Detection of Nucleic Acids (iii) NUMBER OF SEQUENCES: 13 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC comp~til~le (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO) (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) sTRANDFnNEss: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(ix) FEATURE:
(A) NAMEfKEY: Modified-site (B) LOCATION:15 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l-adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:14 43001~NGLDOC
, (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l-~hyll~illyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) ~EATURE:
(A) NAME/KEY: Modified-site ~3) LOCATION:13 (D) OTHERINFORMATION:/product= "OTHER"
/note= "Xaa is N-((1 -cytosyl)acetyl)-N-(2-arninoethyl)-beta-alanine"
(ix) F~ATllRE:
(A) NA~JKEY: Modified-site (13) LOCATION:11..12 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l-adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:10 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((1-cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (13) LOCATION:9 ~D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-(( 1 -adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATI~RE:
(A) NAME/KEY: Modified-site ~3) LOCATION:8 ~D) OTEIER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((1 -cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAMEIKEY: Modified-site (B) LOCATION:7 (D) OTHERINFORMATION:/product= "OTHER"
/note= "Xaa is N-((1 -thyminyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site 4300E~GL.DOC
(B) LOCATION:6 ~D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l -guaninyl)acetyl)-N-(z-~minoettlyl)-beta-alanine (ix) ~EATI~
(A) NAMEtKEY: Modified-site Q3) LOCATION:5 (D) OTE~R INFORMATION:/product= "OTHER"
/note= "Xaa is N-((1-cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATI~E:
(A) NAMElKEY: Modified-site (13) LOCATION:4 (D) OTHER lNFORMATION:/product= "OT~R"
/note= "Xaa is N-((l-adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:3 (D) OTHER INFORMATION:/product= "OTE~ER"
/note= "Xaa is N-((l-Lhy~ yl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:2 ~D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l -guaninyl)acetyl)-N-(~-aminoethyl)-beta-alanine"
(ix) ~EATURE:
(A) NAMEIKEY: Modified-site ~B) LOCATION: 1 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((1 -thyminyl)acetyl)-N-(2-amino-N'-(hexa(8-amino-3 ,6-dioxa-oc tano-l-yl)-ethyl)-beta-alanine"
.
(lX) FEATllRE:
(A) NAMEIKEY: Modified-site Q3) LOCATION:16 ~D) Ol~RINFORMATION:/product= "Ol~R"
/note= "Amide"
.~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly (2) INFORMATION FOR SEQ ID NO: 2:
Cl) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECUIE TYPE: peptide (iii) HYPOTHETICAL: NO
(ix) FEATURE:
(A) NAME/KEY: Modified-site Q3) LOCATION:15 (D) OTHER~FORMATION:/product= "OTHER"
/note= "Xaa is N~ adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
~ (A) NAME/KEY: Modified-site ~13) LOCATION: 14 (D) OTHER ~FORMATION:/product= "OTHER"
/note= "Xaa is N-((1-thy~ yl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (13) LOCATION: 13 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((1 -cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:11..12 ~D) OTHERINFORMATION:/product= "OTHER"
/note= "Xaa is N-((1 -adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:10 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l-cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) ~EATURE:
(A) NA~/K~Y: Modified-site (B) LOCATION:9 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l -adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (13) LOCATION:8 (D) OTEIER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l -guaninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:7 (D) O l~R ~IFORMATION:/product= "OT~R"
/note= "Xaa is N-((l -thyminyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:6 (:D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l -guaninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) ~EATURE:
(A) N~¢/KEY: Modified-site (B) LOCATION:5 (D) Ol~RINFORMATION:/product= "OTHER"
/note= "Xaa is N-((l -cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAMEIKEY: Modified-site (B) LOCATION:4 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-~l-adeninyl)acetyl)-N-(2-arninoethyl)-beta-alanine"
430~1ENGLDOC
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:3 Q~) OTHER INFO~MATION:/product= "OTHER"
/note= "Xaa is N-((l-ll~y~ lyl)acetyl)-N-(2-~min~ethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site ~B) LOCATION:2 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((1 -guaninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION: 1 (D) OTHER INFORMATION:/producP "OT~R"
/note= "Xaa is N-((l -thyminyl)acetyl)-N-(2-amino-N'-(hexa(8-amino-3 ,6-dioxa-oc tano- 1 -yl)-ethyl)-beta-alanine"
(ix) FEATllRE:
(A) NAME/KEY: Modified-site ~B) LOCATION: 16 - (D) OTHER INFORMATION:/product= "OTHER"
/note= "Amide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly (2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
430()ENGL.DOC
x) FEATI~
(A) NAME/KEY: Modified-site ~B) LOCATION:15 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l-adeninyl)acetyl)-N-(2-~minoethyl)-beta-alanine"
~lX) ~ATInRE:
(A) NAME/KEY: Modified-site ~13) LOCATION: 14 ~D) OTE~R INFORMATION:tproduct= "OTHER"
/note= "Xaa is N-((1-LLy~ yl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (13) LOCATION:13 ~D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((1-cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
~ix) ~EATURE:
(A) NA~/KEY: Modified-site ~B) LOCATION: 11 . .12 ~D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l-adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
~ix) ~EATURE:
(A) NAME/KEY: Modified-site (13) LOCATION:10 (D) Ol~RINFORMATION:/product= "OTHER"
/note= "Xaa is N-((1-cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (13) LOCATION:9 (D) Ol~R INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l-adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
~ix) FEATI~E:
(A) NAME/KEY: Modified-site (13) LOCATION:8 (D) Ol~R~FORMATION:/product= "OTHER"
/note= "Xaa is 43~;Nt'.T. T'W
N-((l-cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/~Y: Modified-site (B) LOCATION:7 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l-~hymll~yl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site ~13) LOCATION:6 (D) OTHER INl~ORMATION:/product= "OTHER"
/note= "Xaa is N-((1-adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAMEIKEY: Modified-site (13) LOCATION:5 ~D) OTHERINFORMATION:/product= "OTHER"
/note= "Xaa is N-((1-cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:4 (D) OTHER I~lFORMATION:/product= "OTHER"
/note= "Xaa is N-((l-adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATllRE:
(A) NAME/KEY: Modified-site (13) LOCATION:3 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((1 -~Lylllillyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAMElKEY: Modified-site (B) LOCATION:2 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l -guaninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION: 1 ~D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l-thyminyl)acetyl)-N-(2-amino-N'-(hexa(8-amino-3,6-dioxa-oc - tano-l-yl)-ethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site ~13) LOCATION:16 ~D) OTHER INFORMATION:/product= "OTHER"
/note= "Amid"
.
(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly (2) SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 Base pairs (B) TYPE: Nucleotide (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid (A) DESCRIPTION: /desc = "oligodesoxyribonucleotide"
(iii) HYPOTHETICAL: NO
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION:15 (D) OTHER INFORMATION:/note= "labelled at the 5'-phosphate with digoxigenin via aminolinker (Boehringer ~nnh~im GmbX BRD) or 32-P"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
(2) lNFORMATION FOR SEO ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 Base pairs 4300ENGL.DOC-(B) TYPE: Nucleotide (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid (A) DESCRIPTION: /desc = "oligodesoxyribonucleotide"
(iii) HYPOTHETICAL: NO
(ix) FEATURE:~
(A) NAME/KEY: rnisc_feature (B) LOCATION:15 (D) OTHER INFORMATION:/note= "labelled at the 5'-phosphate with digoxigenin via aminolinker (Boehringer ~nnheim GmbX BRD)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 Base pairs ~13) TYPE: Nucleotide (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid (A) DESCRIPTION: /desc = "oligodesoxyribonucleotide"
(iii) HYPOTHETICAL: NO
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION:15 (D) OTHER INFORMATION:/note= "labelled at the 5'-phosphate with oxigf~nin via arninolinker (Boehringer ~nnhPim GmbX BRD)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
(2) INFORMATION FOR SEQ ID NO: 7:
430(ENGI~DOC
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids ~13) TYPE: amino acid (C) STRANDEDNESS: Single ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(ix) ~EATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:15 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((1 -adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:14 ~D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l -lhyll~illyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION: 13 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l-cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site ~3) LOCATION: 11..12 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l -adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site Q3) LOCATION: 10 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((1-cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site 4~ t'.T T10~
(B) LOCATION:9 ~D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l-adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (13) LOCATION:8 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l-cytosyl)acetyl)-N-(2 -aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:7 ~) OTHER INFORMATIO~:/product= "OTHER"
/note= "Xaa is N-((l -thyrninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (13) LOCATION:6 ~D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l -guaninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(IX) FEAllJlRE:
(A) NAME/KEY: Modified-site (B) LOCATION:5 (D) OTHERlNFORMATION:/product= "OTHER"
/note= "Xaa is N-((l -cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:4 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-(( 1 -adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site ~13) LOCATION:3 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l-~llyll~ yl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
430QI~NGLDOC
(A) NAME/KEY: Modified-site (B) LOCATION:2 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((1 -guaninyl)acetyl)-N-(2-aminoethyl)-beta-alar~ine"
(ix) FEATURE:
(A) NAME/K~Y: Modified-site (B) LOCATION: 1 (D) OTHER INFORMATION:/product= "OT~R"
/note= "Xaa is N-~l-~ymillyl)acetyl)-N-(2-amino-N'-(tri(8-amino-3 ,6-dioxa-oct ano-1-yl)-ethyl)-beta-alanine"
(ix) FEATURE:
(A) NAMEIKEY: Modified-site (B) LOCATION: 16 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Amide"
(xi) SEQUENCE DESCR~PTION: SEQ ID NO: 7:
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly (2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 Base pairs (13) TYPE: Nucleotide (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid (A) DESCRIPTION: /desc = "oligodesoxyribonucleotide"
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
1111111111 llllllGTACGTCACAACTA 30 (2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
430al3NGL.DOG
(A) LENGTH: 16 Amino acids (}3) TYPE: amino acid (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOT~TICAL: NO
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION:15 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l -adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION: 14 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l-~hylnillyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION: 13 ~D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-(( 1 -cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NA~3/~Y: Modified-site ~13) LOCATION: 11 . .12 (D) OTHER ~FORMATION:/product= "OTHER"
/note= "Xaa is N-((l -adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME~KEY: Modified-site ~B) LOCATION: 10 (D) OTHER INFO:~MATION:/product--"OTHER"
/note= "Xaa is N-((1 -cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) ~EATURE:
(A) NAMEIKEY: Modified-site (B) LOCATION:9 430~XENGI,DOC
-(I)) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l-adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATVRE:
(A) NAME/KEY: Modified-site (B) LOCATION:8 (D) OTHER INFORMATION:/producP "OTHER"
/note= "Xaa is N-((l-cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) ~EATURE:
(A) NAME~KEY: Modified-site (E3) LOCATION:7 ~D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-(( 1 -~11yl~lillyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site ~13) LOCATION:6 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((1-guaninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEAT~RE:
(A) NAMEIKEY: Modified-site (13) LOCATION:S
~D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((l -cytosyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) FEATURE:
(A) NAME/~EY: Modified-site (13) LOCATION:4 (D) OTHER ~IFORMATION:/product= "OTEIER"
/note= "Xaa is N-(( l -adeninyl)acetyl)-N-(2-aminoethyl)-beta-alanine"
(ix) ~EATURE:
(A) NAME/KEY: Modified-site (13) LOCATION:3 ~D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-~l -lhymi~yl)acetyl)-N-(2-aminoethyl)-beta-alanine~
(ix) FEATVRE:
(A) NAME~KEY: Modified-site 430~.Nr.r. noC
(B) LOCATION:2 (D) OTHER INFORMATION:/product= "OTHER"
/note= "Xaa is N-((1 -guaninyl)acetyl)-N-(2-arninoethyl)-beta-alanine"
(~x) FEATURE:
(A) NAME~KEY: Modified-site ~B) LOCATION: 1 ~1)) OTHER INFORMATION:/producP "OTHER"
/note= "Xaa is N-((l-thyminyl)acetyl)-N-(2-amino-Nl-(nona(8-amino-3,6-dioxa-oc tano-1-yl)-ethyl)-beta-alanine"
(ix) FEATI~RE:
(A) NAME/KEY: Modified-site (B) LOCATION:16 (D) OTHERINFORMATION:/product= "OTHER"
/note= "Arnide"
(x~) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly (2) rNFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 Base pairs (B) TYPE: Nucleotide (C) STRANDEDNESS: Single (D) TOPOLOGY: linear ~ (ii) MOLECULE TYPE: Other nucleic acid (A) DESCRIPTION: /desc = "oligodesoxyribonucleotide"
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
1111111111 llllllGTACGTGACAACTA 30 (2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 Base pairs 4300ENGL.DOC
(B) TYPE: Nucleotide (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid (A) DESCRIPTION: /desc = "oligodesoxyribonucleotide"
(iii) HYPOTHETICAL: NO
(xi) S}~QUENCE DESCRIPTION: SEQ ID NO: 11:
l l l l l l l l l L l l l l l lGTAC ATCACAACTA 30 (2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CH.9R~CTERISTICS:
(A) LENGTH: 17 Base pairs ~13) TYPE: Nucleotide (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid (A) DESCRIPTION: /desc= "Oligodesoxyribonucleotide"
(iii) HYPOl~TICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 Base pairs (B) TYPE: Nucleotide (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other nucleic acid (A) DESCRIPTION: /desc = "Oligodesoxyribonucleotide"
(iii? HYPOTHETICAL: NO
(ix) FEATURE:
(A) NAME/KEY: misc_feature 43001~NGL.DOC
-~B) LOCATION: 1 (D) OTHER INFORMATION:/note= "A at the 5'-terminus is bound via aminomodifier ~Boehringer l!~nnh~im GmbH) to digoxigenin"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
43Q~.Nr.r. r~oc
Claims (13)
1. Solid carrier having two or more nucleic acid analogs with different base sequences bound to different sites on its surface.
2. Carrier in accordance with claim 1, characterized in that the nucleic acid analogs are covalently bound.
3. Carrier in accordance with claim 1 or 2, characterized in that the nucleic acid analogs are bound by a linker that is more than 15 atoms and fewer than 200 atoms in length.
4. Carrier in accordance with one of the aforementioned claims, characterized in that the solid carrier has a surface that is not charged and/or is hydrophilic.
5. Method for the sequence-specific detection of a nucleic acid by - bringing a sample containing nucleic acids in contact with the sites on the surface of a carrier as described in one of the claims 1 to 4, wherein at least one of the nucleic acid analogs has a base sequence that is complementary to one base sequence of the nucleic acid to be detected and at least one other nucleic acid analog has a base sequence that is not complementary to a base sequence of the nucleic acid to be detected, said procedure being performed under conditions in which the nucleic acid to be detected binds to the nucleic acid analog, - determining the binding that took place at the predetermined site as an indicator of the presence of the nucleic acid to be detected.
6. Method in accordance with claim 5, characterized in that the conditions under which the nucleic acid to be detected binds with the nucleic analog mean the presence of less than 10 mM salt.
7. Method in accordance with claim 5 or 6, characterized in that the nucleic acid to be detected is the result of a nucleic acid amplification reaction.
8. Method in accordance with one of the claims 5 to 7, characterized in that the nucleic acid is detectably labeled.
9. Method in accordance with one of the claims 5 to 7, characterized in that the binding is detected by means of an intercalating agent.
10. Method in accordance with one of the claims 5 to 7, characterized in that the binding that took place is detected using an antibody against the binding product that is labelled in such a way that it can be detected.
11. Method for the selective detection of mutants of nucleic acids in the presence of a large excess of non-mutant nucleic acids, characterized in that the sample containing nucleic acid is brought in contact with a carrier as described in claim 1, and the binding of the mutant nucleic acid and/or the non-mutant nucleic acids is determined at different sites.
12. Method for the determination of the relative quantity of a mutant and a normal nucleic acid by bringing a sample containing nucleic acid in contact with a solid carrier as described in Claim 1 and deriving an indication of the quantity of mutant nucleic acids by comparing the bound mutant nucleic acid and non-mutant nucleic acid.
13. Use of a solid carrier in accordance with claim 1 for the quantitative determination of nucleic acids.
Applications Claiming Priority (6)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP95103122.8 | 1995-03-04 | ||
| EP95103122 | 1995-03-04 | ||
| EP95118843.2 | 1995-11-30 | ||
| EP95118843 | 1995-11-30 | ||
| DE19548590A DE19548590A1 (en) | 1995-12-23 | 1995-12-23 | Solid carrier with different nucleic acid analogues at different surface positions |
| DE19548590.4 | 1995-12-23 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2214430A1 true CA2214430A1 (en) | 1996-09-12 |
Family
ID=27215779
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002214430A Abandoned CA2214430A1 (en) | 1995-03-04 | 1996-03-04 | Sequence-specific detection of nucleic acids |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0813610A1 (en) |
| JP (2) | JP2000513921A (en) |
| AU (1) | AU5002996A (en) |
| CA (1) | CA2214430A1 (en) |
| WO (1) | WO1996027680A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6365349B1 (en) | 1997-07-22 | 2002-04-02 | Qiagen Genomics, Inc. | Apparatus and methods for arraying solution onto a solid support |
Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7375198B2 (en) | 1993-10-26 | 2008-05-20 | Affymetrix, Inc. | Modified nucleic acid probes |
| US6156501A (en) * | 1993-10-26 | 2000-12-05 | Affymetrix, Inc. | Arrays of modified nucleic acid probes and methods of use |
| US6458530B1 (en) | 1996-04-04 | 2002-10-01 | Affymetrix Inc. | Selecting tag nucleic acids |
| WO1998008981A1 (en) | 1996-08-30 | 1998-03-05 | Life Technologies, Inc. | METHODS FOR IDENTIFICATION AND ISOLATION OF SPECIFIC NUCLEOTIDE SEQUENCES IN cDNA AND GENOMIC DNA |
| US6306588B1 (en) | 1997-02-07 | 2001-10-23 | Invitrogen Corporation | Polymerases for analyzing or typing polymorphic nucleic acid fragments and uses thereof |
| CN1268979A (en) * | 1997-07-22 | 2000-10-04 | 拉普吉恩公司 | Multiple functionilities within an array element and uses thereof |
| WO2000070345A1 (en) | 1999-05-14 | 2000-11-23 | Iris Bio Technologies | Reversible immobilization of ligands onto metal surfaces, their preparation and use in biochemical applications |
| WO2001001144A2 (en) * | 1999-06-30 | 2001-01-04 | Iris Bio Technologies | Hybridization of target dna with immobilized nucleic acid analogs |
| EP1111069A1 (en) * | 1999-12-22 | 2001-06-27 | BioChip Technologies GmbH | Modified nucleic acids and their use |
| US6316608B1 (en) * | 2000-03-20 | 2001-11-13 | Incyte Genomics, Inc. | Combined polynucleotide sequence as discrete assay endpoints |
| DE10152925A1 (en) * | 2001-10-26 | 2003-05-08 | Febit Ag | Asymmetric probes |
| JP2005520554A (en) * | 2002-03-21 | 2005-07-14 | ボストン プローブス，インコーポレイテッド | PNA oligomers, oligomer sets, methods and kits for the detection of Bacillus Anthracis |
| JP4524392B2 (en) * | 2006-04-25 | 2010-08-18 | 国立大学法人 千葉大学 | Method for regenerating carrier for immobilizing probe polynucleotide |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB8810400D0 (en) * | 1988-05-03 | 1988-06-08 | Southern E | Analysing polynucleotide sequences |
| GB8811608D0 (en) * | 1988-05-17 | 1988-06-22 | Animal Health Inst | Detecting disease susceptibility |
| ATE173508T1 (en) * | 1988-05-20 | 1998-12-15 | Hoffmann La Roche | ATTACHMENT OF SEQUENCE-SPECIFIC SAMPLES |
| US5641625A (en) * | 1992-05-22 | 1997-06-24 | Isis Pharmaceuticals, Inc. | Cleaving double-stranded DNA with peptide nucleic acids |
| GB9211979D0 (en) * | 1992-06-05 | 1992-07-15 | Buchard Ole | Uses of nucleic acid analogues |
-
1996
- 1996-03-04 AU AU50029/96A patent/AU5002996A/en not_active Abandoned
- 1996-03-04 EP EP96906732A patent/EP0813610A1/en not_active Withdrawn
- 1996-03-04 JP JP08526599A patent/JP2000513921A/en not_active Ceased
- 1996-03-04 CA CA002214430A patent/CA2214430A1/en not_active Abandoned
- 1996-03-04 WO PCT/EP1996/000893 patent/WO1996027680A1/en not_active Application Discontinuation
-
2003
- 2003-02-26 JP JP2003048630A patent/JP2004000154A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6365349B1 (en) | 1997-07-22 | 2002-04-02 | Qiagen Genomics, Inc. | Apparatus and methods for arraying solution onto a solid support |
Also Published As
| Publication number | Publication date |
|---|---|
| EP0813610A1 (en) | 1997-12-29 |
| WO1996027680A1 (en) | 1996-09-12 |
| JP2000513921A (en) | 2000-10-24 |
| JP2004000154A (en) | 2004-01-08 |
| AU5002996A (en) | 1996-09-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA2399733C (en) | Nucleic acid detection methods using universal priming | |
| US6214587B1 (en) | Isothermal strand displacement nucleic acid amplification | |
| AU783841B2 (en) | Nucleic acid probe arrays | |
| EP1358352B1 (en) | Solid support assay systems and methods utilizing non-standard bases | |
| EP2163653B1 (en) | Nucleic acid detection method having increased sensitivity | |
| US20020197630A1 (en) | Systems and methods for high throughput genetic analysis | |
| US6475721B2 (en) | Sequence specific detection of nucleic acids using a solid carrier bound with nucleic acid analog probes | |
| CA2214430A1 (en) | Sequence-specific detection of nucleic acids | |
| WO2002101358A2 (en) | Multiplexed detection methods | |
| US20170356056A1 (en) | Detection of pna clamping | |
| WO2001004357A2 (en) | Generic sbe-fret protocol | |
| WO2002024960A1 (en) | Detection of dna variation | |
| AU2002345657A1 (en) | Multiplexed detection methods | |
| AU2007200845A1 (en) | Nucleic acid detection methods using universal priming |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Dead |