WO2002046457A2 - Procede de codage de sondes d'hybridation - Google Patents

Procede de codage de sondes d'hybridation Download PDF

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WO2002046457A2
WO2002046457A2 PCT/EP2001/014391 EP0114391W WO0246457A2 WO 2002046457 A2 WO2002046457 A2 WO 2002046457A2 EP 0114391 W EP0114391 W EP 0114391W WO 0246457 A2 WO0246457 A2 WO 0246457A2
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encoder
overhanging end
dna fragment
encoders
sequence
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PCT/EP2001/014391
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WO2002046457A3 (fr
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Achim Fischer
Dieter Newrzella
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Axaron Bioscience Ag
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Publication of WO2002046457A3 publication Critical patent/WO2002046457A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • C12Q1/6855—Ligating adaptors

Definitions

  • the present method is used to encode short, unknown nucleic acid sections of any sequence by means of longer nucleic acid sections which are as different as possible from one another and which can be used as hybridization probes.
  • Another object of the invention is an arrangement of ohgonucleotides on a surface which are essentially complementary to all possible hybridization probes or a part thereof.
  • Preferred areas of application of the method according to the invention are expression analysis and nucleic acid sequencing.
  • SAGE serial analysis of gene expression; Science 1995 Oct 20; 270 (5235): 484-7
  • type IIs restriction endonucleases which can cut double-stranded DNA outside of their recognition sequence, are used to generate short tags become.
  • These short tags are then concatenated and cloned, and the clones obtained are then sequenced.
  • the small length of said tags allows the determination of up to about 25 tag sequences in a single sequencing reaction, which significantly increases the throughput compared to conventional t ⁇ g sequencing.
  • the t ⁇ g length is chosen (for example 9 bp) so that each tag received has a sequence that uniquely represents a particular transcript.
  • a special property of DNA fragment ends which have been generated by the action of a type IIs restriction endonuclease is the fact that on the one hand the position and character of the ends (smooth or overhanging; if overhanging, then also the length of the single-stranded region) are known, on the other hand but the sequence of the ends (terminal bases and, in the case of overhangs, the sequence of the single-stranded region) is not determined by the enzyme.
  • Fok ⁇ which recognizes the sequence GGATG, creates a cut in the strand containing the sequence mentioned at 9 bases away and a further cut at 13 bases away in the opposite strand. In this way, a four-based overhang arises from the choice of the sequence of sequences which are not directly influenced.
  • Such overhangs of known length, but of unknown sequence, generated by type IIs enzymes can be used to “index” fragments via a ligation step.
  • Kato describes a method called “molecular indexing” (Nucleic Acids Res. 1996 Jan 15; 24 (2): 394-5), in which all 64 possible adapters of one type are ligated with their overhanging ends to the overhanging ends of fragments obtained from cDNA molecules by means of type IIs restriction endonucleases.
  • E.cob ' ligase was chosen as the enzyme, which, compared to T4 DNA ligase, has a significantly higher ability to discriminate against "wrong" ligation events (ie overhangs that are not perfectly complementary to each other).
  • Kato teaches the use of "indexed" from Overhang-specific adapters of flanked restriction fragments for expression analysis by comparing the relative frequency of the individual reamplified and electrophoretically separated fragments in different RNA samples.
  • Velculescu et al. described a hybridization conceivable.
  • Pease et al. use the described photolithography technique. Deposition of separately synthesized oligonucleotides on a membrane or on glass, as described, for example, by Schena et al. (Science 1995 Oct 20; 270 (5235): 467-70).
  • a hybridization signal should then only be detectable at that point on the array at which there is an oligonucleotide that is completely complementary to the labeled oligonucleotide.
  • This assumption is based on the concept of sequencing by hybridization (SBH; Drmanac et al., Science 1993 Jun 11; 260 (5114): 1649-52).
  • SBH sequencing by hybridization
  • Sequence G 10 T 10 have a different melting temperature than an oligonucleotide of sequence (GT) 10 .
  • GT oligonucleotide of sequence
  • Experiments of the type outlined above are therefore impaired in that, in addition to the desired signal based on specific hybridization, numerous “false positive” signals which are due to “cross hybridization” nucleic acid molecules which are not completely complementary to one another are detected.
  • the occurrence of cross hybridization events is also favored by the fact that in a collection of all possible 4 n oligonucleotides of length n there are naturally 3 n other oligonucleotides for each individual oligonucleotide, from which the oligonucleotide under consideration differs in only one base and from to whom discrimination by hybridization seems largely impossible.
  • the object of the present invention was to code oligonucleotide tags in such a way that a nucleic acid is provided for each tag of a predetermined length, which firstly clearly defines said tag and secondly to
  • step (c) optionally separating those encoders which were not attached to any DNA fragment in step (b),
  • step (d) Marking the fastening product from step (b), unless marked encoders have already been used for fastening,
  • Encoders with an overhanging end to the first overhanging end of an unknown sequence of the cut attachment product consisting of the DNA fragment and adapter, the overhanging end of the attached first encoder and the first overhanging end of an unknown sequence of the DNA fragment being completely complementary to one another,
  • the DNA fragment with a known end in step (a1) is a double-stranded nucleic acid section of any origin, which has at least one clearly defined end.
  • the end is either smooth or has a single-stranded overhang of known length and possibly known sequence.
  • the DNA fragment can be obtained from genomic DNA or double-stranded cDNA.
  • nucleic acid for example heterohybrids from RNA and DNA or artificial nucleic acids, can also be used in the process according to the invention, provided that they are compatible with the enzymatic steps to be carried out.
  • said known end is preferably produced by treatment with a restriction endonuclease which either produces a single-stranded overhang of known length and, if appropriate, known sequence, or else a smooth end.
  • the end can be generated in a non-sequence-specific manner, for example by mechanical shear or by exonuclease treatment, followed if necessary by repairing the ends to blunt ends.
  • the DNA fragment is a mixture of different double-stranded cDNA molecules cut with a restriction endonuclease.
  • the restriction fragment originating in each case from the 3 'end or from the 5' end of each cDNA molecule can be isolated by coupling to a solid phase, for example magnetic particles.
  • a solid phase for example magnetic particles.
  • an 8mer-t ⁇ g which indicates 5-fold regulation of the associated gene and which shows a signal strength of 40% of the highest measured signal strength, but which is derived from two different genes according to the results of a database search
  • the results of a parallel experiment carried out with another restriction enzyme could be examined to determine which second tag (which in this case very likely has a different, unknown sequence) has the same properties with regard to signal strength and apparent regulatory factor.
  • Both tags are most likely derived from the same gene and now allow its unique identification.
  • SBH sequencing by hybridization
  • the adapter in step (b1) is generally a hybrid of two at least partially complementary nucleic acid strands. This can be about two synthetic oligonucleotides, but also about a plasmid or phage vector.
  • the adapter preferably has a first end which can be attached to the known end of the DNA fragment from (a1), in particular by ligation, and a second end which cannot be attached under the given conditions.
  • the first end can have an overhang that is complementary to the end of the DNA fragment, while the second end is smooth or is characterized by a single- or multi-base mismatch.
  • the adapter has recognition sites for at least two type IIs restriction endonucleases which produce overhanging ends offset from their recognition sites.
  • both recognition sites are arranged in such a way that when the product resulting from attachment of the adapter is incubated with at least one of the two restriction endonucleases, at least one, preferably both, strands of the region of the treated nucleic acid molecule originating from the DNA fragment are cut. It is preferred that the unknown first and second overhangs resulting from said cuts do not overlap with one another, and particularly preferred that the overhangs directly adjoin one another. "Do not overlap” and “adjoin” refers to the position of the cuts on each of the two strands of the double strand and is illustrated in FIG.
  • the encoders from steps (dl) and (fl) are characterized by a double-stranded area and a single-stranded overhang, the single-stranded overhang generally having the same length as the unknown first or first step (cl) or (el) second single-stranded overhang of the DNA fragments.
  • the orientation of the overhang in the same direction is also important: if the overhang of the DNA fragments is a 3 'overhang, then the encoders should also have a 3' overhang, the fragments have a 5 'overhang, so must the encoders are also characterized by a 5 'overhang.
  • one or both strands of the encoder can be marked, for example by fluorescent dyes.
  • the encoders can have sequences required for amplification, for example the sequence for a promoter of a DNA-dependent RNA polymerase such as T7 RNA polymerase or primer binding sequences for PCR primers.
  • the fasteners The encoders are preferably supplied by enzymatic ligation, the ligation conditions (temperature, buffer composition, duration of the ligation, concentrations of enzyme and nucleic acids to be linked to one another) being selected such that “non-specific” ligation events (that is, the ligation is not completely compatible with one another) complementary overhangs) can be suppressed as much as possible.
  • ligase for example the DNA ligases from E.coli as well as from Thermus aquaticus and Thermus thermophilus are known for a higher ligation accuracy than the far Widespread phage T4 DNA ligase.Furthermore, as described above for the adapter, it is possible to first hybridize an ligation conditions (temperature, buffer composition, duration of the
  • the term "clearly distinguish" here means that under suitable hybridization conditions, no strand of one encoder shows marked double-strand formation with a strand of another encoder.
  • All encoders forming a mixture will generally have the same length and the same G / C content, for example 50%.
  • Single-stranded oligonucleotides which have such properties have been described, for example, by Brenner in US Pat. No. 5,635,400 ,
  • step (el) a second overhanging end of unknown sequence is generated, which lies essentially between the adapter fastened in step (bl) and the first unknown overhanging end generated in step (cl). It is possible for said second overhanging end to overlap with the sequence of the adapter and / or the sequence of the overhanging end from step (cl) or to be removed from these sequences by one or more bases. However, it is preferred that the second overhanging end from step (e1), as shown in FIG. 1, borders directly on the sequence of the first overhanging end from step (c1) and does not overlap with the sequence of the adapter.
  • the double-stranded dicoders formed in (fl) consist of a residue of the DNA fragment from step (a1), usually about 4-16 base pairs long, which is flanked by the first and second encoders. Part of the remainder of the DNA fragment is preferably formed by the first single-stranded overhang from step (cl), which was added to the double strand in the course of the attachment of the first encoder in step (dl), and the entire remaining part by that in the course of the attachment of the second Encoders formed in step (fl) to the double strand supplemented second single-stranded overhang from step (el).
  • the optional proofreading of the dicoders serves to suppress hybridization of overhangs which are not completely complementary to one another in steps (dl) and (fl) of declining dicoder species.
  • a known method for proofreading in the sense of eliminating partially mismatched sequences is, for example, the use of MutS, a protein which recognizes mismatches, followed by exonuclease treatment (Mutat. Res. 1997 Mar 21; 374 (2): 277-85).
  • Another option for proofreading is one Treatment with a mismatching endonuclease followed by selective removal of cut DNA strands. The latter could take place, for example, by exonucleolytic degradation starting on the single-strand break generated by the endonuclease.
  • the proofreading step can be dispensed with if the fastening specificity achieved in steps (dl) and (fl) is sufficiently large. If, on the other hand, the fastening specificity is so low that after proofreading has taken place, a portion of all the dicoders formed which is no longer sufficient for the hybridization in step (II) is available, then an amplification of the dicoders which have no mismatches can be carried out.
  • the amplification is preferably carried out by means of an RNA polymerase by repeated rewriting of the dicoder double strand into single-stranded (and generally labeled, see step (hl)) RNA. However, amplification can also be carried out using PCR or other methods.
  • the dicoders can be marked in step (hl) by attaching at least one detectable marking group to one or both strands of the dicoders.
  • Fluorescent labeling groups are preferably used, but the use of other non-radioactive or radioactive labeling groups is also conceivable.
  • An alternative to attaching the labeling groups to already existing nucleic acid strands is to replicative new synthesis of nucleic acid strands, in which the existing nucleic acid strands serve as templates and labeled nucleic acid building blocks (primer molecules or nucleotides) are used.
  • Such a new synthesis can be catalyzed by a DNA polymerase (for example DNA polymerase I, Klenow enzyme or a thermostable DNA polymerase) or an RNA polymerase (for example T7 RNA polymerase); when using an RNA polymerase, care must be taken to ensure that the dicoders to be labeled have a suitable promoter sequence. Otherwise, it is also possible to use encoders already marked for attachment, which have already been provided with fluorescent groups during their synthesis, for example. It would be obvious to encode information about the choice of the marking group, for example the nature of a selected base of the overhang.
  • a DNA polymerase for example DNA polymerase I, Klenow enzyme or a thermostable DNA polymerase
  • RNA polymerase for example T7 RNA polymerase
  • the array of ohgonucleotides in (il) is a collection of nucleic acids capable of hybridization (DNA, RNA, PNA, or derivatives thereof) present in a known arrangement on a surface, which collection can be generated on the surface or deposited there. Said collection comprises all sequences complementary to a strand of the possible dicoder or a known part thereof.
  • hybridization and determination of those oligonucleotides on the array with which labeled encoder strands have hybridized can be carried out in a known manner (cf. Schena et al.).
  • quantification of the hybridization signals is preferred.
  • quantification is usually not necessary, although this can, if desired, be used to distinguish between true-positive signals and false-positive signals, for example, caused by errors in the encoder attachment.
  • the assignment of the information obtained via the hybridization events to the unknown overhanging fragment ends generated in (cl) and in (el) represents a decoding, namely the reversal of the coding of the fragment ends carried out by the step of the encoder attachment.
  • Each specific hybridization event of a certain marked dicoder represents a certain first overhang attached to a first encoder and a certain second overhang attached to a second encoder and thus a partial sequence of the DNA fragment provided in (al), so that the result is a Hybridization finally provides information about all overhanging fragment ends generated in (cl) or (el) and subsequently attached to the associated encoders.
  • the respective first overhang can be assigned to the respective second overhang for each fragment species, so that characteristic sequence information is obtained for each DNA fragment.
  • the partial sequences recognized by means of the first encoder and the means of the second encoder directly adjoin one another for each DNA fragment, so that a coherent sequence, which characterizes the respective fragment, can be assigned to each individual hybridization signal.
  • Will one Hybridization signal assigns the sequence 5'-AACC-3 'recognized by the first encoder and the sequence 5'-CCTT-3' recognized by the second encoder and directly adjoins both sequences in the rest of the DNA fragment contained in the dicoder 5'-AACCAAGG-3 'is the t g sequence characterizing the corresponding DNA fragment.
  • sequences obtained in this way can always be used when DNA fragments are to be recognized on the basis of shorter partial sequences. This is the case, for example, when the relative frequency of transcripts in different biological samples is to be examined as part of an expression analysis.
  • the method according to the invention permits the identification and quantification of transcripts via the tags which represent them, the relative quantification being carried out by measuring signal strengths after the hybridization.
  • additional cleaning steps or steps of removing unreacted starting materials or undesired reaction products can be carried out between individual of the above-described method steps in a manner familiar to the person skilled in the art, for example cleaning adapters which have remained unsecured in step (b1) or encoders which have remained unsecured in step (b1). dl) or (fl).
  • cleaning adapters which have remained unsecured in step (b1) or encoders which have remained unsecured in step (b1).
  • One way to do this is by a final replenishment reaction using biotinylated nucleotides, followed by the coupling of biotinylated molecules to streptavidin bound to the solid phase.
  • biotinylated nucleotides followed by the coupling of biotinylated molecules to streptavidin bound to the solid phase.
  • Adapters or encoders that have remained ligated can also be removed by binding the ligated DNA fragment to a solid phase, followed by one or more washing steps. It would also be conceivable to melt the non-ligated encoders under conditions in which the ligation products are still double-stranded, followed by physical separation of double and single strands, or by enzymatic degradation of single strands.
  • a method consisting of the following steps (see FIG. 2): (a2) provision of at least one DNA fragment with a first overhanging end of known length and unknown sequence,
  • the overhanging end of the attached first encoder and the first overhanging end of the DNA fragment are completely complementary to one another and wherein said first encoder has a recognition site for an overhanging end-producing type IIs restriction endonuclease which is oriented and positioned such that upon incubation of the fixation product from the first encoder and DNA fragment with said type IIs restriction endonuclease, a second overhanging end is generated which contains at least among others bases of the DNA fragment which were not part of the overhanging end in step (a2) and their complement as well was not part of the overhanging end in step (a2),
  • step (c2) incubation with a type IIs restriction endonuclease which recognizes the recognition site contained in the first encoder in step (b2) and which cuts in the manner described in step (b2),
  • step (a2) the DNA fragment to be examined could be treated with a type IIs restriction endonuclease as described in US Pat. No. 5,710,000.
  • a type IIs restriction endonuclease as described in US Pat. No. 5,710,000.
  • the procedure described there has the disadvantage that the orientation and thus the relative position of the detection point to the interface remains unknown. It is therefore preferred to first attach an adapter to a known end according to step (a1) as described under step (b1), which in this case, however, only has to have at least one recognition site for a first type IIs restriction endonuclease. Incubation of the attachment product with this type IIs restriction endonuclease then produces the end to be provided in step (a2).
  • the DNA fragment can be obtained from genomic DNA or double-stranded cDNA.
  • Other types of nucleic acid for example heterohybrids from RNA and DNA or artificial nucleic acids, can also be used in the method according to the invention, provided that they are compatible with the enzymatic steps to be carried out.
  • the DNA fragments are a mixture of cDNA molecules cut with a restriction endonuclease or appropriately prepared clones of a cDNA or genomic library. To uniquely identify the interface, this can be from the 3 'end or the 5' end of one each cDNA molecule-derived restriction fragment can be isolated by coupling to a solid phase, for example magnetic particles.
  • a method is provided, consisting of the following steps (see FIG. 3):
  • (b.3) Assignment of the information about hybridization events obtained in (g3) on the basis of the sequence information about the first encoders used in (b3) to the overhanging fragment end provided in (a3).
  • the optional separation of all or part of the DNA fragment is preferably carried out by means of a restriction endonuclease. This can have its detection point, for example, in the encoder attached in step (b3) (cf. FIG. 3).
  • the separation serves to increase the hybridization specificity in step (f3), since this avoids the risk of undesired cross-hybridization of the DNA fragment with ohgonucleotides of the array.
  • the ligated encoder is marked analogously to what was said above by marking the dicoder in step (hl).
  • an array of nucleic acids complementary to all possible coding nucleic acids is further provided, preferably on a solid support such as glass or plastic.
  • Another object of the invention is the use of nucleic acids encoded as described for the identification of tags via hybridization.
  • the method according to the invention is used for expression analysis.
  • the method according to the invention for sequencing by hybridization is used.
  • the invention further relates to an array of nucleic acids which comprise all or part of all possible combinations of at least two part-sequences.
  • the method for the detection of mutations in particular point mutations (SNPs), is used.
  • Fig. 1 shows the t ⁇ g coding by means of two type IIs recognition sites in a "tandem arrangement" on an adapter
  • Fig. 2 shows the t ⁇ g coding by means of two separate type IIs detection points, one of which is located on an adapter and the other on the first encoder
  • FIG. 2 illustrates the t ⁇ g coding by means of two separate type IIs recognition sites, one of which is located on an adapter and the other on the first encoder, in detail
  • Fig. 3 shows the t ⁇ g coding by means of a single type IIs recognition site, which lies on an adapter, in detail
  • FIG. 4 shows the t ⁇ g coding by means of a single type IIs recognition site, which is located on the DNA molecule to be analyzed, in detail
  • the second strand synthesis was carried out according to Ausubel et al., Current Protocols in Molecular Biology (1999), John Wiley & Sons. 96 ⁇ l of second-strand buffer, 300 ⁇ l of H 2 O, 7 ⁇ l of 10 mM dNTPs, 2.4 ⁇ l of RNaseH (1.5 U / ⁇ l, Promega GmbH, Mannheim) and 12 ⁇ l of DNA polymerase I (10 U / ⁇ l, New England Biolabs GmbH, Schwalbach) was added, mixed, and the reactions were incubated at 22 ° C. for 2 hours. It was extracted with 150 ⁇ l phenol and 150 ⁇ l chloroform and precipitated with 0.1 vol. Sodium acetate pH 5.2 and 2.5 vol.
  • Ethanol The pellet was dissolved in a restriction mixture of 15 ⁇ l 10 ⁇ universal buffer, 2 ⁇ l Mbol (4 U / ⁇ l; Stratagene, Heidelberg) and 84 ⁇ l H 2 O and the reaction was incubated at 37 ° C. for 1 hour. It was extracted with 100 ⁇ l phenol and with 100 ⁇ l chloroform and, after adding 0.1 vol. Sodium acetate pH 5.2, it was precipitated with 2.5 vol. Ethanol.
  • the pellet was placed in 15 ⁇ l of a ligation mixture consisting of 2 ⁇ l adapter MBF2420 (1 ⁇ g / ⁇ l), 1.3 ⁇ l 10 ⁇ ligation buffer (Röche Molecular Biochemicals), 2 ⁇ l 10 mM ATP (Röche Molecular Biochemicals) and 1 ⁇ l T4 DNA ligase (1 U / ⁇ l, Röche Molecular Biochemicals) dissolved and the ligation carried out overnight at 16 ° C. 10 ⁇ l of water and 25 ⁇ l of 2 ⁇ binding and washing buffers (Dynal, Oslo) were added.
  • a ligation mixture consisting of 2 ⁇ l adapter MBF2420 (1 ⁇ g / ⁇ l), 1.3 ⁇ l 10 ⁇ ligation buffer (Röche Molecular Biochemicals), 2 ⁇ l 10 mM ATP (Röche Molecular Biochemicals) and 1 ⁇ l T4 DNA ligase (1 U / ⁇ l, Röche Molecular Bio
  • the two oligonucleotides (ARK, Darmstadt) belonging to each other were dissolved in water and adjusted to a total DNA concentration of 1 ⁇ g / ⁇ l in 1 ⁇ T4 DNA ligation buffer. Then the heating block was heated to 95 ° C., the heating block was switched off and slowly cooled to room temperature. The double-stranded DNA produced in this way was stored at -20 ° C.
  • the oligonucleotides were:
  • Adapter MBF2420 MBF24: 5 '-TGAATCACTAGGATGGCAGCGATC-3'
  • FBM20 5 '-GCTGCCATCCTAGTGATTCA-3'
  • Encoder2 T7 GCGA
  • the streptavidin dyna beads obtained in Example 1 and coated with cDNA fragments were prewarmed in 100 ⁇ l of 1 ⁇ NEBuffer 2 (supplemented with BSA to 200 ⁇ g / ⁇ l) for 10 min at 37 ° C. on an overhead mixer ( Rotation speed approx. 2 rpm). After the addition of 15 U Rbvl (New England Biolabs), the mixture was incubated at 37 ° C. for a further hour while rotating slowly. The reaction vessel was transferred to a magnetic stand and the supernatant was removed after sedimentation of the Dynabeads. After extraction with phenol and chloroform (see above) it was precipitated with ethanol.
  • the pellet was dissolved on ice in 10 ⁇ l of a ligation mixture consisting of 0.5 ⁇ l Taq DNA ligase (4 U / ⁇ l; New England Biolabs), 10 ng each of the encoders 1-ACAG, 1-CGAC, 1-GCAC, 1 -TACG, in 1 x Taq DNA ligase buffer (New England Biolabs).
  • the ligation was carried out for 10 min. Incubated at 16 ° C, stopped on ice, made up to 50 ⁇ l with water and extracted with phenol, then with chloroform.
  • the precipitated encoder ligation products from Examples 3 and 4 were taken up in 17 ⁇ l 1 ⁇ in v / tro transliteration buffer (Ambion, Austin, Tx), each containing 7.5 mM ATP, CTP and GTP as well as 5 mM dTTP and 2 , 5 mM Cy3-UTP (Amersham-Pharmacia, Freiburg) contained. 2 ⁇ l enzyme mix (MEGAscript kit, Ambion) and 1 ⁇ l T7 RNA polymerase (1000 U / ⁇ l; Epicenter, Madison, WI) were added and transcribed at 37 ° C. for 12 h. Then 1 ⁇ l DNase I (2 U / ⁇ l; Ambion) was added and 15 min. incubated at 37 ° C. It was made up to 100 ⁇ l with water, extracted with phenol / chloroform and precipitated with ethanol.
  • the amino modified oligonucleotides 1-16 (see below) at the 5 'end were dissolved in water and adjusted to a concentration of 100 pmol / ⁇ l. 1 ⁇ l of each of these solutions was mixed with 49 ⁇ l binding buffer (150 mM Na phosphate, pH 8.5) and used for array production on 3-D linkslides (Surmodics, Eden Prairie, MN); 0.5 ⁇ l each Oligonucleotide applied in a 4 x 4 matrix with a pipette. After application, the arrays were placed in a reaction chamber saturated with water vapor at room temperature and the coupling reaction was carried out for 14 h.
  • the slides were rinsed thoroughly with 5 ⁇ SSC. Then, to inactivate the surface, it was briefly rinsed with 50 mM ethanolamine / 0.1 M Tris pH 9 and with fresh solution for 15 min. treated at 50 ° C. The slides were washed with water, then 25 ⁇ l of prehybridization solution (5 ⁇ SSC, 5 ⁇ Denhardt's, 0.1% SDS, 0.1 ⁇ g / ⁇ l herring sperm DNA) were added to the arrays and covered with a coverslip. It was 20 min. Pre-hybridized at 50 ° C. The prehybridization solution was rinsed off with water and the slides were briefly air-dried.
  • prehybridization solution was rinsed off with water and the slides were briefly air-dried.
  • the hybridization samples obtained in Example 5 were taken up in 10 ul prehybridization solution, 2 min. denatured in a boiling water bath, placed on the arrays and covered with a coverslip.
  • the hybridization was carried out in a water vapor-saturated chamber for 12 h at 37 ° C. (probes obtained with an encoder according to Example 3) or 45 ° C. (probes obtained with two encoders according to Example 4).
  • the cover slip was removed and briefly rinsed with 5 x SSC / 0.1% SDS. Then 2 x 5 min at room temperature. with 2 x SSC / 0.1% SDS, then 1 min. washed with 0.2 x SSC / 0.1% SDS.
  • the arrays were air dried and examined for detection in a ScanArray 5000 scanner (GSI Lumonics, Billerica, MA). It was scanned with an excitation wavelength of 546 nm and was detected at 570 nm. The hybridization signals were evaluated using the QuantArray, Version 2.0 program.
  • oligonucleotides were:
  • CGAC-GCGA 5 '-Amino-TTAGTCACTGGTAACAGTCGGCGAATTCTGTTCACAAf AG-3'
  • GCAC-GCGA 5 '-Amino-ACGCACACTCCGATCTGTGCGCGAATTCTGTTCACAATAG-3'
  • TACG-CAGG 5 '-Amino-C AGCAAGCATCCTTCCCGTAC AGGGTGATAGGACTAGAAC-3'

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Abstract

Procédé d'identification d'acides nucléiques, caractérisé en ce qu'il comprend les étapes suivantes : (a) préparation d'au moins un fragment d'ADN ayant une extrémité pendante de longueur connue et de séquence inconnue, (b) fixation d'un codeur déterminé, formé d'un mélange de codeurs ayant une extrémité également pendante à l'extrémité pendante de la séquence inconnue du fragment d'ADN, l'extrémité pendante du codeur fixé et l'extrémité pendante de séquence inconnue du fragment d'ADN étant totalement complémentaires, (c) le cas échéant, séparation des codeurs qui, à l'étape (b), n'ont été fixés à aucun fragment d'ADN, (d) marquage du produit de fixation de l'étape (b), dans la mesure où des codeurs non déjà marqués pour la fixation ont été utilisés, (e) hybridation du produit de fixation marqué avec un ensemble d'oligonucléotides qui sont sensiblement complémentaires de l'un des brins de tous les codeurs possibles du mélange de codeurs ou d'une sélection de ceux-ci.
PCT/EP2001/014391 2000-12-07 2001-12-07 Procede de codage de sondes d'hybridation WO2002046457A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2002216078A AU2002216078A1 (en) 2000-12-07 2001-12-07 Method for encoding hybridization probes

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE10060827.2 2000-12-07
DE10060827A DE10060827A1 (de) 2000-12-07 2000-12-07 Verfahren zur Codierung von Hybridisierungssonden

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WO2002046457A2 true WO2002046457A2 (fr) 2002-06-13
WO2002046457A3 WO2002046457A3 (fr) 2002-12-19

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AU (1) AU2002216078A1 (fr)
DE (1) DE10060827A1 (fr)
WO (1) WO2002046457A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115058469A (zh) * 2022-04-25 2022-09-16 深圳大学 一种短dna片段3’端生物素标记的方法

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Publication number Priority date Publication date Assignee Title
US5710000A (en) * 1994-09-16 1998-01-20 Affymetrix, Inc. Capturing sequences adjacent to Type-IIs restriction sites for genomic library mapping
WO1998010095A1 (fr) * 1996-09-05 1998-03-12 Brax Genomics Limited Caracterisation d'un adn
WO2000009756A1 (fr) * 1998-08-17 2000-02-24 The Perkin-Elmer Corporation Analyse d'expression dirigee a l'aide d'un adaptateur
WO2000060124A2 (fr) * 1999-04-06 2000-10-12 Yale University Analyse d'adresses fixes de séquences étiquetées
WO2001012855A2 (fr) * 1999-08-13 2001-02-22 Yale University Etiquette de sequence a codage binaire

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5710000A (en) * 1994-09-16 1998-01-20 Affymetrix, Inc. Capturing sequences adjacent to Type-IIs restriction sites for genomic library mapping
WO1998010095A1 (fr) * 1996-09-05 1998-03-12 Brax Genomics Limited Caracterisation d'un adn
WO2000009756A1 (fr) * 1998-08-17 2000-02-24 The Perkin-Elmer Corporation Analyse d'expression dirigee a l'aide d'un adaptateur
WO2000060124A2 (fr) * 1999-04-06 2000-10-12 Yale University Analyse d'adresses fixes de séquences étiquetées
WO2001012855A2 (fr) * 1999-08-13 2001-02-22 Yale University Etiquette de sequence a codage binaire

Non-Patent Citations (1)

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Title
BRENNER SYDNEY ET AL: "Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays" NATURE BIOTECHNOLOGY, NATURE PUB. CO, NEW YORK, NY, US, Bd. 18, Nr. 6, Juni 2000 (2000-06), Seiten 630-634, XP002199492 ISSN: 1087-0156 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115058469A (zh) * 2022-04-25 2022-09-16 深圳大学 一种短dna片段3’端生物素标记的方法

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DE10060827A1 (de) 2002-06-13
AU2002216078A1 (en) 2002-06-18
WO2002046457A3 (fr) 2002-12-19

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