WO2005010184A1 - Methode servant a detecter une mutation - Google Patents

Methode servant a detecter une mutation Download PDF

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Publication number
WO2005010184A1
WO2005010184A1 PCT/JP2004/010893 JP2004010893W WO2005010184A1 WO 2005010184 A1 WO2005010184 A1 WO 2005010184A1 JP 2004010893 W JP2004010893 W JP 2004010893W WO 2005010184 A1 WO2005010184 A1 WO 2005010184A1
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Prior art keywords
dna
microbeads
sequence
target dna
tag
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PCT/JP2004/010893
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English (en)
Japanese (ja)
Inventor
Nobuto Koyama
Junichi Mineno
Masanori Takayama
Hiroyuki Izu
Shusaku Yamashita
Hiroaki Sagawa
Ikunoshin Kato
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Takara Bio Inc.
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Publication of WO2005010184A1 publication Critical patent/WO2005010184A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

  • the present invention relates to a method for determining a DNA base sequence.
  • the present invention also relates to a method for detecting a mutation using the method for determining a base sequence.
  • DNA sequencing is an essential technique for obtaining genetic information of organisms.
  • Conventionally, widely used nucleotide sequencing techniques are described in the Chemical Chemistry [Maxam et al., Proceedings of Nationala 1 Accady of Sciences USA, Vol. 74, 1258-1262, 1977] and the enzymatic synthesis method [Sanger et al., Bulletin of the American Academy of Sciences, Vol. 74, pp. 5463-5467, 1977].
  • the former is called the Maxam-Gilbert method
  • the latter is called the dideoxy method or the Sanger method.
  • the automatic sequencer based on the principle has been widely spread and has become the mainstream of DNA sequencing. Since these methods require at least one reaction and electrophoresis per sample, the maximum number of sequences that can be obtained at one time using the latest sequencer is 384. .
  • Matsushiburi Parallel Signature Sequencing Method (MPSS method, Brenner et al., Nature Biotechnology, Volume 18, 630-634, 2000, Special Edition Table 1-111 507528] is a technique for determining a base sequence based on a completely different principle from the above-described base sequence determination method.
  • the DNA to be sampled is bound to the microbeads, the microbeads are two-dimensionally filled in a flow cell, and a series of reactions and detections are performed. You can decide.
  • the genomic DNA base sequence differs among individuals, and the site where these phenomena are different is called a genetic polymorphism.
  • a gene that occurs at a frequency of 1% or more in a population is defined as a genetic polymorphism.
  • the following three types of polymorphism markers are mainly used.
  • restriction fragment length polymorphism (restrictionfra gme ntlengthpol ymo r ph i sm ⁇ RFLP), simple arrangement polymorphism (s imp lese qu encel en gthpol ymo rphi sm, SS LP) and single nucleotide polymorphism (si ng lenucleotidepol ymo rphi sm (SNP).
  • SNP single nucleotide polymorphism
  • microsatellite There are many repetitive sequences of 1 to 6 bases in the genome, which are called microsatellite.
  • the number of microsatellite repetitions may vary from individual to individual, and is referred to as SSLP.
  • SSLP The sequence around the microsatellite is different for each repetitive sequence.
  • PCR polymerase chain reaction
  • the number of repetitions can be easily known.
  • a higher density of polymorphic markers than S SLP is required.
  • SNP is the most frequently found polymorphism and is present in humans at a rate of about 1 kb. SNP analysis is useful for understanding the relationship between side effects and individual differences, and for performing more effective and safe medical treatment.Screening and tying of SNPs are conducted on a large scale worldwide. Has been done.
  • the most direct method for detecting a new SNP is to compare the genomic DNA sequences of multiple individuals.
  • the sequence reaction and analysis using an automatic sequencer require a great deal of cost and labor. It costs.
  • An object of the present invention is to provide a method for inexpensively and accurately determining the DNA base sequence of a large number of samples. It is also an object of the present invention to provide a method for detecting mutations at a low cost and with high accuracy using the base rooster sequence determination method.
  • the present inventors have conducted intensive studies, fragmented DNA, ligated the fragmented DNA to a tag vector, prepared a tag library, and bounded a DNA fragment whose base sequence was to be determined to a microphone opening bead.
  • the present inventors have found that a DNA sequence can be determined efficiently by determining a nucleotide sequence by an operation comprising the same steps as the MPSS method, and that the present invention has been found that mutation can be detected by a similar method. completed. That is, the first invention of the present invention is a method for determining a DNA base sequence, which comprises the following steps. ----
  • step (3) (4) a step of binding the DNA obtained in step (3) to microbeads
  • step (8) hybridizing the microbeads obtained in step (8) with a decoder probe to identify an encoded adapter ligated to the target DNA;
  • a second invention of the present invention is a method for detecting a mutation, comprising the following steps.
  • step (10) a step of performing hybridization of the microbeads obtained in step (9) and a decoder probe to identify an encoded adapter ligated to the target DNA
  • step (10) Remove the decoder probe from the microbeads in step (10) to obtain Using the obtained microbeads in the following step (8), and
  • DNA Microphone Bead Array Technology is a technology disclosed in Japanese Patent Application Publication No. 11-507528 and Brena et al., Bulletin of the National Academy of Sciences, Vol. 97, pp. 165-1670, 2000 It refers to technology for analyzing gene expression and gene structure by applying it.
  • a library of DNA immobilized on the microbeads can be prepared, and one type of D ⁇ A is bound to one bead of the microphone mouth.
  • Tag library refers to a library of DNAs that is bound to the microbeads in the DNA microbead array technology. Each clone in the tag library is DNA containing the tag sequence and the DNA to be bound to the microbeads in the same molecule, and a set of these clones is the tag library.
  • a “tag” is an oligonucleotide that is covalently linked to DNA to be attached to the microbeads in DNA microbead array technology, and is used to place a single-sequence DNA on each microbead.
  • the repertoire of tags must be sufficiently larger than the number of clones in the tag library.
  • an “anti-tag” is an oligonucleotide that is covalently bonded to a microbead in DNA microbead array technology and has a sequence that is complementary to the tag.
  • a single array of anti-tags is bound to one microphone mouth bead.
  • the repertoire of multi-tags is substantially the same size as the repertoire of tags.
  • the “MPSS method” is a technology disclosed in Japanese Patent Application Laid-Open No. 2000-501500 and Nayya Biotechnology, Vol. 18, pp. 60-634, 2000. Means.
  • microbeads to which DNA has been bound prepared by DNA microbead array technology, are two-dimensionally packed in a flow cell, digested with type liS restriction enzymes, and the resulting protruding ends are connected to an adapter and ligated. This is a technique for identifying the adapters using fluorescent probes in a flow cell.
  • encoded adapter refers to a mixture of partially double-stranded oligonucleotides used for ligation to a protruding end of a target DNA in the MPSS method.
  • One end of the adapter (hereinafter referred to as A-terminal) may be a protruding end having a sequence to which a decoder probe described below can hybridize.
  • the other end (hereinafter, referred to as B-terminus) may be a protruding end satisfying the following conditions.
  • the type used in the “step of determining the base sequence of the target DNA fragment” in the present invention a 5, 1 or '3, one protruding end capable of ligating to the end of the target DNA generated by the type ⁇ Is restriction enzyme. is there. That is, when the above-mentioned restriction enzyme generates a 5'- (or 3'-) end protruding N base, the A-terminal is also a 5'- (or 3'-) end protruding N base.
  • the n-th base (1 ⁇ n ⁇ N) at the B-terminus is any of A, G, C or T; A mixture of four bases.
  • the central double-stranded portion of the enzyme adapter has the type II s restriction enzyme recognition sequence described above.
  • This restriction enzyme recognition sequence is adjacent to the protruding end sequence used for ligation when DNA obtained by ligating an encoded adapter to a target DNA fragment is digested with the restriction enzyme. It suffices if the sequence of the target DNA is designed to be generated as a protruding end.
  • the encoded adapters in each group are a mixture of 4 N_1 B-terminals, and the entire encoded adapter is 4 XN groups.
  • NX 4 consists of N types of arrays.
  • Suitable adapters that can be suitably used include those described in American Academy of Sciences, Vol. 97, pp. 165-1670, 2000.
  • the enzyme adapter is applicable when using BbVI as a type II s restriction enzyme.
  • the A-terminus has a 3-base overhang with 10 bases, and the B-end has a 5-overhang structure with 4 bases. are doing.
  • Decoder probe is a fluorescently labeled oligonucleotide used to detect which group of the encoded adapter belongs to the MPSS method, and a sequence complementary to the A-terminal sequence of the encoded adapter is used. You only need to have it.
  • fluorescent dye used for the decoder probe, and proteins such as phycoerythrin, low molecular weight compounds such as fluorescein, and quantum dots can be used.
  • Type II s restriction enzyme is a restriction enzyme that recognizes an asymmetric sequence and cuts a site different from the recognition sequence.
  • Type II s restriction enzymes used in step "of determining the nucleotide sequence of I 7 target DN A fragment of the present invention need only include the following conditions. i) Generate 5, 1, or 3 'overhanging ends.
  • the distance between the cleavage site and the recognition sequence is N bases or more, where N is the length of the protruding base at the generated end.
  • a preferred example of the type IIs restriction enzyme used in the "step of determining the nucleotide sequence of the target DNA fragment" is BbvI. As shown below, this enzyme recognizes the GCAG C sequence and generates a 5,1 terminal with four bases protruding.
  • Target DNA refers to DNA whose nucleotide sequence is to be determined. It also refers to the DNA for which the mutation is to be detected. There is no particular limitation on the method of preparing the target DNA.For example, genomic DNA, cDNA, DNA amplified by PCR from these DNAs, or these DNAs are cloned into a vector, for example, a plasmid vector or a bacterio phage vector. NA, a mixture of cloned DNA, and those obtained by removing the vector portion from these cloned DNAs by restriction enzyme digestion or the like.
  • mutation means that genomic DNA sequences are different between individuals, between alleles of the same individual, between cells, or between alleles of the same cell, and at different sites. That is, it includes genetic polymorphisms, mutations, mutations caused by mutagenic agents or electromagnetic waves, mutations caused by artificial gene recombination, and changes in the genomic DNA sequence caused by infection with a virus or the like.
  • mutant includes various forms of mutation such as “base substitution”, “deletion mutation”, “insertion mutation” and the like.
  • base substitution means that at a specific site on a nucleic acid, a part of the base is replaced by another base.
  • the number of bases substituted in the “base substitution” described in the present specification is not particularly limited, and one or more bases may be substituted.
  • Substitutions found in one base in the nucleotide sequence are called “Single Nucleotide Substitution Polymorphisms (SNPs) J.
  • SNPs Single Nucleotide Substitution Polymorphisms
  • deletion mutation means that at a specific site on a nucleic acid, a part of the nucleotide sequence is deleted.
  • the deleted base sequence may be a single base or a plurality of bases.
  • deletions of these nucleotide sequences include those occurring at a plurality of positions in a specific region on a nucleic acid.
  • “deletion mutations” include deletion of a specific region of a gene, for example, but not limited to, for example, the region of exon and Z or intron, and deletion of the full length of the gene. Further, deletion of a base sequence artificially introduced into a nucleic acid is also included in the “deletion mutation” in the present specification.
  • the “insertion mutation” described in this specification means that a base sequence is inserted at a specific site on a nucleic acid.
  • the inserted base sequence may be a single base, a plurality of bases, or an arbitrary chain length.
  • these base sequences The insertion of a row includes those occurring at a plurality of positions in a specific region on the nucleic acid.
  • insertion of a base sequence artificially introduced into a nucleic acid is also included in the “insertion mutation” in this specification.
  • the present invention is suitable for detecting genomic polymorphisms and variations, particularly for detecting and screening base substitutions on genes, for example, SNPs.
  • the first embodiment of the present invention is the determination of a DNA base sequence, which is performed by the following steps.
  • the target DNA is first fragmented.
  • preparation of DNA having an internal sequence of the target DNA at the end is referred to as fragmentation.
  • the method of fragmentation is not particularly limited, but a method using an enzyme, a physical method such as sonication shearing, a chemical method such as an acid treatment, or a method combining these methods can be used.
  • Deoxyribonuclease (DNase) can be used as the enzyme.
  • restriction enzymes and endo-type enzymes such as DNaseI can be suitably used.
  • DNA having an internal sequence at the end of the target DNA can be obtained by partially digesting the target DNA with an exonuclease, for example, BAL31 exonuclease, exonuclease III, exonuclease, T4 DNA polymerase, or the like.
  • exonuclease for example, BAL31 exonuclease, exonuclease III, exonuclease, T4 DNA polymerase, or the like.
  • exonuclease for example, BAL31 exonuclease, exonuclease III, exonuclease, T4 DNA polymerase, or the like.
  • exonuclease for example, BAL31 exonuclease, exonuclease III, exonuclease, T4 DNA polymerase, or the like.
  • E. coli nuclease is also included in the fragmentation herein.
  • the fragmentation method can be appropriately selected depending on whether the sequence to be determined is the whole sequence of the target DNA, a part of the force, and if so, which part.
  • a method of randomly fragmenting DNA for example, DNaseI treatment, ultrasonic treatment, acid treatment and the like can be suitably used.
  • the sequence of the target portion can be determined efficiently by fragmentation using a restriction enzyme that recognizes the sequence.
  • the degree of fragmentation may be appropriately selected depending on the length of the sequence to be determined and the fragmentation method.However, in the base sequencing step described below, a sequence of about 20 bases can be decoded. It is desirable to set the conditions as described above.
  • the following method can be suitably used.
  • the target DNA is amplified by PCR.
  • one or both primers are biotinylated.
  • the target DNA is fragmented by sonication, and the terminal phosphate group is removed by alkaline phosphatase, the terminal is blunted by DNA polymerase, and phosphorylation of the one end by polynucleotide kinase is performed.
  • a first adapter having a MmeI recognition site at this end is ligated, and DNA containing biotin is bound to magnetic beads on which avidin has been immobilized.
  • the DNA is digested with MmeI to cut out a DNA consisting of the first adapter and a target DNA of about 20 base pairs adjacent thereto.
  • a second adapter is ligated to the end generated by MmeI digestion of this DNA.
  • the DNA thus obtained is used in the following steps for preparing a doug library.
  • the fragmented target DNA prepared in step (1) is ligated to a tag vector.
  • a tag vector As the tag sequence contained in the tag vector, those disclosed in Japanese Translation of PCT International Publication No. 11'1-507528- can be used. Among them, the Bulletin of the National Academy of Sciences, Vol. 97, No. 1665-1670 Pages, 2000, are particularly preferably used. It is also desirable that the tag vector contains a selection marker for drug resistance and the like. When E. coli is used as a host, genes such as ampicillin resistance, chloramphenicol resistance, kanamycin resistance, and streptomycin resistance can be used as markers.
  • the ends of the DNA fragment ligated to the tag vector may be blunt-ended by enzymatic treatment, for example, treatment with BAL31 exonuclease, T4 DNA polymerase, Klenow enzyme, or a combination of these enzymes. .
  • the DNA fragment thus prepared can be efficiently ligated to a tag vector that has been linearized by treatment with a restriction enzyme that generates blunt ends. Fragmentation by physical or chemical treatment
  • the end shapes of the DNA fragments are irregular, and usually contain a mixture of 5, 10 ⁇ , 5'-phosphate, 3, 1 O, 3'-phosphate. Of these, only DNA having 5, monophosphate and 3, 1OH groups can be used as substrates for DNA ligases such as T4 DNA ligase.
  • a protruding end can be obtained by ligating a linker to the blunted end of the fragmented DNA and digesting with a restriction enzyme that recognizes the sequence of the linker, or ligating an adapter. This DNA fragment can be more efficiently linked to a linear tag vector having complementary ends.
  • the tag vector linked to the fragmented DNA is introduced into an appropriate host, preferably E. coli.
  • DNA can be introduced into a host by a known method, and there is no particular limitation.
  • an electroporation method or a method using a competent cell can be used.
  • the transformant is cultured under the selection pressure corresponding to the marker gene of the tag vector, and a tag library, which is a mixture of fragmented DNA and DNA linked to the tag vector, is prepared from the cells.
  • the preparation of the tag library may be performed by preparing the DNA by a known method.
  • the tag vector is a plasmid vector, for example, the Arikari-SDS method can be used.
  • Independent clones appropriately set Surebayore the purpose, but, 1 0 4 or more in consideration of the sequencing capabilities of MP SS, preferably 1 0 5 or more, more preferably be set to 1 0 6 or more Guess. It is effective to prepare a tag library as a plurality of pools and obtain the desired number of clones by selecting the number of pools to be used. In this case, a tag library having the desired number of clones can be easily prepared by setting the number of clones per pool to 10,000 to several hundred thousand and the number of pools to be prepared from 2 to 100.
  • the tag vector of Tokuheihei 1 1-5 0 7 5 2 8 is a mixture of about 1.70 million types of plasmids with different tag sequences. Sequence analysis by MPSS method In this method, it is necessary to bind one type of DNA to one microphone mouth bead, and therefore, one type of tag sequence must correspond to one type of target DNA sequence. By setting the number of clones included in each pool of the tag library to 170,000 to 100,000 and binding the target DNA to the microphone bead for each pool, two or more target DNAs bind Tags can be reduced to a low value of 0.5 to several percent.
  • a fragment containing the target DNA fragment and the tag is prepared from the tag library.
  • the method is not particularly limited.For example, a method of removing the vector portion from the DNA by digestion with one or more appropriate restriction enzymes, followed by molecular weight fractionation by gel electrophoresis or gel filtration, and a method by PCR Is mentioned.
  • the PCR method has advantages such as being simple when preparing a large amount of DNA containing a target DNA and a tag, and being able to amplify only the desired portion, so that the contamination of the vector portion can be suppressed to a small amount.
  • the subsequent operation can be performed efficiently and easily by labeling the primer on the target DNA side with a fluorescent group and the primer on the tag side with a group capable of specific selection such as biotin.
  • the tag portion of this DNA is made single-stranded by, for example, the following method.
  • the tag for 2000 consists of A, T, and G bases, and the tag chain consists of T, A, and C.
  • digest the DNA with a restriction enzyme that cuts between the tag and the terminus on the tag side, remove the terminal fragment, and then use (4 DNA polymerase in the presence of 10 to 4 tags to remove only the tag portion). Can be single-stranded.
  • microbeads anti-tagged micro-mouth beads
  • Microbeads can be prepared, for example, by the method described in JP-T-11-507528 and the bulletin of the National Academy of Sciences, Vol. 97, pp. 1665-1670, 2000. Mix target DNA with single-stranded tag and microbeads and incubate. I The conditions of the composition and temperature of the incubation solution are not particularly limited as long as the anti-tag on the microbeads and the single-stranded tag bound to the fragmented target DNA specifically hybridize. Vol. 97, pp.
  • a covalent bond is formed between the anti-tag and the target DNA fragment bound to the microbeads by DNA ligase.
  • the DNA ligase to be used is not particularly limited.
  • T4 DNA ligase and Escherichia coli DNA ligase can be suitably used.
  • Performing DNA polymerase in the presence of dATP, dGTP, dCTP, or dTTP prior to the reaction with DNA ligase may increase the efficiency of the DNA ligase reaction.This reaction may be performed if necessary.
  • the DNA polymerase to be used is not particularly limited, but for example, T4 DNA polymerase is preferably used.
  • the base sequence of the DNA immobilized on the microbeads prepared in the above step (4) can be determined according to the following operation.
  • start adapter 1 for analyzing four bases from the end of the target DNA and a target sequence Starter adapter (start adapter 2) for analysis of 4 bases at a distance of 2 bases from the end of the primer may be used, or only one starter adapter may be used. Good.
  • the recognition sequence of the type II s restriction enzyme to be used in the next step is previously inserted into the tag vector used in step (2) near the site where the target DNA is to be cloned. Removal of the DNA fragment containing the fluorescent group and ligation of the starting adapter can be omitted.
  • the microbead prepared in this way is two-dimensionally filled in a flow cell.
  • the flow cell is composed of glass plates stacked at intervals slightly wider than the diameter of the microphone bead, and there is a dam near the outlet to prevent the beads from flowing out. Perform the following reaction in a flow cell to determine the nucleotide sequence of the target DNA.
  • the starting adapter has a type lis restriction enzyme recognition sequence, and is designed so that the end of the target DNA becomes a protruding end when digested with this enzyme.
  • the type II s restriction enzyme is passed through the flow cell filled with beads and reacted with DNA on the beads to make the end of the target DNA a protruding end.
  • the type lis restriction enzyme is not particularly limited as long as it generates a protruding end, but an enzyme generating a 4-base protruding end, for example, BbVI can be suitably used.
  • the adapter is a partially double-stranded synthetic DNA having a 4 ′ 5 ′ overhang at one end and a 10 ′ 3,1 overhang at the other end.
  • the sequence of the double-stranded portion of the dead adapter is common, and is divided into 16 groups by the sequence at the protruding end. Enco and dead adapters belonging to the same group have one 5'-protruding end and one 3'-protruding end sequence in common, and the remaining three bases of one protruding end have four types of bases. It is a mixture.
  • the structure of the encoder adapter is not particularly limited as long as it can perform MPSS. That is, if it has a single-stranded portion linked to the protruding end of the target DNA, a double-stranded portion having a type lis restriction enzyme recognition sequence, and a single-stranded portion for hybridizing a decoder probe, Good.
  • the type II s restriction enzyme recognition sequence and its position can be recognized by the type II s restriction enzyme used here, as long as it can generate a protruding end from which the base to be determined next protrudes.
  • the decoder-one probe only needs to have a sequence complementary to the decoder probe binding sequence of the encoded adapter and a fluorescent group.
  • the fluorescent group for example, phycoerythrin can be suitably used.
  • the base adjacent to the base sequence determined above becomes a protruding end. From here on, the base sequence is determined by repeating the cycle of ligation of the adapter ( e ), hybridization with the decoder probe, recording with the CCD camera, and removal of the decoder probe (f). Yes, as described above, several hundred thousand to 100,000 DNA bases consisting of about 20 bases A row is obtained.
  • MPDS Massively Parallel Diced-DNA Sequencing
  • a second embodiment of the present invention is the detection of a mutation.
  • the size (A), the number of individuals (B), the redundancy (C), and the sequence length (D) determined by the MPDS method described above, of the target DNA to be detected And the number of sequences (E) obtained by the MPDS method has the following relationship.
  • a target DNA to be detected is amplified, and its base sequence is determined.
  • the size of the target DNA to be detected may be appropriately selected depending on the size of the gene to be analyzed, the number of individuals, and the required degree of redundancy. From the standpoint of ease of amplification, it is desirable that the number be several kb or less or ten and several kb or less. If you want to cover a wider area than this, you can divide it into several areas and amplify them separately.
  • the amplification method is not particularly limited, and a method of amplifying in vitro or a method of amplifying by i ⁇ can be used. For example,?
  • DNA may be cloned and amplified in a host cell such as Escherichia coli, or a combination of these methods may be used.
  • the nucleotide sequence of the target DNA thus amplified is determined according to the first embodiment of the present invention, and the mutation is detected.
  • samples from each individual are mixed, but there is no particular limitation on the stage at which the samples are mixed.
  • genomic DNA or cDNA from each individual who becomes type III may be mixed and then amplified, or the target DNA amplified for each individual may be mixed.
  • the fragmented target DNA may be mixed, the tag library prepared for each individual may be mixed, or migrobeads may be prepared for each individual, mixed, and then mixed with the MPDS method.
  • the nucleotide sequence may be deciphered by the above method, or may be mixed at other stages.
  • a homology search is performed between the known sequence of the target sequence and the sequence obtained by the MPDS method.
  • Known programs such as BLAST can be used as the homology search program.
  • alignment can be performed by performing homology search on this sequence, and the sequence differs.
  • the presence and frequency of the mutation can be known from the base and its frequency.
  • --Target DNA is prepared from a plurality of groups, for example, a normal group and a patient group, and the mutation is detected according to the second embodiment of the present invention, thereby screening for a genetic polymorphism associated with a disease. can do.
  • the number of individuals in each group can be set as appropriate depending on how often a gene polymorphism that appears should be screened, and the size of the target DNA and the required redundancy. For example, when screening polymorphisms present in a target DNA region of 10 kb from 119 individuals and obtaining 700,000 sequences of 17 bases, the redundancy is 1.0. Become.
  • the average number of sequences containing a certain base is 1190. Assuming that this base is G in the sequence of 1133 and A in the sequence of 57, there is a gene polymorphism consisting of two bases, G and A, at this site. It turns out that the frequency is 4.8%.
  • the tying of the child polymorphism can be performed. Since it is necessary to identify the individual from which each sequence is derived, the ability to type one individual per MPDS or the microbeads derived from each individual by dividing the flow cell area Fill and perform MP DS. Since the number of individuals is smaller than that of gene polymorphism typing, it is possible to increase the redundancy and improve the accuracy and / or increase the size of the target DNA to perform tying on many genes at once. . While many of the conventional diving methods target only known gene polymorphisms, according to the method of the present invention, there is a possibility that a novel gene polymorphism can be discovered simultaneously with tying.
  • Example 1 target only known gene polymorphisms, according to the method of the present invention, there is a possibility that a novel gene polymorphism can be discovered simultaneously with tying.
  • yeast extract 0.5% NaCl, pH 7.0
  • LB-chloramfecole plate solidified by adding 1.5% agar to the above medium at 37 ° C. The culture was performed aerobically.
  • HL-60 cells ATCC CCL 240
  • KATO III cells H SRRB J CRB061 1
  • DMEM Dulbecco's modified method Igu Le medium
  • HL—60 fine RNA prepared from vesicles and KATO III cells was converted into type I, the primers represented by SEQ ID NO: 1 in the Sequence Listing, the reverse primers represented by SEQ ID NO: 2 in the Sequence Listing, and the On Step RNA PCR Kit.
  • RT_PCR was performed using (AMV) (manufactured by Takara Paio) to amplify cDNA encoding human aldehyde dehydrogenase 2.
  • the PCR reaction product was separated by agarose gel electrophoresis, a gel block containing 2.4 kb DNA was cut out, and DNA was purified therefrom using EAS YTRAP Ver. 2 (Takara Bione; h).
  • the tag vector pLCV2 was constructed according to the method described in the Bulletin of the American Academy of Sciences, Vol. 97, pp. 165-1670, 2000. After pLCV2 was digested with BamH'I and Bb sl (both manufactured by YouEngland Piorap (NEB)), dephosphorylation was performed using ⁇ small intestine alkaline phosphatase (CIAP, manufactured by Takara Bio Inc.). The ends were blunted using DN AB lintng Kit (manufactured by Takara Bio Inc.).
  • Samples A, B, and C were mixed at a weight ratio of 10: 1 and 100: 1, respectively.
  • the following operation was performed for each sample. 5 g each of sample A, sample B, and sample C were dissolved in 400 ⁇ l of TE buffer (1 OmM Tris—HC1 pH7: 5, ImMEDTA), and UR-20P (Tomy Seie) The ultrasonic treatment for 15 seconds was performed 5 times according to). DNA that has been dephosphorylated with CIAP is purified by phenol treatment, chloroform treatment, and ethanol precipitation, and the DNA ends are blunted using the Takara Blunting Kination Ligation Kit (Takara Bionet; fcII). And phosphoric acid.
  • the fragmented DNA and the above-described linearized pLCV2 were ligated using a DNA Ligation Kit (manufactured by Takara Bio Inc.), and E. coli TOP 10 was excised by electroporation using the obtained recombinant plasmid. Transformed.
  • the number of independent clones was calculated from the number of colonies formed by inoculating a part of the transformant on an LB-chloramphenicol plate, and another part was transferred to six 50 ml LB medium containing chloramphenicol. Inoculate and cultivate, QI AGEN P 1 a sm id Plasmid DNA was purified from the culture using Midi Kit (Qiagen).
  • the number of independent clones per LB medium containing 50 ml of chloramphenicol was about 160,000. Plasmid DNA obtained from each culture was used as a tag library pool, and a tag library consisting of six pools was obtained.
  • the tag library pool was subjected to PCR using type I, FAM-labeled PCR-R primer (SEQ ID NO: 3) and biotinylated PCR-F primer (SEQ ID NO: 4) to amplify the target DNA fragment and the portion containing the tag. .
  • the operation is performed for each pool, and in this example, one pool is described.
  • T4 DNA polymerase manufactured by Takara Bio Inc. was allowed to act in the presence of dGTP to make the tag portion single-stranded.
  • a covalent bond was formed between the target DNA fragment and the anti-tag by the action of T4 DNA ligase on the sorted microphone-mouth beads, and then quenched with Dpnil. Further, after T4 DNA polymerase was allowed to act on the microbeads in the presence of dGTP, the starting adapter was ligated using T4 DNA ligase.
  • the starting adapter is composed of the oligonucleotide represented by SEQ ID NO: 5 in the sequence listing whose 5'-end is labeled with FAM, and the nucleotide sequence represented by SEQ ID NO: 6 in the sequence listing phosphorylated at one end. The oligonucleotides shown are annealed. Microphone beads with fluorescence by FAM were sorted using a MoFlo cytometer (second sorting).
  • a microbead to which an initiation adapter is connected is filled in a flow cell, and the base sequence of a DNA fragment on a microphone mouth bead according to the operation of the MPSS method described in Nayya Biotechnology, Vol. 18, pp. 630-634, 2000 It was determined. As a result, about 800,000 sequences consisting of 20 bases were obtained.
  • D aildi cells (ATCC CCL-213), HT-29 cells (ATCC HTB-38), A431 cells (ATCC CRL-1555) and SW480 cells (ATCC CCL-228) each containing 10% fetal serum DM
  • E aildi cells (ATCC CCL-213), HT-29 cells (ATCC HTB-38), A431 cells (ATCC CRL-1555) and SW480 cells (ATCC CCL-228) each containing 10% fetal serum DM
  • the cells were cultured in M in the presence of 5% CO 2 at 37 ° C for 7 days. These cells were collected, and RNA was prepared from each of them using a Trizol reagent. CDNA was synthesized from these RNAs by using Reverse Transcriptase M—MLV (RNase H-1) (Takarapaio). Next, the above-mentioned cDNA was designated as type II, and a primer p53 1L represented by SEQ ID NO: 8 in the sequence listing, a primer p53-2R represented by SEQ ID NO: 9 in the sequence listing, and Takara Ex Taq Hot Start PCR was performed using Version (manufactured by Takara Bio Inc.) to amplify the cDNA encoding p53.
  • telomere sequence Of the published nucleotide sequence of p53c DNA (NM 000546), amplified by the above RT-PCR SEQ ID NO: 10 in the sequence listing.
  • the PCR product was separated by agarose gel electrophoresis, a gel block containing 1.9 kb DNA was cut out, and the RT-PCR product was purified using QIAquick Gel Extraction Kit (Qiagen). did.
  • Samples were prepared by mixing equal amounts of RT-PCR products prepared from the four types of cells. This sample was subjected to sonication, dephosphorylation treatment, blunt-end blunting, ligation to pLCV2, transformation of TOP10, and preparation of a plasmid in the same manner as in Example 1 to obtain a tag library.
  • This sample was subjected to sonication, dephosphorylation treatment, blunt-end blunting, ligation to pLCV2, transformation of TOP10, and preparation of a plasmid in the same manner as in Example 1 to obtain a tag library.
  • microphone mouth beads to which the cDNA fragment on the tag library was bound were prepared in the same manner as in Example 1.
  • the nucleotide sequence of the DNA fragment on the microbeads was determined. As a result, about 940,000 sequences consisting of 20 bases were obtained. These sequences were clustered to remove sequences with an appearance frequency of less than 50, and then compared with the published base sequence of p53 cDNA (NM-000546). As a result, about 790,000 sequences were mapped on p5.3cDNA as a completely matched sequence or a sequence containing a single base mismatch. For each base site of p53 cDNA, the total appearance frequency of the mapped sequence and the appearance frequency of each base were counted.
  • Example 2 direct sequencing was performed on the RT-PCR product obtained in Example 2 (1), and sites where base substitution was observed between cells (three sites) were identified.
  • Table 1 shows the frequency of occurrence of sequences corresponding to each base substitution in DNA on microbeads for these sites.
  • the appearance frequency of the base at the base substitution site corresponded to the base content expected from the mixing ratio of the RT-PCR product in the sample.
  • the present invention provides a method for determining a DNA base sequence and a method for detecting a mutation.
  • a large amount of DNA nucleotide sequence can be analyzed at lower cost than by a conventional method such as an automatic sequencer using the dideoxy method.
  • the use of the mutation detection method of the present invention makes it easy to detect mutations from a large number of samples, so that infrequent mutations can be found at low cost. As a result, effective and safe medical treatment by applying polymorphic tying such as SNP becomes possible. Sequence listing free text
  • SEQ ID NO: 1 PCR primer to amplify a gene encoding numan
  • SEQ ID NO: 2 PCR primer to amplify a gene encoding human
  • SEQ ID NO: 3 PCR primer PCR-R to amplify a DNA fragment in tag library pool.
  • SEQ ID NO: 4 PCR primer PCR-F to amplify a DNA fragment in tag library pool.
  • SEQ ID NO: 6 Oligonucleotide thick initiating adapter.
  • SEQ ID N0: 8 Oligonucleotide thick initiating adapter.
  • SEQ ID N0: 9 PCR primer to amplify a gene encoding human p53 cDNA.

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Abstract

Méthode servant à déterminer des séquences de base d'ADN par rapport à une multiplicité de spécimens, de façon économique et extrêmement précise. Méthode servant à détecter des mutations de manière économique et extrêmement précise, au moyen de la première méthode de détermination des séquences de base.
PCT/JP2004/010893 2003-07-25 2004-07-23 Methode servant a detecter une mutation WO2005010184A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007147168A2 (fr) * 2006-06-16 2007-12-21 The Children's Mercy Hospital Atténuation de la mort cellulaire induite par hyperoxie à l'aide de l'aldéhyde déhydrogénase mitochondriale

Citations (2)

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Publication number Priority date Publication date Assignee Title
WO1995027080A2 (fr) * 1994-04-04 1995-10-12 Lynx Therapeutics Inc Sequençage d'adn par ligature et clivage par etapes
WO1997046704A1 (fr) * 1996-06-06 1997-12-11 Lynx Therapeutics, Inc. Signatures par ligature d'adaptateurs codes

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Publication number Priority date Publication date Assignee Title
WO1995027080A2 (fr) * 1994-04-04 1995-10-12 Lynx Therapeutics Inc Sequençage d'adn par ligature et clivage par etapes
WO1997046704A1 (fr) * 1996-06-06 1997-12-11 Lynx Therapeutics, Inc. Signatures par ligature d'adaptateurs codes

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Title
BRENNER, S. ET AL.: "Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays", NATURE BIOTECHNOLOGY, vol. 18, 2000, pages 630 - 634, XP002199492 *
BRENNER, S. ET AL.: "In vitro cloning of complex mixtures of DNA on microbeads: Physical separation of differentially expressed cDNAs", PNAS, vol. 97, no. 4, 2000, pages 1665 - 1670, XP002183229 *
KOYAMA, N. ET AL.: "DNA Microbeads Array Gijutsu o Mochiita Idenshi Hatsugen Kaiseki", NIPPON NOGEI KAGAKU KAISHI, vol. 76, no. 5, 2002, pages 474 - 479, XP002984907 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007147168A2 (fr) * 2006-06-16 2007-12-21 The Children's Mercy Hospital Atténuation de la mort cellulaire induite par hyperoxie à l'aide de l'aldéhyde déhydrogénase mitochondriale
WO2007147168A3 (fr) * 2006-06-16 2008-08-07 Childrens Mercy Hospital Atténuation de la mort cellulaire induite par hyperoxie à l'aide de l'aldéhyde déhydrogénase mitochondriale

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