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WO2007064022A1 - Procede pour confirmation de preparation d'acide nucleique - Google Patents

Procede pour confirmation de preparation d'acide nucleique Download PDF

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Publication number
WO2007064022A1
WO2007064022A1 PCT/JP2006/324317 JP2006324317W WO2007064022A1 WO 2007064022 A1 WO2007064022 A1 WO 2007064022A1 JP 2006324317 W JP2006324317 W JP 2006324317W WO 2007064022 A1 WO2007064022 A1 WO 2007064022A1
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WIPO (PCT)
Prior art keywords
nucleic acid
probe
prepared
microarray
rna
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PCT/JP2006/324317
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English (en)
Japanese (ja)
Inventor
Hiroyuki Ooshima
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Mitsubishi Rayon Co., Ltd.
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Filing date
Publication date
Application filed by Mitsubishi Rayon Co., Ltd. filed Critical Mitsubishi Rayon Co., Ltd.
Priority to JP2006554379A priority Critical patent/JP5296318B2/ja
Publication of WO2007064022A1 publication Critical patent/WO2007064022A1/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
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

Definitions

  • the present invention relates to a method for confirming the success or failure of preparation of a nucleic acid, preferably an miRNA.
  • miRNA is a short single-stranded RNA of 15-25 bases.
  • RNA fraction of the extra chain length in order to improve the labeling efficiency.
  • 200 bases including miRNA from total RNA in advance for the purpose of suppressing cross hypridation. It is necessary to collect only the short RNA fraction of the following grade, label it, and use it for hybridization.
  • Kit protocols for fractionating short RNAs are available from several companies including Ambion and Invitrogen.
  • Methods for fractionating short-chain RNAs include a method of fractionating short-chain RNA from a gel according to the mobility of nucleic acids after electrophoresis using a gel, a method using a silica gel membrane, etc.
  • a method for extracting only short-chain RNA based on the time difference required for passage through an acrylic gel is known.
  • the fractionation range may vary depending on the concentration of the salt solution, the EtOH content, temperature, time, and the like.
  • a method for determining the success or failure of the fractionation operation a method of performing electrophoresis using a high-concentration acrylamide gel and confirming the bun and the condensate can be considered.
  • the bands stained with intercalators etc. in the gel after electrophoresing them are mainly rRNA and tRNA. Since the amount of miRNA of about 20 bases contained in total RNA is very small, its presence cannot often be confirmed as a band unless a large amount of sample is used.
  • An object of the present invention is to provide a method for confirming the success or failure of an operation for preparing a nucleic acid such as miRNA from a sample.
  • the present inventor has conducted intensive research.
  • the present inventor prepared a short-chain nucleic acid containing miRNA from a sample such as total RNA in the presence of a control nucleic acid having approximately the same length as miRNA.
  • a control nucleic acid having approximately the same length as miRNA.
  • the present invention is as follows.
  • a method for confirming the success or failure of a nucleic acid preparation operation comprising the following steps:
  • a method for confirming the success or failure of a nucleic acid preparation operation comprising the following steps:
  • nucleic acid from a sample comprising a control nucleic acid having a length equivalent to the length of the nucleic acid to be prepared and a control nucleic acid having a length of 10 to 100 times the length of the nucleic acid to be prepared;
  • the nucleic acid to be prepared includes, for example, miRNA.
  • the step of preparing the nucleic acid includes, for example, purifying the nucleic acid and labeling the nucleic acid.
  • nucleic acid to be prepared when a nucleic acid to be prepared is prepared from a sample, it is possible to confirm the success or failure of the preparation operation by allowing a control nucleic acid having a length equivalent to the nucleic acid to be present in the sample. .
  • FIG. 1 is a view showing an arrangement fixing device for producing a hollow fiber bundle.
  • FIG. 2 is a diagram showing an image of the microarray after the hybridization in Example 2.
  • the box image shows the positions of probe contl, probe cont2, probe cont4, probe coht5, probe cont6, probe cont7, YLR377C 21W and YLR377C 21b'2 (upper), and YLR377C 21b-3 (lower) in order of left force (Upper panel, lower panel).
  • the present invention is a method for confirming the success or failure of a nucleic acid preparation operation. Specifically, in the present invention, first, a nucleic acid preparation operation is performed in the presence of a control nucleic acid (hereinafter also referred to as “short-chain spike control”) having substantially the same length as the nucleic acid to be prepared. Next, using a microphone array with a captive probe having a sequence complementary to the spike control, the prepared nucleic acid is hybridized, and then the signal of the short spike control exceeds a certain level. If the value can be detected, it is confirmed that there is no problem in the nucleic acid preparation operation, or if the signal is not detected, it is confirmed that the nucleic acid has been lost in the pre-hybridization preparation operation. It is.
  • a control nucleic acid hereinafter also referred to as “short-chain spike control”
  • a nucleic acid preparation operation if an appropriate preparation operation is performed, it is empirically clear that the sample is not mixed with the prepared sample (hereinafter referred to as “long”). There may also be additional chain spike control). In this embodiment, if the signal of the long chain spike control is not detected, it is confirmed that there is no problem in the preparation operation itself, or if the signal of the long chain spike control is detected at a certain value or more. During the preparation operation, it is confirmed that a nucleic acid longer than the length to be fractionated is mixed in the prepared nucleic acid.
  • nucleic acid to be prepared or “nucleic acid to be prepared” means a nucleic acid that is targeted to be obtained from a starting sample by a preparation operation.
  • the present invention provides a means for confirming whether or not a nucleic acid to be prepared has actually been prepared.
  • a nucleic acid preparation operation can be performed from the spike control and the detection result of the microarray using the probe mounted on the microarray. It becomes possible to confirm whether or not. For example, if the signal cannot be confirmed in the microarray analysis of the target nucleic acid (for example, miRNA) by the method of the present invention, the target nucleic acid can be detected in the original starting sample. It can be confirmed whether or not the nucleic acid for preparation was lost in the preparation operation. '
  • a spita control is a nucleic acid added to a starting sample.
  • the spike control of the present invention may be either DNA or RNA, but those skilled in the art can appropriately select the type of nucleic acid in consideration of the nucleic acid to be prepared, the preparation conditions, and the like.
  • the spike control of the present invention is preferably the same type as the nucleic acid in terms of matching efficiency with the nucleic acid to be prepared.
  • the spike control is preferably DNA.
  • the spike control may be either RNA or DNA.
  • RNA is preferable in that the labeling efficiency coincides with the nucleic acid to be prepared, and the signal intensity is strong, and DNA is preferable in terms of production stability and ease of operation.
  • a person skilled in the art can appropriately select the type of nucleic acid to be used for spike control from DNA and RNA.
  • the spike chondrol of the present invention can be synthesized by a known method.
  • it can be synthesized using a commercially available nucleic acid synthesizer such as Applied Biosystems 3400 DNA synthesizer.
  • the spike control of the present invention can also be obtained by using PCR in which a nucleic acid containing the spike control sequence is in the form of a cage.
  • a nucleic acid containing the spike control sequence is in the form of a cage.
  • Fowa one de primer one used for PCR 5 'end by using the primer phosphorylated, one may obtain single-stranded DNA by exonuclease treatment
  • the resulting PCR products were obtained c may chains DNA can be used as a spike control.
  • the spike control of the present invention is RNA, a transcript is used. It is also possible. ⁇ '
  • the spita control added to the starting sample may be one type of nucleic acid or a plurality of types of nucleic acid.
  • each control may be added in the same amount, or different amounts may be added for each control.
  • Each spike control may be added separately to the starting sample, or mixed prior to the starting sample. .
  • a short spike control may be added to the starting sample.
  • a long chain spike control may be further added to the starting sample. The short chain spike control and long chain spike control are described below.
  • a control nucleic acid having the same or almost the same length as the nucleic acid to be prepared is also referred to as a “short spike control”. '
  • “equivalent length” or “approximately the same length” means the same length as the length of the nucleic acid to be prepared or the length of the nucleic acid ⁇ 100 to 0 bases, preferably ⁇ 10 ⁇ 0 bases, more preferably ⁇ 6-0 bases, more preferably ⁇ 1-0 bases in length
  • the short spike control is prepared with the nucleic acid to be prepared and analyzed by microarray.
  • the short spike control is added to the starting sample to determine the presence or absence of the nucleic acid to be prepared in the sample to be analyzed by microarray. Therefore, it is desirable that the short spike control is a sequence that does not cross-hybridize with the probe corresponding to the nucleic acid to be prepared mounted on the array in the microarray analysis. That is, it is preferable to design the short spike control tool so as to have a low homology with the nucleic acid to be prepared in consideration of the homology with the nucleic acid to be prepared. Homology can be obtained by obtaining known sequence information from an existing database and using a known homology search software. wear.
  • the short spike control role with low homology to the nucleic acid to be prepared is designed based on the sequence of a gene that is specifically expressed in a species different from the species from which the starting sample is derived.
  • short-chain spike control is also possible for nucleic acids that are highly hybridized under stringent conditions with nucleic acids containing a base sequence complementary to the spike control as described above, and that have low homology to the nucleic acids that are to be prepared. Can be used as
  • the stringent conditions are the conditions at the time of washing after hybridization, and the salt concentration is 100 to 1000 mM, the temperature is 40 to 75 ° C., preferably the salt concentration is 100 to 500 mM, This means that the temperature is between 40 ° C and 60 ° C.
  • conditions such as 50 ° C with 2xSSC can be mentioned.
  • a short spike control can be designed based on either or both of the following conditions (i) and (ii).
  • a sequence that meets the conditions (i) and (ii) may be searched from a random 21-base sequence composed of A, T, G, and C.
  • a homology search may be performed with miRBase, a miRNA database, to confirm that the similarity to miRNA is sufficiently low.
  • miRBase a miRNA database
  • a long control nucleic acid which is empirically clear that it will not be mixed into the prepared sample if it is appropriately prepared, is also referred to as “long-chain spike control”.
  • Appropriate preparation operations include, for example, operations according to the instructions attached to the kit used for the preparation operation.
  • Nucleic acid lengths that are empirically apparent not to be contaminated can be, for example, nucleic acid lengths outside the fractional range described in the instructions attached to the kit.
  • the long spike control is a control having a length of 10 to 100 times, preferably 10 to 5 times, more preferably 10 to 20 times the length of the nucleic acid to be prepared. It is a nucleic acid.
  • Long-chain spike control is used to determine whether or not a nucleic acid of a length that is empirically clear from the appropriate preparation procedure in the process of preparing the nucleic acid to be prepared has been mixed by actual operation. Is added to the sample to determine Therefore, the base length of the long-chain spike control needs to be sufficiently longer than the expected length to be fractionated by the preparation operation, or in some cases, sufficiently short.
  • Nucleic acid lengths that are not contaminated by appropriate preparation operations depend on the nature of the preparation operation. For example, when performing a preparative operation using a column, if the amount of ethanol input is less than expected, longer RNA strands may be mixed into short RNA. The temperature during the preparation operation and the time required for the preparation may also affect the fractionation range.
  • the long-chain spike control is a sequence that does not cross-hybridize with the probe corresponding to the nucleic acid to be prepared mounted on the array in the microarray analysis. That is, the long-chain spike control is preferably designed to have low homology with the nucleic acid to be prepared in consideration of homology with the nucleic acid to be prepared. Homology can be determined as described above.
  • the long spike control with low homology to the nucleic acid to be prepared is designed based on the sequence of a gene that is specifically expressed in a species different from the species from which the starting sample is derived.
  • nucleic acid that is hybridized under stringent conditions with a nucleic acid containing a complementary base sequence to the spike control as described above, and a nucleic acid with low homology to the nucleic acid to be prepared should also be used as a long-chain spike control. Can do.
  • a long-chain spita control can be designed based on the sequence information of yeast YLR377C.
  • yeast YLR377C When the genomic DNA of yeast YLR377C is amplified by PCR using the following primer, a 300 base pair PCR product can be obtained. At this time, if a forward primer whose 5 'end is phosphorylated is used, the resulting PCR product is treated with strandase TM ⁇ Exonuclease (manufactured by Novagen) etc. to make a single strand. Troll (ssYLR377C 300b).
  • Reverse primer CTACTGTGACTTGCCAATATGGTCTAAAAA (SEQ ID NO: 1 4)
  • nucleic acid to be prepared means a nucleic acid targeted for acquisition from a starting sample by a preparation operation.
  • the nucleic acid to be prepared is not limited as long as it can be fractionated from the whole nucleic acid contained in the sample using a certain nucleic acid length as an index, but includes short-chain nucleic acids such as short-chain RNA. be able to.
  • the short-chain nucleic acid include a nucleic acid having 150 to 15 bases, preferably 50 to 15 bases, more preferably 25 to 15 bases.
  • the nucleic acid to be prepared is, for example, miRNA.
  • the above spike control is added to the starting sample, and nucleic acid is prepared from the sample containing the spike control.
  • the origin of the starting sample is not particularly limited, and examples thereof include mammals, plants, insects, nematodes, and other derived samples.
  • the starting sample used in the present invention may be a tissue, a cell, a cell extract or the like as long as it is derived from the above, and, like the total RNA obtained from the cell extract, an intermediate stage of nucleic acid purification operation It may be a sample.
  • total RNA derived from a mouse can be extracted by a known method such as AGPC (acid guanidinium thiocyanate-phenol chloroform) method. ',
  • the amount of spike control added to the starting sample is appropriately determined by those skilled in the art in consideration of the amount or concentration of the nucleic acid to be prepared in the starting sample, the detection limit of the detection device used, the dynamic range, etc. can do.
  • an amount that can be sufficiently detected after hybridization is added based on the performance of the detection device used.
  • the long chain control should be added so that the amount of the labeling substance involved in the detection can be sufficiently detected by a predetermined method. ⁇ . If too much spike control is added, non-specific signals may be generated, or long spike control may be mixed into the short nucleic acid fraction. When these phenomena are resolved as a result of hybridization, the amount of spike control added can be adjusted.
  • both short-chain spike control and long-chain spike control are 0.1 pg to 10000 ng / 50/1, preferably lpg to: l000 ng / 50 1, more preferably 10 pg to 100 ng / 50 / l can be added to the starting sample.
  • the starting sample is 12.5 g of total RNA and the purification scale is 50 ⁇ l start
  • the amount of each short spike control added is 50 pg to 500 pg
  • the amount of each long spike control is 5 ng. (Example 2).
  • the spike concentration of each spike control can be changed as appropriate.
  • the nucleic acid preparation operation includes a nucleic acid purification operation and a Z or nucleic acid labeling operation.
  • the order of the nucleic acid purification operation and the nucleic acid labeling operation is not particularly limited, and any operation may be performed first or simultaneously.
  • Nucleic acid This preparation operation may include a purification operation or a labeling operation multiple times, such as when a labeling operation is performed subsequent to the purification operation and a further purification operation is performed.
  • a known nucleic acid purification method can be used for the purification operation.
  • the nucleic acid to be prepared can be purified from the sample by the guanidine hydrochloride method or the phenol extraction method.
  • a nucleic acid to be prepared can be purified using a commercially available kit.
  • a total RNA extraction kit such as ISOGEN, Trizol, or RNeasy or an RNA purification kit can be purchased and used for the purification operation of the present invention.
  • the nucleic acid to be prepared can be purified according to the instructions attached to the kit.
  • the prepared nucleic acid is supplied to a microarray, the prepared nucleic acid is used in a cross-hyperpreparation from the viewpoint of improving the detection sensitivity of the microarray and by reducing the number of self-rows contained in the sample. From the viewpoint of suppressing dialysis, it is preferable to obtain a nucleic acid within the range of at least ⁇ 500 bases from the length of the target nucleic acid by fractionation. In order to obtain a nucleic acid having a desired length, column fractionation or ethanol precipitation can be used. Nucleic acids can be fractionated into short-chain nucleic acids and long-chain nucleic acids by the method described in Example 2 if they are arranged.
  • the nucleic acid to be prepared is miRNA
  • the known guanidine hydrochloride method X can be converted to low molecular weight RNA of 200 bases or less by column fractionation after phenol extraction.
  • it can be prepared with mirVana miRNA Isolation kit (Ambion).
  • nucleic acid labeling method can be used for the labeling operation.
  • nucleic acid samples such as a method that incorporates aminoallyl-dUTP at the time of reverse transcription using a random primer, a method that incorporates a labeling substance via aminoallyl, a method that uses a T4 RNA Ligase to label nucleic acid, and a method that uses end-labeling with piotin hydrazide.
  • the nucleic acid to be prepared can be labeled using a commercially available kit.
  • nucleic acids may be directly labeled using ULYSIS Alexa Fluor from Invitrogen or PlatinaBright from KREATECH Biotechnology BV,
  • a kit such as mirVana miRNA Labeling Kit manufactured by Ambion may be purchased and used for the labeling operation of the present invention.
  • the nucleic acid to be prepared can be labeled according to the instructions attached to the kit.
  • the nucleic acid is labeled with a radioisotope, a fluorescent label, an enzyme label or the like.
  • fluorescent labeling for example, labeling with Cy3, Cy5, FAM, Alexa Flour (registered trademark), DY-647, PacificBlue TM can be mentioned.
  • the microarray is used to immobilize oligonucleotide probes, cDNA probes (hereinafter also referred to as “probes”) in a plurality of sections, monitor gene expression patterns, and novel genes.
  • probes cDNA probes
  • Various forms of microarrays are known. For example, a microarray in which probes are fixed at high density on a substrate using optical lithography technology, a microarray in which probes are fixed on a glass substrate by spotting, and the like are known.
  • a fiber type microarray is also known (see Japanese Patent No. 3 5 10 8 8 2).
  • microarray a microarray in which a plurality of through holes exist in the substrate and the probes are fixed by using gels in the through holes is preferably used.
  • examples of such a microarray include a microarray described in Japanese Patent Application Laid-Open No. 20:00 — 6055 4 and a fiber type microarray.
  • the fiber-type microarray can be produced, for example, through the following steps a) to d). a) A step of producing an array by arranging a plurality of hollow fibers in three dimensions so that the longitudinal directions of the hollow fibers are the same.
  • a gel precursor polymerizable solution containing a nucleic acid probe is introduced into the hollow part of each hollow fiber of the block body, a polymerization reaction is performed, and the gel-like substance containing the nucleic acid probe is held in the hollow part.
  • hollow fiber materials include nylon 6, nylon 66, polyamide hollow fibers such as aromatic polyamide, polyethylene terephthalate, polybutylene terephthalate, polylactic acid, polyglycolic acid, and polycarbonate.
  • Polyester hollow fibers acrylic hollow fibers such as polyacrylonitrile, polyolefin hollow fibers such as polyethylene polypropylene, polymethacrylate hollow fibers such as methyl polymethacrylate, and polyvinyl alcohol hollow fibers
  • Polyvinylidene chloride hollow fiber, polyvinyl chloride hollow fiber, polyurethane hollow fiber, phenol 3 ⁇ 4 hollow fiber, polyvinylidene fluoride is a fluorine-based hollow fiber made of polytetrafluoroethylene, polyalkylene, etc. Paraoxybenzoate hollow fiber Etc.
  • the hollow fibers are arranged three-dimensionally so that their longitudinal directions are the same.
  • an arrangement method for example, a method in which a plurality of hollow fibers are arranged in parallel at a predetermined interval on a sheet-like material such as an adhesive sheet to form a sheet, and then the sheet is wound spirally (Japanese Patent Laid-Open No. 11). 1 0 8 9 2 8).
  • two perforated plates each having a plurality of holes provided at a predetermined interval are overlapped so that the hole portions coincide with each other, hollow fibers are passed through the hole portions, and the interval between the two perforated plates is increased.
  • There is a method of opening and filling the periphery of the hollow fiber between the two porous plates with a curable resin raw material and curing see Japanese Patent Application Laid-Open No. 2000-133-4553).
  • the array is then embedded so that the array is not disturbed.
  • Examples of the embedding method include a method of pouring a polyurethane resin, an epoxy resin or the like into a gap between fibers, and a method of bonding fibers by heat fusion.
  • a gel precursor polymerizable solution containing a nucleic acid probe is introduced into the hollow part of each hollow fiber of the embedded array, and a polymerization reaction is performed in the hollow part. As a result, the gel is held in the hollow portion, and the nucleic acid probe is fixed to the gel.
  • gel precursor solutions include acrylic amide, N, N-dimethyl attaly Noreamide, N-isopropylacrylamide, N-acryloylaminoethoxy alcohol, N-acryloylaminobromanol, N-methyl linoleoamide, N_vinylpyrrolidone, hydroxyl
  • monomers such as shetyl methacrylate, (meth) acrylic acid, allyldextrin, and methylene bis (meth) allylamide, polyethylene dairy coal (crosslinkable monomer) Meta) Atallate is included.
  • the nucleic acid probe is chemically bonded to the gel structure by copolymerizing with the gel precursor via the end of the nucleic acid probe (Japanese Patent Laid-Open No. 20-210). 4-1 6 3 2 1 1).
  • the block is cut into thin pieces in the direction crossing the longitudinal direction of the hollow fiber.
  • the slices produced here can be used as micro and array.
  • the thickness of the microarray is about 0.1 mm to 1 mm. The cutting can be performed by, for example, a microtome or a laser.
  • the probe is fixed to the hollow portion of the hollow fiber, that is, the gel in the through hole of the microarray. Therefore, since the probe is fixed to the three-dimensional structure, the probe is fixed in a certain space.
  • the surrounding environment, where the probe is fixed has spatial freedom, and different types of probes are fixed across a physical partition. Therefore, the above microarray is different from a microarray in which probes are fixed at a high density on a flat substrate, and the probes mounted on it function sufficiently as probes, and further, with different types of adjacent probes. There is no non-specific reaction. Therefore, the probe can be designed and fixed to the microarray without the need to bind extra sequences that are not involved in the hyper-precipitation such as linkers and spacers.
  • the probe mounted on the microarray includes a nucleic acid which is a preparation object, for example, a nucleic acid containing a sequence complementary to miRNA.
  • a microarray equipped with a probe containing a sequence complementary to the nucleic acid to be prepared When the nucleic acid to be prepared is miRNA, the sequence is complementary to miRNA, particularly mature miRNA. My with a probe containing a complementary sequence It is preferable to use a chromoarray. '''
  • nucleic acid sequence information can be obtained from Genbank.
  • miRNA sequence information can be obtained by accessing miRBase (http://microrna.sanger.ac.uk/).
  • miRBase http://microrna.sanger.ac.uk/.
  • a person skilled in the art can appropriately design and synthesize a probe to be mounted on the microarray of the present invention from known sequence information.
  • a nucleic acid having a random sequence can also be used as the probe.
  • a nucleic acid having a random sequence can be appropriately designed and synthesized by those skilled in the art.
  • the probe mounted on the microarray includes a nucleic acid containing a sequence complementary to the spike control of the present invention as a probe for detecting the spike control of the present invention.
  • the probe sequence may be any sequence that specifically hybridizes to the spike control in the microphone array analysis. That is, the probe only needs to be a nucleic acid that hybridizes only with the spiter control in the prepared nucleic acid sample used for hybridization.
  • probe contl ATACTACGGTAACGGGCCTGT (SEQ ID NO: 7) probe cont2 GGAAGAAACGTCGACCCAAAG (-SEQ ID NO: 8) probe cont4 TACGGAAGGTCCGGCATACAA (SEQ ID NO: 9) probe cont5 TGCTCGACAACGATTGGCAAG (SEQ ID NO: 10) probe cont6 TTGCGGTACGACCGACGCGTGTT 2) Long-chain spike control when the nucleic acid to be prepared is miRNA
  • Examples of the probe for detecting include the following DNAs containing a sequence complementary to ssYLR377C.
  • the chain length of the probe mounted on the microarray is 10 to 100 bases, preferably 10 to 40 bases.
  • the probe may be a nucleic acid such as DNA or RNA, but is preferably DNA.
  • the probe can be synthesized by a known method.
  • it can be synthesized by a method such as a phosphodiester method, a phosphotriester method, or a solid phase phosphoramidide method.
  • it can be synthesized by using a DNA automatic synthesizer sold by Toyo.
  • the probe is fixed to a plurality of sections of the microarray. Only one type of probe may be fixed to each section, or multiple types of probes may be fixed.
  • the fixing method differs depending on the form of the microarray.For example, if the microarray is manufactured using a photomask, it is not necessary to synthesize and prepare the probe in advance as described above. It can be synthesized sequentially. If the microarray is made by spotting, prepare probes in advance, attach appropriate functional groups to the probes, and spot the solution containing the probes on the substrate. At this time, the probe is fixed by a chemical bond between the functional group introduced into the probe and the substrate (the surface is modified if necessary). If necessary, a linker having an appropriate chain length can be bound to a probe complementary to miRNA.
  • RNA examples include endogenous small molecule RNA such as ribosomal RNA (rRNA), transfer RNA (tRNA), small nucleolar ribonuleoprotein particle RNA (snoRNA), and small nuclear RNA (Ul to Ul8 snRNA).
  • endogenous small molecule RNA such as ribosomal RNA (rRNA), transfer RNA (tRNA), small nucleolar ribonuleoprotein particle RNA (snoRNA), and small nuclear RNA (Ul to Ul8 snRNA).
  • rRNA ribosomal RNA
  • tRNA transfer RNA
  • snoRNA small nucleolar ribonuleoprotein particle RNA
  • small nuclear RNA Ul to Ul8 snRNA
  • 5SrRNA, 5.8SrRNA, UlsnRNA, U2snRNA, U3snRNA, U4snRNA, U5snRNA, U6snRNA, and Arg-tRNA are preferably used as the endogenous small RNA.
  • the prepared solution containing the nucleic acid sample is applied to the above microarray, and hybridization between the nucleic acid sample and the probe is performed. After the hybridization operation, necessary operations such as washing are performed to detect the labeled compartment on the microarray, that is, the compartment forming a hybrid with the probe.
  • the detection method the device and the detection method are appropriately selected according to the labeled sample.
  • the time of cleaning it may be changed step by step from a weak condition to a strong condition (step cleaning).
  • the washing strength means the strength of the action of dissociating the hybridization between the nucleic acid sample and the probe.
  • the cleaning conditions are adjusted to the probe with the lowest stringency among the probes mounted on the microarray, and cleaning and detection are performed.
  • the probe is washed step by step until the washing condition of the probe having the highest stringency, and the hybrid is detected each time.
  • the cleaning effect depends on the salt concentration of the cleaning solution, the cleaning temperature, the amount of the cleaning solution, the cleaning time, etc.
  • the influence of the salt concentration and temperature on the cleaning effect is determined by the relationship with the nucleic acid composition of the probe.
  • the salt concentration is kept constant and the temperature of the cleaning solution is changed stepwise.
  • the salt concentration is arbitrarily selected (however, a value within the applicable range of the Tm (melting temperature) calculation formula is selected).
  • the probe having the minimum Tm is calculated from the Tm calculation formula.
  • the minimum Tm value preferably a temperature of the cleaning solution which is lower than the minimum value by c or more, is selected as the first stage cleaning condition.
  • the maximum Tm value of the probes also mounted on the array preferably at least 2 steps to the temperature higher than the maximum value by 1 ° C or more, cleaning solution temperature Change the cleaning procedure. .
  • the temperature of the cleaning liquid is kept constant and the salt concentration in the cleaning liquid is changed stepwise.
  • the cleaning temperature is arbitrarily selected first (however, a value that can be obtained from the salt concentration within the applicable range of the Tm calculation formula is selected).
  • the probe with the smallest Tm is calculated from the Tm calculation formula, and the minimum Tm value is the same as or better than the specified cleaning solution temperature.
  • the salt concentration of the cleaning solution calculated to be higher than 1 ° C was selected as the first-stage cleaning condition, and then the maximum Tm value of the probe mounted on the microarray was specified.
  • This is a method in which the cleaning operation is performed by changing the salt concentration of the cleaning solution at least in two or more stages until the salt concentration is calculated to be 1 ° C or more lower than the cleaning solution temperature.
  • the preparation operation itself such as the purification operation and the labeling operation, should be judged successful. Can do.
  • the signal is not detected (or if it is detected at a value less than a certain value), it can be determined that the preparation operation of the nucleic acid before hyperpridation was not successful. Possible causes of the failure of the preparation operation include the loss of nucleic acid during the purification operation, or the fact that the labeling operation was not performed as expected.
  • “a value above a certain value” is, for example, the value obtained by adding the signal average value of multiple background spots to the signal standard deviation of the background spot 3 times. .
  • the long-chain nucleic acid to the prepared nucleic acid It can be judged that the preparation operation was successful even though the contamination of the mixture was sufficiently suppressed.
  • long chain spike control port If the signal is detected at a certain level or higher, it can be determined that the preparation operation was not successful.
  • the cause of the failure of the preparation operation may be that a nucleic acid longer than the standard to be fractionated was mixed in the short RNA during the purification operation.
  • the present invention can also be provided as a kit for confirming the success or failure of nucleic acid preparation operations.
  • This kit includes reagents necessary for confirming the success or failure of nucleic acid preparation operations, such as short-chain spike control, long-chain spike control, and complementary to these control tools.
  • reagents necessary for confirming the success or failure of nucleic acid preparation operations such as short-chain spike control, long-chain spike control, and complementary to these control tools.
  • Some or all of microarrays on which probes containing sequences are mounted, reagents for nucleic acid preparation, reagents for microarrays, instructions for use, etc. are included, but are not limited thereto.
  • the kit may include a container necessary for confirming the success or failure of the nucleic acid preparation operation.
  • the nucleic acid to be prepared is a short-chain nucleic acid (for example, 15 to 25 bases), it is difficult to obtain an internal control for comparing the signal intensity between samples after the purification operation.
  • This short spike control can also be used as a control to correct errors in operations such as purification, labeling, and hybridization.
  • one probe was prepared to capture Arg-tRNA.
  • the miRNA detection array was equipped with the nine types of probes described in (2), and a microarray equipped with all 190 probes was used.
  • oligo DNAs were mounted on a miRNA detection microarray as follows.
  • the oligonucleotides that serve as probes were synthesized using an automated DNA synthesizer.
  • aminolink TM manufactured by PE Biosystems
  • oligonucleotide is reacted with the oligonucleotide, followed by deprotection, whereby aminohexyl is added to the end of each oligonucleotide.
  • a 5'-0-aminohexyligonucleotide into which a group was introduced was prepared. .
  • a hollow fiber bundle was manufactured using the array fixing device shown in FIG.
  • X, y, and z are orthogonal three-dimensional axes, and the X axis coincides with the longitudinal direction of the fiber.
  • a porous plate with a thickness of 0.1 mm provided with a total of 144 pieces in each of 1 and 2 rows in a vertical and horizontal direction, with a hole having a diameter of 0.32 mm and a distance between the centers of the holes being 0 and 42 mm.
  • 2 1 Two sheets were prepared. These perforated plates were overlapped, and polycarbonate hollow fibers 3 1 (Mitsubishi Engineering Plastics Co., Ltd., 1 mass% added carbon black) were passed through all the holes one by one.
  • the resin raw material was poured into the container from the upper part of the container.
  • the resin is 2.5 masses based on the total weight of polyurethane resin adhesive (Nippon Polyurethane Industry Co., Ltd. Nipponran 4276, Coronate 4403). /. Use the one with the power of Bon Black did.
  • the resin was cured by standing at 25 ° C for 1 week. Next, the porous plate and the plate-like material were removed to obtain a hollow fiber bundle.
  • a gel precursor polymerizable solution containing the probe nucleic acid was placed in a desiccator. After reducing the pressure inside the desiccator, one end of the hollow fiber bundle where the fiber bundle was not fixed was immersed in this solution. Nitrogen gas was sealed in the desiccator, and a gel precursor polymerizable solution containing a capillary probe was introduced into the hollow portion of the hollow fiber. Next, the inside of the container was brought to 70 ° C., and a polymerization reaction was carried out over 3 hours.
  • the obtained hollow fiber bundle was sliced in a direction perpendicular to the longitudinal direction of the fiber using a microtome to obtain 50 thin sheet (DNA microarray) having a thickness of 0: 5 mm.
  • probe contl ATACTACGGTAACGGGCCTGT (SEQ ID NO: 7) probe cont2 GGAAGAAACGTCGACCCAAAG (SEQ ID NO: 8) probe cont4 TACGGAAGGTCCGGCATACAA (SEQ ID NO: 9) probe cont5 TGCTCGACAACGATTGGCAAG (SEQ ID NO: 1 0) probe cont6 TTGCGGTACGACCGATCAGTT (SEQ ID NO: 1 1) probe cont7 TCGCTTTGACACGTAATGCGG (SEQ ID NO: 1 2) DNA was synthesized using an automatic DNA synthesizer. '
  • a 300 base pair PCR product was obtained from the yeast YLR377C gene using the following primer designed based on the yeast YLR377C gene sequence.
  • Reverse primer CTACTGTGACTTGCCAATATGGTCTAAAAA (SEQ ID NO: 1 4)
  • the forward primer uses a primer that is phosphorylated at the 5 'end.
  • the resulting PCR product is treated with strandase TM ⁇ Exonuclease (Novagen) to make a single strand, and spiked.
  • Control The name of this control is ssYLR377C 300b.
  • the sequence of ssYLR377C 300b is SEQ ID NO:
  • the probe was synthesized using an automatic DNA synthesizer.
  • Example 2 miRNA microarray analysis
  • the spike control synthesized in Example 1 was mixed as follows. Contl 5 ⁇ 1
  • RNA was eluted with the labeling buffer 251 attached to ULYSIS Alexa Fluor 647 labeling kit (Invitrogen). The obtained RNA was designated as short RNA.
  • RNA was eluted with 25 ⁇ l of labeling buffer attached to ULYSIS Alexa Fluor 647 labeling kit (Invitrogen). The obtained RNA was designated as long RNA.
  • the long RNA was labeled in the same manner as the labeling of the short RNA of (4).
  • the short RNA and long RNA labeled in (4) and (5) were prepared so that the final concentration was 2xSSC, 0.2% SDS, and the amount of solution was 1501. After the preparation, the mixture was heated at 70 ° C. for 2 minutes, centrifuged at lOOOOrpm for 2 minutes, and subjected to hybridization using the miRNA array prepared in Example 1.
  • Hybridization was carried out at 50 ° C for 16 hours under light-shielded conditions.
  • Washing was performed at 2xSSC, 0.2% SDS, 50 ° C for 40 minutes, and then at 2xSSC, 50 ° C for 10 minutes.
  • each probe spot of the microarray after hybridization was imaged and digitized using a cooled CCD camera type fluorescence detector.
  • Figure 2 shows the microarray image after hybridization.
  • the upper panel in Fig. 2 shows the image obtained when labeling and hybridizing long RNA.
  • the lower panel in Figure 2 is an image obtained when hybridizing short RNA.
  • the white solid box indicates the position of the probe (probe contl, 2, 4-7) to detect the short chain spike control
  • the white dashed box indicates The position of the probe (YLR377C 21b-l to 3) for detecting the long-chain spike control is shown.
  • RNA When long RNA is hybridized, a signal is detected in the probe for the long spike control (white broken part), and no signal is detected in the probe for the short spike control (white solid line part). (Upper panel). On the other hand, when short-stranded RNA is hybridized, a signal is detected in the probe for the short spike control (white solid line portion), and a signal is detected in the probe for the long spike control (white dashed line portion). None (lower panel). Therefore, in this example, it can be determined that the fractionation operation of long RNA and short RNA functioned effectively.
  • the signal from the probe for miRNA detection when hybridizing short RNA was sufficiently strong compared to the result of hybridization of long RNA (lower panel in Fig. 2). Compared to the probe signal corresponding to trawl, it was sufficiently strong. Therefore, it is considered that the miRNA detection probe is functioning sufficiently.
  • the miRNA probe spot in which a signal was detected in long-chain RNA (Fig. 2, upper panel) is considered to detect a pre-stressed miRNA.
  • SEQ ID Nos: 1 to 18 synthetic DNA

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Abstract

La présente invention concerne un procédé pour confirmer le succès ou l'échec d'une procédure pour la préparation d'un acide nucléique. Le procédé comprend les étapes consistant à : (a) préparer un acide nucléique à partir d'un échantillon contenant un échantillon témoin qui a la même longueur que celle d'un acide nucléique à préparer ; et (b) causer l'hybridation entre l'acide nucléique et une sonde disposée sur une puce à ADN et détecter la présence d'un signal de l'acide nucléique témoin qui s'est hybridé avec une sonde ayant une séquence complémentaire de l'acide nucléique témoin.
PCT/JP2006/324317 2005-11-29 2006-11-29 Procede pour confirmation de preparation d'acide nucleique WO2007064022A1 (fr)

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BENES V. ET AL.: "Standardization of protocols in cDNA microarray analysis", TRENDS BIOCHEM. SCI., vol. 28, no. 5, 2003, pages 244 - 249, XP003014037 *
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