JP2011239708A - Design method for probe for nucleic acid standard substrate detection, probe for nucleic acid standard substrate detection and nucleic acid detecting system having the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To detect a new nucleic acid standard sample with high level of accuracy.SOLUTION: The nucleic acid standard sample having a specific base sequence to be objected is detected by using a probe for standard substrate detection including oligonucleotide comprising at least continuous 15 bases of one of the specific base sequences.

Description

  The present invention relates to a probe design method for detecting a nucleic acid standard used in a system for detecting and / or quantifying nucleic acid, a nucleic acid standard detection probe designed by the design method, and the nucleic acid standard detection The present invention relates to a nucleic acid detection system provided with a probe.

  In recent years, with the rapid increase in needs for tailor-made medical care, discrimination of genetic polymorphisms related to DNA / RNA such as SNPs and transcriptomes, and nucleic acid quantification techniques for them have been actively introduced in the medical field. Nucleic acid quantification targeting multiple subjects is indispensable for infectious disease diagnosis by quantitative detection of nucleic acids (genes) derived from pathogenic viruses and bacteria, and prognosis after drug administration. In the fields of food, agriculture, forestry and fisheries, and the environment, nucleic acid quantification techniques for microbial quantitative inspection and varietal contamination evaluation using nucleic acids as an index are beginning to be introduced.

  At present, various quantitative nucleic acid evaluation methods such as a quantitative PCR method and a DNA microarray (DNA chip) technique are used for quantification of a nucleic acid having a specific sequence. In quantitative PCR, PCR is performed using primers for detecting a target nucleic acid, and the amount of the target present is quantitatively evaluated. In addition, in order to quantitatively evaluate gene expression levels and the like, DNA microarrays are designed to relatively quantify gene expression levels by arranging a large number of probes that specifically hybridize with each mRNA as a target.

  In these nucleic acid quantification techniques, a nucleic acid standard is used as a reference for determining that there is no problem in the quantification operation itself. When performing relative quantification by DNA microarray, competitive PCR, or the like, a nucleic acid having a base sequence different from the test nucleic acid is added as an internal standard substance, and the measurement value is standardized. Traditionally, some of the naturally occurring gene sequences have been used as the nucleic acid standard used here, but these conventional nucleic acid standards compare the quantitative results in common among various methods, devices, and kits. Did not have enough performance to do.

  In order to solve these problems, a method for designing a DNA comprising a non-natural and non-ordered base sequence has been developed. In this method, DNA sequences that are unlikely to cause mishybridization with each other are efficiently designed. In this method, when a DNA sequence having a predetermined length is represented by a bit string (template) consisting of 0 and 1 for G or C and A or T, these are shifted between templates and between reverse sequences of templates. Select a template in which the Hamming distance between sequences and between these linked sequences is equal to or greater than a predetermined value, and the DNA sequences maintain at least the Hamming distance k from the set of DNA sequences represented by the template. A set of DNA sequences is selected (see Patent Documents 1 and 2).

  Furthermore, the present inventors have developed a method for designing a DNA consisting of a base sequence that has no bias in the base sequence and that does not have a higher order structure by utilizing these principles (Patent Literature). 3). In this method, a plurality of DNAs designed by combining a plurality of DNAs having a certain length and a certain CG content are excluded from those designed to match an existing base sequence registered in a database, and the DNAs are created in combination. . Specifically, using the method of Patent Document 1, a plurality of base sequences having a GC content of 50% and a length of 12 mer are designed, and these sequences are joined together to design a base sequence of about 600 bases. Among the plurality of candidate sequences designed in such a manner, a sequence whose nucleic acid base sequence and translation product sequence do not match 60% or more of the existing nucleic acid / protein database record is selected.

  By the way, as a main problem caused by using a known gene sequence as a nucleic acid standard, for example, the following can be considered.

1) In the case of a nucleic acid standard having a base sequence that exists in nature, there is a risk of hybridization with DNA or RNA that is unexpectedly mixed into the nucleic acid quantification system, which affects the amplification efficiency of PCR and the detection system. Given, the quantitative value may not be accurate.

2) When the nucleotide sequence, GC content, and ease of formation of higher order structures in the nucleic acid standard affect the amplification efficiency such as PCR, and the target sequence in the nucleic acid standard to be used is different There is a risk of differences in quantitative values.

3) Conventionally, different reference materials have been used for each quantification device and quantification method for the purpose of calibration and the like. Since primers and probes are designed for different types of standard substances with different base sequences and physical properties, the efficiency of amplification and hybridization differs for each nucleic acid quantification apparatus and quantification method. For this reason, it is difficult to objectively compare and calibrate quantitative values. Compare the quantitative superiority and inferiority of each method, device, kit, etc., and determine the quantitative values obtained by different methods, devices, kits, etc. It was very difficult to evaluate and compare.

  The technique of Patent Document 3 is a technique developed by the present inventors in order to solve the above problems, and the DNA sequence obtained by the technique is a non-natural sequence, and the above 1) to 3) Part of the problem is a nucleic acid standard that can be avoided. However, since the DNA sequence still contains a plurality of sequences that match 20 or more bases continuously with the naturally occurring sequence, in order to prepare a nucleic acid probe or primer for detection of the DNA sequence, If a partial sequence in a DNA sequence or its complementary sequence is to be used, there is a high possibility of selecting the similar sequence portion, and as a result, there remains a problem that a naturally occurring sequence is simultaneously detected. In addition, the DNA sequence is at most 600 bases long, and when a longer sequence is used as a standard substance at the time of measurement using a DNA myloarray or the like, or in a multiplex nucleic acid quantification system, multiple types of nucleic acid standards having the same performance are used. There was also a problem that it was not possible to cope with the case where substances were required at the same time.

  In order to solve such problems, the present inventors are able to calibrate quantitation values obtained by various nucleic acid quantification devices to objective values with accurate quantitation values of nucleic acids. Therefore, we filed a patent application regarding a new nucleic acid standard that can compare and evaluate the quantification ability between each nucleic acid quantification device, or can objectively evaluate and compare the quantitative values obtained by different nucleic acid quantification devices. (Japanese Patent Application No. 2008-295338).

JP 2004-355294 A WO2003 / 038091 JP 2007-60966 A

  An object of the present invention is to provide a detection probe design method capable of detecting a novel nucleic acid standard sample disclosed in Japanese Patent Application No. 2008-295338 with high accuracy, and detection of a nucleic acid standard substance designed by the design method. Another object of the present invention is to provide a nucleic acid detection system including a probe for nucleic acid and a probe for detecting the nucleic acid standard substance.

  The present invention includes the following.

  (1) A probe for detecting a standard substance comprising an oligonucleotide consisting of at least 15 consecutive base sequences of any one of SEQ ID NOs: 1 to 40.

  (2) The oligonucleotide is composed of at least 15 consecutive base sequences selected from the group consisting of SEQ ID NOs: 2-18, 20-22, 24-36, 38 and 40. Standard substance detection probe.

  (3) The probe for detecting a nucleic acid standard substance according to (1) or (2), wherein the oligonucleotide is composed of 60 bases.

  (4) A nucleic acid detection system comprising the nucleic acid standard substance detection probe according to any one of (1) to (3).

  (5) The nucleic acid detection system according to (4), which is a microarray in which the probe for detecting a nucleic acid standard substance is fixed on a substrate.

(6) identifying a plurality of detection probe candidate sequences having a predetermined base length based on the base sequence of the nucleic acid standard substance to be detected;
Search for off-target of each detection probe candidate sequence specified in the above step, calculate the binding rate between the oligonucleotide having the detection probe candidate sequence and the off-target, and a plurality of detection probe candidate sequences specified in the above step Removing the identified off-target whose calculated coupling ratio exceeds a threshold value from among the following:
A method for designing a probe for detecting a nucleic acid standard substance, comprising calculating a Tm value for each detection probe candidate sequence not excluded in the above step and selecting a nucleic acid standard substance detection probe based on the calculated Tm value .

  (7) The method further comprises a step of calculating a dimer formation ratio and / or a secondary structure formation ratio in each molecule for each detection probe candidate sequence, and the calculated dimer formation ratio and / or secondary structure formation. The method for designing a probe for detecting a nucleic acid standard substance according to (6), further comprising the step of excluding those having a ratio exceeding a predetermined threshold.

  (8) The nucleic acid standard according to (6), wherein the off-target is specified by searching a database storing base sequence information based on the base sequences of the specified detection probe candidate sequences. How to design a probe for detection.

  (9) The binding rate between the oligonucleotide having the detection probe candidate sequence and the off-target is the binding rate at a temperature when the detection probe candidate sequence and the nucleic acid standard substance to be detected are hybridized at a predetermined ratio. The method for designing a probe for detecting a nucleic acid standard according to (6), wherein the probe is calculated.

  (10) In the step of selecting a probe for detecting a nucleic acid standard substance based on the Tm value, the probe for detecting a nucleic acid standard substance is assumed to have a higher rank as the Tm value approaches a predetermined value among a plurality of detection probe candidate sequences. (6) The method for designing a probe for detecting a nucleic acid standard substance according to (6).

  (11) The method further comprises a step of performing a hybridization experiment between the dilution series of the nucleic acid standard substance to be detected and the selected nucleic acid standard substance detection probe, and verifying the detectability of the selected nucleic acid standard substance detection probe. (6) The method for designing a probe for detecting a nucleic acid standard substance according to (6).

  According to the probe for detecting a nucleic acid standard according to the present invention, the quantification ability between the nucleic acid quantification apparatuses can be compared and evaluated, or the quantitative values obtained by different nucleic acid quantification apparatuses can be objectively evaluated and compared. It becomes possible to detect a novel nucleic acid standard with high accuracy.

It is a characteristic view which shows the result of having detected nucleic acid standard substance 500_1 using DNA microarray A. FIG. It is a characteristic view which shows the result of having detected nucleic acid standard substance 500_2 using DNA microarray A. FIG. It is a characteristic view which shows the result of having detected nucleic acid standard substance 500_3 using DNA microarray A. FIG. It is a characteristic view which shows the result of having detected nucleic acid standard substance 500_4 using DNA microarray A. FIG. It is a characteristic view which shows the result of having detected nucleic acid standard substance 500_5 using DNA microarray A. FIG. It is a characteristic view which shows the result of having detected nucleic acid standard substance 1000_1 using DNA microarray A. FIG. It is a characteristic view which shows the result of having detected nucleic acid standard substance 1000_2 using DNA microarray A. FIG. It is a characteristic view which shows the result of having detected nucleic acid standard substance 1000_3 using DNA microarray A. FIG. It is a characteristic view which shows the result of having detected nucleic acid standard substance 1000_4 using DNA microarray A. FIG. It is a characteristic view which shows the result of having detected nucleic acid standard substance 1000_5 using DNA microarray A. FIG. It is a characteristic view which shows the result of having detected nucleic acid standard substance 500_1 using six types of DNA microarrays AF. It is a characteristic view which shows the result of having detected nucleic acid standard substance 500_2 using six types of DNA microarrays AF. It is a characteristic view which shows the result of having detected nucleic acid standard substance 500_3 using six types of DNA microarrays AF. It is a characteristic view which shows the result of having detected nucleic acid standard substance 500_4 using six types of DNA microarrays AF. It is a characteristic view which shows the result of having detected nucleic acid standard substance 500_5 using six types of DNA microarrays AF. It is a characteristic view which shows the result of having detected nucleic acid standard substance 1000_1 using six types of DNA microarrays AF. It is a characteristic view which shows the result of having detected nucleic acid standard substance 1000_2 using six types of DNA microarrays AF. It is a characteristic view which shows the result of having detected nucleic acid standard substance 1000_3 using six types of DNA microarrays AF. It is a characteristic view which shows the result of having detected nucleic acid standard substance 1000_4 using six types of DNA microarrays AF. It is a characteristic view which shows the result of having detected nucleic acid standard substance 1000_5 using six types of DNA microarrays AF.

Hereinafter, the present invention will be described in detail.
1. Definition of Terms In the present invention, “nucleic acid” refers to a nucleotide or polynucleotide in which a plurality of nucleosides are ester-linked in a straight chain via a phosphate group, including a short chain such as an oligonucleotide, in a single-stranded state. In addition, a state in which a double strand is formed with a complementary strand is also included. The type of nucleotide is usually DNA or RNA, but when some nucleotides are modified with a methyl group or the like, and oxygen is substituted with sulfur, artificial nucleic acids such as PNA and LNA are also included. In addition, when the base sequence of the nucleic acid of the present invention is indicated by each sequence number, the base sequence of the single-stranded DNA described is read as the corresponding base sequence. For example, in the case of double-stranded DNA, it is read as a pair with a complementary sequence, and in the case of single-stranded RNA, thymine (T) is read as uracil (U).

  In the present invention, the “probe” includes an oligonucleotide formed by arranging the nucleic acids defined above in a predetermined order, and may be referred to as a nucleic acid probe. The “probe” allows the oligonucleotide to hybridize to a nucleic acid construct having a predetermined sequence by complementary binding of nucleic acids. In other words, the “probe” can supplement the nucleic acid construct by hybridizing to the target nucleic acid construct. For example, when detecting the presence or absence of the target nucleic acid construct, It can be used in measuring the abundance of the nucleic acid construct. Here, the target nucleic acid construct is not particularly limited, and is synthesized from mRNAs expressed in cells such as cells derived from mammals including humans, microorganisms including bacteria and fungi, plant cells, insect cells or the like. CDNA. Examples of the target nucleic acid construct include DNA and RNA extracted from foods and environmental samples, or nucleic acid fragments amplified using these DNA or RNA as a template. Furthermore, the target nucleic acid construct is meant to include a “nucleic acid standard substance” whose details will be described later.

  When the nucleic acid construct to be hybridized is a “nucleic acid standard substance”, the “probe” is particularly referred to as a “nucleic acid standard substance detection probe”. In addition to the nucleic acid standard substance detection probe, the “probe” includes a detection probe for detecting a nucleic acid construct for the purpose described above.

  In addition to the oligonucleotide that hybridizes to the target nucleic acid construct, the “probe” detects hybridization with the linker molecule for fixing one end of the oligonucleotide to the substrate and the target nucleic acid construct. Therefore, it may contain a labeling substance such as a fluorescent substance, a quenching substance, or any other molecule or substance. The “probe” does not contain molecules and substances such as the above-described linker molecules, labeling substances, and quenching substances, and may consist of the above-described oligonucleotides.

  Here, the term “oligonucleotide” means that the number of nucleotides is not limited at all, and includes a molecule composed of any number of nucleotides. Specifically, for example, the oligonucleotide can be designed as a molecule having a length of 15 to 500 bases.

  In particular, the oligonucleotide in the standard substance detection probe is designed to have a predetermined base sequence by a design method described in detail later. Specifically, in the probe for detecting a standard substance according to the present invention, the oligonucleotide consists of at least 15 consecutive base sequences of any one of SEQ ID NOs: 1 to 40. That is, in the present invention, 40 types of probes for detecting a standard substance containing oligonucleotides consisting of at least 15 consecutive base sequences of any one of SEQ ID NOS: 1 to 40 are provided.

  Furthermore, the base length of the oligonucleotide in the probe for detecting a nucleic acid standard substance can be, for example, 15 to 60 bases, preferably 20 to 60 bases, and preferably 40 to 60 bases. More preferably, it is more preferably 50 to 60 bases, and most preferably 60 bases. In addition, the base length of the oligonucleotide in the nucleic acid standard substance detection probe can be designed together with the base length of the detection probe described above, for example.

  On the other hand, in the present invention, the term “nucleic acid standard substance” refers to the following 2. Means a nucleic acid construct having a base sequence without bias in GC content and having a non-natural base sequence that does not have a higher order structure, obtained according to the design method of In particular, in the prior application (Japanese Patent Application No. 2008-295338), as a “nucleic acid standard substance”, a nucleic acid comprising a base sequence of 10,000 bases or more, and a partial sequence of the nucleic acid having a length of 500 bases or 1000 bases A plurality of nucleic acids consisting of the base sequences are designed. In the present invention, as the nucleic acid standard, in particular, a nucleic acid standard consisting of a base sequence of 500 bases shown in any of SEQ ID NOs: 41 to 45, and a base sequence of 1000 bases shown in any of SEQ ID NOs: 46 to 50 The nucleic acid standard substance which consists of can be mentioned.

  In addition, the “nucleic acid standard substance” may be one that has been modified to immobilize the above-described nucleic acid construct on the surface of a substrate, a bead or the like, or a tag for labeling. May be. In particular, the “nucleic acid standard substance” consisting of the base sequence shown in any of SEQ ID NOs: 41 to 50 is a combination of oligonucleotides having a constant GC content, and the entire nucleic acid base sequence as well as a partial region thereof. Is designed to be different from the base sequence of a naturally occurring nucleic acid so that the GC content remains constant and does not form a higher order structure. Therefore, when this nucleic acid standard is used, PCR amplification or the like is not affected by variations in GC content in the sequence or the presence or absence of higher-order structures, and the bases differ from those of natural nucleic acids. Since it has a sequence, even if DNA other than the target existing in nature is unexpectedly mixed in the quantification system of the calibration experiment, it has little influence on PCR amplification and the like. Due to this, when this nucleic acid standard is used, the target nucleic acid construct can be detected and quantified with high accuracy. Furthermore, this nucleic acid standard does not have 20 or more consecutive bases that match the base sequence of a naturally occurring nucleic acid. For this reason, the above-described probe for detecting a nucleic acid standard substance is prevented from simultaneously hybridizing a naturally occurring sequence, even if it hybridizes to any part of these nucleic acid standard substances.

2. Method for designing “nucleic acid standard” In order to prepare the nucleic acid standard defined in (1), a base sequence is designed according to the procedures of the following steps (1) to (6), and DNA synthesis or RNA synthesis is performed according to the obtained base sequence.

(1) A step of randomly generating a plurality of types of oligonucleotides having a length of 36 bases, the GC content of which is set to a constant value in the range of 30 to 70%,
(2) Among the oligonucleotides, a step of excluding oligonucleotides corresponding to the following (a) to (c):
(A) a sequence showing similarity to an existing base sequence registered in a public base sequence database (a sequence that matches a known sequence of 18 bases or more out of 36 bases),
(B) the nucleotide sequences of the oligonucleotides are compared with each other, and a sequence that matches 18 or more bases with other sequences,
(C) a sequence in which four or more identical bases are continuous,
(3) A step of joining a plurality of oligonucleotides remaining in the step (2) in an arbitrary order to obtain a polynucleotide having a length of several thousand to several tens of thousands of bases;
(4) searching for a sequence corresponding to the following (d) to (g) in the base sequence of the polynucleotide obtained in the step (3), and removing the corresponding region;
(D) a repetitive sequence having a matched sequence of 15 bases or more out of 20 bases,
(E) A sequence that may form a double-stranded structure (secondary structure) in the sequence, that is, a sequence that binds 15 bases or more out of 20 bases (complementary sequence), and a possibility that the bases bind continuously for 10 bases or more. With a sequence (complementary sequence),
(F) A sequence showing similarity to an existing base sequence registered in the public base sequence database, that is, a sequence that matches a known sequence of 50 bases or more out of 100 bases, and a sequence that matches 20 or more bases in succession with another sequence Array,
(G) a sequence in which four or more bases are consecutive,
(5) A step of confirming that the base sequence and the corresponding amino acid sequence of the polynucleotide obtained in the step (4) do not match 50% or more of the existing database record.
(6) A step of cutting the obtained long-chain sequence into an arbitrary length as necessary.

  That is, such a nucleic acid standard design method is similar to an existing base sequence registered in a database from a large number of DNAs designed by combining a plurality of DNAs having a certain length and a certain CG content. After deleting them and combining them, a sequence similar to a known base sequence existing in nature is further deleted from the binding sequence, and the sequence of a certain length selected does not have a higher-order structure. That is, the corresponding sequence that is supposed to promote the formation of the higher order structure is deleted, and it is further confirmed that there is no repetitive sequence in the nucleic acid sequence.

  In addition, uracil in the RNA sequence is read as thymine when compared with the base sequence in the existing database, such as the above steps (a) and (f).

  The designed nucleic acid standard substance can be suitably used as a standard DNA sample used in a DNA quantification apparatus and a standard RNA sample used in an RNA quantification apparatus. By using a standard RNA sample between different RNA quantification devices and comparing them, performance comparison such as sensitivity and accuracy can be performed, and calibration of each quantification device is also possible.

  Here, examples of the DNA quantification apparatus include a quantitative PCR apparatus and a DNA microarray apparatus. In addition, examples of the RNA quantification apparatus include a quantitative PCR apparatus and a DNA microarray apparatus that also target RNA molecules.

  In order to prepare a standard DNA sample and a standard RNA sample, it can be used as a solution in which a predetermined amount of a nucleic acid standard substance is dissolved in sterile water or a buffer solution. The sample can also be used by mixing a plurality of types of nucleic acid standards.

  In these nucleic acid quantification techniques, the above-described nucleic acid standard substance can be used as a reference for determining that there is no problem in the quantification operation itself. In addition, when the relative quantification of nucleic acids is performed by DNA microarray, quantitative PCR, or the like, the above-described nucleic acid standard substance can be added as an internal standard substance to standardize the measurement value.

  Furthermore, in the DNA microarray apparatus, when the above-mentioned nucleic acid standard substance is immobilized on the same or different substrate or bead surface as the target test nucleic acid sample in a single strand state, various analysis of the test nucleic acid sample is performed. As a control sample. Even if the nucleic acid corresponding to the base sequence at any position of the full length is selected, there is almost no possibility of detecting a naturally occurring sequence even if it is immobilized at a high density on the substrate surface. It can also be used for background correction with a chip immobilized on a substrate or for quality control of a DNA microarray.

3. Method for designing probe for detecting nucleic acid standard substance In order to prepare the probe for detecting a nucleic acid standard substance defined in 1), a base sequence is designed according to the following steps 1 to 3, and DNA synthesis or RNA synthesis is performed according to the obtained base sequence.

<Step 1>
Based on the base sequence of the nucleic acid standard to be detected, a plurality of probe candidate sequences are designed so as to cover the entire nucleic acid standard. Specifically, for example, first, the base length of the probe candidate sequence to be designed is fixed to a predetermined length. Next, the predetermined length is assigned to the base sequence of the nucleic acid standard substance so as to include one end thereof to be the first probe candidate sequence, and then the predetermined sequence is shifted by one base to the other end. Assign the length of to other probe candidate sequences. The predetermined length is assigned so as to include the other end of the base sequence of the nucleic acid standard substance, and this is used as the last probe candidate sequence. That is, when the base sequence of the nucleic acid standard substance to be detected is X base length and the probe candidate sequence to be designed is n base length, (X−n + 1) probe candidate sequences are designed.

  At this time, the nucleic acid standard substance to be detected may be divided into a plurality, and a plurality of probe candidate sequences may be designed according to the above-described method for each divided region. For example, the nucleic acid standard substance to be detected can be divided into a 3 ′ terminal region and a 5 ′ terminal region, and probe candidate sequences can be designed for each of the 3 ′ terminal region and the 5 ′ terminal region. .

<Step 2>
In step 2, the plurality of probe candidate sequences designed in step 1 are narrowed down based on the cross-hybridization rate with the off target.

  In Step 2, first, for each probe candidate sequence, an off-target is searched from a known database by applying a homology search program. Here, the homology search program is not particularly limited, but known programs such as SSEARCH, FASTA, BLAST, and PSI-BLAST can be applied. Further, publicly known databases are not particularly limited, but databases such as Genbank (NCBI), DDBJ (Japan DNA Data Bank), EMBL (EBI), Unigene and TIGR Gene Indeces that store nucleic acid sequences may be used. it can.

  More specifically, the database is searched using the base sequence of the probe candidate sequence as a query sequence. As a result of the search, an entry including a base sequence having a region having a high homology value with respect to the base sequence of the probe candidate sequence is specified. Depending on the database used for the search, the entry may include mRNAs actually expressed in cells, genes based on genomic analysis, information on splicing variants, RNA information such as snRNA, miRNA, rRNA, Mt_tRNA, etc. Contains sequence information.

  As an example, 100 types of entries can be specified as the off target in descending order of homology. In the following processing, as the information regarding the off target, only the base sequence included in the specified entry may be used. Therefore, the base sequence included in the specified entry may be acquired.

Next, a temperature (denoted as T ₉₉ ) at which the ratio (binding rate) at which each probe candidate sequence hybridizes to the nucleic acid standard substance to be detected reaches a predetermined value (for example, 99%) is calculated. At this time, as an example, the following formula disclosed in Nucleic Acid Res. 2006, 34, W665-W669 can be used.

当該式において左辺fは結合率である。また、当該式において、ΔG_Tは温度Tにおける結合自由エネルギー変化であり、C_Pはプローブ候補配列の濃度であり、Rは気体定数であり、Tは絶対温度である。

In the formula, the left side f is a coupling rate. Further, in the formula, .DELTA.G _T is the binding free energy change at a temperature T, C _P is the concentration of probe candidate sequences, R is the gas constant, T is the absolute temperature.

When calculating the temperature described above, the C _P can be assigned, for example, 0.5 [mu] M, the f can be calculated value of T as 99. Incidentally, the above formula, .DELTA.G _T is, Nearest Neighbor method for nucleotide sequence of the nucleic acid calibrator to be detected (PNAS, 1998, 95: 1460-1465 ) can be calculated based on. In addition, hybrid-min can be used as software to calculate ΔG by Nearest Neighbor method (Bioinformatics, Volume II. Structure, Functions and Applications (Method in Molecular Biology) 2008, chapter 1, pages 3-31) .

By this process, the temperature (T ₉₉ ) at which each probe candidate sequence identified in Step 1 above and the nucleic acid standard substance hybridize with a binding rate of 99%, for example, can be calculated.

Next, in the step 2, the specified off-target as described above, the binding rate at T ₉₉ calculated as described above is calculated according to the above equation. Even at this stage, ΔG _T can be calculated based on the Nearest Neighbor method (PNAS, 1998, 95: 1460-1465) for the off-target base sequence. As described above, all of the probes candidate sequence designed in step 1, to calculate the binding rate and off-target in the T _99.

  Next, in this step 2, from among the plurality of probe candidate sequences designed in the above step 1, those for which an off target whose binding rate exceeds a threshold value (for example, 0.1%) is specified. That is, according to this step 2, the plurality of probe candidate sequences designed in the above step 1 can be narrowed down to only probe candidate sequences that generate cross-hybridization with an off target with a very low probability.

<Step 3>
In Step 3, Tm values are calculated for the probe candidate sequences narrowed down in Step 2. The Tm value is a temperature at which the coupling rate becomes 50%. Therefore, it can be calculated from the above formula (T is determined with f = 0.5). At this time, the speed can be increased by using the binary search algorithm. Further, the method of calculating the Tm value for the probe candidate sequences narrowed down in step 2 is not limited to such a method, and a conventionally known method may be adopted.

  In this step 3, Tm values are calculated for each of the plurality of probe candidate sequences narrowed down in step 2, and therefore, the plurality of probe candidate sequences are sorted in the order close to the desired Tm value (for example, Tm = 80 ° C.). Can do. That is, in this step 3, among the plurality of probe candidate sequences narrowed down in step 2, the Tm value can be ranked higher as it is closer to a predetermined value.

  For example, when designing a probe for detecting a nucleic acid standard substance for each of a plurality of nucleic acid standard substances, the probe for nucleic acid standard substance detection can hybridize well under the same temperature and salt concentration. It is preferable to approximate. According to this step 3, for each of a plurality of nucleic acid standards, probe candidate sequences having Tm values close to each other can be specified. In addition, as a desired Tm value, it can set suitably in the range of 60 to 90 degreeC, for example.

  In Step 3, the dimer formation ratio may be calculated for the probe candidate sequences narrowed down in Step 2 by a known program such as Hybrid-min. In step 3, the formation ratio of secondary structure in the molecule may be calculated for the probe candidate sequences selected in step 2 by a known program such as Hybrid-ss. Thresholds may be set in advance for the dimer formation ratio and secondary structure formation ratio, and those exceeding the threshold may be excluded from the probe candidate sequences.

<Step 4>
In this step 4, the probe candidate sequences narrowed down to the above step 3 are actually synthesized as oligonucleotides and hybridized with a separately synthesized nucleic acid standard, and the performance as a probe is evaluated.

  At this time, as a hybridization experiment, it is preferable to perform an experiment using various platforms related to the DNA microarray. Examples of DNA microarrays include DNA microarray technology provided by Agilent Technologies, Inc., Toray Industries, Inc., Mitsubishi Rayon Co., Ltd., DNA Chip Laboratories, Inc., Roche Nimblegen Co., Ltd., and Kurashiki Spinning Co., Ltd. (Kurabo). Can be used. In addition, about the DNA microarray of each company, a protocol can be obtained from each company, and those skilled in the art can implement suitably based on these protocols.

  Specifically, as a hybridization experiment, a sample solution (including a gene serving as a positive control) in which a nucleic acid standard substance to be detected is diluted stepwise is prepared, and an oligonucleotide consisting of a candidate probe sequence to be evaluated. A DNA microarray in which is fixed is used. The sample solution may contain a nucleic acid fragment amplified by PCR or RT-PCR using a synthesized nucleic acid standard as a template, or may contain a synthesized nucleic acid standard as it is. Moreover, as a label | marker at the time of detecting hybridization, it may differ with various platforms, and you may use the same label | marker.

  As a result of the hybridization experiment, the signal value derived from the oligonucleotide composed of the probe candidate sequence to be evaluated is normalized, and it is verified whether the normalized signal value changes depending on the concentration of the nucleic acid standard. The normalization method is not particularly limited, and examples thereof include a process of subtracting the background value from the raw signal value in the same DNA microarray and dividing the result by the median value of a plurality of types of positive control-derived signals. The background value is a signal when a region where the oligonucleotide is not fixed is measured. Further, the positive control is not particularly limited, and examples thereof include 10 types of genes such as ACTB gene, B2M gene, GAPDH gene, GUSB gene, HPRT1 gene, PGK1 gene, PPIA gene, RPLP0 gene, TBP gene, and YWHAZ gene. be able to.

  In this step 4, it is possible to select a plurality of probe candidate sequences further narrowed down in step 3 that can generate a signal depending on the concentration of the nucleic acid standard based on the result of the hybridization experiment. it can. In particular, in this Step 4, it is preferable to select a probe candidate sequence capable of generating a signal depending on the concentration of the nucleic acid standard substance in all of the plurality of platforms related to the DNA microarray.

  Specifically, a nucleic acid substance detection probe was designed for the nucleic acid standard substance consisting of the base sequence shown in any of SEQ ID NOs: 41 to 50 by executing the above steps 1 to 4. Specifically, these 10 kinds of nucleic acid standards were divided into 5 ′ terminal region and 3 ′ terminal region, respectively, and two probes for detecting nucleic acid standard materials were designed for each of the 5 ′ terminal region and the 3 ′ terminal region. In addition, for the nucleic acid standard substance having a length of 500 bases shown in SEQ ID NOs: 41 to 45, the 5 ′ end region from the 5 ′ end to the 300 th end was the 5 ′ end region, and the 301 th to 3 ′ end was the 3 ′ end region. In Step 2, SSARCH was used as a homology search program with default settings, and an Ensemble (rel.49) human cDNA sequence database was used as the database.

  A list of designed probes for detecting nucleic acid standards is shown in the table below.

なお、上記表において、配列番号４１〜５０に示した塩基配列からなる１０種類の核酸標準物質は、それぞれ順に「５００＿１」、「５００＿２」、「５００＿３」、「５００＿４」、「５００＿５」、「１０００＿１」、「１０００＿２」、「１０００＿３」、「１０００＿４」及び「１０００＿５」と表記した。表１に示すように、配列番号４１〜５０に示した塩基配列からなる１０種類の核酸標準物質について、それぞれ４種類の核酸標準物質検出用プローブ（合計４０種類）を設計することができた。

In the above table, 10 types of nucleic acid standard substances consisting of the base sequences shown in SEQ ID NOS: 41 to 50 are respectively “500_1”, “500_2”, “500_3”, “500_4”, “500_5”, “1000_1”. ”,“ 1000_2 ”,“ 1000_3 ”,“ 1000_4 ”, and“ 1000_5 ”. As shown in Table 1, four types of nucleic acid standard substance detection probes (total of 40 types) could be designed for each of the ten types of nucleic acid standard substances consisting of the base sequences shown in SEQ ID NOs: 41 to 50.

4). Use of probe for detecting nucleic acid standard substance 3. The probe for detecting a nucleic acid standard substance designed in (1) can be widely used in nucleic acid detection systems and nucleic acid quantification systems based on hybridization between probes such as DNA microarrays and DNA bead arrays and nucleic acids to be detected. When used in a DNA microarray, the probe for detecting a nucleic acid standard substance is fixed at a predetermined position on the surface of the substrate. When used in a DNA bead array, a probe for detecting a nucleic acid standard substance is immobilized on a predetermined bead surface.

  In such a system, one type of nucleic acid standard substance detection probe may be used, but a plurality of types of nucleic acid standard substance detection probes may be used. All or part of the 40 types of nucleic acid standard substance detection probes (SEQ ID NOs: 1 to 40) designed as described above can be used. In particular, the above-described 40 types of nucleic acid standard substance detection probes are excellent in detection sensitivity on various platforms, and it has been demonstrated that nucleic acid standard substances can be quantified with high accuracy. It is.

  Among these, probe names displayed in the oligo_id column in Table 1 are excluded: 500_1_1 (SEQ ID NO: 1), 500_5_3 (SEQ ID NO: 19), 1000_1_3 (SEQ ID NO: 23), 1000_5_1 (SEQ ID NO: 37), and 1000_5_3 (SEQ ID NO: 39). It has been demonstrated that other nucleic acid standard substance detection probes can quantitate nucleic acid standard substances with particularly high sensitivity (see Examples described later). Therefore, except for the probe names displayed in the oligo_id column in Table 1, 500_1_1 (SEQ ID NO: 1), 500_5_3 (SEQ ID NO: 19), 1000_1_3 (SEQ ID NO: 23), 1000_5_1 (SEQ ID NO: 37) and 1000_5_3 (SEQ ID NO: 39) It is more preferable to use a probe for detecting a nucleic acid standard.

  The probe for detecting a nucleic acid standard substance according to the present invention can be detected with high sensitivity regardless of whether the nucleic acid to be detected is DNA or RNA. If the nucleic acid to be detected is a DNA microarray, a nucleic acid standard detection probe may be prepared as a DNA molecule. If the nucleic acid to be detected is RNA, a nucleic acid standard detection probe is prepared as an RNA molecule. do it.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, the technical scope of this invention is not limited to these Examples.

Example 1
DNA microarray In this example, DNA microarrays of six companies, Agilent Technology Co., Ltd., Toray Co., Ltd., Mitsubishi Rayon Co., Ltd., DNA Chip Laboratories Co., Ltd., Roche Nimblegen Co., Ltd., and Kurashiki Spin Co., Ltd. (Kurabo Co., Ltd.) Was used for gene expression analysis according to the standard protocol of each company. For these 6 companies, DNA microarrays comprising 40 nucleic acid standard substance detection probes having 40 types of oligonucleotides having the base sequences shown in SEQ ID NOs: 1 to 40 were produced according to the specifications specific to each company. Hereinafter, in the description of this example, the six types of DNA microarrays with different specifications manufactured by the above six companies are denoted as A to F in random order.

  Each DNA microarray was provided with a probe that specifically hybridizes to 10 types of genes as a positive control for utilizing signal normalization. The 10 types of positive control genes were ACTB gene, B2M gene, GAPDH gene, GUSB gene, HPRT1 gene, PGK1 gene, PPIA gene, RPLP0 gene, TBP gene and YWHAZ gene.

Preparation of Sample In order to evaluate the detectability of the above 40 types of nucleic acid standard substance detection probes, a dilution series of 10 types of nucleic acid standard substances consisting of the base sequences shown in SEQ ID NOs: 41 to 50 was prepared as samples. In addition, 10 types of nucleic acid standard substances consisting of the base sequences shown in SEQ ID NOS: 41 to 50 are respectively “500_1”, “500_2”, “500_3”, “500_4”, “500_5”, “1000_1”, “1000_2”. ”,“ 1000 — 3 ”,“ 1000 — 4 ”, and“ 1000 — 5 ”.

  First, six types of mixed liquids having the compositions shown in Table 2 were prepared.

  For dilution, Human Reference RNA (50 ng / uL) was used to prevent adsorption to the inner surface of the tube. Human Reference RNA (50 ng / uL) was prepared from commercially available HURR (pool) (1241.78 ng / uL) (24.2 uL HURR mixed with 575.8 uL D.W.).

  Next, a standard amount of 6 types (S-1, S-2, S-3, S-4, S-5, S-6) is obtained by separating and mixing a predetermined amount of these 6 types of liquid mixture. A substance stock solution was prepared. Table 3 shows the concentrations of 10 kinds of nucleic acid standard substances contained in the standard substance stock solution.

  Next, 22.55 ul (28 ug) of HURR (pool) was used against the obtained 6 standard stock solutions (S-1, S-2, S-3, S-4, S-5, S-6). ) (1241.78 ng / uL) was added, and the concentration was adjusted to use 6.56 ul (1 microgram) per reaction to prepare 6 types of samples.

DNA Microarray Measurement Using the samples prepared as described above, signals in 40 types of nucleic acid standard substance detection probes were measured according to the protocols of each company. The measured signals were normalized and compared by the following method. That is, the signal value in the nucleic acid standard substance detection probe was normalized by subtracting the background value from the signal value in the nucleic acid standard substance detection probe and dividing it by the median value of the signal value in the 10 positive controls.

result
The result of detecting the nucleic acid standard substance 500_1 using the DNA microarray A is shown in FIG. FIG. 1 is a graph showing the detectability of four types of nucleic acid standard substance detection probes 500_1_1, 500_1_2, 500_1_3, and 500_1_4 prepared for the nucleic acid standard substance 500_1. In FIG. 1, the horizontal axis represents the logarithm of the concentration of the nucleic acid standard substance, and the vertical axis represents the logarithm of the median value of the signal value after normalization.

  Similarly, the detection results of the nucleic acid standard substances “500_2”, “500 — 3”, “500 — 4”, “500 — 5”, “1000 — 1”, “1000 — 2”, “1000 — 3”, “1000 — 4” and “1000 — 5” are shown in FIGS. It was shown to.

  Moreover, the result of having detected nucleic acid standard substance 500_1 using six types of DNA microarrays AF is shown in FIG. FIG. 11 is a graph showing the detectability of four types of nucleic acid standard substance detection probes 500_1_1, 500_1_2, 500_1_3, and 500_1_4 prepared for the nucleic acid standard substance 500_1 in all six types of DNA microarrays A to F. In FIG. 11, the horizontal axis represents the logarithm of the concentration of the nucleic acid standard substance, and the vertical axis represents the logarithm of the median value of the signal value after normalization.

  Similarly, the detection results of the nucleic acid standard substances “500_2”, “500 — 3”, “500 — 4”, “500 — 5”, “1000 — 1”, “1000 — 2”, “1000 — 3”, “1000 — 4” and “1000 — 5” are shown in FIGS. It was shown to.

  As shown in FIGS. 1-20, it became clear that 40 types of probes for nucleic acid standard substance detection prepared in the present Example can detect the nucleic acid standard substance to be detected with high accuracy. Moreover, as shown in FIGS. 1-20, the signal value resulting from these 40 types of nucleic acid standard substance detection probes and the density | concentration of a nucleic acid standard substance show the linear relationship. Therefore, by using these 40 types of nucleic acid standard substance detection probes and a dilution series of nucleic acid standard substances, for example, when quantifying mRNA expressed in cells, the nucleic acid to be detected contained in the sample is greatly reduced. Can be accurately quantified.

Claims (11)

  A probe for detecting a standard substance comprising an oligonucleotide consisting of at least 15 consecutive base sequences of any one of SEQ ID NOs: 1 to 40.   The nucleic acid standard according to claim 1, wherein the oligonucleotide comprises at least 15 consecutive bases of a base sequence selected from the group consisting of SEQ ID NOs: 2-18, 20-22, 24-36, 38 and 40. Probe for substance detection.   The probe for detecting a nucleic acid standard substance according to claim 1 or 2, wherein the oligonucleotide is composed of 60 bases.   A nucleic acid detection system comprising the probe for detecting a nucleic acid standard substance according to any one of claims 1 to 3.   5. The nucleic acid detection system according to claim 4, which is a microarray in which the probe for detecting a nucleic acid standard substance is fixed on a substrate. Identifying a plurality of detection probe candidate sequences having a predetermined base length based on the base sequence of the nucleic acid standard substance to be detected;
Search for off-target of each detection probe candidate sequence specified in the above step, calculate the binding rate between the oligonucleotide having the detection probe candidate sequence and the off-target, and a plurality of detection probe candidate sequences specified in the above step Removing the identified off-target whose calculated coupling ratio exceeds a threshold value from among the following:
A method for designing a probe for detecting a nucleic acid standard substance, comprising calculating a Tm value for each detection probe candidate sequence not excluded in the above step and selecting a nucleic acid standard substance detection probe based on the calculated Tm value .   For each of the detection probe candidate sequences, the method further comprises a step of calculating a dimer formation ratio and / or a secondary structure formation ratio in the molecule, and the calculated dimer formation ratio and / or secondary structure formation ratio is predetermined. 7. The method for designing a probe for detecting a nucleic acid standard substance according to claim 6, further comprising the step of excluding those exceeding the threshold.   7. The probe for detecting a nucleic acid standard substance according to claim 6, wherein the off-target is specified by searching a database storing base sequence information based on the base sequences of the specified detection probe candidate sequences. Design method.   The binding rate between the oligonucleotide having the detection probe candidate sequence and the off-target is calculated as the binding rate at a temperature when the detection probe candidate sequence and the nucleic acid standard substance to be detected are hybridized at a predetermined ratio. The method for designing a probe for detecting a nucleic acid standard substance according to claim 6.   In the step of selecting a nucleic acid standard substance detection probe based on the Tm value, the nucleic acid standard substance detection probe is selected as a higher rank as the Tm value is closer to a predetermined value among a plurality of detection probe candidate sequences. The method for designing a probe for detecting a nucleic acid standard substance according to claim 6.   The method further comprises the step of performing a hybridization experiment between the dilution series of the nucleic acid standard substance to be detected and the selected nucleic acid standard substance detection probe, and verifying the detectability of the selected nucleic acid standard substance detection probe. Item 7. A method for designing a probe for detecting a nucleic acid standard according to Item 6.

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