JP2004065125A - Method for determining number of iterations for target nucleic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for determining the number of iterations of repeating sequences included in a nucleic acid. <P>SOLUTION: The method for determining the number of iterations for the target nucleic acid, a method for determining the number of iterations for the target nucleic acid including a target sequence x1-(y)<SB>n</SB>-x2, comprises (1) extending the nucleic acid under the conditions of properly affording extension reaction using a primer X1-(Y)<SB>m</SB>-X2( wherein, m is an integer of ≥1, being equal to the number of iterations n predicted in advance ) including a repeating sequence comprising a sequence X1 complementary to a sequence x1 and a sequence Y complementary to a fundamental unit y in any number of iterations m and a sequence X2 complementary to a sequence x2 and (2) judging the presence/absence of the extension in the reaction product. <P>COPYRIGHT: (C)2004,JPO

Description

【図面の簡単な説明】
【図１】本発明の第１の態様に従う反復数決定方法を示す図。
【図２】本発明の第２の態様に従う反復数決定方法を示す図。
【図３】本発明の第３の態様に従う反復配列数決定方法により得られる反応産物を示す図。
【図４】本発明の態様において使用され得る核酸プローブ固定化基体の第１の例を示す図。
【図５】本発明の態様において使用され得る核酸プローブ固定化基体の第２の例を示す図。
【図６】第１の実施例の結果を示す電気泳動用ゲルの写真。
【図７】実施例２に係る反復配列数決定方法に使用した核酸プローブ固定化基体の平面図。
【図８】実施例２の結果を示すグラフ。
【図９】実施例３に係る反復配列数決定方法に使用した核酸プローブ固定化基体の平面図。
【図１０】実施例３の結果を示すグラフ。
【符号の説明】
１．ターゲット核酸　　２．反復配列　　３．ターゲット配列　　４．プライマー　　５．反応産物　　６．タグ　　７．プライマー　　８．ターゲット核酸　９．プライマー　　１１．核酸プローブ固定化基体　　１２．基体　　１３．電極　　１４．核酸プローブ　　１５．パット　　１６．核酸プローブ固定化基体　　１７．基体　　１８．核酸プローブ　　２０．核酸プローブ固定化基体　２１．基体　　２２．電極　　２３．パット　　３０．核酸プローブ固定化基体　　３１．基体　　３２．核酸プローブ固定化領域[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for determining the number of repeats of a repetitive sequence present in a nucleic acid.
[0002]
[Prior art]
Many repetitive sequences have been found in the genome. These are often used as genetic markers in gene analysis and the like. As a method of determining the number of repetitions of a repetitive sequence, electrophoresis has been widely used so far (Japanese Patent Application Laid-Open No. 8-510910). However, the conventional method using electrophoresis requires cleavage with a restriction enzyme and other complicated operations, and takes a considerable amount of time to determine. Therefore, a new method with excellent operability is required.
[0003]
As an example of means for solving the conventional problem, there is a method utilizing analysis on a solid substrate. For example, in such a method, a plurality of fluorescently labeled probes (secondary probes) corresponding to the number of repetitions are prepared, and a sandwich hybridization reaction and a ligation reaction are performed using the probes. In such a method, a fluorescent signal is detected in a corresponding portion only when a secondary probe corresponding to the number of repeated sequences is added. Since such a method does not require the use of restriction enzymes or electrophoresis, there is a merit that the operability is improved, but the specificity of the ligation reaction is not necessarily high, and the reaction efficiency is reduced on a solid phase. . Under such circumstances, it is said that there is a problem in detection accuracy.
[0004]
[Problems to be solved by the invention]
In view of the circumstances described above, an object of the present invention is to provide a method for determining the number of repetitive sequences contained in a nucleic acid.
[0005]
[Means for Solving the Problems]
Means for solving the above-mentioned problems include:
A target sequence x1- (y) consisting of a repetitive sequence containing a basic unit y with an arbitrary number of repetitions n, a sequence x1 on the 3 'side thereof, and a sequence x2 on the 5' side of the repetitive sequence _n A method for determining the number of repetitions of a target nucleic acid comprising -x2,
(1) A primer X1- (Y containing a sequence X1 complementary to the sequence x1, a repetitive sequence containing the sequence Y complementary to the basic unit y at an arbitrary repetition number m, and a sequence X2 complementary to the sequence x2. ) _m Using -X2 (where m is an integer greater than or equal to 1 and an integer equal to the number n of repeats predicted in advance) to elongate the nucleic acid under conditions that allow an appropriate elongation reaction;
(2) determining the number of repetitions of the repetitive sequence of the target nucleic acid by determining the presence or absence of extension in the reaction product obtained by the extension of (1);
This is a method for determining the number of repetitions of a target nucleic acid comprising:
[0006]
BEST MODE FOR CARRYING OUT THE INVENTION
The method for determining the number of repetitions according to an embodiment of the present invention is a method for determining the number of repetitions of a basic unit constituting a repetitive sequence included in a target sequence.
[0007]
1. Explanation of terms
As used herein, the term "repeated sequence" refers to a sequence in which two or more identical or extremely similar nucleotide sequences are repeated on a nucleic acid (eg, genome), and is generally referred to as a "repeated sequence." It is also called an array. Elements repeated in the repetitive sequence are referred to as "basic units", and the base sequence constituting the basic unit is referred to as a unit sequence. The number of repetitions of the basic unit is referred to as “repetition number”.
[0008]
The term “nucleic acid” as used herein includes RNA and DNA, peptide nucleic acids (ie, PNA), nucleic acid analogs such as methylphosphonate nucleic acids and S-oligos, cDNA and cRNA, and oligonucleotides and polynucleotides, and the like. Is a term generally indicating a substance represented by a base sequence. Such a nucleic acid may be a naturally occurring one or an artificially synthesized one.
[0009]
The repeat sequence whose number of repeats is determined by the method according to the embodiment of the present invention is not particularly limited, and its unit sequence consists of a sequence of about 2 to about 15 bases, preferably about 2 to about 10 bases. It may be a repetitive sequence. The number of repetitions is not particularly limited, but is preferably about 2 to about 30.
[0010]
For example, a repetitive sequence whose number of repeats is determined by a method according to an embodiment of the present invention includes a microsatellite sequence (generally also referred to as microsatellite DNA), a minisatellite sequence, a short tandem repeat (STR), and a tandem repeat. It may be a repetitive sequence such as a sequence, tandem repeat and inverted repeat.
[0011]
In particular, for example, sequences showing genetic polymorphisms represented by, for example, a CA repeat sequence containing C and A as a unit sequence and a GT repeat sequence containing G and T as a unit sequence, and CAG which is a causative gene of Huntington's disease Determining the number of repeats in a microsatellite sequence, such as a polymorphism associated with a disease, such as a three-base sequence, is useful in clinical and basic medicine, as well as in biochemistry. Here, according to general notation, each base is described as "C" for cytosine, "T" for thymine, "A" for adenine, and "G" for guanine.
[0012]
The method according to the invention may be performed on a sample nucleic acid comprising a target nucleic acid. As used herein, the term "sample nucleic acid" refers to a sample containing a target nucleic acid that is the subject of a method according to an embodiment of the present invention. The sample nucleic acid is known per se from a sample collected from an individual, for example, a sample containing nucleic acids such as blood such as peripheral venous blood, leukocytes, serum, urine, feces, semen, saliva, cultured cells, organs and tissues. May be prepared by the method described in The term "individual" as used herein may include humans, non-human animals and plants, and microorganisms such as viruses, bacteria, bacteria, yeasts and mycoplasmas. The method according to the present invention may be performed on a sample containing an artificially synthesized nucleic acid. The sample may be subjected to any necessary pretreatment, such as homogenate and extraction, if necessary. Such a pretreatment could be selected by a person skilled in the art depending on the sample.
[0013]
As used herein, the terms "complementary", "complementary", and "complementary" may be in the range of 50% to 100%, preferably 100% complementary. As used herein, the terms "homology", "homologous" and "homology" may be 50% to 100% homologous, preferably 100% homologous.
[0014]
2. How to determine the number of iterations
(1) First aspect
First, the basic concept of the method for determining the number of repetitions according to the present invention will be described using the first embodiment. The first embodiment will be described with reference to FIG.
[0015]
The target nucleic acid 1 is a nucleic acid containing a repetitive sequence 2 and is a target nucleic acid whose repeat number n is to be determined. Here, one basic unit is indicated by “y”. The sequences at sites adjacent to both ends of the repetitive sequence 2 consisting of y are x1 and x2, respectively. The site where the repetitive sequence 2, the sequence x1, and the sequence x2 are combined is the target sequence 3 to which a specific primer binds. The target sequence 3 contained in the target nucleic acid 1 according to the present invention can be represented by the general formula “x1- (y) _n −x2 ”. Here, “n” is an integer of 1 or more. For example, the target sequence 3 used in this embodiment, which is one aspect of the present invention, has "x1- (y)" as shown in FIG. ₄ -X2 ".
[0016]
In order to determine the number of repetitions n contained in such a target nucleic acid 1, a sequence comprising a complementary basic unit Y complementary to the basic unit y at an arbitrary number of repetitions and complementary to x1 so as to sandwich the same. A primer 4 containing X1 and a sequence X2 complementary to x2 is used. When the primer 4 is represented by a general formula, “X1- (Y) _m -X2 ". Here, m is an integer of 1 or more, and there may be a case where m = n and a case where m ≠ n.
[0017]
Here, "y" (here, y is lowercase) contained in the target sequence and "Y" (here, Y is uppercase) contained in the primer sequence are complementary to each other. “X1” (where x is lowercase) contained in the target sequence, “X1” (where X is uppercase) contained in the primer sequence, and “x2” (where x is lowercase) and “X2” (where x is lowercase) x is a capital letter) and are complementary to each other.
[0018]
For example, there are three types of primers used in this embodiment, which is one aspect of the present invention. That is, as shown in FIG. 1, a primer 4a containing three complementary basic units with a repeat number of 3, a primer 4b containing a complementary basic unit with four repeats, and a primer 4c containing a complementary basic unit with five repeats. These primers are each represented by “X1- (Y) ₃ -X2 "," X1- (Y) ₄ -X2 "and" X1- (Y) ₅ -X2 ".
[0019]
An example of a method for determining the number of repetitions in a target nucleic acid using the above primers will be described below.
[0020]
First, a first reaction system involving the target nucleic acid 1 and the primer 4a, a second reaction system involving the target nucleic acid 1 and the primer 4b, and a third reaction system involving the target nucleic acid 1 and the primer 4c are prepared. I do. In each reaction system, an extension reaction is performed under conditions under which appropriate amplification occurs. Next, nucleic acids present in each reaction system after the reaction are analyzed.
[0021]
The following events occur in each reaction system after the reaction. That is, in the first reaction system, the target nucleic acid 1 and the primer 4a can partially hybridize. However, even if such hybridization occurs, it is not appropriate for the extension reaction. Therefore, the expected extension reaction does not occur. Therefore, the reaction product 5a is not a reaction product obtained when a desired extension reaction occurs. A similar event occurs in the third reaction system. Therefore, the reaction product 5c is not a reaction product obtained when a desired extension reaction occurs.
[0022]
On the other hand, in the second reaction system, the number of repetitions of the target nucleic acid 1 and the number of repetitions of the primer 4b match. Thus, the hybridization that occurs there is suitable for extension. Therefore, the desired extension is achieved and the resulting reaction product 5b is a reaction product obtained when the desired extension reaction occurs.
[0023]
Since the extension in each reaction system causes the above-mentioned events, the number of repetitions of the target nucleic acid 1 can be determined by analyzing the nucleic acid present after the reaction and detecting the presence or absence of the extension.
[0024]
That is, in the present embodiment shown in FIG. 1, the target sequence 1 is obtained by repeating the unit sequence four times (ie, n = 4) (a) ₄ It becomes clear that it contains the repetitive sequence 2 shown by.
[0025]
As described above, the target nucleic acid targeted in the present invention is “x1- (y) _n -X2 ", for which the general primer used is" X1- (Y) _m -X2 ". In the primers used according to the embodiments of the present invention, m = n primers may be used alone, m ≠ n primers may be used alone, m = n primers and m ≠ n primers May be used in combination.
[0026]
When a plurality of m = n primers and m ≠ n primers are used in combination, the m = n primers and the m ≠ n primers are used separately in independent reaction vessels and the reaction is performed. And may be used and the reaction performed in one reaction vessel. The “reaction vessel” here may be any space for performing a reaction, such as a vessel having a capacity therein and a surface of a substrate for performing the reaction therein.
[0027]
For example, if elongation is detected as a result of the reaction, m = n and the number of repeats of the target nucleic acid is determined to be the number of repeats of the particular primer used. If no elongation is detected as a result of the reaction, m ≠ n, and it is determined that the number of repetitions of the target nucleic acid is not the number of repetitions of the primer used.
[0028]
X1 length and (Y) _m And the length of X2 may be about 50 bases or more, preferably 0 to about 50 bases, and more preferably about 15 to about 30 bases. In order to correspond to a long repetitive sequence, it is preferable that the full length is long, but if the full length is long, the discrimination ability is reduced. Therefore, the optimal chain length is constituted in the above-mentioned range.
[0029]
X1 may have a length of 0 to about 40 bases, preferably about 1 to about 20 bases, and more preferably about 2 to about 10 bases. In order to correspond to a repetition sequence as long as possible, it is preferable that X1 is short, but if it is too short, the discrimination ability decreases. Therefore, the optimal chain length is constituted in the above-mentioned range.
[0030]
(Y) _m Has a length of 2 to about 40 bases, preferably about 4 to about 30 bases, and more preferably about 6 to about 20 bases. It is preferable that the total length is long in order to correspond to a long repetitive sequence, but if the total length is long, the discrimination ability decreases. Therefore, the optimal chain length is constituted in the above-mentioned range.
[0031]
The length of X2 may be 0 to about 40 bases, preferably about 1 to about 20 bases, and more preferably about 2 to about 10 bases. In order to correspond to a repetition sequence as long as possible, it is preferable that X2 is short, but if it is too short, the discrimination ability decreases. Therefore, the optimal chain length is constituted in the above-mentioned range.
[0032]
Further, it is preferable that the total length of the primer is 0 to 50 bases, and the length of X2 contained in the primer is 1 to 20 bases. More preferably, the total length of the primer is 15 to 30 bases, and the length of X2 contained in the primer is 2 to 10 bases. In order to correspond to a long repetitive sequence, it is preferable that the full length is long, but if the full length is long, the discrimination ability is reduced. Further, in order to correspond to a repetitive sequence as long as possible, it is preferable that X1 and X2 are short, but if it is too short, the discrimination ability decreases. Therefore, the optimal chain length is constituted in the above-mentioned range.
[0033]
Further, the reaction between the primers 4a, 4b and 4c and the target nucleic acid 1 may be performed in one reaction vessel, and the reaction between several primers and the target nucleic acid 1 may be performed in one reaction vessel. The reaction between all the primers and the target nucleic acid 1 may be performed in one reaction vessel.
[0034]
In the above-described embodiment, an example in which three types of primers are used has been described. However, the present invention is not limited to this, and one or more types of primers may be used. For example, when it is desired to determine whether or not the number of repeats is a specific number, a primer having Y of a desired number of repeats may be used. Further, the number of repetitions of Y is not limited to “3”, “4” and “5” as used in the method described above, and may be arbitrarily selected and used by the practitioner. For example, the number of repeats predicted for the target nucleic acid may be used.
[0035]
X1- (y) as described above _n X1- (Y) for detecting the number of repeats of the repetitive sequence contained in the target nucleic acid containing -x2 _m A primer consisting of -X2 is also included in the scope of the present invention.
[0036]
As used herein, the term "extend a nucleic acid" refers to extending a nucleic acid of interest by causing a polymerase to act using a template nucleic acid and a primer complementary to a partial base sequence thereof. And further indicates both such extension and amplification in which such extension is performed repeatedly.
[0037]
The extension reaction that may be utilized in accordance with aspects of the present invention may be the means commonly used to extend nucleic acids, known per se, or the means used to amplify nucleic acids. Any of such means may be used. Although not limited thereto, for example, a method utilizing a polymerase chain reaction (generally called PCR, hereinafter referred to as PCR), reverse transcription PCR, or the like can be used. Furthermore, Nucleic acid strand amplification (NASBA), Transcription mediated amplification (TMA), Ligase chain reaction (LCR), Strand displacement amplification (SDA), Isothermaland Chimeric primer-initiated Amplification of Nucleic acids (ICANN) and Rolling circle amplification (RCA Means) can also be used in accordance with aspects of the present invention.
[0038]
As used herein, "conditions under which an appropriate extension reaction can be obtained" refers to various conditions for the polymerase to synthesize a nucleic acid chain, for example, conditions such as temperature, pH, salt concentration, and dNTP, which make the activity of the polymerase appropriate. Indicate a condition that is sufficient to obtain
[0039]
Furthermore, a tag consisting of a base sequence, a fluorescent substance such as Cy5 and Cy3, a dye substance, a magnetic substance, and a pair of substances that specifically bind to each other such as avidin and biotin are added to the 5 ′ end of the primer. One may be provided as a labeling substance. Further, an enzyme such as hapten, oxidase or phosphatase, or ferrocene or quinones may be provided. Primers with such modifications may also be used in the method according to the invention.
[0040]
The detection of the extension may be performed using a means for analyzing the length of the base sequence known per se. For example, any electrophoresis known per se may be used. Examples of such electrophoresis are, for example, agarose gel electrophoresis and acrylamide gel electrophoresis, microfluidic chips and the like. Further, a nucleic acid probe-immobilized substrate provided with a nucleic acid probe may be used, and such a nucleic acid probe-immobilized substrate is generally known as a DNA chip or a DNA microarray or a DNA macroarray. Any device may be used. Further, microbeads, microtiter plates, and the like can be used as a substrate for immobilizing probes. Alternatively, a sequencer known per se may be used.
[0041]
When a nucleic acid probe-immobilized substrate is used, detection may be performed by performing an extension reaction or an amplification reaction and then hybridizing with a nucleic acid probe having complementarity to a part of the reaction product. Alternatively, when a tag is included in the primer, a nucleic acid probe-immobilized substrate provided with a nucleic acid probe having a sequence complementary to the tag may be used. For the detection of elongation, the obtained reaction product may be used as it is as a double strand, or may be used after being converted into a single strand. Further, any one of the above-described primers may be immobilized on a nucleic acid probe-immobilized substrate as a nucleic acid probe, and an elongation or amplification reaction may be performed in that state. Thereafter, the double strand obtained on the substrate may be detected.
[0042]
(2) Second aspect
Next, a method of determining the number of repetitions according to the second embodiment will be described with reference to FIG. The second embodiment uses primers 7a to 7c, which additionally have tags 6a to 6c at their 5 'end, to detect the same number of repeats of the target nucleic acid 1 as targeted in the first embodiment. Otherwise, primers 7a to 7c are equal to primers 4a to 4c of the first embodiment.
[0043]
The tag 6 is preferably a nucleic acid consisting of the base sequence Z, and the type of base sequence or its length may be changed for each primer used. The length of the base sequence Z is not limited to this, but may be about 1 base to about 50 bases or less, preferably 15 bases to 30 bases. In addition, it is desirable to use an orthonormal array in which a secondary structure is difficult to be obtained (Sakakibara, Genome Informatics, 11, 33-42 (2000)).
[0044]
By using the tags 6a to 6b, the extension can be detected more easily. That is, it is possible to easily detect by using a nucleic acid probe-immobilized substrate provided with a primer having a sequence complementary to the base sequence of each tag.
[0045]
Except for the features described above, the method for determining the number of repetitions according to the second aspect can be performed in the same manner as the method described in the first aspect, and various changes are made according to the first aspect. Is also good.
[0046]
(3) Third aspect
FIG. 3 is a diagram showing reaction products obtained by the third method for determining the number of repeated sequences. The third embodiment is an example in which three types of target nucleic acids having different numbers of repetitions are targeted. The target nucleic acid 8a has four basic unit repeats, the target nucleic acid 8b has five repeats, and the target nucleic acid 8c has five repeats.
[0047]
In this embodiment, three types of primers are used. The basic unit of the primer 9a has four repetitions, the primer 9b has five repetitions, and the primer 9c has six repetitions. All of the target nucleic acids 8a to 8c may be contained in one sample nucleic acid, any one target nucleic acid may be contained in one sample nucleic acid, and any two target nucleic acids may be contained in one sample nucleic acid. It may be contained in one sample nucleic acid.
[0048]
Except for the above features, the method for determining the number of repetitive sequences may be carried out in the same manner as in the first and second embodiments. Although an example in which three types of target nucleic acids are identified using three types of primers is shown here, the present invention is not limited to this, and the number of repetitive sequences of the present invention can be similarly determined for two or more types of target nucleic acids. It is possible to implement a decision method. Also, in that case, one or more primers can be used as desired.
[0049]
In any case, the target elongation is achieved only when the number of repetitions is the same as in the first and second embodiments described above. Therefore, the number of repetitions of the target nucleic acid can be determined by analyzing the reaction product. Is possible.
[0050]
3. Nucleic acid probe-immobilized substrate
The nucleic acid probe-immobilized substrate that can be used in the embodiment of the present invention basically comprises a substrate and a nucleic acid probe immobilized on the substrate. Preferably, the nucleic acid probe is immobilized on the substrate. The nucleic acid probe-immobilized substrate used in the embodiment of the present invention will be described below using examples.
[0051]
Here, the “nucleic acid probe” is a nucleic acid containing a base sequence complementary to a target sequence, and refers to a nucleic acid fragment to be immobilized on a substrate. The nucleic acid probe has a sequence that is complementary to the target sequence of interest, so that it can hybridize with the target sequence under appropriate conditions.
[0052]
Here, the “target sequence” refers to a base sequence whose presence is to be detected or a sequence to be captured by the base sequence of a nucleic acid probe. A nucleic acid containing such a target sequence is called a target nucleic acid. For example, when the base sequence of the tag provided in the primer is used as the target sequence, the sequence of the nucleic acid probe may be a sequence complementary to the sequence of the desired tag.
[0053]
As used herein, the terms "complementary", "complementary", and "complementary" may be in the range of 50% to 100%, preferably 100% complementary. As used herein, the terms "homology", "homologous" and "homology" may be 50% to 100% homologous, preferably 100% homologous.
[0054]
As used herein, the term "detecting hybridization" refers to detecting double-stranded nucleic acid generated by hybridization, or pre-labeling a sample nucleic acid with any labeling substance, and after hybridization. This is a general term for detecting a signal derived from the labeling substance, or detecting, by other means known per se, the presence of double-stranded nucleic acid or the occurrence of hybridization by the reaction. Yes, detection may be achieved by any means.
[0055]
In addition, when a sequence within 5 bases from the 3 ′ end of X2 is substituted with a base different from the complementary sequence of x2, the specificity of identification can be improved. The number of substitution is preferably one or more and two or less. The position of substitution is preferably within 3 bases from the 3 'end. Further, T or C when the base before substitution is A, C or A when the base before substitution is T, C or T when the base before substitution is G, and G or G when the base before substitution is C. When it is replaced with, the discrimination ability can be further improved.
[0056]
(1) First example
A first example of the nucleic acid probe-immobilized substrate that can be used in the embodiment of the present invention will be described with reference to FIG. The nucleic acid probe-immobilized substrate 11 of the first example includes a nucleic acid probe 14 immobilized on an electrode 13 provided on a substrate 12 (FIG. 4). The electrode 13 is connected to a pad 15 for extracting electrical information.
[0057]
Such a nucleic acid probe-immobilized substrate 11 can be produced, for example, by arranging electrodes on a substrate such as a silicon substrate by means known per se, and immobilizing the nucleic acid probe on the electrode surface. is there.
[0058]
In this embodiment, the number of electrodes is six, but the number of electrodes arranged on one base is not limited to this. Further, the arrangement pattern of the electrodes is not limited to that shown in FIG. 4, and a person skilled in the art can appropriately change the design as needed. A reference electrode and a counter electrode may be provided as necessary. Such a nucleic acid probe-immobilized substrate is also within the scope of the present invention.
[0059]
(2) Second example
FIG. 5 schematically shows a second example of the nucleic acid probe-immobilized substrate that can be used according to the embodiment of the present invention. The nucleic acid probe-immobilized substrate 16 according to the second example has a nucleic acid probe 18 immobilized on a substrate 17 (FIG. 5).
[0060]
Such a nucleic acid probe-immobilized substrate 16 can be manufactured, for example, by immobilizing the nucleic acid probe 18 on a substrate such as a silicon substrate by means known per se.
[0061]
In this embodiment, the number of nucleic acid probes arranged on one substrate is not limited to this, and may be changed as desired, and a nucleic acid probe having a plurality of types of base sequences may be added to one substrate. It may be arranged. The solid phase pattern of a plurality and / or a plurality of types of nucleic acid probes on a substrate can be appropriately changed by a person skilled in the art as needed. Such a nucleic acid probe-immobilized substrate is also within the scope of the present invention.
[0062]
In the case of a nucleic acid probe-immobilized substrate for performing fluorescence detection as described in the second example, the nucleic acid probe may be immobilized on any of the above substrates. Further, in the case of a nucleic acid probe-immobilized substrate for performing electrochemical detection as described in the first example, an electrode is attached to any of the substrates so that electrochemical detection is possible. It is sufficient to dispose and immobilize the nucleic acid probe on the electrode.
[0063]
Electrodes that can be used in the present invention are not particularly limited, for example, graphite, glassy carbon, pyrolytic graphite, carbon paste, carbon electrode such as carbon fiber, platinum, platinum black, gold, palladium, Noble metal electrodes such as rhodium, oxide electrodes such as titanium oxide, tin oxide, manganese oxide and lead oxide, Si, Ge, ZnO, CdS, TiO ₂ , A semiconductor electrode such as GaAs, and titanium. These electrodes may be coated with a conductive polymer or a monomolecular film, and may be treated with another surface treating agent as desired.
[0064]
The nucleic acid probe may be fixed by any means known per se. For example, the nucleic acid probe may be directly immobilized on a treated or untreated substrate or electrode surface by covalent bonding, ionic bonding, physical adsorption, or the like. Alternatively, a linker agent that assists in fixing the nucleic acid probe may be used, and the nucleic acid probe may be fixed to the substrate or the electrode via a spacer. Further, a blocking agent for preventing non-specific binding of the sample nucleic acid to the electrode may be treated on the electrode together with a linker agent. Further, the linker agent and the blocking agent used here may be, for example, substances for performing electrochemical detection advantageously.
[0065]
Further, nucleic acid probes having different base sequences may be respectively immobilized on different electrodes, and a plurality of types of nucleic acid probes having different base sequences are immobilized on one electrode in a mixed state. You may.
[0066]
(3) Detection
The nucleic acid probe-immobilized substrate according to the present invention comprises an electrochemical method as a means for detecting the presence of a double strand resulting from a hybridization reaction between a nucleic acid probe immobilized on the substrate and a target nucleic acid. And fluorescence detection methods are available.
[0067]
(A) Electrochemical detection
The double-stranded nucleic acid can be detected electrochemically using, for example, a double-stranded recognition substance known per se.
[0068]
The double-stranded recognizer used here is not particularly limited, but for example, bisintercalators such as Hoechst 33258, acridine orange, quinacrine, dounomycin, metallointercalators, and bisacridines, tris intercalators and polyintercalators Etc. can be used. Furthermore, these intercalators can be modified with an electrochemically active metal complex such as ferrocene, viologen, or the like. Further, any other known double-stranded recognition substance is preferably used in the present invention.
[0069]
In the nucleic acid probe-immobilized substrate according to the present invention, the nucleic acid probe is immobilized on the electrode. Detection of a double-stranded nucleic acid using such an electrode may further use a counter electrode or a reference electrode, as in other general electrochemical detection methods. When a reference electrode is provided, a general reference electrode such as a silver / silver chloride electrode or a mercury / mercury chloride electrode may be used.
[0070]
For example, when detecting whether extension has been achieved by a target nucleic acid and a primer in a sample nucleic acid, the test may be performed as follows.
[0071]
The optionally pretreated sample nucleic acid is extended using a primer according to an embodiment of the present invention. The obtained extension product is converted into a single strand by a method known per se, and is brought into contact with a nucleic acid probe immobilized on a nucleic acid probe-immobilized substrate. For example, it is also possible to use a nucleic acid probe complementary to a part of the extended single strand including the primer used, or to use a nucleic acid probe having a sequence complementary to the tag as described above. Good. However, the sequence of the nucleic acid probe is not limited to these. Further, a nucleic acid probe-immobilized substrate on which a nucleic acid probe for use in the detection of elongation of the present invention is immobilized is also within the scope of the present invention.
[0072]
Subsequently, the reaction is performed under conditions that allow appropriate hybridization. Such appropriate conditions can be determined by those skilled in the art depending on various conditions such as the type of base contained in the target sequence, the type of nucleic acid probe provided on the nucleic acid probe-immobilized substrate, the type of sample nucleic acid and their state. If so, it can be selected appropriately. Although not limited thereto, for example, the reaction may be performed under the following conditions.
[0073]
That is, the hybridization reaction solution is performed in a buffer having an ionic strength of 0.01 to 5 and a pH of 5 to 10. To this solution, dextran sulfate as a hybridization accelerator, salmon sperm DNA, bovine thymus DNA, EDTA, a surfactant and the like may be added. The sample nucleic acid obtained here is added and denatured at 90 ° C. or higher. Insertion of the nucleic acid probe-immobilized substrate into the heat-denatured sample nucleic acid may be performed immediately after denaturation or after quenching to 0 ° C. Further, it is also possible to carry out a hybridization reaction by dropping a solution on a substrate.
[0074]
During the reaction, the reaction rate may be increased by an operation such as stirring or shaking. The reaction temperature may be, for example, in the range of 10 ° C. to 90 ° C., and the reaction time may be about 1 minute to about 1 night. After the hybridization reaction, the electrodes are washed. For washing, for example, a buffer solution having an ionic strength of 0.01 to 5 and a pH of 5 to 10 may be used. When the target nucleic acid containing the target sequence is present in the sample nucleic acid, it hybridizes with the nucleic acid probe, thereby generating a double-stranded nucleic acid.
[0075]
Subsequently, double-stranded nucleic acid generated by the following procedure is detected by electrochemical means. Generally, after the hybridization reaction, the substrate is washed, a double-stranded recognizer is allowed to act on the double-stranded portion formed on the electrode surface, and a signal generated thereby is electrochemically measured.
[0076]
The concentration of the double-stranded recognizer varies depending on its type, but is generally used in the range of 1 ng / mL to 1 mg / mL. In this case, a buffer solution having an ionic strength of 0.001 to 5 and a pH of 5 to 10 may be used.
[0077]
For example, in the electrochemical measurement, a potential equal to or higher than the potential at which the double-stranded recognizer reacts electrochemically may be applied, and the reaction current value derived from the double-stranded recognizer may be measured. At this time, the potential may be swept at a constant speed, applied as a pulse, or a constant potential may be applied. At the time of measurement, for example, the current and voltage may be controlled using a device such as a potentiostat, a digital multimeter, and a function generator. For example, the concentration of the target nucleic acid may be calculated from a calibration curve based on the obtained current value.
[0078]
In addition, electrochemical detection means known per se, for example, means disclosed in the following documents (Hashimoto et al. 1994, Wang et al. 1998) can also be preferably used in the method of the present invention. In that article, Hashimoto et al. Reported sequence-specific gene detection using a gold electrode modified with a DNA probe and an electrochemically active dye. The anodic current from the dye correlates with the concentration of the target DNA. Wang et al. Also reported indicator-free electrochemical DNA hybridization. The configuration of this biosensor involves immobilization of an inosine-substituted probe (without guanine) on a carbon paste electrode and chronopotentiometric detection of duplex formation due to the presence of the guanine oxidation peak of the label. The detection means described in these documents may preferably be used in the present invention.
[0079]
(B) Fluorescence detection method
In the case of using a fluorescent labeling substance, the antisense primer may be labeled with a substance capable of obtaining a fluorescent activity such as a fluorescent dye such as FITC, Cy3, Cy5 or rhodamine. Alternatively, detection may be performed by using a second probe labeled with the above-mentioned substance instead of the label. A plurality of labeling substances may be used simultaneously.
[0080]
In some embodiments, a hybridization reaction between a nucleic acid component extracted from a sample and a nucleic acid probe immobilized on a nucleic acid probe-immobilized chip is performed, for example, as follows. That is, the hybridization reaction solution is performed in a buffer having an ionic strength of 0.01 to 5 and a pH of 5 to 10. To this solution, dextran sulfate as a hybridization accelerator, salmon sperm DNA, bovine thymus DNA, EDTA, a surfactant and the like may be added. The extracted nucleic acid component is added thereto, and denatured at 90 ° C. or higher. Insertion of the nucleic acid probe-immobilized chip may be performed immediately after denaturation or after rapid cooling to 0 ° C. Further, it is also possible to carry out a hybridization reaction by dropping a solution on a substrate. During the reaction, the reaction rate may be increased by an operation such as stirring or shaking. The reaction temperature may be, for example, in the range of 10 ° C. to 90 ° C., and the reaction time may be about 1 minute to about 1 night. After the hybridization reaction, washing is performed. For washing, for example, a buffer solution having an ionic strength of 0.01 to 5 and a pH of 5 to 10 is used.
[0081]
In the case of fluorescence detection, the detection of the hybridization reaction is performed by detecting the labeled base sequence in the sample or the label in the secondary probe using an appropriate detection device according to the type of the label. When the label is a fluorescent substance, the label may be detected using, for example, a fluorescence detector.
[0082]
4. Assay kit
In the present invention, the primer according to any of the embodiments of the present invention as described above, the nucleic acid probe-immobilized substrate, and the nucleic acid probe containing a sequence complementary to the tag sequence may be provided as an appropriate kit depending on the purpose of use. Good.
[0083]
For example, in the case of a primer according to the present invention, a kit may be provided in combination with a reaction vessel and / or salts for buffer solution and / or other necessary reagents such as a polymerase and / or a substrate. For example, a nucleic acid probe containing a sequence complementary to a tag sequence, a primer and an extension product or a part thereof may be used as a substrate and / or various reagents and / or labeling substances for producing a desired nucleic acid probe-immobilized substrate. A kit may be provided in combination with the above. The primers and the desired nucleic acid probe and / or nucleic acid probe-immobilized substrate may be combined as needed to provide a kit. However, the kit provided by the present invention is not limited to the above combinations, and may be provided as a kit in combination with various ones as necessary. The purpose of use is not limited to this, and kits for any of the above-mentioned purposes can be formed. Such a kit is also included in the scope of the present invention.
[0084]
【Example】
Example 1
In Example 1, a target nucleic acid was prepared by using three types of primers, a first primer (SEQ ID NO: 1), a second primer (SEQ ID NO: 2), and a third primer (SEQ ID NO: 3), as sense primers. The number of repeats of the microsatellite sequence present in the human genome fragment (SEQ ID NO: 7) was determined.
[0085]
The number of repeats of the basic unit of each primer used was different, the first primer had 4 repeats, the second primer had 5 repeats, and the third primer had 6 repeats.
[0086]
The PCR reaction conditions were as follows. That is, a PCR reaction was first performed at 94 ° C. for 5 minutes, then 30 cycles of 94 ° C. for 30 seconds and 74 ° C. for 60 seconds, and further at 72 ° C. for 7 minutes. Thereafter, electrophoresis was performed to perform fluorescent staining.
[0087]
Using the human genomic fragment shown in SEQ ID NO: 7 as a template, and the nucleic acid described in SEQ ID NO: 7 as an antisense primer, three different primers of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 An amplification reaction was performed in the reaction vessel. FIG. 6 shows the result. As is clear from FIG. 6, as a result of the electrophoresis, a band having the highest fluorescence intensity was observed when the second primer of SEQ ID NO: 2 was used. That is, it was revealed that amplification was hardly performed unless primers corresponded to the number of repetitions of the target nucleic acid. In the case of this example, the number of repetitions of the target nucleic acid in this sample nucleic acid is determined to be 5.
[0088]
This provided a method for simply, inexpensively, and rapidly determining the number of repetitive sequences contained in a nucleic acid in a sample nucleic acid.
[0089]
Example 2
In Example 2, a target nucleic acid was amplified using a primer having a tag, and then hybridized to the primer immobilized on the nucleic acid probe-immobilized substrate, and the resulting hybridization was electrochemically detected. An example of performing a method for determining the number of repetitions will be described.
[0090]
FIG. 7 is a plan view showing a pattern of the nucleic acid probe-immobilized substrate 20 used in the method for determining the number of repetitive sequences according to Example 2 of the present invention. The nucleic acid probe-immobilized substrate 20 includes electrodes 22a, 22b, and 22c arranged on the substrate 21. On the electrode 22a, a nucleic acid having the base sequence of SEQ ID NO: 4 was immobilized as a first nucleic acid probe. A nucleic acid having the nucleotide sequence of SEQ ID NO: 5 was immobilized on the electrode 22b as a second nucleic acid probe. A nucleic acid having the base sequence of SEQ ID NO: 6 was immobilized on the electrode 22c as a third nucleic acid probe.
[0091]
The nucleic acid probe-immobilized substrate was manufactured as follows. First, a probe consisting of the nucleic acid represented by SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6 having the thiol at the 5 'end was immobilized on an electrode obtained by patterning gold on a substrate.
[0092]
Here, the nucleic acid probe of SEQ ID NO: 4 is composed of a sequence complementary to the tag sequence on the 5 ′ side of the primer of SEQ ID NO: 1. Similarly, the nucleic acid probe of SEQ ID NO: 5 is The nucleic acid probe of No. 6 consists of a sequence complementary to the tag of the primer of SEQ ID No. 3.
[0093]
First, using a human genomic fragment consisting of the nucleic acid represented by the nucleotide sequence of SEQ ID NO: 8 as a template, a mix of primers of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 as a sense primer, and SEQ ID NO: Amplification reaction was carried out by PCR using No. 7.
[0094]
The PCR reaction conditions were as follows. That is, a PCR reaction was first performed at 94 ° C. for 5 minutes, then 30 cycles at 94 ° C. for 30 seconds and 74 ° C. for 60 seconds, and further performed at 72 ° C. for 7 minutes. Then, the PCR reaction solution obtained by this PCR was reacted. The hybridization reaction was carried out at 35 ° C. for 1 hour using the PCR reaction solution after heat denaturation. Thereafter, a reaction with the intercalating agent Hoechst 33258 was performed, and hybridization was detected by an electrode. The result is shown in FIG. As shown in FIG. 8, the highest current value was obtained from the electrode on which the second nucleic acid probe was immobilized.
[0095]
This provided a method for simply, inexpensively, and rapidly determining the number of repetitive sequences contained in a nucleic acid in a sample nucleic acid.
[0096]
Example 3
Example 3 is to amplify a target nucleic acid using a primer having a tag, then hybridize to a primer immobilized on a nucleic acid probe-immobilized substrate, and detect the resulting hybridization by fluorescence intensity. The example which performs the method of determining the number of repetitions is shown.
[0097]
FIG. 9 is a plan view showing the configuration of the nucleic acid probe-immobilized substrate used in the method for determining the number of repeated sequences according to Example 3 of the present invention. As the nucleic acid probe-immobilized substrate, a slide glass coated with polylysine was used as a substrate, and the nucleic acid probes of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 were immobilized thereon. Each immobilization was performed on the nucleic acid probe immobilization regions 32a, 32b and 33c provided on the substrate. That is, a nucleic acid having the nucleotide sequence of SEQ ID NO: 4 was immobilized on the immobilization region 32a as a first nucleic acid probe. A nucleic acid having the nucleotide sequence of SEQ ID NO: 5 was immobilized on the immobilization region 32b as a second nucleic acid probe. A nucleic acid having the nucleotide sequence of SEQ ID NO: 6 was immobilized on the immobilization region 32c as a third nucleic acid probe.
[0098]
Here, the nucleic acid probe of SEQ ID NO: 4 is composed of a sequence complementary to the tag sequence on the 5 ′ side of the primer of SEQ ID NO: 1. Similarly, the nucleic acid probe of SEQ ID NO: 5 is The nucleic acid probe of No. 6 consists of a sequence complementary to the tag of the primer of SEQ ID No. 3.
[0099]
First, using a human genomic fragment consisting of the nucleic acid represented by the nucleotide sequence of SEQ ID NO: 8 as a template, a mix of primers of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 as a sense primer, and a cy5 label as an antisense primer An amplification reaction was performed by PCR using the nucleic acid of SEQ ID NO: 7 obtained above.
[0100]
The PCR reaction conditions were as follows. That is, a PCR reaction was first performed at 94 ° C. for 5 minutes, then 30 cycles at 94 ° C. for 30 seconds and 74 ° C. for 60 seconds, and further performed at 72 ° C. for 7 minutes. Then, the PCR reaction solution obtained by this PCR was reacted after heat denaturation. The hybridization reaction was performed at 35 ° C. for 1 hour using the PCR reaction solution as it was. Thereafter, hybridization between the PCR product contained in the PCR reaction solution and the nucleic acid probe was detected by measuring the fluorescence intensity. The results are shown in FIG. As shown in FIG. 10, the highest current value was obtained from the electrode on which the second nucleic acid probe was immobilized.
[0101]
This provided a method for simply, inexpensively, and rapidly determining the number of repetitive sequences contained in a nucleic acid in a sample nucleic acid.
[0102]
【The invention's effect】
According to the present invention as described above, a method for determining the number of repeats of a repetitive sequence contained in a nucleic acid is provided.
[0103]
[Sequence list]

[Brief description of the drawings]
FIG. 1 is a diagram showing a method for determining the number of iterations according to a first embodiment of the present invention.
FIG. 2 is a diagram showing a method for determining the number of repetitions according to a second embodiment of the present invention.
FIG. 3 is a diagram showing a reaction product obtained by the method for determining the number of repetitive sequences according to the third embodiment of the present invention.
FIG. 4 is a diagram showing a first example of a nucleic acid probe-immobilized substrate that can be used in an embodiment of the present invention.
FIG. 5 is a diagram showing a second example of a nucleic acid probe-immobilized substrate that can be used in an embodiment of the present invention.
FIG. 6 is a photograph of an electrophoresis gel showing the results of the first example.
FIG. 7 is a plan view of a nucleic acid probe-immobilized substrate used in the method for determining the number of repeated sequences according to Example 2.
FIG. 8 is a graph showing the results of Example 2.
FIG. 9 is a plan view of a nucleic acid probe-immobilized substrate used in the method for determining the number of repeated sequences according to Example 3.
FIG. 10 is a graph showing the results of Example 3.
[Explanation of symbols]
1. 1. Target nucleic acid 2. repetitive sequence 3. Target sequence Primer5. Reaction product 6. Tags 7. Primer 8. 8. Target nucleic acid Primer 11. Nucleic acid probe-immobilized substrate 12. Substrate 13. Electrode 14. Nucleic acid probe 15. Pat16. Nucleic acid probe-immobilized substrate 17. Substrate 18. Nucleic acid probe 20. Nucleic acid probe-immobilized substrate 21. Substrate 22. Electrode 23. Pat 30. Nucleic acid probe-immobilized substrate 31. Substrate 32. Nucleic acid probe immobilization region

Claims (10)

A target sequence x1- (y) _n − comprising a repetitive sequence containing a basic unit y with an arbitrary number of repetitions n, a sequence x1 on the 3 ′ side thereof, and a sequence x2 on the 5 ′ side of the repetitive sequence. A method for determining the number of repetitions of a target nucleic acid comprising x2,
(1) A primer X1- (Y) including a sequence X1 complementary to the sequence x1, a repetitive sequence including the sequence Y complementary to the basic unit y at an arbitrary repetition number m, and a sequence X2 complementary to the sequence x2. Using _m- X2 (where m is an integer greater than or equal to 1 and an integer equal to the predicted number of iterations n) to elongate the nucleic acid under conditions that provide for an appropriate elongation reaction; ,
(2) determining the number of repetitions of the repetitive sequence of the target nucleic acid by determining the presence or absence of extension in the reaction product obtained by the extension of (1);
A method for determining the number of repetitions of a target nucleic acid, comprising: M in the primer X1- (Y) _m -X2 is an integer equal to the predicted number of repeats n, and there are a plurality of the predicted number of repeats n; 2. The method for determining the number of repetitions of a target nucleic acid according to claim 1, wherein corresponding primers are used. 3. The method for determining the number of repetitions of a target nucleic acid according to claim 2, wherein the primers used are used in different reaction vessels for each sequence. When the primer is a primer Z-X1- (Y) _m -X2 further having a tag sequence Z at its 5 'end, and the presence or absence of extension in (2) is determined, the tag sequence Z The hybridization is performed by hybridizing the reaction product to a nucleic acid probe-immobilized substrate having a nucleic acid probe having a sequence complementary to that under conditions capable of obtaining appropriate hybridization, and detecting the resulting hybridization. The method for determining the number of repetitions of a target nucleic acid according to any one of claims 1 to 3, characterized in that: The method for determining the number of repetitions of a target nucleic acid according to claim 4, wherein detecting the hybridization is performed by electrochemical detection. The method for determining the number of repetitions of a target nucleic acid according to claim 4, wherein the detecting of the hybridization is performed by detecting a fluorescence intensity. The method for determining the number of repetitions of a target nucleic acid according to any one of claims 1 to 6, wherein the extension in (1) is amplification. The target according to any one of claims 1 to 7, wherein the total length of the primer is 10 to 50 bases, and the length of X2 contained in the primer is 1 to 20 bases. A method for determining the number of repeats of a nucleic acid. The total length of the primer is 15 to 30 bases, and the length of X2 contained in the primer is 2 to 10 bases, The primer according to any one of claims 1 to 7, wherein A method for determining the number of repeats for a target nucleic acid. A primer consisting of X1- (Y) m-X2 for detecting the number of repeats of a repetitive sequence contained in a target nucleic acid containing x1- (y) nx2.

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