JP2005013122A - Method for stabilizing reagent for amplifying gene - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To extremely improve the stability, activity and convenience of using operation of a reagent used for isothermal amplification of a nucleic acid by solving the problems in which the amplification reaction proceeds under operation of the reagent and under preservation. <P>SOLUTION: This method involves carrying out the gene amplification in the coexistence of at least one of the following oligonucleotides having sequences competing with a target gene sequence against a chimera oligonucleotide primer used in the amplification: The oligonucleotide has the ratio of the Tm value between the chimera oligonucleotide primer and a nucleic acid sequence of the target gene substantially complementary to the base sequence of the primer, to the Tm value between the oligonucleotide and the chimera oligonucleotide primer, regulated within the range of 1.2-3.3, and also has its 3' terminus blocked so as not to be extended. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

【００６１】
この増幅試薬に４０μｌの９０ｍＭ酢酸マグネシウム液および２０μｌの内部標準（Ｉｎｔｅｒｎａｌ ｃｏｎｔｒｏｌ）ＤＮＡ（０．９ｐｇ／μｌ）を加え、室温に７０分間放置した。その後、１反応あたり２５μｌずつ分注し、等容量のテンプレートＤＮＡを加え、変性・アニーリング操作をすることなく５５℃、１時間の増幅反応を行った。増幅後、固層プレート発光法により増幅産物を検出した。以下の表２に結果を示す。
【００６２】
【表２】

【００６３】
表２に示したように、増幅試薬Ａ（従来法）では増幅不良を起こし目的の遺伝子を検出することができなかった。一方、増幅試薬Ｂでは安定した増幅反応を示し、目的遺伝子を検出することが可能であった。すなわち、本発明の方法が増幅反応の安定化に寄与することが確認できた。
【００６４】
（２）クラミジア／リン菌遺伝子のＩＣＡＮ法による増幅における冷蔵での増幅試薬ミックスチャーの安定化の検討
上記実施例１―（１）と同様の方法で、増幅試薬Ａ、Ｂそれぞれに４０μｌの９０ｍＭ酢酸マグネシウム液および２０μｌの内部標準ＤＮＡ（０．９ｐｇ／μｌ）を加え、４℃、１週間保存し、冷蔵保存試験を行った。以下の表３に結果を示す。
【００６５】
【表３】

【００６６】
表３に示したように、増幅試薬Ａにおいては目的遺伝子ＣＴおよびＮＧの検出は可能であったが、ＩＣ（内部標準）の増幅不良を起こし、０コピー区画においてさえＩＣは検出されなかった。一方、増幅試薬Ｂにおいては変性・アニーリング操作をすることなく安定した増幅を示し、全て目的遺伝子を検出することができた。すなわち、本発明の方法が、低温、長期期間の保存・安定化に好適であることが確認できた。
【００６７】
実施例２ クラミジア／リン菌遺伝子のＩＣＡＮ法による増幅における再現性の改善
上記実施例１―（１）と同様の方法で増幅試薬Ａと増幅試薬Ｂそれぞれに４０μｌの９０ｍＭ酢酸マグネシウム液および２０μｌの内部標準ＤＮＡ（０．９ｐｇ／μｌ）を加え、低コピー数のＣＴ，ＮＧテンプレートを用いて変性・アニーリング操作をすることなく１０回の反応を行い同時再現性を比較した。以下の表４に結果を示す。
【００６８】
【表４】

【００６９】
表４に示したように、本発明のＳｅｎｓｉｂｌｅ ｄｅｃｏｙを含む増幅試薬Ｂの方が良好な再現性が得られた。
【００７０】
実施例３ ＨＰＶ６、１１遺伝子ＩＣＡＮ増幅における増幅の安定化
ＨＰＶ６遺伝子より、５’末端をビオチン標識したセンスプライマー（配列番号９）およびアンチセンスプライマー（配列番号１０）を設計、合成した。また、Ｓｅｎｓｉｂｌｅ ｄｅｃｏｙとして、３’末端をアミノ修飾したセンスプライマー（配列番号１２）および３’末端をアミノ修飾したアンチセンスプライマー（配列番号１３）を設計、合成した。
ＨＰＶ１１遺伝子より、アンチセンスプライマー（配列番号１１）を設計、合成した。センスプライマーは、ＨＰＶ６遺伝子の５’末端をビオチン標識したセンスプライマー（配列番号９）を用いた。また、Ｓｅｎｓｉｂｌｅ ｄｅｃｏｙとして、３’末端をアミノ修飾したアンチセンスプライマー（配列番号１４）を設計、合成した。センスプライマーは、ＨＰＶ６遺伝子の３’末端をアミノ修飾したセンスプライマー（配列番号１２）を用いた。
【００７１】
上記実施例１−（１）と同様の方法で、Ｓｅｎｓｉｂｌｅ ｄｅｃｏｙを含まない増幅試薬Ａ、 Ｓｅｎｓｉｂｌｅ ｄｅｃｏｙを含む増幅試薬Ｂを調製し、それぞれに４０μｌの９０ｍＭ酢酸マグネシウム液および２０μｌの内部標準ＤＮＡ（０．９ｐｇ／μｌ）を加え、変性・アニーリング操作することなく等温増幅させた。以下の表５に結果を示す。
【００７２】
【表５】

【００７３】
表５に示したようにＨＰＶ６、１１ＤＮＡの増幅を比較した結果、本発明の方法を用いることにより低コピー数での増幅に改善が認められた。
【００７４】
【発明の効果】
上記の、本発明のオリゴヌクレオチド（Ｓｅｎｓｉｂｌｅ ｄｅｃｏｙ ）を使用する遺伝子増幅方法は、中程度高温における等温遺伝子増幅方法に有用である。また、本発明の方法は、増幅試薬の保存・安定化方法として有効である。
【００７５】
【配列表フリーテキスト】
ＳＥＱ ＩＤ ＮＯ：１ ； Ｃｈｉｍｅｒｉｃ ｏｌｉｇｏｎｕｃｌｅｏｔｉｄｅ ｔｏ ｄｅｔｅｃｔ Ｃｈｌａｍｙｄｉａ ｔｒａｃｈｏｍａｔｉｓ ｃｒｉｐｔｉｃ ｐｌａｓｍｉｄ ｇｅｎｅ． ”Ｎｕｃｌｅｏｔｉｄｅｓ １９ ｔｏ ２２ ａｒｅ ｒｉｂｏｎｕｃｌｅｏｔｉｄｅｓ− ｏｔｈｅｒ ｎｕｃｌｅｏｔｉｄｅｓ ａｒｅ ｄｅｏｘｙｒｉｂｏｎｕｃｌｅｏｔｉｄｅｓ．”
ＳＥＱ ＩＤ ＮＯ：２ ； Ｃｈｉｍｅｒｉｃ ｏｌｉｇｏｎｕｃｌｅｏｔｉｄｅ ｔｏ ｄｅｔｅｃｔ Ｃｈｌａｍｙｄｉａ ｔｒａｃｈｏｍａｔｉｓ ｃｒｉｐｔｉｃ ｐｌａｓｍｉｄ ｇｅｎｅ． ”Ｎｕｃｌｅｏｔｉｄｅｓ １６ ｔｏ １８ ａｒｅ ｒｉｂｏｎｕｃｌｅｏｔｉｄｅｓ− ｏｔｈｅｒ ｎｕｃｌｅｏｔｉｄｅｓ ａｒｅ ｄｅｏｘｙｒｉｂｏｎｕｃｌｅｏｔｉｄｅｓ， ａｎｄ ５’ ｅｎｄ ｉｓ ｌａｂｅｌｅｄ ｂｙ ｂｉｏｔｉｎ．”
ＳＥＱ ＩＤ ＮＯ：３ ； Ｔｈｅ ３’−ＯＨ ｇｒｏｕｐ ｏｆ ｎｕｃｌｅｏｔｉｄｅ ａｔ ３’ ｅｎｄ ｉｓ ｐｒｏｔｅｃｔｅｄ ｗｉｔｈ ａｍｉｎｏ ｇｒｏｕｐ．
ＳＥＱ ＩＤ ＮＯ：４ ； Ｔｈｅ ３’−ＯＨ ｇｒｏｕｐ ｏｆ ｎｕｃｌｅｏｔｉｄｅ ａｔ ３’ ｅｎｄ ｉｓ ｐｒｏｔｅｃｔｅｄ ｗｉｔｈ ａｍｉｎｏ ｇｒｏｕｐ．
ＳＥＱ ＩＤ ＮＯ：５ ； Ｃｈｉｍｅｒｉｃ ｏｌｉｇｏｎｕｃｌｅｏｔｉｄｅ ｔｏ ｄｅｔｅｃｔ Ｎｅｉｓｓｅｒｉａ ｇｏｎｏｒｒｈｏｅａ ｃｐｐＢ ｇｅｎｅ． ”Ｎｕｃｌｅｏｔｉｄｅｓ １８ ｔｏ ２０ ａｒｅ ｒｉｂｏｎｕｃｌｅｏｔｉｄｅｓ− ｏｔｈｅｒ ｎｕｃｌｅｏｔｉｄｅｓ ａｒｅ ｄｅｏｘｙｒｉｂｏｎｕｃｌｅｏｔｉｄｅｓ．”
ＳＥＱ ＩＤ ＮＯ：６ ； Ｃｈｉｍｅｒｉｃ ｏｌｉｇｏｎｕｃｌｅｏｔｉｄｅ ｔｏ ｄｅｔｅｃｔ Ｎｅｉｓｓｅｒｉａ ｇｏｎｏｒｒｈｏｅａ ｃｐｐＢ ｇｅｎｅ． ”Ｎｕｃｌｅｏｔｉｄｅｓ １５ ｔｏ １７ ａｒｅ ｒｉｂｏｎｕｃｌｅｏｔｉｄｅｓ− ｏｔｈｅｒ ｎｕｃｌｅｏｔｉｄｅｓ ａｒｅ ｄｅｏｘｙｒｉｂｏｎｕｃｌｅｏｔｉｄｅｓ， ａｎｄ ５’ ｅｎｄ ｉｓ ｌａｂｅｌｅｄ ｂｙ ｂｉｏｔｉｎ．”
ＳＥＱ ＩＤ ＮＯ：７ ； Ｔｈｅ ３’−ＯＨ ｇｒｏｕｐ ｏｆ ｎｕｃｌｅｏｔｉｄｅ ａｔ ３’ ｅｎｄ ｉｓ ｐｒｏｔｅｃｔｅｄ ｗｉｔｈ ａｍｉｎｏ ｇｒｏｕｐ．
ＳＥＱ ＩＤ ＮＯ：８ ； Ｔｈｅ ３’−ＯＨ ｇｒｏｕｐ ｏｆ ｎｕｃｌｅｏｔｉｄｅ ａｔ ３’ ｅｎｄ ｉｓ ｐｒｏｔｅｃｔｅｄ ｗｉｔｈ ａｍｉｎｏ ｇｒｏｕｐ．
ＳＥＱ ＩＤ ＮＯ：９ ； Ｃｈｉｍｅｒｉｃ ｏｌｉｇｏｎｕｃｌｅｏｔｉｄｅ ｔｏ ｄｅｔｅｃｔ ＨＰＶ６ ｇｅｎｅ． ”Ｎｕｃｌｅｏｔｉｄｅｓ １８ ｔｏ ２０ ａｒｅ ｒｉｂｏｎｕｃｌｅｏｔｉｄｅｓ− ｏｔｈｅｒ ｎｕｃｌｅｏｔｉｄｅｓ ａｒｅ ｄｅｏｘｙｒｉｂｏｎｕｃｌｅｏｔｉｄｅｓ， ａｎｄ ５’ ｅｎｄ ｉｓ ｌａｂｅｌｅｄ ｂｙ ｂｉｏｔｉｎ．”
ＳＥＱ ＩＤ ＮＯ：１０ ； Ｃｈｉｍｅｒｉｃ ｏｌｉｇｏｎｕｃｌｅｏｔｉｄｅ ｔｏ ｄｅｔｅｃｔ ＨＰＶ６ ｇｅｎｅ． ”Ｎｕｃｌｅｏｔｉｄｅｓ ２０ ｔｏ ２２ ａｒｅ ｒｉｂｏｎｕｃｌｅｏｔｉｄｅｓ− ｏｔｈｅｒ ｎｕｃｌｅｏｔｉｄｅｓ ａｒｅ ｄｅｏｘｙｒｉｂｏｎｕｃｌｅｏｔｉｄｅｓ．”
ＳＥＱ ＩＤ ＮＯ：１１ ； Ｃｈｉｍｅｒｉｃ ｏｌｉｇｏｎｕｃｌｅｏｔｉｄｅ ｔｏ ｄｅｔｅｃｔ ＨＰＶ１１ ｇｅｎｅ． ”Ｎｕｃｌｅｏｔｉｄｅｓ ２０ ｔｏ ２２ ａｒｅ ｒｉｂｏｎｕｃｌｅｏｔｉｄｅｓ− ｏｔｈｅｒ ｎｕｃｌｅｏｔｉｄｅｓ ａｒｅ ｄｅｏｘｙｒｉｂｏｎｕｃｌｅｏｔｉｄｅｓ．”
ＳＥＱ ＩＤ ＮＯ：１２ ； Ｔｈｅ ３’−ＯＨ ｇｒｏｕｐ ｏｆ ｎｕｃｌｅｏｔｉｄｅ ａｔ ３’ ｅｎｄ ｉｓ ｐｒｏｔｅｃｔｅｄ ｗｉｔｈ ａｍｉｎｏ ｇｒｏｕｐ．
ＳＥＱ ＩＤ ＮＯ：１３ ； Ｔｈｅ ３’−ＯＨ ｇｒｏｕｐ ｏｆ ｎｕｃｌｅｏｔｉｄｅ ａｔ ３’ ｅｎｄ ｉｓ ｐｒｏｔｅｃｔｅｄ ｗｉｔｈ ａｍｉｎｏ ｇｒｏｕｐ．
ＳＥＱ ＩＤ ＮＯ：１４ ； Ｔｈｅ ３’−ＯＨ ｇｒｏｕｐ ｏｆ ｎｕｃｌｅｏｔｉｄｅ ａｔ ３’ ｅｎｄ ｉｓ ｐｒｏｔｅｃｔｅｄ ｗｉｔｈ ａｍｉｎｏ ｇｒｏｕｐ．
【００７６】
【配列表】

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an improvement in the stability and operating conditions of reagents and kits in gene amplification reactions, and in particular to an improvement in isothermal gene amplification.
[0002]
[Prior art]
In recent years, many techniques for amplifying a target gene have been reported.
For example, PCR (Polymerase Chain Reaction) method (for example, refer to Patent Documents 1 to 3), LCR (Ligase Chain Reaction) method (for example, refer to Patent Document 4), and more recently, temperature cycling is not required, and gene is maintained at a constant temperature. The SDA (Strand Displacement Amplification) method (for example, see Patent Document 5), the improved SDA method (for example, see Patent Documents 6 to 8), the LAMP (Loop-mediated isometric Amplification) method (for example, , Patent Document 9), ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleica ids) method (for example, Patent Document 10 or 11 reference) and various methods have been reported.
[0003]
In the gene amplification method, various solutions have been taken in order to solve the problem that nonspecific amplification frequently occurs. For example, in the PCR method, as one of them, there is a method of solving non-specific amplification by using a nucleic acid polymerase only at a high temperature called a hot start method. This method includes a method using an antibody against a polymerase (for example, see Non-Patent Document 1), a method for trapping magnesium in a wax that melts at a high temperature (for example, see Patent Document 12 or 13), and a certain nucleic acid. There is a method of reversibly inhibiting a nucleic acid polymerase (AmpliTaq Gold (Perkin Elmer)) and the like.
These methods are useful improvements in the PCR method using a denaturation / amplification temperature of 65 ° C. to 98 ° C., but the gene amplification method described above performs a gene amplification reaction at a moderately high temperature of 40 ° C. to 65 ° C. It was a technology that could not be used for such purposes.
[0004]
In addition, as a method for increasing the specificity in the amplification reaction, that is, inhibiting unwanted side reactions, a method in which a non-extendible oligo blocker is set between amplification primers and a PCR reaction is carried out in the presence of the primers (for example, patents) Reference 14), PCR methods using peptide nucleic acids (for example, see Non-Patent Document 2 or Patent Document 15) and the like are disclosed. However, these methods are difficult to adapt to the isothermal gene amplification method at moderately high temperatures. The improved methods listed above are limited to inhibiting non-specific amplification in the PCR method in particular, and do not allow improvement of the stability of the reagent mixture and complicated operation in the isothermal gene amplification method. There wasn't.
[0005]
As another method for increasing the specificity in the amplification reaction, there is a PCR method using a primer that self-anneales (self-looping) at a temperature equal to or lower than the extension reaction temperature (for example, see Non-Patent Document 3). However, in order to prepare such a loop primer, it is necessary to additionally synthesize a 10-mer loop-forming oligonucleotide part in addition to the primer sequence itself. In addition, the primer used in the method requires 30 to 40 mer, and when combined with the loop portion, the total length is 40 to 50 mer, which is not suitable for the use conditions of commonly used primers and is not industrial. . Furthermore, the method requires an annealing temperature of 60 to 72 ° C., and the annealing temperature must be increased by 2 ° C. for each PCR cycle. Thus, this method is very complicated and cannot be used for the isothermal gene amplification method.
[0006]
There is also a PCR method using a double-stranded primer pair and a strand displacement type DNA polymerase (see, for example, Patent Document 16). This method is a homogeneous gene amplification method and is a method in which at least one energy-decay oligonucleotide that cannot be extended to inhibit non-specific binding of a primer to a template strand coexists. However, nothing is said about the stability of the reagent mixture.
[0007]
Furthermore, the nucleic acid polymerase used in the isothermal gene amplification method at moderately high temperatures has an optimum temperature from 40 ° C. to 65 ° C., but exhibits a reasonable primer extension activity even at 5 ° C. to 37 ° C. Therefore, when performing isothermal gene amplification at moderately high temperatures, the amplification mixture is handled on ice and a sample containing nucleic acid is added to the amplification mixture to prevent non-specific amplification from occurring. Later, they were handled on ice, and the reaction was started by putting them in a heater or warm bath heated to a suitable temperature in advance.
Also, when measuring multiple samples at the same time, it takes a considerable amount of time to prepare reaction solutions for all the samples. The reaction solution prepared at the beginning takes the planned reaction start, that is, incubation at a predetermined temperature. In some cases, the reaction progressed before the start or the side reaction progressed, resulting in poor reproducibility.
Furthermore, even if the reagent once prepared is stored in a normal refrigerator, an amplification reaction occurs in several hours to several days, and there is a problem that the storage stability of the prepared reagent is poor.
In order to improve reproducibility, in many of these amplification methods, a target gene and a primer are preliminarily subjected to denaturation annealing, and thus, it is necessary to carry out troublesome pretreatment such as cooling after heating or neutralizing after alkali treatment.
[0008]
[Patent Document 1]
US Pat. No. 4,683,195
[0009]
[Patent Document 2]
U.S. Pat.No. 4,683,202
[0010]
[Patent Document 3]
US Pat. No. 4,800,159
[0011]
[Patent Document 4]
European Patent Application No. 320308
[0012]
[Patent Document 5]
Japanese Patent Publication 7-114718
[0013]
[Patent Document 6]
US Pat. No. 5,196,777
[0014]
[Patent Document 7]
WO95 / 25180 pamphlet
[0015]
[Patent Document 8]
WO99 / 49081 pamphlet
[0016]
[Patent Document 9]
International Publication No. 00/28082 Pamphlet
[0017]
[Patent Document 10]
International Publication No. 00/56877 Pamphlet
[0018]
[Patent Document 11]
WO 02/16639 pamphlet
[0019]
[Patent Document 12]
US Pat. No. 5,599,660
[Patent Document 13]
US Pat. No. 6,403,341
[Patent Document 14]
US Pat. No. 5,849,497
[Patent Document 15]
US Pat. No. 5,891,625
[Patent Document 16]
US Pat. No. 5,348,853
[Non-Patent Document 1]
Biotechniques, 25, 716-722 (1998)
[Non-Patent Document 2]
Nucleic Acids Research, Volume 20, pages 2493-2496 (1992)
[Non-Patent Document 3]
Biotechniques, Vol. 29, pp. 1018-1024 (2000)
[0020]
[Problems to be solved by the invention]
The object of the present invention is to overcome the problem that the amplification reaction proceeds even at a low temperature of 4 ° C. during the operation and storage of the reagent, and the stability and performance of the reagent used for nucleic acid amplification at an isothermal temperature. In addition, it is to greatly improve the ease of use operation.
[0021]
[Means for Solving the Problems]
[0022]
The present inventors are oligonucleotides having a sequence that competes with a target gene sequence for a chimeric oligonucleotide primer used for amplification, and the Tm value between the oligonucleotide and the chimeric oligonucleotide primer, The ratio of the Tm value between the chimeric oligonucleotide primer and the nucleic acid sequence of the target gene substantially complementary to the base sequence of the primer is in the range of 1.2 to 3.3; It has been found that the above problem can be solved by coexisting at least one oligonucleotide blocked so that the terminal cannot be extended in the gene amplification reaction solution.
[0023]
Furthermore, the present inventors have bound an oligonucleotide that cannot be extended to the optimal temperature of the nucleic acid polymerase to the chimeric oligonucleotide primer, and when the optimal temperature is reached, the oligonucleotide dissociates from the primer, It was found that the primer is bound to the target gene, extended by nucleic acid polymerase, and amplified, so that the stability of the reagent is improved even when the target gene and the primer coexist at room temperature. Completed.
[0024]
A first invention of the present invention is a method for stabilizing a gene amplification reagent, wherein an oligonucleotide having a base sequence substantially complementary to a chimeric oligonucleotide primer used in a gene amplification reaction is used. The ratio of the Tm value between the chimeric oligonucleotide primer and the nucleic acid sequence of the target gene substantially complementary to the base sequence of the primer to the Tm value between the chimeric oligonucleotide primer and the chimeric oligonucleotide primer is 1.2 to The present invention also relates to a method characterized in that at least one oligonucleotide that is in the range of 3.3 and is blocked so that the 3 ′ end of the oligonucleotide cannot be extended is allowed to coexist in the gene amplification reaction solution.
[0025]
In the first invention of the present invention, the oligonucleotide may be a DNA or RNA oligonucleotide, or a DNA-RNA chimeric oligonucleotide.
[0026]
The second invention of the present invention relates to a kit for the method of the first invention of the present invention.
[0027]
DETAILED DESCRIPTION OF THE INVENTION
As used herein, deoxyribonucleotide (also referred to herein as dN) refers to a nucleotide whose sugar moiety is composed of D-2-deoxyribose. For example, the base moiety includes adenine, cytosine, guanine, The thing which has thymine is mentioned. Furthermore, deoxyribonucleotide analogs such as deoxyribonucleotides and deoxyinosine nucleotides having modified bases such as 7-deazaguanosine are also included in the above deoxyribonucleotides.
[0028]
In the present specification, ribonucleotide (also referred to as N in the present specification) refers to a nucleotide whose sugar moiety is composed of D-ribose, and whose base moiety has adenine, cytosine, guanine, uracil. Can be mentioned. Furthermore, the ribonucleotide includes a modified ribonucleotide. For example, a modified ribonucleotide in which the oxygen atom of the phosphate group at the α-position is replaced with a sulfur atom [(α-S) ribonucleotide, (α-S) N is also described. And other derivatives.
[0029]
In the present specification, the 3 ′ terminal side refers to a part of a nucleic acid, for example, a primer from the center to the 3 ′ terminal. Similarly, the 5 ′ end side refers to a portion of the nucleic acid from the center to the 5 ′ end.
[0030]
In this specification, DNA polymerase refers to an enzyme that synthesizes a new DNA strand using a DNA strand as a template, and includes naturally occurring DNA polymerase and a mutant enzyme having the above activity. Examples of the enzyme include a DNA polymerase having strand displacement activity, a DNA polymerase not having 5 ′ → 3 ′ exonuclease activity, and a DNA polymerase having both reverse transcriptase activity and endonuclease activity.
[0031]
In this specification, the term “strand displacement activity” means that when DNA replication is performed according to a nucleic acid sequence as a template, it proceeds while replacing the DNA strand to release the complementary strand annealed to the template strand, ie, strand displacement (strand substitution). It refers to an activity that can be displaced. In the present specification, a DNA chain released from a nucleic acid sequence serving as a template by strand replacement is referred to as a “substitution strand”.
[0032]
As used herein, “Tm value” refers to the temperature at which a double strand of DNA is heat denatured to become a single strand. In other words, it refers to the theoretical limit temperature at which the template DNA and oligonucleotide form a double strand. The Tm value can be calculated. Although not particularly limited, for example, the nearest neighbor method, the Wallance method, or the GC% method is exemplified.
[0033]
In the closest base pair method,
Tm = (1000ΔH) / (− 10.8 + ΔS + Rln (Ct / 4)) − 273.15 + 16.6 log [Na +]
ΔH: Sum of nearest enthalpy changes in hybrid [kcal / mol]
−10.8; ΔS (initiation) [cal / mol · K]
ΔS: the sum of nearest entropy changes in the hybrid [cal / mol · K]
R: Gas constant (1.987 cal / deg · mol)
Ct: total molar concentration of oligo [mol / l]
Na ⁺ Na ⁺ Molar concentration of [mol / l]
Although there is no particular limitation, the parameters are the total molar concentration of the oligo of 0.5 μM, Na ⁺ The case where the molar concentration of is set to 50 mM is exemplified.
[0034]
In the Wallance method,
Tm = 2 (A + T) +4 (G + C)
(A, T, G, C: Number of occurrences of each nucleotide)
It can be calculated by
[0035]
In the GC% method,
Tm = 81.5 + 16.6log [Na +] + 41 (XG + XC) −500 / L−0.62F
Na +; molar concentration of Na + [mol / l]
XG, XC; mole fraction of G and C
L: Length of the oligonucleotide forming the double strand
F: molar concentration of formamide
It can be calculated by
[0036]
In the method of the present invention, the Tm value is adjusted by, for example, (1) giving a mutation to the sequence of Sensitive Decoy, (2) introducing a nucleic acid analog, (3) setting a sequence shorter than the primer, (4) use A method such as adjusting the salt concentration of the buffer to be used can be used.
[0037]
Hereinafter, the present invention will be described in detail.
(1) Method of the present invention
The method of the invention is characterized by the use of oligonucleotides for the stabilization of gene amplification reagents. That is, an oligonucleotide having a base sequence substantially complementary to a chimeric oligonucleotide primer used in a gene amplification reaction, the chimeric oligonucleotide primer for the Tm value between the oligonucleotide and the chimeric oligonucleotide primer The ratio of the Tm value between the nucleotide sequence of the primer and the nucleic acid sequence of the target gene substantially complementary to the primer is in the range of 1.2 to 3.3, and the 3 ′ end of the oligonucleotide is used It is preferable that an oligonucleotide modified so that the nucleic acid chain is not extended under the conditions to be present coexist in the gene amplification reaction. As used herein, “substantially complementary nucleic acid sequence” means a nucleic acid sequence that can be annealed to DNA as a template under the reaction conditions used. Hereinafter, the oligonucleotide of the present invention is referred to as “Sensible decay”.
[0038]
The Sensitive Decoy is preferably an oligonucleotide composed of deoxyribonucleotides. The Sensitive Decoy is not particularly limited as long as it is a sequence that can be annealed to the chimeric oligonucleotide primer used in the nucleic acid amplification reaction, and the length may be equal to, greater than or less than the primer length. Good. Preferably, the target oligonucleotide is shorter than the primer length to be used and substantially complementary to the base sequence of the chimeric oligonucleotide primer and the primer with respect to the Tm value between the Sensitive Decoy and the chimeric oligonucleotide primer. Ratio of Tm value between nucleic acid sequence of gene [(Tm value between chimeric oligonucleotide primer and nucleic acid sequence of target gene substantially complementary to base sequence of the primer) / (Sensible decoy and chimeric oligo The Tm value between the nucleotide primer)] is preferably in the range of 1.2 to 3.3, more preferably in the range of 1.5 to 2.5. The Tm value can be calculated by the closest base pair method, the Wallance method, or the GC% method, and the closest base pair method and the Wallance method are particularly preferable.
[0039]
The Sensitive Decoy of the present invention can contain one or more nucleic acid analogs as long as the Sensitive Decay function is not lost, and the nucleic acid analogs can be used in combination of a plurality of types. The nucleic acid analog is not particularly limited. For example, a deoxyribonucleotide analog having a modified base such as deoxyinosine nucleotide, deoxyuracil nucleotide, or 7-deazaguanine, a nucleic acid analog having a ribose derivative, etc. can be used. it can.
[0040]
In addition, the Sensitive Decoy of the present invention is modified so that the nucleic acid chain is not extended under the condition that the 3 ′ end is used. Although the modification method is not particularly limited, a known chemical synthesis method can be used, and methods such as amination, biotinylation, and phosphorylation can be used.
[0041]
The Sensitive Decoy used in the present invention can be synthesized by the phosphoamidite method using, for example, a DNA synthesizer type 394 of Applied Biosystems Inc. (ABI, Applied Biosystems Inc.) so as to have an arbitrary nucleic acid sequence. As another method, there are a phosphoric acid triester method, an H-phosphonate method, a thiophosphonate method, and the like, but they may be synthesized by any method.
[0042]
In the method of the present invention, the above Sensitive Decoy has a nucleotide sequence substantially complementary to a primer used when replicating or amplifying a nucleic acid fragment of a target gene, and therefore can be annealed with a primer. In addition, during the replication or amplification reaction, Sensitive Decay competes with the nucleic acid chain of the target gene to which the primer hybridizes. As a result, in the method of the present invention, the nucleic acid fragment of the target gene can be specifically suppressed, that is, non-specific replication or amplification can be suppressed.
[0043]
In the method of the present invention, as the DNA polymerase, a DNA polymerase having a DNA strand displacement activity can be used. Those having substantially no 5 ′ → 3 ′ exonuclease activity can be particularly preferably used.
The DNA polymerase used in the present invention is not particularly limited as long as it has the above-mentioned strand displacement activity. For example, Bacillus caldotenax (hereinafter referred to as B.ca) or Bacillus stearothermophilus. (Bacillus stearothermophilus, hereinafter referred to as B.st) and other mutants lacking the 5 ′ → 3 ′ exonuclease activity of DNA polymerases derived from thermophilic Bacillus bacteria, and Escherichia coli (hereinafter referred to as E. coli) And a large fragment (Klenow fragment) of the derived DNA polymerase I. In addition, as the DNA polymerase that can be used in the present invention, any of room temperature to heat resistant can be suitably used.
[0044]
B. ca is a thermophilic bacterium having an optimum growth temperature of about 70 ° C. Bca DNA polymerase derived from this bacterium has DNA-dependent DNA polymerase activity, RNA-dependent DNA polymerase activity (reverse transcription activity), 5 ′ → 3 ′ exo It is known to have nuclease activity, 3 ′ → 5 ′ exonuclease activity. The enzyme may be either purified and obtained from its original origin or a recombinant protein produced by genetic engineering. In addition, the enzyme may be modified by substitution, deletion, addition, insertion or the like by genetic engineering or other methods. Examples of such an enzyme include 5 ′ → 3 ′ exonuclease. Examples thereof include Bca DNA polymerase (manufactured by Takara Bio Inc.), which is a Bca DNA polymerase deficient in activity.
[0045]
Moreover, RNaseH in this invention will not be specifically limited if it can be used for the method of this invention, For example, any of various viruses, phages, prokaryotes, and eukaryotes may be sufficient. Further, it may be either cellular RNase H or viral RNase H. For example, E. coli RNaseHI is exemplified as the cellular RNaseH, and HIV-1-derived RNaseH is exemplified as the viral RNaseH. In the method of the present invention, RNase H can be any of type I, type II, and type III. Although not particularly limited, for example, RNaseHI derived from Escherichia coli, Pyrococcus genus, Arkaeoglobus genus bacterium or Thermococcus genus RNaseHII, Bacillus caldotenax, Thermotoga maritima, Thermotoga maritima, Ribonuclease H from Methanococcus jannashi and Thermococcus litoralis can be suitably used.
[0046]
In addition, the efficiency of the cleavage reaction of an endonuclease used in the method of the present invention, for example, RNase H, depends on the base sequence near the 3 ′ end of the primer and may affect the amplification efficiency of the desired DNA. It is natural to design an optimal primer for RNaseH.
[0047]
As a reaction buffer used in the present invention, a buffer containing a buffer component, a magnesium salt, other metal salt, or dNTP is used. In addition, it is natural to optimize the type and concentration of the salt according to the metal requirement of the enzyme used. The buffer component is not particularly limited, and for example, bicine, tricine, hepes, tris, phosphate (sodium phosphate, potassium phosphate, etc.) can be preferably used. In particular, a buffer containing bicine, tricine, hepes, or phosphate as a buffer component is suitable for the present invention. Therefore, an optimal buffer may be selected depending on the reaction temperature, the endonuclease or DNA polymerase to be used.
[0048]
The final concentration of the buffer component is in the range of 5 mM to 100 mM, particularly preferably in the range of 15 mM to 50 mM, and pH 6.0 to 9.5, particularly preferably in the range of pH 7.0 to 9.2 is used. The For example, a pH 7.5-9.2 buffer containing 22 mM-46 mM tricine, a pH 7.0-8.0 buffer containing 25 mM-50 mM potassium phosphate, 16 mM-40 mM Hepes-potassium hydroxide. The buffer of pH 7.0-8.0 which contains can be used conveniently. Further, the magnesium salt is not particularly limited, but for example, magnesium chloride, magnesium acetate or magnesium sulfate can be suitably used, and its concentration is in the range of 1 mM to 20 mM, particularly preferably 2 mM to 10 mM in the final concentration. . Moreover, the potassium salt may be included, for example, in the case of potassium acetate, the range of 80 mM-120 mM is preferable at final concentration. Furthermore, dNTPs (dATP, dCTP, dGTP, dTTP mixture) serving as a substrate for the DNA elongation reaction are each in the final concentration range of 0.1 mM to 3.0 mM, particularly preferably 0.2 mM to 1.2 mM. The amount of the primer to be used is in the range of 1 pmol to 1000 pmol per 50 μl of the reaction solution, and particularly preferably in the range of 10 pmol to 150 pmol. Furthermore, additives intended to stabilize the amplification reaction and the like can coexist in the reaction solution, each having a final concentration of 0.1% or less bovine serum albumin (BSA), 10% or less dimethyl sulfoxide ( DMSO) 4 mM or less putrescine dihydrochloride or 0.01% or less propylenediamine may be added. In addition, NMP (1-methyl-2-pyrrolididinone), glycerol, polyethylene glycol, dimethyl sulfoxide and / or formamide may be included, and the addition of these organic solvents reduces non-specific annealing of the oligonucleotide primer. It is expected that
[0049]
In this specification, the nucleic acid used as a template, that is, DNA or RNA, may be prepared or isolated from any sample that may contain the nucleic acid. Furthermore, the above sample may be directly used for the nucleic acid amplification reaction of the present invention. The sample containing such nucleic acid is not particularly limited, and examples thereof include whole blood, serum, buffy coat, urine, feces, cerebrospinal fluid, semen, saliva, tissue (eg, cancer tissue, lymph node, etc.), cell, and the like. Biological samples such as cultures (eg mammalian cell cultures and bacterial cultures), nucleic acid containing samples such as viroids, viruses, bacteria, molds, yeasts, plants and animals, microorganisms such as viruses or bacteria May be contaminated or infected (food, biological preparations, etc.), or samples that may contain organisms such as soil and wastewater. Moreover, the nucleic acid containing preparation obtained by processing the said sample etc. by a well-known method may be sufficient. As the preparation, for example, a cell disrupted product, a sample obtained by fractionating the same, a nucleic acid in the sample, or a specific nucleic acid molecule group, for example, a sample enriched with mRNA can be used in the present invention. Furthermore, a nucleic acid such as DNA or RNA obtained by amplifying the nucleic acid contained in the sample by a known method can be suitably used.
[0050]
The preparation of the nucleic acid-containing preparation from these materials is not particularly limited, and can be performed, for example, by dissolution treatment with a surfactant, ultrasonic treatment, shaking and stirring using glass beads, use of a French press, or the like. In some instances, it may be advantageous to further manipulate and purify the nucleic acid (eg, when an endogenous nuclease is present). In these examples, nucleic acid purification is performed by known methods such as phenol extraction, chromatography, ion exchange, gel electrophoresis, or density gradient centrifugation.
[0051]
The nucleic acid amplification reaction used in the present invention is not particularly limited, but a known nucleic acid amplification method such as a PCR method, an LCR method, an SDA method, an improved SDA method, a LAMP method, or an ICAN method may be used. Preferably, the ICAN method can be used.
[0052]
When it is desired to amplify a nucleic acid having a sequence derived from RNA, the method of the present invention may be carried out using as a template cDNA synthesized by reverse transcription using the RNA as a template. The RNA that can be applied to the method of the present invention is not particularly limited as long as the primer used for the reverse transcription reaction can be prepared. In addition to the total RNA in the sample, mRNA, tRNA, rRNA, etc. An RNA molecule group or a specific RNA molecular species can be mentioned.
[0053]
(2) Kit of the present invention
The present invention provides a kit for use in a method for amplifying or detecting a nucleic acid of a target gene. In one embodiment, the kit is characterized in that it contains instructions for use of DNA polymerase and endonuclease in the method of the invention in packaged form. Furthermore, a kit containing a DNA polymerase having strand displacement activity, an endonuclease, and a strand displacement reaction buffer is preferably used in the method of the present invention. Alternatively, a commercially available DNA polymerase and / or endonuclease having strand displacement activity may be selected and used according to the instructions. Furthermore, a reagent for reverse transcription reaction using RNA as a template may be included.
[0054]
The above “instructions” are printed materials that describe how to use the kit, for example, how to prepare reagent solutions for the method of the present invention, recommended reaction conditions, etc. In addition, the label attached to the kit, the one written on the package containing the kit, etc. are included. Furthermore, information disclosed and provided through an electronic medium such as the Internet is also included.
[0055]
Further, the kit of the present invention may contain a reaction buffer containing bicine, tricine, hepes, phosphate, or a tris buffer component, and an annealing solution. Moreover, DNA polymerase and RNaseH which have strand displacement ability may be contained. Further, it may contain a modified deoxyribonucleotide or deoxynucleotide triphosphate analog.
[0056]
Furthermore, the kit used for the target nucleic acid detection method includes the above-mentioned instructions, reagents for amplification reaction, chimeric oligonucleotide primer suitable for target nucleic acid amplification, Sensitive decoy, and amplified target nucleic acid. It may contain a reagent for detection, such as a probe.
The kit of the present invention also includes a kit containing the chimeric oligonucleotide primer, Sensitive decoy and / or the probe of the present invention used in the present invention.
[0057]
【Example】
EXAMPLES The present invention will be specifically described below with reference to examples, but the scope of the present invention is not limited to these examples.
In addition, the synthetic DNA used in the following examples was produced by a synthesizer manufactured by ABI.
The ICAN method was carried out by the method described in WO 02/16639 pamphlet. In addition, Afu (Archaeoglobus fulgidus) -derived RNase H used in the present invention was prepared by the method described in WO 02/22831.
[0058]
Example 1 Stabilization of ICAN Amplification Reagent Mixture in ICAN Method by Sensitive Decoy
Example 1
(1) Examination of stabilization of amplification reagent mixture at room temperature in the amplification of Chlamydia / phosphorus gene by ICAN method
A sense primer (SEQ ID NO: 1) and an antisense primer (SEQ ID NO: 2) labeled with biotin at the 5 ′ end were designed and synthesized from the chlamydia (CT) cryptoplasmid region. In addition, a sense primer (SEQ ID NO: 3) in which the 3 ′ end was amino-modified and an antisense primer (SEQ ID NO: 4) in which the 3 ′ end was amino-modified were designed and synthesized as a sensitive decoy.
A sense primer (SEQ ID NO: 5) and an antisense primer (SEQ ID NO: 6) labeled with biotin at the 5 ′ end were designed and synthesized from the gonococcal (NG) cppB gene. In addition, a sense primer (SEQ ID NO: 7) in which the 3 ′ end was amino-modified and an antisense primer (SEQ ID NO: 8) in which the 3 ′ end was amino-modified were designed and synthesized as a sensitive decoy.
[0059]
An amplification reagent mixture containing only the above primer and an amplification reagent mixture containing the primer and Sensitive Decoy were prepared. The composition is shown in Table 1 below. (Displays the volume per 18 reactions)
[0060]
[Table 1]

[0061]
To this amplification reagent, 40 μl of 90 mM magnesium acetate solution and 20 μl of internal control DNA (0.9 pg / μl) were added and left at room temperature for 70 minutes. Thereafter, 25 μl was dispensed per reaction, an equal volume of template DNA was added, and an amplification reaction was carried out at 55 ° C. for 1 hour without denaturation / annealing operation. After amplification, the amplification product was detected by solid-layer plate luminescence. The results are shown in Table 2 below.
[0062]
[Table 2]

[0063]
As shown in Table 2, amplification reagent A (conventional method) caused amplification failure and failed to detect the target gene. On the other hand, amplification reagent B showed a stable amplification reaction and was able to detect the target gene. That is, it was confirmed that the method of the present invention contributes to stabilization of the amplification reaction.
[0064]
(2) Examination of stabilization of amplification reagent mix in refrigeration in the amplification of Chlamydia / phosphorus gene by ICAN method
In the same manner as in Example 1- (1) above, 40 μl of 90 mM magnesium acetate solution and 20 μl of internal standard DNA (0.9 pg / μl) were added to each of amplification reagents A and B, and stored at 4 ° C. for 1 week. A refrigerated storage test was conducted. The results are shown in Table 3 below.
[0065]
[Table 3]

[0066]
As shown in Table 3, the amplification reagent A was able to detect the target genes CT and NG, but caused an IC (internal standard) amplification failure, and no IC was detected even in the 0 copy section. On the other hand, amplification reagent B showed stable amplification without any denaturation / annealing operation, and all target genes could be detected. That is, it was confirmed that the method of the present invention is suitable for storage and stabilization at a low temperature and for a long period.
[0067]
Example 2 Improvement of reproducibility in amplification of Chlamydia / phosphorus gene by ICAN method
In the same manner as in Example 1- (1) above, 40 μl of 90 mM magnesium acetate solution and 20 μl of internal standard DNA (0.9 pg / μl) were added to amplification reagent A and amplification reagent B, respectively, and low copy number CT, Using the NG template, the reaction was repeated 10 times without denaturation / annealing, and the simultaneous reproducibility was compared. The results are shown in Table 4 below.
[0068]
[Table 4]

[0069]
As shown in Table 4, better reproducibility was obtained with the amplification reagent B containing the Sensitive Decoy of the present invention.
[0070]
Example 3 Stabilization of amplification in HPV6, 11 gene ICAN amplification
From the HPV6 gene, a sense primer (SEQ ID NO: 9) and an antisense primer (SEQ ID NO: 10) labeled with biotin at the 5 ′ end were designed and synthesized. In addition, a sense primer (SEQ ID NO: 12) in which the 3 ′ end was amino-modified and an antisense primer (SEQ ID NO: 13) in which the 3 ′ end was amino-modified were designed and synthesized as a sensitive decoy.
An antisense primer (SEQ ID NO: 11) was designed and synthesized from the HPV11 gene. As the sense primer, a sense primer (SEQ ID NO: 9) in which the 5 ′ end of the HPV6 gene was labeled with biotin was used. In addition, an antisense primer (SEQ ID NO: 14) in which the 3 ′ end was amino-modified was designed and synthesized as a sensitive decoy. As the sense primer, a sense primer (SEQ ID NO: 12) obtained by amino-modifying the 3 ′ end of the HPV6 gene was used.
[0071]
In the same manner as in Example 1- (1) above, amplification reagent A not containing Sensitive Decoy and amplification reagent B containing Sensitive Decoy were prepared, and 40 μl of 90 mM magnesium acetate solution and 20 μl of internal standard DNA (0 .9 pg / μl) and isothermal amplification without denaturation / annealing operation. The results are shown in Table 5 below.
[0072]
[Table 5]

[0073]
As shown in Table 5, as a result of comparing the amplification of HPV6 and 11DNA, improvement in amplification at a low copy number was recognized by using the method of the present invention.
[0074]
【The invention's effect】
The above gene amplification method using the oligonucleotide of the present invention is useful for an isothermal gene amplification method at moderately high temperatures. Further, the method of the present invention is effective as a method for storing and stabilizing an amplification reagent.
[0075]
[Sequence Listing Free Text]
SEQ ID NO: 1; Chimeric oligomeric to detect Chlamydia trachomatis cryptoplasmid gene. “Nucleotides 19 to 22 are ribonucleotides—other nucleosides are deoxyribonucleotides.”
SEQ ID NO: 2; Chimeric oligomeric to detect Chlamydia trachomatis cryptographic plasmid gene. “Nucleotides 16 to 18 are ribonucleotides—other nucleosides are deoxyribonucleotides, and 5 ′ end is labeled by biotin.”
SEQ ID NO: 3; The 3′-OH group of nucleotides at 3 ′ end is protected with amino group.
SEQ ID NO: 4; The 3′-OH group of nucleotides at 3 ′ end is protected with amino group.
SEQ ID NO: 5; Chimeric oligonucleotide to detect Neisseria gonorrhoea cppB gene. “Nucleotides 18 to 20 are ribonucleotides—other nucleosides are deoxyribonucleotides.”
SEQ ID NO: 6; Chimeric oligonucleotide to detect Neisseria gonorrhoea cppB gene. “Nucleotides 15 to 17 area ribonucleotides—other nucleosides are deoxyribonucleotides, and 5 ′ end is labeled by biotin.”
SEQ ID NO: 7; The 3′-OH group of nucleotides at 3 ′ end is protected with amino group.
SEQ ID NO: 8; The 3′-OH group of nucleotides at 3 ′ end is protected with amino group.
SEQ ID NO: 9; Chimeric oligonucleotide to detect HPV6 gene. “Nucleotides 18 to 20 are ribonucleotides—other nucleosides are deoxyribonucleotides, and 5 'end is labeled by biotin.”
SEQ ID NO: 10; Chimeric oligonucleotide to detect HPV6 gene. “Nucleotides 20 to 22 area ribonucleotides—other nucleosides are deoxyribonucleotides.”
SEQ ID NO: 11; Chimeric oligonucleotide to detect HPV11 gene. “Nucleotides 20 to 22 area ribonucleotides—other nucleosides are deoxyribonucleotides.”
SEQ ID NO: 12; The 3′-OH group of nucleotides at 3 ′ end is protected with amino group.
SEQ ID NO: 13; The 3′-OH group of nucleotides at 3 ′ end is protected with amino group.
SEQ ID NO: 14; The 3′-OH group of nucleotides at 3 ′ end is protected with amino group.
[0076]
[Sequence Listing]

Claims (3)

A method for stabilizing a gene amplification reagent, wherein an oligonucleotide having a base sequence substantially complementary to a chimeric oligonucleotide primer used in a gene amplification reaction is used, and between the oligonucleotide and the chimeric oligonucleotide primer The ratio of the Tm value to the Tm value between the chimeric oligonucleotide primer and the nucleic acid sequence of the target gene substantially complementary to the base sequence of the primer is in the range of 1.2 to 3.3; A method comprising coexisting at least one oligonucleotide blocked so that the 3 ′ end of the oligonucleotide cannot be extended in a gene amplification reaction solution. The method according to claim 1, wherein the oligonucleotide is a DNA or RNA oligonucleotide or a DNA-RNA chimeric oligonucleotide. Kit for the method according to claim 1 or 2.