JP6896620B2 - ロックされた核酸を有する配列変換およびシグナル増幅dnaならびにそれを用いた検出方法 - Google Patents
ロックされた核酸を有する配列変換およびシグナル増幅dnaならびにそれを用いた検出方法 Download PDFInfo
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Description
配列表
本出願は配列表を含み、この配列表はその内容が参照により組み込まれ、「12350USL1_SeqList.TXT」というファイル名のテキストファイルとして本出願の申請と共に提出される。配列表ファイルは2014年10月7日に作成され、サイズは17,464バイトである。
本明細書で開示される研究の少なくとも一部は、日本政府機関である国立研究開発法人科学技術振興機構(JST)の補助金による支援を受けた。
一態様において、本開示は試料中の標的核酸を検出する方法に関するものであって、前記方法は5’から3’方向に、シグナルDNA生成配列(A)、エンドヌクレアーゼ認識部位(B)、および標的核酸の3’末端と相補的な配列であって、少なくとも1つの化学的に修飾されたヌクレオチドを有する配列(C)を含むオリゴヌクレオチド(配列変換DNAまたはSC DNA)、ポリメラーゼ、ならびにニッキング反応のためのエンドヌクレアーゼと前記試料を接触させることを含む。本態様の実施形態において、本方法は、シグナルDNAの存在が試料中の標的核酸の存在を示す、シグナルDNAの有無を決定することも含む。
反応は、ヒトhsa−miR−24(配列番号33)と同一のDNA配列の標的DNAのさまざまな量(1nM、100pM、10pM、1pM、100fM、10fMまたは1fM)の存在下または非存在下におけるシグナルDNAの生成を検出するために、SC DNA #246
反応は、ヒトhsa−miR−24(配列番号33)と同一のDNA配列の標的DNAの存在下(1nM、100pM、10pM、1pM、100fM)または非存在下におけるシグナルDNAの生成を検出するために、SC DNA #246(配列番号32)およびSA DNA #339(配列番号25)を用いて、さまざまな温度(20℃、25℃、30℃、37℃、45℃および50℃)において実行された。SC DNA #246およびSA DNA #339の両方は、イオン交換HPLCを用いた精製された。
さらなる反応は、ヒトhsa−miR−24(配列番号33)と同一のDNA配列の標的DNAの存在下(100pM)または非存在下におけるシグナルDNAの生成を検出するために、多数の異なるSA DNA(下記表3に記載)と共にSC DNA # 246(配列番号32)を用いて実行された。
さらなる反応は、ヒトhsa−miR−24(配列番号33)と同一のDNA配列の標的DNAの存在下(1nM、100pM、10pM、1pMおよび100fM)または非存在下におけるシグナルDNAの生成を検出するために、多数の異なるSA DNA(下記表4に記載)と共にSC DNA #246(配列番号32)を用いて実行された。表4に示されるように、実験は未精製またはHPLC精製SA DNAのいずれかを用いて実行された。
Claims (19)
- 試料中の標的核酸を検出する方法であって、
5’から3’方向に、シグナルDNA生成配列、エンドヌクレアーゼ認識部位、および標的核酸の3’末端と相補的な配列であって、ロックされた核酸を含む配列を含む第1のオリゴヌクレオチド、
5’から3’方向に、第1のオリゴヌクレオチドのシグナルDNA生成配列によって生成したシグナルDNA配列に相補的な第1のシグナルDNA生成配列、エンドヌクレアーゼ認識部位、および第1のオリゴヌクレオチドのシグナルDNA生成配列によって生成したシグナルDNA配列に相補的な配列であって、ロックされた核酸を含む第2の配列を含む第2のオリゴヌクレオチド、
ポリメラーゼならびに
ニッキング反応のためのエンドヌクレアーゼ
と試料を接触させること、並びに
シグナルDNAの存在が試料中の標的核酸の存在を示す、シグナルDNAの有無を決定すること
を含む方法。 - ロックされた核酸がオリゴヌクレオチドの3’末端から1、2、3、4、5、6、7、8、9、10位またはそれらの組合せの位置にある、請求項1に記載の方法。
- 第2のオリゴヌクレオチドが2、3または4つのロックされた核酸を含む、請求項1に記載の方法。
- ロックされた核酸が第2のオリゴヌクレオチドの3’末端から3および6位の位置にある、請求項3に記載の方法。
- +/−2℃または+/−5℃の範囲内の実質的に定温で実行される、請求項1に記載の方法。
- 約20℃から約42℃の温度で実行される、請求項1から5のいずれかに記載の方法。
- 標的がマイクロRNAであるか、または感染性因子に由来する、請求項1から5のいずれかに記載の方法。
- 5’から3’方向に、既知のシグナルDNA配列と相補的な第1の配列、エンドヌクレアーゼ認識部位、および第1の配列と同一の既知のシグナルDNA配列と相補的な第2の配列を含む化学的に修飾されたオリゴヌクレオチドであって、第2の配列はオリゴヌクレオチドの3’末端から1、2、3、4、5、6、7、8、9、10位またはそれらの組合せの位置にあるロックされた核酸を含む、化学的に修飾されたオリゴヌクレオチド。
- ロックされた核酸がオリゴヌクレオチドの3’末端から3および6位の位置にある、請求項8に記載の化学的に修飾されたオリゴヌクレオチド。
- a)オリゴヌクレオチドの3’末端から6位と他の1−5および7−10位のいずれかとの組合せ、
b)オリゴヌクレオチドの3’末端から1および2位、
c)オリゴヌクレオチドの3’末端から1および10位、
d)オリゴヌクレオチドの3’末端から2および10位、
e)オリゴヌクレオチドの3’末端から4および8位、
f)オリゴヌクレオチドの3’末端から5および9位、
g)オリゴヌクレオチドの3’末端から1および8位、
h)オリゴヌクレオチドの3’末端から2および8位、
i)オリゴヌクレオチドの3’末端から3および8位ならびに
j)オリゴヌクレオチドの3’末端から2および7位
からなる群から選択される位置にロックされた核酸を含む、請求項8に記載の化学的に修飾されたオリゴヌクレオチド。 - 第2の配列が3つのロックされた核酸を含む、請求項8に記載の化学的に修飾されたオリゴヌクレオチド。
- ロックされた核酸が、
a)オリゴヌクレオチドの3’末端から6位と1−5および7−10位のいずれか2つとの組合せ、ならびに
b)オリゴヌクレオチドの3’末端から1、2および10位
からなる群から選択される位置にある、請求項11に記載の化学的に修飾されたオリゴヌクレオチド。 - 第2の配列が4つのロックされた核酸を含む、請求項8に記載の化学的に修飾されたオリゴヌクレオチド。
- ロックされた核酸がオリゴヌクレオチドの3’末端から6位、ならびに1−5および7−10位の3つの位置のその他の組合せである、請求項13に記載の化学的に修飾されたオリゴヌクレオチド。
- 3’末端修飾をさらに含む、請求項8から14のいずれかに記載の化学的に修飾されたオリゴヌクレオチド。
- 試料中の標的核酸を検出する組成物であって、
5’から3’方向に、シグナルDNA生成配列、エンドヌクレアーゼ認識部位、および標的核酸の3’末端と相補的な配列であって、ロックされた核酸を含む配列を含む第1のオリゴヌクレオチド、
5’から3’方向に、第1のオリゴヌクレオチドのシグナルDNA生成配列によって生成したシグナルDNA配列に相補的な第1のシグナルDNA生成配列、エンドヌクレアーゼ認識部位、および第1のオリゴヌクレオチドのシグナルDNA生成配列によって生成したシグナルDNA配列に相補的な配列であって、ロックされた核酸を含む第2の配列を含む第2のオリゴヌクレオチド、
ポリメラーゼならびに
ニッキング反応のためのエンドヌクレアーゼ
を含む組成物。 - ロックされた核酸が第2のオリゴヌクレオチドの3’末端から1、2、3、4、5、6、7、8、9、10位またはそれらの組合せの位置にある、請求項16に記載の組成物。
- ロックされた核酸が第2のオリゴヌクレオチドの3’末端から3および6位の位置にある、請求項16に記載の組成物。
- 試料中の標的核酸を検出するキットであって、
5’から3’方向に、シグナルDNA生成配列、エンドヌクレアーゼ認識部位、および標的核酸の3’末端と相補的な配列であって、ロックされた核酸を含む配列を含む第1のオリゴヌクレオチド、
5’から3’方向に、第1のオリゴヌクレオチドのシグナルDNA生成配列によって生成したシグナルDNA配列に相補的な第1のシグナルDNA生成配列、エンドヌクレアーゼ認識部位、および第1のオリゴヌクレオチドのシグナルDNA生成配列によって生成したシグナルDNA配列に相補的な配列であって、ロックされた核酸を含む第2の配列を含む第2のオリゴヌクレオチド、
ポリメラーゼならびに
ニッキング反応のためのエンドヌクレアーゼ
を含むキット。
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JP6773651B2 (ja) | 2020-10-21 |
CN107075585A8 (zh) | 2019-02-15 |
US20160102339A1 (en) | 2016-04-14 |
CN107109473A (zh) | 2017-08-29 |
JP2017530715A (ja) | 2017-10-19 |
EP3207159A2 (en) | 2017-08-23 |
WO2016059473A2 (en) | 2016-04-21 |
EP3207159A4 (en) | 2018-05-30 |
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