JP7443251B2 - 核酸の合成後修飾のための試薬及び方法 - Google Patents
核酸の合成後修飾のための試薬及び方法 Download PDFInfo
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- JP7443251B2 JP7443251B2 JP2020566283A JP2020566283A JP7443251B2 JP 7443251 B2 JP7443251 B2 JP 7443251B2 JP 2020566283 A JP2020566283 A JP 2020566283A JP 2020566283 A JP2020566283 A JP 2020566283A JP 7443251 B2 JP7443251 B2 JP 7443251B2
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Description
別段の定義がない限り、本明細書で使用される全ての技術用語及び科学用語は、本発明が属する技術分野の当業者によって一般的に理解されている意味と同じ意味を有する。本明細書に記載されるものと同様である本質的に任意の方法及び材料が本発明の実施又は試験で使用され得るが、例示的な方法及び材料のみを記載する。本発明の目的のために、次の用語を以下のように定義する。
式中、aは0~4の整数である。好ましくは、aは2であり、リン酸エステルはリボース、例えば、リボース3’-三リン酸、2’-デオキシリボース3’-三リン酸、リボース5’-三リン酸、2’-デオキシリボース5’-三リン酸、3’-ハロリボース5’-三リン酸、3’-アルキルリボース5’-三リン酸、2’,3’-ジデオキシリボース5’-三リン酸等の3’又は5’炭素に結合している。
用語「制限エンドヌクレアーゼ」及び「制限酵素」とは、特定のヌクレオチド配列で又は特定のヌクレオチド配列の近傍で、二本鎖DNAを切断する酵素、典型的には細菌由来の酵素を指す。
b)CS6 DNAポリメラーゼ
c)Thermotoga maritimaDNAポリメラーゼ
d)Thermus aquaticusDNAポリメラーゼ
e)Thermus thermophilusDNAポリメラーゼ
f)Thermus flavusDNAポリメラーゼ
g)Thermus filiformisDNAポリメラーゼ
h)Thermus sp.sps17DNAポリメラーゼ
i)Thermus sp.Z05 DNAポリメラーゼ
j)Thermotoga neapolitanaDNAポリメラーゼ
k)Termosipho africanusDNAポリメラーゼ
l)Thermus caldophilusDNAポリメラーゼ
T-G-R-L-S-S-Xb7-Xb8-P-N-L-Q-Nのモチーフを有する変異型DNAポリメラーゼであってもよく;ここで、Xb7は、S又はTから選択されるアミノ酸であり、Xb8は、G、T、R、K、又はLから選択されるアミノ酸であり、ポリメラーゼは、3’~5’エキソヌクレアーゼ活性を含み、野生型DNAポリメラーゼと比較して核酸伸長率及び/又は逆転写効率が改善されており、該野生型DNAポリメラーゼにおいて、Xb8は、D、E、又はNから選択されるアミノ酸である。
DNA前処理:標準的な固相DNA合成から得られた未精製DNAオリゴヌクレオチド(クルード)を、標準的な方法で脱保護し、脱塩して、残留アミンの任意の痕跡を除去した。オリゴヌクレオチド(合成スケール1μmol)を凍結乾燥し、温浴で補助しながら水(100μL)に再溶解した。塩化リチウム(80μL、10M)の水溶液を加え、混合物を10分間激しく振盪した。氷冷エタノール(3.3mL)を加え、チューブを数回穏やかに反転させた。混合物を-20℃で20分間冷却し、予め冷却した遠心機で7℃にて30分間遠心分離した(2500rpm)。無色の上清を注意深くデカントし、廃棄した。残った固体を乾燥エタノールで洗浄し、高真空で乾燥し、水性緩衝液(1mL、50mM、pH8.0~9.0)に再溶解した。緩衝液は、ホウ酸塩、炭酸塩、HEPBS、HEPPS、又はTEABから選択した。DNAの分析量をUPLCで分析して、アミノ修飾標的配列の量を決定した。260nmにおける標的DNA配列の算出された吸光係数を用いて、分光光度計で全核酸濃度を決定した。
DNAプローブの品質及び機能性は、DMT-MM試薬を用いて調製されたプローブと、従来のNHSエステル化学を用いて調製されたプローブとのqPCR性能を直接比較することにより検討した。qPCRはRoche cobas(登録商標)LIAT(登録商標)システムで行った。標的DNA配列はTrichomonas vaginalis及びMycoplasma genitalium病原菌に基づいた。
DNAオリゴヌクレオチド出発物質は、15μmolスケールの標準固相合成から得られ、精製することなく標識反応に使用された。
Claims (13)
- 検出可能な標識による核酸の合成後修飾のための方法であって、前記方法が、以下:
(a)1つ以上のアミノ修飾を含む、アミノ修飾核酸分子を調製する工程;と、
(b)弱配位性又は非配位性アニオンである対アニオンの存在下において、4-(4,6-ジメトキシ-1,3,5-トリアジン-2-イル)-4-メチルモルホリニウム(DMT-MM)カチオンによりカルボキシ修飾標識を活性化する工程、ここで、前記弱配位性又は非配位性アニオンがカルボン酸ではない;と、
(c)前記アミノ修飾核酸分子を前記活性化カルボキシ修飾標識と反応させて、標識された核酸分子を生成する工程と、
を含み、
前記検出可能な標識が、蛍光色素、発光色素、消光色素、リン光色素、及びアフィニティタグからなる群より選択され、そして
前記弱配位性又は非配位性アニオンが、テトラフルオロホウ酸イオン(BF 4 - )、ヘキサフルオロリン酸イオン(PF 6 - )、ベンゼンスルホン酸イオン(CH 5 SO 3 - )、ビス(トリフルオロメタンスルホニル)イミド((N[SO 2 (CF 3 )] 2 ) - )、臭化物イオン(Br - )、10-カンファースルホン酸イオン(10-camphorosulfonate)、ヨウ化物イオン(I - )、メタンスルホン酸イオン(メシラート、CH 3 SO 3 - )、過塩素酸イオン(ClO 4 - )、リン酸イオン(PO 4 3- )、硫酸イオン(SO 4 2- )、テトラクロロアルミン酸イオン(AlCl 4 - )、テトラキス[3,5-ビス(トリフルオロメチル)フェニル]ホウ酸イオン(B[3,5-(CF 3 ) 2 C 6 H 3 ] 4 - )、テトラキス(ヘキサフルオロイソプロピル)アルミン酸イオン(Al[OC(CF 3 ) 3 ] 4 - )、テトラキス(ペンタフルオロフェニル)ホウ酸イオン[B(C 6 F 5 ) 4 - ]、p-トルエンスルホン酸イオン(トシラート、CH 3 C 6 H 4 SO 3 - )、及びトリフルオロメタンスルホン酸イオン(トリフラート、CF 3 SO 3 - )からなる群より選択される、方法。 - 前記弱配位性又は非配位性アニオンが、テトラフルオロホウ酸イオン(BF4 -)又はヘキサフルオロリン酸イオン(PF6 -)である、請求項1に記載の方法。
- 前記核酸分子が、オリゴヌクレオチドである、請求項1又は2に記載の方法。
- 前記オリゴヌクレオチドが、5’末端又は3’末端でアミノ修飾されている、請求項3に記載の方法。
- 前記オリゴヌクレオチドが、内部でアミノ修飾されている、請求項3に記載の方法。
- 前記オリゴヌクレオチドが、ホスホロアミダイトに基づくDNA合成を用いて合成される、請求項3~5のいずれか一項に記載の方法。
- 前記標識が、蛍光色素である、請求項1~6のいずれか一項に記載の方法。
- 前記蛍光色素が、ホスホロアミダイトに基づくDNA合成と本質的に不適合である、請求項7に記載の方法。
- 前記蛍光色素が、スルホン酸塩部分を含む、請求項1~8のいずれか一項に記載の方法。
- 前記工程がオリゴヌクレオチド合成中に行われ、ここで、前記オリゴヌクレオチドが固体支持体に結合している、請求項1~9のいずれか一項に記載の方法。
- 前記核酸分子が、DNA、RNA、ロックド核酸(LNA)、ペプチド核酸(PNA)、核酸類似体、並びにDNA、RNA、LNA、PNA、及び核酸類似体のハイブリッド混合物からなる群より選択される、請求項1~10のいずれか一項に記載の方法。
- 請求項1~11のいずれか一項に記載の方法を実施することを含む、標識される標識を有する、合成後修飾を含む核酸を製造するための方法。
- 請求項1~11のいずれか一項に記載の方法を実施することを含む、標識される標識を有する、合成後修飾を含む核酸を備えるキットを製造するための方法。
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