NUCLEIC ACID DETECTION METHOD, NUCLEIC ACID DETECTION APPARATUS, AND NUCLEIC ACID DETECTION KIT

• Main Authors: YAMOTO RIE, YONEKAWA YUKI, YUMOTO MASAYUKI, SUZUKI SACHIE, MAEDA NORIO
• Format: Patent
• Language: English; Japanese
• Published: 19.09.2019
• Subjects: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; BEER; BIOCHEMISTRY; CHEMISTRY; COMPOSITIONS OR TEST PAPERS THEREFOR; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL ORENZYMOLOGICAL PROCESSES; ENZYMOLOGY; MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEICACIDS OR MICROORGANISMS; METALLURGY; MICROBIOLOGY; MUTATION OR GENETIC ENGINEERING; PROCESSES OF PREPARING SUCH COMPOSITIONS; SPIRITS; VINEGAR; WINE

To provide a nucleic acid detection method with which it may simply and quantitatively detected whether all cytosines in a target nucleic acid have been unmethylated or not.SOLUTION: There is provided a nucleic acid detection method comprising: a conversion step of converting unmethylated cytosines in a target nucleic acid into uracil to obtain a nucleic acid sample; an amplification step of amplifying a target site containing at least one of uracil or methylated cytosine from the nucleic acid sample to obtain amplified nucleic acid fragments; a circularization step of circularizing the amplified nucleic acid fragments so as to obtain circularized nucleic acid fragments; a nucleic acid extension step of subjecting the circularized nucleic acid fragments to (a) a nucleic acid extension reaction with a sense strand extension primer complementary to a site in a sense strand of the circularized nucleic acid fragments and bases for obtaining extended nucleic acid fragments, or (b) a nucleic acid extension reaction with an antisense strand extension primer complementary to a site in an antisense strand of the circularized nucleic acid fragments and bases for obtaining extended nucleic acid fragments; and a detection step of detecting the extended nucleic acid fragments.SELECTED DRAWING: None 【課題】標的核酸中の全てのシトシンが非メチル化されているか否かを簡便かつ定量的に検出することができる核酸検出方法の提供。【解決手段】標的核酸中の非メチル化シトシンをウラシルに変換して核酸試料を得る変換工程と、核酸試料からウラシル及びメチル化シトシンの少なくともいずれかを含む標的箇所を増幅して増幅核酸断片を得る増幅工程と、増幅核酸断片を環状化して環状化核酸断片を得る環状化工程と、環状化核酸断片に対し、（ａ）環状化核酸断片のセンス鎖の一部に相補的な塩基配列を有するセンス鎖用伸長反応プライマー、並びに基質を用いる核酸伸長反応により伸長核酸断片を得る、（ｂ）環状化核酸断片のアンチセンス鎖の一部に相補的な塩基配列を有するアンチセンス鎖用伸長反応プライマー、並びに基質を少なくとも用いる核酸伸長反応により伸長核酸断片を得る、核酸伸長工程と、伸長核酸断片を検出する検出工程と、を含む核酸検出方法。【選択図】なし