JP5689061B2 - 逆転写ポリメラーゼ連鎖反応用の組成物 - Google Patents
逆転写ポリメラーゼ連鎖反応用の組成物 Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
なお、本願は、2009年9月1日出願の日本国特許出願第2009−201561号に対して優先権を主張するものであり、日本国特許出願第2009−201561号の全内容を本願に組み込むものである。
本発明の組成物は、耐熱性DNAポリメラーゼ、逆転写酵素、色素マーカー、比重増加剤、及び反応緩衝剤を含む。
本発明のcDNAの合成方法は、本発明のRT−PCR用の反応液を逆転写ポリメラーゼ連鎖反応に供する工程を含む。当該逆転写ポリメラーゼ連鎖反応は、逆転写反応終了後にそのままポリメラーゼ連鎖反応を実施するワンステップ逆転写ポリメラーゼ連鎖反応であることが好ましい。
本発明のRNAの検出方法は、(A)本発明のRT−PCR用の反応液を逆転写ポリメラーゼ連鎖反応に供する工程、及び(B)工程(A)において増幅されたcDNAを、電気泳動により検出する工程を含む。上記の工程(A)における逆転写ポリメラーゼ連鎖反応は、「(2)本発明のcDNAの合成方法」と同様の条件で実施することができる。
本発明の逆転写ポリメラーゼ連鎖反応用のキットは、耐熱性DNAポリメラーゼ及び逆転写酵素を含有する酵素溶液、並びに色素マーカー及び比重増加剤を含有する反応緩衝液
を含む。
ワンステップRT−PCR用反応液への、色素マーカー及び比重増加剤の添加を検討した。RT−PCR用反応液の調製には、逆転写酵素及び耐熱性DNAポリメラーゼとしてPrimeScript(登録商標) One Step RT−PCR Kit Ver.2(タカラバイオ社製)に含まれるPrimeScript(登録商標) 1 step Enzyme Mixを用いた。鋳型として、HL60細胞から取得した全RNAを用い、プライマーとして、CCND2遺伝子の2.8kbの領域を増幅するためのプライマー対、CCND2F(配列番号:1)及びCCND2R(配列番号:2)を用いた。
色素マーカー及び比重増加剤を含むワンステップRT−PCR用反応液の比重増加剤として、ポリエチレングリコールを使用した場合の効果を検討した。
色素マーカー及び比重増加剤を含むワンステップRT−PCR用反応液の比重増加剤として、エチレングリコールを使用した場合の効果を検討した。
種々の色素マーカーが1ステップRT−PCRに及ぼす影響について検討した。
色素マーカーの含有量が1ステップRT−PCRに及ぼす影響について検討した。
−30℃の保存条件下でも凍結しない、操作性に優れたワンステップRT−PCR用の5倍濃度のプレミックス試薬について検討した。
10v/v%、15v/v%、20v/v%、25v/v%及び30v/v%のグリセロールを含むワンステップRT−PCR用の5倍濃度のプレミックス試薬(以下、5×プレミックス)をそれぞれ2mLずつ調製した。当該5×プレミックスのグリセロール以外の成分を、表6に示す。
−30℃の保存条件下でも凍結しない、操作性に優れたワンステップRT−PCR用の5倍濃度のプレミックス試薬について検討した。
実施例6−(1)において、−30℃の保存条件下での凍結が認められたグリセロール濃度が20v/v%又は25v/v%の5×プレミックス(RT−PCR反応時のグリセロール濃度がそれぞれ4v/v%及び5v/v%)について、エチレングリコール及びポリエチレングリコールの添加を検討した。
実施例6−(3)において認められた、グリセロール、ポリエチレングリコール、及びエチレングリコールを共存させることによる効果について、ポリエチレングリコールの濃度による影響を確認した。
SEQ ID NO:2 ; Primer CCND2R to amplify a 2.8k bp fragment of CCND2 gene.
Claims (7)
- 逆転写ポリメラーゼ連鎖反応用の1.5〜5倍濃度のプレミックス試薬であって、耐熱性DNAポリメラーゼ、逆転写酵素、色素マーカー、及び比重増加剤を含むプレミックス試薬。
- 比重増加剤が、グリセロール、エチレングリコール、ポリエチレングリコール及びその組合せからなる群より選択される、請求項1に記載のプレミックス試薬。
- 色素マーカーが、タートラジン、アシッドレッド18、キシレンシアノール及びその組合せからなる群より選択される、請求項1に記載のプレミックス試薬。
- 比重増加剤として、20〜30容量%のグリセロール、及び2.5〜7.5重量/容量%のポリエチレングリコールを含む、請求項2に記載のプレミックス試薬。
- 比重増加剤として5〜7.5容量%のエチレングリコールを更に含む、請求項4に記載のプレミックス試薬。
- −20℃〜−30℃の保存条件下でも凍結しない、請求項1記載のプレミックス試薬。
- RNAの検出方法であって、
(A)請求項1〜6のいずれか一項に記載のプレミックス試薬、並びに鋳型として用いるRNA、及び少なくとも1種のオリゴヌクレオチドプライマーを含む逆転写ポリメラーゼ連鎖反応用の反応液を調製する工程、
(B)工程(A)の反応液を逆転写ポリメラーゼ連鎖反応に供する工程、及び
(C)工程(B)において増幅されたcDNAを、電気泳動により検出する工程、
を含む方法。
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