JPWO2019136413A5 - - Google Patents
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- JPWO2019136413A5 JPWO2019136413A5 JP2020537593A JP2020537593A JPWO2019136413A5 JP WO2019136413 A5 JPWO2019136413 A5 JP WO2019136413A5 JP 2020537593 A JP2020537593 A JP 2020537593A JP 2020537593 A JP2020537593 A JP 2020537593A JP WO2019136413 A5 JPWO2019136413 A5 JP WO2019136413A5
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- 150000007523 nucleic acids Chemical class 0.000 claims description 138
- 108020004707 nucleic acids Proteins 0.000 claims description 136
- OIVLITBTBDPEFK-UHFFFAOYSA-N Dihydrouracil Chemical compound O=C1CCNC(=O)N1 OIVLITBTBDPEFK-UHFFFAOYSA-N 0.000 claims description 51
- RWQNBRDOKXIBIV-UHFFFAOYSA-N Thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 claims description 46
- 229940113082 Thymine Drugs 0.000 claims description 23
- 229940104302 Cytosine Drugs 0.000 claims description 18
- OPTASPLRGRRNAP-UHFFFAOYSA-N Cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 claims description 18
- 230000000903 blocking Effects 0.000 claims description 11
- 229910000085 borane Inorganic materials 0.000 claims description 8
- 229910000090 borane Inorganic materials 0.000 claims description 8
- DMJZZSLVPSMWCS-UHFFFAOYSA-N diborane Chemical compound B1[H]B[H]1 DMJZZSLVPSMWCS-UHFFFAOYSA-N 0.000 claims description 8
- 239000003638 reducing agent Substances 0.000 claims description 8
- UORVGPXVDQYIDP-UHFFFAOYSA-N trihydridoboron Substances B UORVGPXVDQYIDP-UHFFFAOYSA-N 0.000 claims description 8
- FHSISDGOVSHJRW-UHFFFAOYSA-N 5-formylcytosine Chemical compound NC1=NC(=O)NC=C1C=O FHSISDGOVSHJRW-UHFFFAOYSA-N 0.000 claims description 6
- 108090000790 Enzymes Proteins 0.000 claims description 5
- 102000004190 Enzymes Human genes 0.000 claims description 5
- BLQMCTXZEMGOJM-UHFFFAOYSA-N 5-carboxycytosine Chemical compound NC=1NC(=O)N=CC=1C(O)=O BLQMCTXZEMGOJM-UHFFFAOYSA-N 0.000 claims description 4
- 101700043716 TET1 Proteins 0.000 claims description 4
- 101700048164 TET2 Proteins 0.000 claims description 4
- 101700085225 TET3 Proteins 0.000 claims description 4
- QHXLIQMGIGEHJP-UHFFFAOYSA-N boron;2-methylpyridine Chemical group [B].CC1=CC=CC=N1 QHXLIQMGIGEHJP-UHFFFAOYSA-N 0.000 claims description 4
- 238000009585 enzyme analysis Methods 0.000 claims description 4
- 238000002493 microarray Methods 0.000 claims description 4
- 108091007521 restriction endonucleases Proteins 0.000 claims description 4
- AGGHKNBCHLWKHY-UHFFFAOYSA-N sodium;triacetyloxyboron(1-) Chemical compound [Na+].CC(=O)O[B-](OC(C)=O)OC(C)=O AGGHKNBCHLWKHY-UHFFFAOYSA-N 0.000 claims description 4
- RYVNIFSIEDRLSJ-UHFFFAOYSA-N 5-Hydroxymethylcytosine Chemical compound NC=1NC(=O)N=CC=1CO RYVNIFSIEDRLSJ-UHFFFAOYSA-N 0.000 claims description 3
- LRSASMSXMSNRBT-UHFFFAOYSA-N 5-Methylcytosine Chemical compound CC1=CNC(=O)N=C1N LRSASMSXMSNRBT-UHFFFAOYSA-N 0.000 claims description 3
- 241000283690 Bos taurus Species 0.000 claims description 2
- 241000224436 Naegleria Species 0.000 claims description 2
- 102100003997 TET1 Human genes 0.000 claims description 2
- 102100003998 TET2 Human genes 0.000 claims description 2
- 102100004000 TET3 Human genes 0.000 claims description 2
- 101700066455 TRN2 Proteins 0.000 claims description 2
- OPCPRUQQEJNFIV-UHFFFAOYSA-N disodium;cyanoboron(1-) Chemical compound [Na+].[Na+].[B-]C#N.[B-]C#N OPCPRUQQEJNFIV-UHFFFAOYSA-N 0.000 claims description 2
- 229910000033 sodium borohydride Inorganic materials 0.000 claims description 2
- YOQDYZUWIQVZSF-UHFFFAOYSA-N sodium borohydride Substances [BH4-].[Na+] YOQDYZUWIQVZSF-UHFFFAOYSA-N 0.000 claims description 2
- ODGROJYWQXFQOZ-UHFFFAOYSA-N sodium;boron(1-) Chemical compound [B-].[Na+] ODGROJYWQXFQOZ-UHFFFAOYSA-N 0.000 claims description 2
- 229920001850 Nucleic acid sequence Polymers 0.000 claims 2
- 240000004997 Coprinus comatus Species 0.000 claims 1
- 235000004439 Coprinus comatus Nutrition 0.000 claims 1
- 238000000034 method Methods 0.000 claims 1
- 230000004048 modification Effects 0.000 claims 1
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- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 4
- XCCTYIAWTASOJW-XVFCMESISA-N Uridine-5'-Diphosphate Chemical compound O[C@@H]1[C@H](O)[C@@H](COP(O)(=O)OP(O)(O)=O)O[C@H]1N1C(=O)NC(=O)C=C1 XCCTYIAWTASOJW-XVFCMESISA-N 0.000 description 2
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 2
- OAKJQQAXSVQMHS-UHFFFAOYSA-N hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 2
- 150000002443 hydroxylamines Chemical class 0.000 description 2
- 239000007800 oxidant agent Substances 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- LPGWNCNRGQANGC-UHFFFAOYSA-N pyridin-1-ium-1-ylboranuide Chemical compound [BH3-][N+]1=CC=CC=C1 LPGWNCNRGQANGC-UHFFFAOYSA-N 0.000 description 2
- 101700034878 setA Proteins 0.000 description 2
- 241001515965 unidentified phage Species 0.000 description 2
- 229920000160 (ribonucleotides)n+m Polymers 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N D-Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- QRXWMOHMRWLFEY-UHFFFAOYSA-N Isoniazid Chemical class NNC(=O)C1=CC=NC=C1 QRXWMOHMRWLFEY-UHFFFAOYSA-N 0.000 description 1
- AQFWNELGMODZGC-UHFFFAOYSA-N O-ethylhydroxylamine Chemical group CCON AQFWNELGMODZGC-UHFFFAOYSA-N 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
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- 229920003013 deoxyribonucleic acid Polymers 0.000 description 1
- QUSNBJAOOMFDIB-UHFFFAOYSA-N ethyl amine Chemical compound CCN QUSNBJAOOMFDIB-UHFFFAOYSA-N 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 150000002429 hydrazines Chemical class 0.000 description 1
- -1 hydroxylamine compound Chemical class 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N potassium Chemical group [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
Description
本発明の実施形態では、修飾標的核酸の配列を決定する工程は、鎖終結配列決定、マイクロアレイ、高処理量配列決定、および制限酵素分析を含む。
本発明の実施形態において、例えば以下の項目が提供される。
（項目１）
標的核酸中の５－メチルシトシン（５ｍＣ）を特定するための方法であって、前記方法が、
ａ． 前記標的核酸を含む核酸試料を提供する工程、
ｂ． 前記核酸を修飾する工程であって、前記修飾する工程が、
ｉ． 核酸試料中の５－ヒドロキシメチルシトシン（５ｈｍＣ）にブロッキング基を添加する工程と、
ii． 前記核酸試料中の前記５ｍＣを５－カルボキシルシトシン（５ｃａＣ）および／または５－ホルミルシトシン（５ｆＣ）に変換する工程と、
iii． 修飾標的核酸を含む修飾核酸試料を提供するために、前記５ｃａＣおよび／
または５ｆＣをジヒドロウラシル（ＤＨＵ）に変換する工程と、を含む、修飾する工程、および
ｃ． 前記配列におけるＤＨＵの存在を検出すること、または前記ＤＨＵをチミン（Ｔ）に変換して前記配列における前記チミン（Ｔ）の存在を検出することを含む、前記修飾標的核酸の配列を検出する工程、を含み、
前記標的核酸と比較した前記修標的核酸の配列におけるシトシン（Ｃ）のＤＨＵへの移行またはシトシン（Ｃ）のチミン（Ｔ）への移行が、前記標的核酸中の５ｍＣの位置を提供する、方法。
（項目２）
各移行位置でのＤＨＵまたはＴのパーセンテージが、前記標的核酸中の各位置での５ｍＣの定量レベルを提供する、項目１に記載の方法。
（項目３）
標的核酸中の５ｍＣまたは５ｈｍＣを特定する方法であって、前記方法が、
ａ． 前記標的核酸を含む核酸試料を提供する工程、
ｂ． 前記核酸を修飾する工程であって、前記修飾する工程が、
ｉ． 前記核酸試料中の前記５ｍＣおよび５ｈｍＣを５－カルボキシルシトシン（５ｃａＣ）および／または５ｆＣに変換する工程と、
ii． 修飾標的核酸を含む修飾核酸試料を提供するために、前記５ｃａＣおよび／または５ｆＣをジヒドロウラシル（ＤＨＵ）に変換する工程と、を含む、修飾する工程、および
ｃ． 前記配列におけるＤＨＵの存在を検出すること、または前記ＤＨＵをチミン（Ｔ）に変換して前記配列における前記チミン（Ｔ）の存在を検出することを含む、前記修飾標的核酸の配列を検出する工程、を含み、
前記標的核酸と比較した前記修飾標的核酸の配列におけるシトシン（Ｃ）のＤＨＵへの移行またはシトシン（Ｃ）のチミン（Ｔ）への移行が、前記標的核酸中の５ｍＣまたは５ｈｍＣのいずれかの位置を提供する、方法。
（項目４）
各移行位置でのＤＨＵまたはＴのパーセンテージが、前記標的核酸中の各位置での５ｍＣまたは５ｈｍＣの定量レベルを提供する、項目３に記載の方法。
（項目５）
標的核酸中の５ｍＣを特定するための、および５ｈｍＣを特定するための方法であって、前記方法が、
ａ． 前記標的核酸中の５－メチルシトシン（５ｍＣ）を特定する工程であって、前記特定する工程が、
ｉ． 前記標的核酸を含む第一核酸試料を提供する工程、
ii． 前記第一試料中の前記核酸を修飾する工程であって、前記修飾する工程が、
（１）． 前記第一核酸試料中の、５－ヒドロキシメチルシトシン（５ｈｍＣ）にブロッキング基を添加することと、
（２）． 前記第一核酸試料中の前記５ｍＣを、５ｃａＣおよび／または５ｆＣに変換することと、
（３）． 修飾標的核酸を含む修飾第一核酸試料を提供するために、前記５ｃａＣおよび／または５ｆＣをジヒドロウラシル（ＤＨＵ）に変換することと、を含む、修飾する工程、
iii． 前記配列におけるＤＨＵの存在を検出すること、または前記ＤＨＵをチミン
（Ｔ）に変換して前記配列における前記チミン（Ｔ）の存在を検出することを含む、前記修飾標的核酸の配列を検出する工程であって、前記標的核酸と比較した前記修飾標的核酸の配列におけるシトシン（Ｃ）のＤＨＵへの移行またはシトシン（Ｃ）のチミン（Ｔ）への移行が、前記標的核酸中の５ｍＣの位置を提供する、検出する工程、を含む、特定する工程、
ｂ． 前記標的核酸中の５ｍＣまたは５ｈｍＣを特定する工程であって、前記特定する工程が、
ｉ． 前記標的核酸を含む第二核酸試料を提供する工程、
ii． 前記第二試料中の前記核酸を修飾する工程であって、前記修飾する工程が、
（１）． 前記第二核酸試料中の５ｍＣおよび５ｈｍＣを、５ｃａＣおよび／または５ｆＣに変換することと、
（２）． 修飾標的核酸を含む修飾第二核酸試料を提供するために、前記５ｃａＣおよび／または５ｆＣをジヒドロラシル（ＤＨＵ）に変換することと、を含む、修飾する工程、および
iii． 前記配列におけるＤＨＵの存在を検出すること、または前記ＤＨＵをチミン
（Ｔ）に変換して前記配列における前記チミン（Ｔ）の存在を検出することを含む、前記第二試料から前記修飾標的核酸の配列を検出する工程であって、前記標的核酸と比較した前記修飾標的核酸の配列におけるシトシン（Ｃ）のＤＨＵへの移行またはシトシン（Ｃ）のチミン（Ｔ）への移行が、前記標的核酸中の５ｍＣまたは５ｈｍＣのいずれかの位置を提供する、検出する工程、を含む、特定する工程、ならびに、
ｃ． 工程（ａ）および（ｂ）の結果を比較する工程であって、工程（ｂ）で存在するが工程（ａ）では存在しないＣからＤＨＵまたはＣからＴへの移行が、前記標的核酸中の５ｈｍＣの位置を提供する、比較する工程、を含む、方法。
（項目６）
工程（ａ）において、各移行位置でのＤＨＵまたはＴのパーセンテージが、前記標的核酸中の各位置で５ｍＣの定量レベルを提供し、工程（ｂ）において、各移行位置でのＤＨＵまたはＴのパーセンテージが、前記標的核酸中の各位置で５ｍＣ、または５ｈｍＣの定量レベルを提供し、および工程（ｃ）において、工程（ｂ）で、工程（ａ）ではなく、特定されたＣからＤＨＵまたはＴへの移行についてのパーセンテージの差異が、前記標的核酸中の各位置で５ｈｍＣの定量レベルを提供する、項目５に記載の方法。
（項目７）
前記５ｈｍＣに添加される前記ブロッキング基が糖である、項目１～２または項目５～６のいずれか一項に記載の方法。
（項目８）
前記糖がグルコースまたは修飾グルコースである、項目７に記載の方法。
（項目９）
前記ブロッキング基が、グルコシルトランスフェラーゼ酵素の存在下で、糖に連結されたウリジン二リン酸（ＵＤＰ）に前記核酸試料を接触させることにより、前記５ｈｍＣに添加される、項目７に記載の方法。
（項目１０）
前記グルコシルトランスフェラーゼ酵素が、Ｔ４バクテリオファージβ－グルコシルトランスフェラーゼ（βＧＴ）、Ｔ４バクテリオファージα－グルコシルトランスフェラーゼ（αＧＴ）、ならびにその誘導体および類似体からなる群から選択される、項目９に記載の方法。
（項目１１）
前記核酸試料中の前記５ｍＣを、５ｃａＣ及び／または５ｆＣに変換する工程、ならびに前記核酸試料中の前記５ｍＣ及び５ｈｍＣを、５ｃａＣ及び／または５ｆＣに変換する工程は、テンイレブントランスロケーション（ＴＥＴ）酵素と前記核酸試料を接触させることを含む、項目１～６のいずれか一項に記載の方法。
（項目１２）
前記ＴＥＴ酵素が、ヒトＴＥＴ１、ＴＥＴ２、及びＴＥＴ３、マウスＴｅｔ１、Ｔｅｔ２、及びＴｅｔ３、ネグレリアＴＥＴ（ＮｇＴＥＴ）、ウシグソヒトヨタケ（ＣｃＴＥＴ）、ならびにその誘導体または類似体からなる群から選択される、項目１１に記載の方法。
（項目１３）
前記ＴＥＴ酵素がＮｇＴＥＴまたはマウスＴＥＴである、項目１２に記載の方法。
（項目１４）
標的核酸中の５ｃａＣ、または５ｆＣを特定するための方法であって、前記方法が、
ａ． 前記標的核酸を含む核酸試料を提供する工程、
ｂ． 修飾標的核酸を含む修飾核酸試料を提供するために、前記５ｃａＣおよび５ｆＣをジヒドロウラシル（ＤＨＵ）に変換する工程、
ｃ． 前記修飾標的核酸のコピー数を増幅する工程、および
ｄ． 前記修飾標的核酸の配列を検出する工程、を含み、
前記標的核酸と比較した前記修飾標的核酸の配列におけるシトシン（Ｃ）のチミン（Ｔ）への移行が、前記標的核酸中の５ｃａＣ、または５ｆＣのいずれかの位置を提供する、方法。
（項目１５）
各移行位置でのＴのパーセンテージが、前記標的核酸中の各位置で、５ｃａＣ、または５ｆＣの定量レベルを提供する、項目１４に記載の方法。
（項目１６）
標的核酸中の５ｃａＣを特定するための方法であって、前記方法が、
ａ． 前記標的核酸を含む核酸試料を提供する工程、
ｂ． 前記核酸試料中の５ｆＣにブロッキング基を添加する工程、
ｃ． 修飾標的核酸を含む修飾核酸試料を提供するために、前記５ｃａＣをジヒドロウラシル（ＤＨＵ）に変換する工程、および
ｄ． 前記配列におけるＤＨＵの存在を検出すること、または前記ＤＨＵをチミン（Ｔ）に変換して前記配列における前記チミン（Ｔ）の存在を検出することを含む、前記修飾標的核酸の配列を検出する工程、を含み、
前記標的核酸と比較した前記修飾標的核酸の配列におけるシトシン（Ｃ）のＤＨＵへの移行またはシトシン（Ｃ）のチミン（Ｔ）への移行が、前記標的核酸中の５ｃａＣの位置を提供する、方法。
（項目１７）
各移行位置でのＤＨＵまたはＴのパーセンテージが、前記標的核酸の各位置で５ｃａＣの定量レベルを提供する、項目１６に記載の方法。
（項目１８）
前記５ｆＣにブロッキング基を添加する工程が、ヒドロキシルアミン誘導体、ヒドラジン誘導体、およびヒドラジド誘導体から選択されるアルデヒド反応性化合物と、前記核酸試料を接触させることを含む、項目１６に記載の方法。
（項目１９）
前記ヒドロキシルアミン誘導体が、Ｏ－エチルヒドロキシルアミンである、項目１８に記載の方法。
（項目２０）
標的核酸中の５ｆＣを特定するための方法であって、前記方法が、
ａ． 前記標的核酸を含む核酸試料を提供する工程、
ｂ． 前記核酸試料中の５ｃａＣにブロッキング基を添加する工程、
ｃ． 修飾標的核酸を含む修飾核酸試料を提供するために、前記５ｆＣをジヒドロウラシル（ＤＨＵ）に変換する工程、および
ｄ． 前記配列におけるＤＨＵの存在を検出すること、または前記ＤＨＵをチミン（Ｔ）に変換して前記配列における前記チミン（Ｔ）の存在を検出することを含む、前記修飾標的核酸の配列を検出する工程、を含み、
前記標的核酸と比較した前記修飾標的核酸の配列におけるシトシン（Ｃ）のＤＨＵへの移行またはシトシン（Ｃ）のチミン（Ｔ）への移行が、前記標的核酸中の５ｆＣの位置を提供する、方法。
（項目２１）
各移行位置でのＤＨＵまたはＴのパーセンテージが、前記標的核酸中の各位置で５ｆＣの定量レベルを提供する、項目２０に記載の方法。
（項目２２）
５ｃａＣにブロッキング基を添加する前記工程が、前記核酸試料をカルボン酸誘導体化試薬、及びアミン、ヒドラジン又はヒドロキシルアミン化合物と接触させることを含む、項目２０に記載の方法。
（項目２３）
５ｃａＣにブロッキング基を添加する前記工程が、前記核酸試料を１－エチル－３－（３－ジメチルアミノプロピル）カルボジイミド（ＥＤＣ）と接触させることと、エチルアミンと接触させることと、を含む、項目２２に記載の方法。
（項目２４）
標的核酸中の５ｈｍＣを特定するための方法であって、前記方法が、
ａ. 前記標的核酸を含む核酸試料を提供する工程、
ｂ. 前記試料中の前記核酸を修飾する工程であって、
ｉ 前記核酸試料中の前記５ｈｍＣを５ｃａＣおよび／または５ｆＣに変換する工程と
ii 前記５ｃａＣおよび／または５ｆＣをジヒドロウラシル（ＤＨＵ）に変換して修飾標的核酸を含む修飾核酸試料を提供する工程と、を含む、修飾する工程、および
ｃ. 前記配列におけるＤＨＵの存在を検出すること、または前記ＤＨＵをチミン（Ｔ
）に変換して前記配列におけるチミン（Ｔ）の存在を検出することを含む、前記修飾標的核酸の配列を検出する工程であって、前記標的核酸と比較した前記修飾標的核酸の配列におけるシトシン（Ｃ）のＤＨＵへの移行またはシトシン（Ｃ）のチミン（Ｔ）への移行が、前記標的核酸中の５ｈｍＣの位置を提供する、検出する工程、を含む、方法。
（項目２５）
各移行位置でのＤＨＵまたはＴのパーセンテージが、前記標的核酸中の各位置で５ｈｍＣの定量レベルを提供する、項目２４に記載の方法。
（項目２６）
前記５ｈｍＣを５ｃａＣおよび／または５ｆＣに変換する工程が、前記核酸試料を酸化剤と接触させることを含む、項目２４に記載の方法。
（項目２７）
前記酸化剤が、過ルテニウム酸カリウムまたはＣｕ（ＩＩ）／ＴＥＭＰＯである、項目２６に記載の方法。
（項目２８）
前記方法が、前記修飾標的核酸のコピー数を増幅する前記工程をさらに含む、項目１～６、および１４～２７のいずれか一項に記載の方法。
（項目２９）
前記修飾標的核酸のコピー数を増幅する前記工程が、ポリメラーゼ連鎖反応（ＰＣＲ）、またはプライマー伸長を行うことを含む、項目２８に記載の方法。
（項目３０）
前記５ｃａＣおよび／または５ｆＣをＤＨＵへ変換する前記工程が、前記核酸試料を還元剤に接触させることを含む、項目１～６、および１４～２９のいずれか一項に記載の方法。
（項目３１）
前記還元剤が、ピリジンボラン、２－ピコリンボラン（ｐｉｃ－ＢＨ
３
）、ボラン、水素化ホウ素ナトリウム、シアノホウ水素化ナトリウム、およびナトリウムトリアセトキシボロヒドリドからなる群から選択される、項目３０に記載の方法。
（項目３２）
前記還元剤が、ピリジンボラン、または２－ピコリンボランである、項目３１に記載の方法。
（項目３３）
前記修飾標的核酸の配列を特定する前記工程が、鎖終結配列決定、マイクロアレイ、高処理量配列決定、および制限酵素分析のうちの一つ以上を含む、項目１～６、および１４～２７のいずれか一項に記載の方法。
（項目３４）
前記核酸がＤＮＡである、項目１～３３のいずれか一項に記載の方法。
（項目３５）
前記核酸がＲＮＡである、項目１～３３のいずれか一項に記載の方法。
In embodiments of the invention, the steps of sequencing the modified target nucleic acid include chain termination sequencing, microarray, high throughput sequencing, and restriction enzyme analysis.
In the embodiment of the present invention, for example, the following items are provided.
(Item 1)
A method for identifying 5-methylcytosine (5 mC) in a target nucleic acid, wherein the method is:
a. The step of providing a nucleic acid sample containing the target nucleic acid,
b. The step of modifying the nucleic acid, wherein the modifying step is
i. A step of adding a blocking group to 5-hydroxymethylcytosine (5 hmC) in a nucleic acid sample, and
ii. A step of converting the 5 mC in the nucleic acid sample to 5-carboxylcytosine (5caC) and / or 5-formylcytosine (5fC).
iii. 5caC and / above to provide a modified nucleic acid sample containing the modified target nucleic acid.
Or a step of converting 5fC to dihydrouracil (DHU), including, a step of modifying, and a step.
c. A step of detecting the sequence of the modified target nucleic acid, which comprises detecting the presence of DHU in the sequence or converting the DHU to thymine (T) to detect the presence of thymine (T) in the sequence. , Including
A method in which the transfer of cytosine (C) to DHU or the transfer of cytosine (C) to thymine (T) in the sequence of the repaired target nucleic acid compared to the target nucleic acid provides a position of 5 mC in the target nucleic acid. ..
(Item 2)
The method of item 1, wherein the percentage of DHU or T at each transition position provides a quantitative level of 5 mC at each position in said target nucleic acid.
(Item 3)
A method for identifying 5 mC or 5 hmC in a target nucleic acid, wherein the method is:
a. The step of providing a nucleic acid sample containing the target nucleic acid,
b. The step of modifying the nucleic acid, wherein the modifying step is
i. The step of converting the 5mC and 5hmC in the nucleic acid sample to 5-carboxycytosine (5caC) and / or 5fC, and
ii. A step of converting the 5caC and / or 5fC to dihydrouracil (DHU) to provide a modified nucleic acid sample containing the modified target nucleic acid, and a step of modifying.
c. A step of detecting the sequence of the modified target nucleic acid, which comprises detecting the presence of DHU in the sequence or converting the DHU to thymine (T) to detect the presence of thymine (T) in the sequence. , Including
The transfer of cytosine (C) to DHU or the transfer of cytosine (C) to thymine (T) in the sequence of the modified target nucleic acid compared to the target nucleic acid is at either the 5 mC or 5 hmC position in the target nucleic acid. How to provide.
(Item 4)
The method of item 3, wherein the percentage of DHU or T at each transition position provides a quantitative level of 5 mC or 5 hmC at each position in said target nucleic acid.
(Item 5)
A method for identifying 5 mC in a target nucleic acid and for identifying 5 hmC, wherein the method is:
a. The step of identifying 5-methylcytosine (5 mC) in the target nucleic acid, wherein the specifying step is
i. A step of providing a first nucleic acid sample containing the target nucleic acid,
ii. The step of modifying the nucleic acid in the first sample, wherein the modifying step is
(1). Adding a blocking group to 5-hydroxymethylcytosine (5 hmC) in the first nucleic acid sample and
(2). Converting the 5mC in the first nucleic acid sample to 5caC and / or 5fC,
(3). A step of modifying, comprising converting said 5caC and / or 5fC to dihydrouracil (DHU) to provide a modified primary nucleic acid sample comprising the modified target nucleic acid.
iii. Detecting the presence of DHU in the sequence, or thymine the DHU
A step of detecting the sequence of the modified target nucleic acid, which comprises converting to (T) to detect the presence of the thymine (T) in the sequence, wherein the sequence of the modified target nucleic acid is compared with the target nucleic acid. A step of identifying, comprising a step of detecting, where the transfer of cytosine (C) to DHU or the transfer of cytosine (C) to thymine (T) in said provides a position of 5 mC in the target nucleic acid.
b. The step of identifying 5 mC or 5 hmC in the target nucleic acid, wherein the specifying step is
i. The step of providing a second nucleic acid sample containing the target nucleic acid,
ii. The step of modifying the nucleic acid in the second sample, wherein the modifying step is
(1). Converting 5mC and 5hmC in the second nucleic acid sample to 5caC and / or 5fC,
(2). A step of modifying, comprising converting the 5caC and / or 5fC to dihydrorasyl (DHU) to provide a modified second nucleic acid sample comprising the modified target nucleic acid, and
iii. Detecting the presence of DHU in the sequence, or thymine the DHU
A step of detecting the sequence of the modified target nucleic acid from the second sample, which comprises converting to (T) to detect the presence of the thymine (T) in the sequence, wherein the sequence is compared with the target nucleic acid. The step of detecting that the transfer of cytosine (C) to DHU or the transfer of cytosine (C) to thymine (T) in the sequence of the modified target nucleic acid provides the position of either 5 mC or 5 hmC in the target nucleic acid. , Including, and identifying steps, as well.
c. In the step of comparing the results of steps (a) and (b), the transition from C to DHU or C to T, which is present in step (b) but not in step (a), is in the target nucleic acid. A method comprising a step of comparing, providing a position of 5 hmC.
(Item 6)
In step (a), the percentage of DHU or T at each transition position provides a quantitative level of 5 mC at each position in said target nucleic acid, and in step (b) the percentage of DHU or T at each transition position. Provides a quantitative level of 5 mC, or 5 hmC, at each position in said target nucleic acid, and in step (c), from step C to DHU or T, instead of step (a). 5. The method of item 5, wherein the difference in percentages for migration provides a quantitative level of 5 hmC at each position in said target nucleic acid.
(Item 7)
The method according to any one of items 1 to 2 or items 5 to 6, wherein the blocking group added to the 5 hmC is sugar.
(Item 8)
7. The method of item 7, wherein the sugar is glucose or modified glucose.
(Item 9)
7. The method of item 7, wherein the blocking group is added to the 5 hmC by contacting the nucleic acid sample with sugar-linked uridine diphosphate (UDP) in the presence of a glucosyltransferase enzyme.
(Item 10)
9. The method of item 9, wherein the glucosyltransferase enzyme is selected from the group consisting of T4 bacteriophage β-glucosyltransferase (βGT), T4 bacteriophage α-glucosyltransferase (αGT), and derivatives and analogs thereof.
(Item 11)
The step of converting the 5mC in the nucleic acid sample to 5caC and / or 5fC and the step of converting the 5mC and 5hmC in the nucleic acid sample to 5caC and / or 5fC are ten-eleven translocation (TET) enzymes. The method according to any one of items 1 to 6, which comprises contacting the nucleic acid sample with the nucleic acid sample.
(Item 12)
Item 11 The TET enzyme is selected from the group consisting of human TET1, TET2, and TET3, mouse Tet1, Tet2, and Tet3, Naegleria TET (NgTET), bovine inky cap (CcTET), and derivatives or analogs thereof. The method described in.
(Item 13)
12. The method of item 12, wherein the TET enzyme is NgTET or mouse TET.
(Item 14)
A method for identifying 5caC or 5fC in a target nucleic acid, wherein the method is:
a. The step of providing a nucleic acid sample containing the target nucleic acid,
b. The step of converting the 5caC and 5fC to dihydrouracil (DHU) to provide a modified nucleic acid sample containing the modified target nucleic acid.
c. The step of amplifying the copy number of the modified target nucleic acid, and
d. Including the step of detecting the sequence of the modified target nucleic acid.
A method, wherein the transfer of cytosine (C) to thymine (T) in the sequence of the modified target nucleic acid compared to the target nucleic acid provides either a 5caC or 5fC position in the target nucleic acid.
(Item 15)
14. The method of item 14, wherein the percentage of T at each transition position provides a quantitative level of 5caC, or 5fC, at each position in said target nucleic acid.
(Item 16)
A method for identifying 5caC in a target nucleic acid, wherein the method is:
a. The step of providing a nucleic acid sample containing the target nucleic acid,
b. The step of adding a blocking group to 5fC in the nucleic acid sample,
c. A step of converting the 5caC to dihydrouracil (DHU) to provide a modified nucleic acid sample containing the modified target nucleic acid, and
d. A step of detecting the sequence of the modified target nucleic acid, which comprises detecting the presence of DHU in the sequence or converting the DHU to thymine (T) to detect the presence of thymine (T) in the sequence. , Including
A method in which the transfer of cytosine (C) to DHU or the transfer of cytosine (C) to thymine (T) in the sequence of the modified target nucleic acid compared to the target nucleic acid provides the position of 5caC in the target nucleic acid. ..
(Item 17)
16. The method of item 16, wherein the percentage of DHU or T at each transition position provides a quantitative level of 5 caC at each position of said target nucleic acid.
(Item 18)
Item 16. The method of item 16, wherein the step of adding a blocking group to the 5fC comprises contacting the nucleic acid sample with an aldehyde-reactive compound selected from a hydroxylamine derivative, a hydrazine derivative, and a hydrazide derivative.
(Item 19)
18. The method of item 18, wherein the hydroxylamine derivative is O-ethylhydroxylamine.
(Item 20)
A method for identifying 5fC in a target nucleic acid, wherein the method is:
a. The step of providing a nucleic acid sample containing the target nucleic acid,
b. The step of adding a blocking group to 5caC in the nucleic acid sample,
c. A step of converting the 5fC to dihydrouracil (DHU) to provide a modified nucleic acid sample containing the modified target nucleic acid, and
d. A step of detecting the sequence of the modified target nucleic acid, which comprises detecting the presence of DHU in the sequence or converting the DHU to thymine (T) to detect the presence of thymine (T) in the sequence. , Including
A method in which the transfer of cytosine (C) to DHU or the transfer of cytosine (C) to thymine (T) in the sequence of the modified target nucleic acid compared to the target nucleic acid provides a position of 5fC in the target nucleic acid. ..
(Item 21)
20. The method of item 20, wherein the percentage of DHU or T at each transition position provides a quantitative level of 5 fC at each position in said target nucleic acid.
(Item 22)
The method of item 20, wherein the step of adding a blocking group to 5caC comprises contacting the nucleic acid sample with a carboxylic acid derivatizing reagent and an amine, hydrazine or hydroxylamine compound.
(Item 23)
Item 22. The step of adding a blocking group to 5caC comprises contacting the nucleic acid sample with 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and with ethylamine. The method described in.
(Item 24)
A method for identifying 5 hmC in a target nucleic acid, wherein the method is:
a. Step of providing a nucleic acid sample containing the target nucleic acid,
b. A step of modifying the nucleic acid in the sample.
i A step of converting the 5 hmC in the nucleic acid sample to 5 caC and / or 5 fC.
ii A step of converting the 5caC and / or 5fC to dihydrouracil (DHU) to provide a modified nucleic acid sample containing the modified target nucleic acid, and a step of modifying.
c. Detecting the presence of DHU in the sequence, or thymine (T) the DHU.
) To detect the presence of thymine (T) in the sequence, the step of detecting the sequence of the modified target nucleic acid, cytosine (in the sequence of the modified target nucleic acid compared to the target nucleic acid). A method comprising the step of detecting, where the transfer of C) to DHU or the transfer of cytosine (C) to thymine (T) provides a position of 5 hmC in said target nucleic acid.
(Item 25)
24. The method of item 24, wherein the percentage of DHU or T at each transition position provides a quantitative level of 5 hmC at each position in said target nucleic acid.
(Item 26)
24. The method of item 24, wherein the step of converting 5 hmC to 5 caC and / or 5 fC comprises contacting the nucleic acid sample with an oxidizing agent.
(Item 27)
26. The method of item 26, wherein the oxidizing agent is potassium perteniumate or Cu (II) / TEMPO.
(Item 28)
The method according to any one of items 1 to 6 and 14 to 27, wherein the method further comprises the step of amplifying the copy number of the modified target nucleic acid.
(Item 29)
28. The method of item 28, wherein the step of amplifying the copy number of the modified target nucleic acid comprises performing a polymerase chain reaction (PCR) or primer extension.
(Item 30)
The method according to any one of items 1 to 6 and 14 to 29, wherein the step of converting 5caC and / or 5fC to DHU comprises contacting the nucleic acid sample with a reducing agent.
(Item 31)
30. The reducing agent is selected from the group consisting of pyridineborane, 2-picoline borane (pic-BH 3 ), borane, sodium borohydride, sodium cyanoborohydride, and sodium triacetoxyborohydride. Method.
(Item 32)
31. The method of item 31, wherein the reducing agent is pyridineborane or 2-picoline borane.
(Item 33)
Any of items 1-6 and 14-27, wherein the step of identifying the sequence of the modified target nucleic acid comprises one or more of chain termination sequencing, microarray, high throughput sequencing, and restriction enzyme analysis. The method described in item 1.
(Item 34)
The method according to any one of items 1 to 33, wherein the nucleic acid is DNA.
(Item 35)
The method according to any one of items 1 to 33, wherein the nucleic acid is RNA.
Claims (14)
前記標的核酸を含む核酸試料を提供する工程、
前記標的核酸を修飾する工程であって、前記修飾する工程が、
一つ以上の５ｃａＣまたは５ｆＣ残基が生成されるように前記核酸試料をテンイレブントランスロケーション（ＴＥＴ）酵素と接触させることによって、前記核酸試料中の前記５ｍＣおよび５ｈｍＣを５－カルボキシルシトシン（５ｃａＣ）および／または５－ホルミルシトシン（５ｆＣ）に変換する工程と、
修飾標的核酸を含む修飾核酸試料を提供するために、前記標的核酸をボラン還元剤で処理することによって前記５ｃａＣおよび／または５ｆＣをジヒドロウラシル（ＤＨＵ）に変換する工程と、を含む、修飾する工程、および
前記修飾標的核酸の配列を検出する工程、
を含み、
前記標的核酸と比較した前記修飾標的核酸の配列におけるシトシン（Ｃ）のチミン（Ｔ）への移行またはシトシン（Ｃ）のＤＨＵへの移行が、前記標的核酸中の５ｍＣまたは５ｈｍＣのいずれかの位置を提供する、方法。 A method for identifying 5-methylcytosine ( 5 mC ) or 5-hydroxymethylcytosine ( 5 hmC ) in a target nucleic acid, wherein the method is described as described above.
The step of providing a nucleic acid sample containing the target nucleic acid,
The step of modifying the target nucleic acid, wherein the modifying step is
By contacting the nucleic acid sample with ten-eleven translocation (TET) enzyme so that one or more 5caC or 5fC residues are produced, the 5mC and 5hmC in the nucleic acid sample are 5-carboxylcytosine (5caC). And / or the process of converting to 5-formylcytosine ( 5fC ) , and
A step of converting the 5caC and / or 5fC to dihydrouracil (DHU) by treating the target nucleic acid with a borane reducing agent to provide a modified nucleic acid sample comprising the modified target nucleic acid. , And the step of detecting the sequence of the modified target nucleic acid,
Including
The transfer of cytosine (C) to thymine (T) or the transfer of cytosine (C) to DHU in the sequence of the modified target nucleic acid compared to the target nucleic acid is either 5 mC or 5 hmC in the target nucleic acid. How to provide that position.
５－カルボキシルシトシン（５ｃａＣ）および／または５－ホルミルシトシン（５ｆＣ）を含む核酸試料を提供する工程、および A step of providing a nucleic acid sample containing 5-carboxylcytosine (5caC) and / or 5-formylcytosine (5fC), and
修飾核酸を含む修飾核酸試料を提供するために、前記核酸をボラン還元剤で処理することによって前記５ｃａＣおよび／または５ｆＣをジヒドロウラシル（ＤＨＵ）に変換する工程 A step of converting the 5caC and / or 5fC to dihydrouracil (DHU) by treating the nucleic acid with a borane reducing agent to provide a modified nucleic acid sample containing the modified nucleic acid.
を含む、方法。Including, how.
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