四色荧光标记二硫键可逆终端的合成及在DNA合成测序中的应用

doi:10.7503/cjcu20140251

高等学校化学学报 ›› 2014, Vol. 35 ›› Issue (11): 2346.doi: 10.7503/cjcu20140251

四色荧光标记二硫键可逆终端的合成及在DNA合成测序中的应用

汤道年^1,³, 江敏¹, 李小卫², 康亚妮², 伍新燕³, 龚兵^4,⁵(), 邵志峰², 赵小东², 沈玉梅¹()

1. 上海交通大学系统生物医学研究院, 系统生物医学教育部重点实验室,2. 生物医学工程学院, Bio-ID研究中心, 上海 200240
3. 华东理工大学化学与分子工程学院, 先进材料重点实验室和精细化工研究所, 上海 200237
4. 北京师范大学化学学院, 北京 100875
5. 布法罗纽约州立大学化学系, 纽约 14260

收稿日期:2014-03-24 出版日期:2014-11-10 发布日期:2014-10-17
作者简介:联系人简介: 沈玉梅, 女, 博士, 研究员, 博士生导师, 主要从事化学生物学及两亲性嵌段共聚物的合成及应用研究. E-mail:yumeishen@sjtu.edu.cn;龚兵, 男, 博士, 教授, 博士生导师, 主要从事化学生物学及纳米材料的设计与应用研究. E-mail:bgong@bnu.edu.cn
基金资助:
国家自然科学基金(批准号: 81071250, 91227109)和科技重大专项“重大新药创制”项目(批准号: 2011ZX09501-001-05)资助

Synthesis and Application of Four Fluorescence Labeled Nucleotides Through Disulfide as Reversible Terminators in DNA Sequencing by Synthesis

TANG Daonian^1,³, JIANG Min¹, LI Xiaowei², KANG Yani², WU Xinyan³, GONG Bing^4,^5,^*(), SHAO Zhifeng², ZHAO Xiaodong², SHEN Yumei^1,^*()

1. Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine, Ministry of Education,2. School of Biomedical Engineering, Bio-ID Center, Shanghai Jiao Tong University, Shanghai 200240, China
3. Key Laboratory for Advanced Materials and Institute of Fine Chemicals, School of Chemistry & Molecular Engineering,East China University of Science and Technology, Shanghai 200237, China
4. College of Chemistry, Beijing Normal University, Beijing 100875, China
5. Department of Chemistry, University at Buffalo, the State University of New York, Buffalo, NY 14260, USA

Received:2014-03-24 Online:2014-11-10 Published:2014-10-17
Contact: GONG Bing,SHEN Yumei E-mail:bgong@bnu.edu.cn;yumeishen@sjtu.edu.cn
Supported by:
† Supported by the National Natural Science Foundation of China(Nos.81071250, 91227109) and the “Creation of Major New Drugs” Major Projects in National Science and Technology, China(No.2011ZX09501-001-05)

摘要/Abstract

摘要：

分别以5-碘-2'-脱氧尿苷、 5-碘-2'-脱氧胞苷、 7-去氮-7-碘-2'-脱氧腺苷及7-去氮-7-碘-2'-脱氧鸟苷为原料, 以二硫键为可裂解连接单元, 通过多步反应合成了四色荧光标记不同碱基的脱氧核糖核苷酸; 研究了该类荧光标记核苷酸作为可逆终端在DNA合成测序中的应用. 所得产物结构经¹H NMR, ³¹P NMR及HRMS表征, 并对其进行了DNA高通量测序的测试. 结果表明, 该类荧光标记核苷酸作为DNA合成测序的可逆终端能够满足高通量测序的生化反应要求, 具有较好的应用前景.

关键词: DNA合成测序, 可逆终端, 可裂解连接单元, 四色荧光标记

Abstract:

DNA sequencing is one of the important means of biological research. The development of accurate and high-throughput DNA-sequencing methods has a strong impact on biomedical research. Sequencing by synthesis(SBS) approaches is one of the most important techniques of DNA sequencing. A series of SBS methods have been investigated and implemented. In SBS methods, the principal thing is to synthesize four fluorescently labelled cyclic reversible terminators as the sequencing reagents. These cyclic reversible terminators need to connect the fluorescein through a cleavable linker. In chemical biology, disulfide bridges, which are known to be efficiently and rapidly cleaved by mild reducing agents, have been used in a wide range of applications including drug delivery, functional and structural proteomics, DNA and tumor imaging. The disulfide-based cleavable linker is commonly used due to its rapid cleavage and straightforward synthesis. In this work, starting from 5-iodo-2'-deoxyuridine, 5-iodo-2'-deoxycyridine, 7-deazo-7-iodo-2'-deoxyadenosine and 7-deazo-7-iodo-2'-deoxyguanosine, four kinds of fluorescence labelled necleotides with four different bases through disulfide were synthesized and characterized. The synthesized fluorescence labelled nucleotides can be recognized and accepted by an identified DNA polymerase, incorporated faithfully into DNA strand, followed by cleaved completely by DTT within 1 min. These results demonstrate that the fluorescence labelled nucleotides have potential value as reversible terminators in DNA sequencing by synthesis.

Key words: DNA sequencing by synthesis, Reversible terminator, Cleavable linker, Four fluorescence labelled nucleotide

中图分类号:

O629.11

TrendMD:

汤道年, 江敏, 李小卫, 康亚妮, 伍新燕, 龚兵, 邵志峰, 赵小东, 沈玉梅. 四色荧光标记二硫键可逆终端的合成及在DNA合成测序中的应用. 高等学校化学学报, 2014, 35(11): 2346.

TANG Daonian, JIANG Min, LI Xiaowei, KANG Yani, WU Xinyan, GONG Bing, SHAO Zhifeng, ZHAO Xiaodong, SHEN Yumei. Synthesis and Application of Four Fluorescence Labeled Nucleotides Through Disulfide as Reversible Terminators in DNA Sequencing by Synthesis. Chem. J. Chinese Universities, 2014, 35(11): 2346.

图/表 4

参考文献 28

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Compd.	Appearance	ESI-HRMS, m/z	Yield(%)	Purity(%, HPLC)
1a	Red solid	1095.1483[M-H]^-	18	90
1b	Yellow solid	1071.0604[M-H]^-	35	98
		991.0912[M-H₂PO₃]^-
1c	Blue solid	1169.3229[M-H]^-	18	100
1d	Brown solid	1422.2546[M-H]^-	43	93
2	White liquid		67
4a	Pale yellow solid		61
4b	Pale yellow solid		58
4c	Pale white solid		92
4d	White solid		39
5a	White solid	522.0070[M+H]⁺	34	100
5b	White solid	519.0059[M-H]^-	34	98
5c	White solid	542.0222[M-H]^-	30	100
5d	White solid	558.0179[M-H]^-	27	98
6a	White solid	716.0039[M-H]^-	15
6b	White solid	716.0039[M-H]^-	11
6c	White solid	739.0201[M-H]^-	35
6d	White solid	755.0166[M-H]^-	33
7a	Red solid	490.1780[M+H]⁺	95
7b	Yellow solid	465.0565[M-H]^-	80
7c	Blue solid	542.3200[M]	25
7d	Red solid	777.2483[M-H]^-	26
9	White solid		21
10	Yellow solid		86

Compd.	Appearance	ESI-HRMS, m/z	Yield(%)	Purity(%, HPLC)
1a	Red solid	1095.1483[M-H]^-	18	90
1b	Yellow solid	1071.0604[M-H]^-	35	98
		991.0912[M-H₂PO₃]^-
1c	Blue solid	1169.3229[M-H]^-	18	100
1d	Brown solid	1422.2546[M-H]^-	43	93
2	White liquid		67
4a	Pale yellow solid		61
4b	Pale yellow solid		58
4c	Pale white solid		92
4d	White solid		39
5a	White solid	522.0070[M+H]⁺	34	100
5b	White solid	519.0059[M-H]^-	34	98
5c	White solid	542.0222[M-H]^-	30	100
5d	White solid	558.0179[M-H]^-	27	98
6a	White solid	716.0039[M-H]^-	15
6b	White solid	716.0039[M-H]^-	11
6c	White solid	739.0201[M-H]^-	35
6d	White solid	755.0166[M-H]^-	33
7a	Red solid	490.1780[M+H]⁺	95
7b	Yellow solid	465.0565[M-H]^-	80
7c	Blue solid	542.3200[M]	25
7d	Red solid	777.2483[M-H]^-	26
9	White solid		21
10	Yellow solid		86

Compd.	¹H NMR^a, δ	³¹P NMR^b, δ
1a	1.24—1.31(m, 1H), 2.04—2.15(m, 1H), 2.26—2.32(m, 1H), 2.65—2.75(m, 2H), 2.94—2.99(m, 2H), 3.17(t,J=4.8 Hz, 2H), 3.25(d, J=9.6 Hz, 8H), 3.72—3.77(m, 2H), 3.85—3.90(m, 3H), 3.96(s, 2H), 4.10—4.16(m, 2H), 4.49(s, 1H), 5.47(s, 1H), 5.84—5.88(m, 1H), 6.70—6.76(m, 2H), 6.91(d, J=9.6 Hz, 1H), 7.00—7.05(m, 1H), 7.18(d, J=9.6 Hz, 1H), 7.30(d, J=9.6 Hz, 1H), 7.60(d, J=8.0 Hz, 1H), 7.80(s, 1H), 8.18(d, J=7.6 Hz, 1H), 8.29(s, 1H)
1b	2.01—2.14(m, 1H), 2.29—2.39(m, 1H), 2.56—2.70(m, 2H), 2.85—3.01(m, 4H), 3.73—3.82(m, 1H), 3.85—4.25(m, 6H), 4.45(s, 1H), 5.92(t,J=4.0 Hz, 1H), 6.73—6.78(m, 4H), 7.31(dd, J=2.8, 8.8 Hz, 2H), 7.37(s, 1H), 7.62(d, J=8.8 Hz, 1H), 7.79(s, 1H), 7.92(d, J=8.0 Hz, 1H)	-21.22, -11.07, -8.27
2	2.34(t, J=2.8 Hz, 1H), 4.16(dd, J=2.4, 5.2 Hz, 2H), 6.61(s, 1H)
4a	2.11(t, J=5.1 Hz, 2H), 3.56—3.58(m, 2H), 3.78(d, J=4.0 Hz, 1H), 4.21(d, J=5.1 Hz, 3H), 5.08(t, J=5.1 Hz, 1H), 5.23(d, J=4.2 Hz, 1H), 6.09(t, J=6.6 Hz, 1H), 8.18(s, 1H), 10.05(t, J=4.8 Hz, 1H), 11.63(s, 1H)
4b	1.94—2.01(m, 1H), 2.12—2.18(m, 1H), 3.51(s, 1H), 3.55—3.62(m, 2H), 3.79(q,J=3.2, 6.8 Hz, 1H), 4.20(d, J=3.2 Hz, 1H), 4.28(s, 1H), 5.05(t, J=4.8 Hz, 1H), 5.20(d, J=3.6 Hz, 1H), 6.10(t, J=6.4 Hz, 1H), 6.84(s, 1H), 7.81(s, 1H), 8.15(s, 1H), 9.96(s, 1H)
4c	2.12—2.18(m, 1H), 2.41—2.46(m, 1H), 3.45—3.57(m, 2H), 3.78—3.81(m, 1H), 4.28—4.31(m, 3H), 5.06(t, J=5.6 Hz, 1H), 5.25(d, J=4.0 Hz, 1H), 6.43—6.47(m, 1H), 7.74(s, 1H), 8.09(s, 1H), 10.08(t, J=5.2 Hz, 1H)
4d	2.26—2.32(m, 1H), 2.48—2.55(m, 1H), 3.70—3.80(m, 2H), 3.96(dd, J=3.6, 6.8 Hz, 1H), 4.33(s, 2H), 4.47—4.50(m, 1H), 6.38—6.42(m, 1H), 7.25(s, 1H)
5a	2.36—2.47(m, 2H), 4.01(s, 2H), 4.20—4.30(m, 3H), 4.63—4.65(m, 1H), 6.28(t, J=6.0 Hz, 1H), 8.42(s, 1H)	-22.39, -11.35, -9.30
5b	2.27—2.33(m, 1H), 2.44—2.50(m, 1H), 4.05(s, 2H), 4.19—4.31(m, 3H), 4.56—4.59(m, 1H), 6.21(t, J=6.0 Hz, 1H), 8.37(s, 1H)	-22.55, -11.51, -10.31
5c	2.46—2.59(m, 2H), 4.10—4.24(m, 5H), 4.70(s, 1H), 6.49(t, J=6.8 Hz, 1H), 7.78(s, 1H), 8.02(s, 1H)	-21.67, -10.98, -8.50
5d	2.41—2.46(m, 1H), 2.53—2.58(m, 1H), 4.06(s, 2H), 4.11—4.20(m, 3H), 4.73(s, 1H), 6.34(t, J=6.8 Hz, 1H), 7.45(s, 1H)	-22.98, -11.24, -10.59
6a	2.18—2.26(m, 1H), 2.36—2.43(m, 1H), 2.66(t, J=6.4 Hz, 2H), 3.08(t, J=6.8 Hz, 2H), 4.08(s, 1H), 4.17(s, 1H), 4.49—4.52(m, 1H), 6.17(t, J=6.4 Hz, 1H), 7.18—7.21(m, 1H), 7.75(d, J=6.0 Hz, 2H), 8.03(s, 1H), 8.29(d, J=4.8 Hz, 1H)
6b	2.18—2.26(m, 1H), 2.36—2.43(m, 1H), 2.66(t, J=6.4 Hz, 2H), 3.08(t, J=6.8 Hz, 2H), 4.08(s, 1H), 4.17(s, 1H), 4.49—4.52(m, 1H), 6.17(t, J=6.4 Hz, 1H), 7.18—7.21(m, 1H), 7.75(d, J=6.0 Hz, 2H), 8.03(s, 1H), 8.29(d, J=4.8 Hz, 1H)
6c	2.46—2.51(m, 1H), 2.59—2.66(m, 1H), 2.74(t, J=6.4 Hz, 2H), 3.15(t, J=6.0 Hz, 2H), 4.18—4.27(m, 5H), 4.71—4.74(m, 1H), 6.52(t, J=6.8 Hz, 1H), 7.00(t, J=5.2 Hz, 1H), 7.55—7.60(m, 3H), 8.05(s, 1H), 8.10(d, J=4.0 Hz, 1H)
6d	2.35—2.40(m, 2H), 2.68(t, J=6.4 Hz, 2H), 3.12(t, J=6.4 Hz, 2H), 4.12—4.21(m, 5H), 4.71(s, 1H), 6.37(t, J=7.6 Hz, 1H), 7.17—7.21(m, 1H), 7.32(s, 1H), 7.77(s, 2H), 8.32(d, J=5.2 Hz, 1H)	-22.52, -11.13, -9.65
7a	1.26—1.31(m, 12H), 2.70(t, J=7.2 Hz, 2H), 3.53(t, J=6.8 Hz, 2H), 6.97(d, J=2.4 Hz, 2H), 7.04(dd, J=2.4, 9.6 Hz, 2H), 7.13(d, J=9.2 Hz, 2H), 7.81(d, J=1.2 Hz, 1H), 8.19(dd, J=1.6, 8.4 Hz, 1H), 8.39(d, J=8.4 Hz, 1H)
7b	2.69(t, J=6.4 Hz, 2H), 3.69(t, J=6.8 Hz, 2H), 6.69—6.72(m, 2H), 6.84(d, J=2.0 Hz, 2H), 6.91(d, J=8.8 Hz, 2H), 7.56(s, 1H), 7.76—7.83(m, 1H), 7.99(t, J=8.4 Hz, 1H)
Compd.	¹H NMR^a, δ	³¹P NMR^b, δ
9	2.49—2.52(m, 2H), 2.58—2.61(m, 2H), 7.26(t, J=7.2 Hz, 3 H), 7.33(t, J=7.2 Hz, 6 H), 7.45(d, J=7.6 Hz, 6 H)
10	2.42(t, J=6.4 Hz, 2H), 3.34(t, J=5.2 Hz, 2H), 6.52(dd, J=2.4, 8.8 Hz, 2H), 6.63—6.65(m, 4 H), 7.12(t, J=7.2 Hz, 3 H), 7.20(t, J=7.6 Hz, 6 H), 7.31(d, J=7.6 Hz, 6 H), 7.45(s, 1 H), 7.76(dd, J=1.6, 8.4 Hz, 1 H), 7.85(d, J=8.4 Hz, 1 H)

Compd.	¹H NMR^a, δ	³¹P NMR^b, δ
1a	1.24—1.31(m, 1H), 2.04—2.15(m, 1H), 2.26—2.32(m, 1H), 2.65—2.75(m, 2H), 2.94—2.99(m, 2H), 3.17(t,J=4.8 Hz, 2H), 3.25(d, J=9.6 Hz, 8H), 3.72—3.77(m, 2H), 3.85—3.90(m, 3H), 3.96(s, 2H), 4.10—4.16(m, 2H), 4.49(s, 1H), 5.47(s, 1H), 5.84—5.88(m, 1H), 6.70—6.76(m, 2H), 6.91(d, J=9.6 Hz, 1H), 7.00—7.05(m, 1H), 7.18(d, J=9.6 Hz, 1H), 7.30(d, J=9.6 Hz, 1H), 7.60(d, J=8.0 Hz, 1H), 7.80(s, 1H), 8.18(d, J=7.6 Hz, 1H), 8.29(s, 1H)
1b	2.01—2.14(m, 1H), 2.29—2.39(m, 1H), 2.56—2.70(m, 2H), 2.85—3.01(m, 4H), 3.73—3.82(m, 1H), 3.85—4.25(m, 6H), 4.45(s, 1H), 5.92(t,J=4.0 Hz, 1H), 6.73—6.78(m, 4H), 7.31(dd, J=2.8, 8.8 Hz, 2H), 7.37(s, 1H), 7.62(d, J=8.8 Hz, 1H), 7.79(s, 1H), 7.92(d, J=8.0 Hz, 1H)	-21.22, -11.07, -8.27
2	2.34(t, J=2.8 Hz, 1H), 4.16(dd, J=2.4, 5.2 Hz, 2H), 6.61(s, 1H)
4a	2.11(t, J=5.1 Hz, 2H), 3.56—3.58(m, 2H), 3.78(d, J=4.0 Hz, 1H), 4.21(d, J=5.1 Hz, 3H), 5.08(t, J=5.1 Hz, 1H), 5.23(d, J=4.2 Hz, 1H), 6.09(t, J=6.6 Hz, 1H), 8.18(s, 1H), 10.05(t, J=4.8 Hz, 1H), 11.63(s, 1H)
4b	1.94—2.01(m, 1H), 2.12—2.18(m, 1H), 3.51(s, 1H), 3.55—3.62(m, 2H), 3.79(q,J=3.2, 6.8 Hz, 1H), 4.20(d, J=3.2 Hz, 1H), 4.28(s, 1H), 5.05(t, J=4.8 Hz, 1H), 5.20(d, J=3.6 Hz, 1H), 6.10(t, J=6.4 Hz, 1H), 6.84(s, 1H), 7.81(s, 1H), 8.15(s, 1H), 9.96(s, 1H)
4c	2.12—2.18(m, 1H), 2.41—2.46(m, 1H), 3.45—3.57(m, 2H), 3.78—3.81(m, 1H), 4.28—4.31(m, 3H), 5.06(t, J=5.6 Hz, 1H), 5.25(d, J=4.0 Hz, 1H), 6.43—6.47(m, 1H), 7.74(s, 1H), 8.09(s, 1H), 10.08(t, J=5.2 Hz, 1H)
4d	2.26—2.32(m, 1H), 2.48—2.55(m, 1H), 3.70—3.80(m, 2H), 3.96(dd, J=3.6, 6.8 Hz, 1H), 4.33(s, 2H), 4.47—4.50(m, 1H), 6.38—6.42(m, 1H), 7.25(s, 1H)
5a	2.36—2.47(m, 2H), 4.01(s, 2H), 4.20—4.30(m, 3H), 4.63—4.65(m, 1H), 6.28(t, J=6.0 Hz, 1H), 8.42(s, 1H)	-22.39, -11.35, -9.30
5b	2.27—2.33(m, 1H), 2.44—2.50(m, 1H), 4.05(s, 2H), 4.19—4.31(m, 3H), 4.56—4.59(m, 1H), 6.21(t, J=6.0 Hz, 1H), 8.37(s, 1H)	-22.55, -11.51, -10.31
5c	2.46—2.59(m, 2H), 4.10—4.24(m, 5H), 4.70(s, 1H), 6.49(t, J=6.8 Hz, 1H), 7.78(s, 1H), 8.02(s, 1H)	-21.67, -10.98, -8.50
5d	2.41—2.46(m, 1H), 2.53—2.58(m, 1H), 4.06(s, 2H), 4.11—4.20(m, 3H), 4.73(s, 1H), 6.34(t, J=6.8 Hz, 1H), 7.45(s, 1H)	-22.98, -11.24, -10.59
6a	2.18—2.26(m, 1H), 2.36—2.43(m, 1H), 2.66(t, J=6.4 Hz, 2H), 3.08(t, J=6.8 Hz, 2H), 4.08(s, 1H), 4.17(s, 1H), 4.49—4.52(m, 1H), 6.17(t, J=6.4 Hz, 1H), 7.18—7.21(m, 1H), 7.75(d, J=6.0 Hz, 2H), 8.03(s, 1H), 8.29(d, J=4.8 Hz, 1H)
6b	2.18—2.26(m, 1H), 2.36—2.43(m, 1H), 2.66(t, J=6.4 Hz, 2H), 3.08(t, J=6.8 Hz, 2H), 4.08(s, 1H), 4.17(s, 1H), 4.49—4.52(m, 1H), 6.17(t, J=6.4 Hz, 1H), 7.18—7.21(m, 1H), 7.75(d, J=6.0 Hz, 2H), 8.03(s, 1H), 8.29(d, J=4.8 Hz, 1H)
6c	2.46—2.51(m, 1H), 2.59—2.66(m, 1H), 2.74(t, J=6.4 Hz, 2H), 3.15(t, J=6.0 Hz, 2H), 4.18—4.27(m, 5H), 4.71—4.74(m, 1H), 6.52(t, J=6.8 Hz, 1H), 7.00(t, J=5.2 Hz, 1H), 7.55—7.60(m, 3H), 8.05(s, 1H), 8.10(d, J=4.0 Hz, 1H)
6d	2.35—2.40(m, 2H), 2.68(t, J=6.4 Hz, 2H), 3.12(t, J=6.4 Hz, 2H), 4.12—4.21(m, 5H), 4.71(s, 1H), 6.37(t, J=7.6 Hz, 1H), 7.17—7.21(m, 1H), 7.32(s, 1H), 7.77(s, 2H), 8.32(d, J=5.2 Hz, 1H)	-22.52, -11.13, -9.65
7a	1.26—1.31(m, 12H), 2.70(t, J=7.2 Hz, 2H), 3.53(t, J=6.8 Hz, 2H), 6.97(d, J=2.4 Hz, 2H), 7.04(dd, J=2.4, 9.6 Hz, 2H), 7.13(d, J=9.2 Hz, 2H), 7.81(d, J=1.2 Hz, 1H), 8.19(dd, J=1.6, 8.4 Hz, 1H), 8.39(d, J=8.4 Hz, 1H)
7b	2.69(t, J=6.4 Hz, 2H), 3.69(t, J=6.8 Hz, 2H), 6.69—6.72(m, 2H), 6.84(d, J=2.0 Hz, 2H), 6.91(d, J=8.8 Hz, 2H), 7.56(s, 1H), 7.76—7.83(m, 1H), 7.99(t, J=8.4 Hz, 1H)
Compd.	¹H NMR^a, δ	³¹P NMR^b, δ
9	2.49—2.52(m, 2H), 2.58—2.61(m, 2H), 7.26(t, J=7.2 Hz, 3 H), 7.33(t, J=7.2 Hz, 6 H), 7.45(d, J=7.6 Hz, 6 H)
10	2.42(t, J=6.4 Hz, 2H), 3.34(t, J=5.2 Hz, 2H), 6.52(dd, J=2.4, 8.8 Hz, 2H), 6.63—6.65(m, 4 H), 7.12(t, J=7.2 Hz, 3 H), 7.20(t, J=7.6 Hz, 6 H), 7.31(d, J=7.6 Hz, 6 H), 7.45(s, 1 H), 7.76(dd, J=1.6, 8.4 Hz, 1 H), 7.85(d, J=8.4 Hz, 1 H)

四色荧光标记二硫键可逆终端的合成及在DNA合成测序中的应用

Synthesis and Application of Four Fluorescence Labeled Nucleotides Through Disulfide as Reversible Terminators in DNA Sequencing by Synthesis

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