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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2020, Vol. 40 Issue (8): 55-62    DOI: 10.13523/j.cb.2005013
综述     
DNA双链断裂检测技术研究进展 *
戴寒莹,徐克前()
中南大学湘雅医学院医学检验系 长沙 410013
Research Progress on DNA Double-Strand Break Assay
DAI Han-ying,XU Ke-qian()
Department of Laboratory Medicine, Xiangya School of Medicine, Central South University, Changsha 410013, China
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摘要：

细胞DNA的完整性受到不同因素的影响,可分为内源性及外源性因素,这些因素均可引起不同程度的DNA损伤。其中,DNA双链断裂是最严重的一种DNA损伤,若未能进行及时的修复,则会引起一系列的损伤反应。严重的DNA双链断裂甚至可以造成细胞凋亡、肿瘤的发生等严重后果。因此,快速并准确地检测细胞DNA双链断裂程度能帮助评估DNA的完整性、内外环境的遗传毒性效应、临床诊断和放化疗监测。DNA双链断裂检测技术近年来发展迅速,目前可基于物理或化学方法、免疫荧光法和高通量测序技术检测DNA双链断裂程度。对这些检测方法的最新研究进展、应用以及其优缺点进行介绍,旨在为后续DNA双链断裂程度检测的研究和临床提供参考。
关键词： DNA双链断裂分析;  彗星试验;  γ-H2AX;  高通量测序    
Abstract:

Different factors affect DNA integrity, which can be divided into endogenous and exogenous factors, these factors can cause different degrees of DNA damage. Among them, DNA double-strand break is the most serious kind of DNA damage, which is characterized by two DNA strands being cut off. If it is not repaired in time, it will cause a series of damage reactions. Serious DNA double strand break can even cause severe consequences such as cell apoptosis and tumor. Therefore, rapid and accurate detection of the degree of DNA double-strand break in cells is helpful to assess DNA integrity, the effects of genetic toxicity in and out of the environment, clinical diagnosis and chemoradiotherapy monitoring. DNA double-strand break detection technology has developed rapidly in recent years. According to the principle, it can be roughly divided into physical or chemical methods, immunofluorescence method and high-throughput sequencing method. These methods detect the physical and chemical characteristics of the products after DNA double-strand break or specific molecular markers, so as to evaluate the degree of DNA double-strand break. The latest research progress, application and advantages and disadvantages of these methods were introduced in order to provide guidance for the future research and clinic of DNA double-strand break detection.
Key words: DNA double-strand break assay    Comet assay    γ-H2AX    High-throughput sequencing
收稿日期: 2020-05-08 出版日期: 2020-09-10
ZTFLH:  Q819  
基金资助: * 国家自然科学基金(81471499);湖南省自然科学基金(2019JJ40347)
通讯作者: 徐克前     E-mail: xukeqian@csu.edu.cn
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戴寒莹
徐克前

引用本文:

戴寒莹,徐克前. DNA双链断裂检测技术研究进展 *[J]. 中国生物工程杂志, 2020, 40(8): 55-62.

DAI Han-ying,XU Ke-qian. Research Progress on DNA Double-Strand Break Assay. China Biotechnology, 2020, 40(8): 55-62.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2005013    或    https://manu60.magtech.com.cn/biotech/CN/Y2020/V40/I8/55

图1  彗星实验检测DSBs原理图
图2  LM-PCR原理图
图3  BLESS和BLISS检测DSBs原理图
方法 检测 优点 缺点
BLESS 直接 高特异性、原位标记DSBs,可提供DSBs全基因组图 需要大量原料、劳动强度大且半定量
BLISS 直接 可在固体表面上原位反应和洗涤,耗时短、可定量 仅能在基因组中的预定位点检测DSB
DSBCapture 直接 以单核苷酸分辨率高灵敏度地鉴定DSBs 需要大量原料、劳动强度大
End-seq 直接 提供DNA断裂和末端切除的高分辨率图 需要大量细胞、背景高
ChIP-seq 间接 DNA结合蛋白位置分析首选方法 需要原料多、无法达到核苷酸分辨率
GUIDE-seq 间接 可检测RGN诱导的DSBs,可在全基因组范围内识别脱靶位点 难以检测到其他DSBs和5'或3'突出端的DSBs
IDLV 间接 可以检测到频率低至1%的脱靶点 需要高技能、高成本
HTGTS 间接 可应用于易位和复发性DSBs的全基因组筛选 成本较高、灵敏度较低、需要NHEJ活性来标记DSBs
LAM-HTGTS 间接 灵敏、可重现、较便宜、易实施、耗时短,能检测到低水平的断裂,分辨率高和背景率较低 仅检测易位的DSB,只能将猎物DSBs连接到已知的诱饵DSBs上
表1  不同DSBs高通量测序技术的比较
方法 优点 缺点 应用
中性滤膜洗脱法 最早的DSBs检测技术 灵敏度低、稳定性差、假阳性率高 放射生物学研究
脉冲电场凝胶电泳 大片段DNA检测,提供片段长度信息 仪器较昂贵、操作费力、耗时长、不能定量 分离DNA片段,DNA细菌分型
单细胞凝胶电泳 用量少、操作简便、成本低 手工操作较多、通量低、结果判读依赖软件、可重复性差 DNA断裂测定,基因毒性测试
γ-H2AX检测 敏感、时效性、使用最广泛 不能分析高剂量DSBs、仪器要求高 细胞放化疗敏感性
TUNEL法 特异性和敏感性较高,可用于细胞形态学研究 无法区分SSBs和DSBs,不能检测大片段DNA 细胞凋亡、评估精子DNA完整性
测序技术 分辨率高、敏感性高、特异性高 测序技术的实施费用和仪器较贵 全基因组定位检测DSBs
表2  主要DSBs检测技术方法学评价
[1] Chatterjee N, Walker G C. Mechanisms of DNA damage, repair and mutagenesis. Environ Mol Mutagen, 2017,58(5):235-263.
doi: 10.1002/em.22087 pmid: 28485537
[2] Mladenov E, Magin S, Soni A, et al. DNA double-strand-break repair in higher eukaryotes and its role in genomic instability and cancer: cell cycle and proliferation-dependent regulation. Semin Cancer Biol, 2016,37-38:51-64.
pmid: 2133109
[3] Keskin H, Storici F. An approach to detect and study DNA double-strand break repair by transcript RNA using a spliced-antisense RNA template. Methods Enzymol, 2018,601:59-70.
doi: 10.1016/bs.mie.2017.11.026 pmid: 29523242
[4] Lopez-Canovas L, Martinez Benitez M B, Herrera Isidron J A, et al. Pulsed field gel electrophoresis: past, present, and future. Anal Biochem, 2019,573:17-29.
doi: 10.1016/j.ab.2019.02.020 pmid: 30826351
[5] Kawashima Y, Yamaguchi N, Teshima R, et al. Detection of DNA double-strand breaks by pulsed-field gel electrophoresis. Genes Cells, 2017,22(1):84-93.
doi: 10.1111/gtc.12457 pmid: 27976495
[6] Takiguchi Y, Kariyazono R, Ohta K. Detection of DNA damage-induced DSBs by the contour-clamped homogeneous electric field (CHEF) system in mammalian cells. Methods Mol Biol, 2020,2119:101-109.
doi: 10.1007/978-1-0716-0323-9_9 pmid: 31989518
[7] Neoh H M, Tan X E, Sapri H F, et al. Pulsed-field gel electrophoresis (PFGE): a review of the “gold standard” for bacteria typing and current alternatives. Infect Genet Evol, 2019,74:103935.
doi: 10.1016/j.meegid.2019.103935 pmid: 31233781
[8] Lytsy B, Engstrand L, Gustafsson A, et al. Time to review the gold standard for genotyping vancomycin-resistant enterococci in epidemiology: comparing whole-genome sequencing with PFGE and MLST in three suspected outbreaks in Sweden during 2013-2015. Infect Genet Evol, 2017,54:74-80.
doi: 10.1016/j.meegid.2017.06.010 pmid: 28627467
[9] Koppen G, Azqueta A, Pourrut B, et al. The next three decades of the comet assay: a report of the 11 th International Comet Assay Workshop . Mutagenesis, 2017,32(3):397-408.
doi: 10.1093/mutage/gex002 pmid: 28340065
[10] Mondal M, Guo J. Comet-FISH for ultrasensitive strand-specific detection of DNA damage in single cells. Methods Enzymol, 2017,591:83-95.
doi: 10.1016/bs.mie.2017.03.023 pmid: 28645380
[11] Ge J, Chow D N, Fessler J L, et al. Micropatterned comet assay enables high throughput and sensitive DNA damage quantification. Mutagenesis, 2015,30(1):11-19.
doi: 10.1093/mutage/geu063 pmid: 25527723
[12] Moller P. The comet assay: ready for 30 more years. Mutagenesis, 2018,33(1):1-7.
doi: 10.1093/mutage/gex046 pmid: 29325088
[13] Goutham V H, Kamalesh M D, Guruprasad P K, et al. A modified fluorimetric neutral filter elution method for analyzing radiation-induced double strand break and repair. Anal Biochem, 2011,414(2):287-293.
doi: 10.1016/j.ab.2011.03.033 pmid: 21453672
[14] Pan X, Wang J, Zhang Y, et al. Detection of trace amounts of target DNA from massive background of nucleic acids by using the LM-PCR-based preamplification method. Biotechnol Appl Biochem, 2017,64(6):879-887.
doi: 10.1002/bab.1545 pmid: 27859653
[15] Lim J J, Lee J, Kim D H, et al. DNA fragmentation of human sperm can be detected by ligation-mediated real-time polymerase chain reaction. Fertil Steril, 2013,100(6):1564-1571.
doi: 10.1016/j.fertnstert.2013.08.017
[16] Plappert-Helbig U, Libertini S, Frieauff W, et al. Gamma-H2AX immunofluorescence for the detection of tissue-specific genotoxicity in vivo. Environ Mol Mutagen, 2019,60(1):4-16.
doi: 10.1002/em.22238 pmid: 30307065
[17] Nelson B C, Wright C W, Ibuki Y, et al. Emerging metrology for high-throughput nanomaterial genotoxicology. Mutagenesis, 2017,32(1):215-232.
doi: 10.1093/mutage/gew037 pmid: 27565834
[18] Lee Y, Wang Q, Shuryak I, et al. Development of a high-throughput gamma-H2AX assay based on imaging flow cytometry. Radiat Oncol, 2019,14(1):150.
doi: 10.1186/s13014-019-1344-7 pmid: 31438980
[19] Turinetto V, Giachino C. Multiple facets of histone variant H2AX: a DNA double-strand-break marker with several biological functions. Nucleic Acids Res, 2015,43(5):2489-2498.
doi: 10.1093/nar/gkv061 pmid: 25712102
[20] Luczak M W, Zhitkovich A. Monoubiquitinated gamma-H2AX: abundant product and specific biomarker for non-apoptotic DNA double-strand breaks. Toxicol Appl Pharmacol, 2018,355:238-246.
doi: 10.1016/j.taap.2018.07.007 pmid: 30006243
[21] Crowley L C, Marfell B J, Waterhouse N J, et al. Detection of DNA fragmentation in apoptotic cells by TUNEL. Cold Spring Harb Protoc, 2016,2016(10):900-904.
[22] Stubenhaus B, Pellettieri J. Detection of apoptotic cells in planarians by whole-mount TUNEL. Methods Mol Biol, 2018,1774:435-444.
doi: 10.1007/978-1-4939-7802-1_16 pmid: 29916169
[23] Clouaire T, Rocher V, Lashgari A, et al. Comprehensive mapping of histone modifications at DNA double-strand breaks deciphers repair pathway chromatin signatures. Mol Cell, 2018,72(2):250-262.
doi: 10.1016/j.molcel.2018.08.020 pmid: 30270107
[24] Lengert N, Mirsch J, Weimer R N, et al. AutoFoci, an automated high-throughput foci detection approach for analyzing low-dose DNA double-strand break repair. Sci Rep, 2018,8(1):17282.
doi: 10.1038/s41598-018-35660-5 pmid: 30470760
[25] Vignard J, Mirey G, Salles B. Ionizing-radiation induced DNA double-strand breaks: a direct and indirect lighting up. Radiother Oncol, 2013,108(3):362-369.
doi: 10.1016/j.radonc.2013.06.013
[26] Crosetto N, Mitra A, Silva M J, et al. Nucleotide-resolution DNA double-strand break mapping by next-generation sequencing. Nat Methods, 2013,10(4):361-365.
[27] Yan W X, Mirzazadeh R, Garnerone S, et al. BLISS is a versatile and quantitative method for genome-wide profiling of DNA double-strand breaks. Nat Commun, 2017,8:15058.
doi: 10.1038/ncomms15058 pmid: 28497783
[28] Lensing S V, Marsico G, Hänsel-Hertsch R, et al. DSBCapture: in situ capture and sequencing of DNA breaks. Nat Method, 2016,13(10):855-857.
doi: 10.1038/nmeth.3960
[29] Canela A, Sridharan S, Sciascia N, et al. DNA breaks and end resection measured genome-wide by end sequencing. Mol Cell, 2016,63(5):898-911.
doi: 10.1016/j.molcel.2016.06.034 pmid: 27477910
[30] Mirzazadeh R, Kallas T, Bienko M, et al. Genome-wide profiling of DNA double-strand breaks by the BLESS and BLISS methods. Methods Mol Biol, 2018,1672:167-194.
doi: 10.1007/978-1-4939-7306-4_14 pmid: 29043625
[31] Shi L, Tang X, Tang G. GUIDE-seq to detect genome-wide double-stranded breaks in plants. Trends Plant Sci, 2016,21(10):815-818.
doi: 10.1016/j.tplants.2016.08.005 pmid: 27593568
[32] Zhu L J, Lawrence M, Gupta A, et al. GUIDEseq: a bioconductor package to analyze GUIDE-Seq datasets for CRISPR-Cas nucleases. BMC Genomics, 2017,18(1):379.
doi: 10.1186/s12864-017-3746-y pmid: 28506212
[33] Wang X, Wang Y, Wu X, et al. Unbiased detection of off-target cleavage by CRISPR-Cas9 and TALENs using integrase-defective lentiviral vectors. Nat Biotechnol, 2015,33:175-178.
doi: 10.1038/nbt.3127 pmid: 25599175
[34] Frock R L, Hu J, Meyers R M, et al. Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases. Nat Biotechnol, 2015,33(2):179-186.
doi: 10.1038/nbt.3101 pmid: 25503383
[35] Hu J, Meyers R M, Dong J, et al. Detecting DNA double-stranded breaks in mammalian genomes by linear amplification-mediated high-throughput genome-wide translocation sequencing. Nat Protoc, 2016,11(5):853-871.
doi: 10.1038/nprot.2016.043 pmid: 27031497
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