生物技术通报 ›› 2023, Vol. 39 ›› Issue (4): 227-235.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0940
张新博(), 崔浩亮, 史佩华, 高锦春, 赵顺然, 陶晨雨()
收稿日期:
2022-07-28
出版日期:
2023-04-26
发布日期:
2023-05-16
通讯作者:
陶晨雨,女,博士,副教授,研究方向:动物遗传育种与繁殖;E-mail: taochenyuty@163.com作者简介:
张新博,女,硕士研究生,研究方向:动物遗传育种与繁殖;E-mail: zhangxinbo58@163.com
基金资助:
ZHANG Xin-bo(), CUI Hao-liang, SHI Pei-hua, GAO Jin-chun, ZHAO Shun-ran, TAO Chen-yu()
Received:
2022-07-28
Published:
2023-04-26
Online:
2023-05-16
摘要:
将染色质免疫共沉淀技术(chromatin immunoprecipitation assay, ChIP)和第二代测序技术相结合的染色体免疫共沉淀测序技术(co-immunoprecitation followed by sequencing, ChIP-seq)是分析全基因组表观遗传学变化的重要方法,它可以快速、有效地检测到在全基因组范围内DNA与蛋白结合位点、转录因子结合位点(transcription factor binding site, TFBS)、组蛋白翻译后修饰(histone post-translation modifications, hPTMs)、核小体定位和DNA甲基化等。然而,长期以来ChIP-seq需要大量细胞来生成高质量数据集,这限制了ChIP在一些细胞数较低的样本如卵母细胞、早期胚胎细胞等研究领域的应用。近年来,在ChIP的基础上,研究人员提出了一系列降低样品起始量和实验成本、提高测序质量的低起始量ChIP-seq方法,促进了表观基因组学的发展。本文综述了ChIP原理和降低起始量的ChIP的方法学发展,并对其中几种比较重要的方法进行了比较,并总结了低起始量的ChIP-seq在表观遗传学研究中的应用。本文最后对低起始量ChIP技术的应用和发展进行了展望,为低细胞数样品在低起始量ChIP的选择提供了参考。
张新博, 崔浩亮, 史佩华, 高锦春, 赵顺然, 陶晨雨. 低起始量的免疫共沉淀技术研究进展[J]. 生物技术通报, 2023, 39(4): 227-235.
ZHANG Xin-bo, CUI Hao-liang, SHI Pei-hua, GAO Jin-chun, ZHAO Shun-ran, TAO Chen-yu. Research Progress in Low-input Chromatin Immunoprecipitation Assay[J]. Biotechnology Bulletin, 2023, 39(4): 227-235.
方法Method | 传统ChIP Traditional ChIP | ULI-NChIP | TAT-ChIP |
---|---|---|---|
起始细胞量Initial cell volume | 107个细胞 | 103个细胞 | 100个细胞 |
样品处理Sample processing | 甲醛交联 | 无需甲醛交联 | 无需甲醛交联 |
DNA片段化DNA fragmentation | 超声处理 | MNase消化 | Tn5转座酶处理 |
接头序列Adaptor Sequence | 加A,加序列接头 | 加A,加序列接头 | Tn5转座酶直接加序列接头 |
扩增方式Amplification method | PCR | PCR | PCR |
DNA纯化DNA purification | 纯化 | 纯化 | 无需纯化 |
文库构建Library construction | 8-10周期 | 8-10周期 | |
测量项目Measurement items | hPTMs | hPTMs | hPTMs |
表1 ChIP、ULI-NChIP 和 TAT-ChIP 三种技术的比较
Table 1 Comparison of three technologies ChIP, ULI-NChIP and TAT-ChIP
方法Method | 传统ChIP Traditional ChIP | ULI-NChIP | TAT-ChIP |
---|---|---|---|
起始细胞量Initial cell volume | 107个细胞 | 103个细胞 | 100个细胞 |
样品处理Sample processing | 甲醛交联 | 无需甲醛交联 | 无需甲醛交联 |
DNA片段化DNA fragmentation | 超声处理 | MNase消化 | Tn5转座酶处理 |
接头序列Adaptor Sequence | 加A,加序列接头 | 加A,加序列接头 | Tn5转座酶直接加序列接头 |
扩增方式Amplification method | PCR | PCR | PCR |
DNA纯化DNA purification | 纯化 | 纯化 | 无需纯化 |
文库构建Library construction | 8-10周期 | 8-10周期 | |
测量项目Measurement items | hPTMs | hPTMs | hPTMs |
方法Method | 传统ChIP Conventional ChIP | LIFE-ChIP-seq | SurfaceChIP-seq | MOWChIP | mu-CM | PnP-ChIP-seq |
---|---|---|---|---|---|---|
起始细胞量Starting cell volume | 107个细胞 | 50个细胞 | 30-100个细胞 | 100个细胞 | 20个细胞 | 15 000个细胞 |
样品处理Sample processing | 甲醛交联 | 甲醛交联 | 无需甲醛交联 | 甲醛交联 | 无需甲醛交联 | 甲醛交联 |
DNA片段化DNA fragmentation | 超声处理 | 超声处理 | MNase消化 | 超声处理 | MNase酶解和 Tn5转座酶处理 | 超声处理 |
接头序列Adaptor Sequence | 加A,加序列接头 | 加A,加序列接头 | 加A,加序列接头 | 加A,加序列接头 | Tn5转座酶直接加序列接头 | 加A,加序列接头 |
扩增方式Amplification method | PCR | PCR | PCR | PCR | PCR | PCR |
DNA纯化DNA purification | 纯化 | 纯化 | 纯化 | 纯化 | 纯化 | 无需纯化 |
文库构建Library construction | 8-10周期 | 1 d | 10个周期 | |||
测量项目Measurement items | hPTMs | hPTMs | hPTMs | hPTMs | hPTMs | hPTMs |
实验周期Experimental period | 4-5 d | 1 h | 1 h | 2 d | 7 h | 5 h |
一次运行得到的数据集 Data set from one run | 单个 | 4个 | 8个 | 8个 | 8个 | 24个 |
表2 ChIP、MOWChIP、LIFE-ChIP-seq、SurfaceChIP-seq、mu-CM 和 PnP-ChIP-seq 五种技术的比较
Table 2 Comparison of five technologies ChIP, MOWChIP, LIFE-ChIP-seq, SurfaceChIP-seq, mu-CM and PnP-ChIP-seq
方法Method | 传统ChIP Conventional ChIP | LIFE-ChIP-seq | SurfaceChIP-seq | MOWChIP | mu-CM | PnP-ChIP-seq |
---|---|---|---|---|---|---|
起始细胞量Starting cell volume | 107个细胞 | 50个细胞 | 30-100个细胞 | 100个细胞 | 20个细胞 | 15 000个细胞 |
样品处理Sample processing | 甲醛交联 | 甲醛交联 | 无需甲醛交联 | 甲醛交联 | 无需甲醛交联 | 甲醛交联 |
DNA片段化DNA fragmentation | 超声处理 | 超声处理 | MNase消化 | 超声处理 | MNase酶解和 Tn5转座酶处理 | 超声处理 |
接头序列Adaptor Sequence | 加A,加序列接头 | 加A,加序列接头 | 加A,加序列接头 | 加A,加序列接头 | Tn5转座酶直接加序列接头 | 加A,加序列接头 |
扩增方式Amplification method | PCR | PCR | PCR | PCR | PCR | PCR |
DNA纯化DNA purification | 纯化 | 纯化 | 纯化 | 纯化 | 纯化 | 无需纯化 |
文库构建Library construction | 8-10周期 | 1 d | 10个周期 | |||
测量项目Measurement items | hPTMs | hPTMs | hPTMs | hPTMs | hPTMs | hPTMs |
实验周期Experimental period | 4-5 d | 1 h | 1 h | 2 d | 7 h | 5 h |
一次运行得到的数据集 Data set from one run | 单个 | 4个 | 8个 | 8个 | 8个 | 24个 |
方法Method | 传统ChIP Conventional ChIP | lobChIP | FARP-ChIP | ChIPmentation |
---|---|---|---|---|
起始细胞量 Initial cell volume | 107个细胞 | 106个细胞 | 500个细胞 | 104个细胞 |
样品处理 Sample processing | 甲醛交联 | 甲醛交联 | 甲醛交联合成DNA | 甲醛交联 |
DNA片段化 DNA fragmentation | 超声处理 | 超声处理 | 超声处理 | Tn5转座酶处理 |
接头序列 Adaptor Sequence | 加A,加序列接头 | 加A,加序列接头 | 加A,加序列接头。 | Tn5转座酶直接加序列接头 |
扩增方式 Amplification method | PCR | PCR | PCR | PCR |
DNA纯化 DNA purification | 纯化 | 无需纯化 | 纯化 | 纯化 |
文库构建 Library construction | 8-10周期 | 16-18个周期 | 12周期 | |
测量项目 Measurement items | TF和hPTMs | TF和hPTMs | hPTMs | hPTMs、TF、核小体 |
实验周期 Experimental period | 4-5 d | 1 d |
表3 ChIP、lobChIP、FARP-ChIP和ChIPmentation四种技术的比较
Table 3 Comparison of four technologies ChIP, lobChIP, FARP-ChIP and ChIPmentation
方法Method | 传统ChIP Conventional ChIP | lobChIP | FARP-ChIP | ChIPmentation |
---|---|---|---|---|
起始细胞量 Initial cell volume | 107个细胞 | 106个细胞 | 500个细胞 | 104个细胞 |
样品处理 Sample processing | 甲醛交联 | 甲醛交联 | 甲醛交联合成DNA | 甲醛交联 |
DNA片段化 DNA fragmentation | 超声处理 | 超声处理 | 超声处理 | Tn5转座酶处理 |
接头序列 Adaptor Sequence | 加A,加序列接头 | 加A,加序列接头 | 加A,加序列接头。 | Tn5转座酶直接加序列接头 |
扩增方式 Amplification method | PCR | PCR | PCR | PCR |
DNA纯化 DNA purification | 纯化 | 无需纯化 | 纯化 | 纯化 |
文库构建 Library construction | 8-10周期 | 16-18个周期 | 12周期 | |
测量项目 Measurement items | TF和hPTMs | TF和hPTMs | hPTMs | hPTMs、TF、核小体 |
实验周期 Experimental period | 4-5 d | 1 d |
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