CN116891532A - 一种基于生物素:亲合素的二抗:转座酶复合物及其在检测蛋白质与dna相互作用中的应用 - Google Patents
一种基于生物素:亲合素的二抗:转座酶复合物及其在检测蛋白质与dna相互作用中的应用 Download PDFInfo
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Abstract
本发明提供一种基于生物素:亲合素的二抗:转座酶复合物及其在检测蛋白质与DNA相互作用中的应用。
Description
技术领域
本发明涉及生物技术领域。本发明具体涉及一种基于生物素:亲合素的二抗:转座酶复合物及其混合物。本发明具体涉及所述复合物或其混合物在检测蛋白质与DNA相互作用中的应用。本发明具体涉及一种能够提高检测蛋白质和DNA相互作用准确性,有效降低背景并提高真实数据产量的方法及其应用。
背景技术
染色质免疫沉淀(ChIP)是广泛用于研究蛋白质与DNA相互作用的方法,通常用于转录因子结合位点或组蛋白特异性修饰位点的研究。将ChIP与大规模平行DNA测序技术相结合,可以让研究者精确绘制感兴趣蛋白的全局DNA结合位点。ChIP的基本流程是:(1)交联:采用甲醛固定组织或细胞,使DNA与蛋白质紧密结合;(2)片段化:该过程破坏染色质,最终获得用于ChIP分析的DNA片段蛋白复合物;(3)染色质免疫沉淀:通过加入针对目的蛋白的抗体,结合靶蛋白-DNA复合物;(4)DNA回收和纯化:对回收的DNA片段进行纯化富集,通过下游检测技术(定量PCR、基因芯片、测序等)分析靶蛋白特异结合的DNA序列信息(doi:10.1038/nrg2641)(图1)。然而,因ChIP技术需要对组织或细胞进行甲醛交联,然后再进行DNA片段化,这使得该技术需要极高的细胞起始投入量(百万级),并且很可能因为甲醛过度交联使实验结果存在假阳性。
为了弥补ChIP的技术缺陷,研究者通过不断的更新与优化,开发了CUT&Run(Cleavage Under Targets&Release Using Nuclease)技术和CUT&Tag(CleavageUnderTargets&Tagmentation)技术(DOI:10.7554/eLife.21856.001;doi.org/10.1038/s41467-019-09982-5)。CUT&Tag以及CUT&Run是研究生物活体细胞中蛋白质与DNA相互作用的技术,其通过抗体富集Tn5转座酶或MNase核酸酶特异切割目的蛋白附近的DNA,再通过对切割后标记的DNA进行文库构建和测序就可以绘制目的蛋白全局的DNA结合位点(图2)。该技术因为不需要交联和物理打断DNA,一定程度的缓解了CHIP技术假阳性和高细胞投入量的缺点。
虽然CUT&Tag和CUT&Run技术相较ChIP有明显提升,但是CUT&Tag和CUT&Run依赖PA/PG-Tn5/MNase融合蛋白与二抗Fc段进行结合,然后将Tn5/MNase带到目标蛋白区域附近进行DNA切割。此过程一方面受限于PA/PG蛋白与二抗Fc段的结合能力,另一方面PA/PG-Tn5/MNase融合蛋白与二抗的数量关系不好量化,极有可能导致过量的PA/PG-Tn5/MNase融合蛋白孵育。未特异结合的PA/PG-Tn5/MNase融合蛋白会在CUT&Tag/CUT&Run实验过程中非特异性的切割染色质开放区域,从而产生大量的TSS(Transcription Start Sites)区域假阳性信号(图3),这会对CUT&Tag/CUT&Run实验数据的真实性造成很大的干扰。不仅如此,TSS区域假阳性信号的富集也会导致同一测序深度下真实信号数据占比低,降低了有效数据的产出。
发明概述
发明目的
1、解决CUT&Tag和CUT&Run实验中的假阳性问题。
2、优化CUT&Tag和CUT&Run实验流程,减少洗涤步骤,不进行模板提取就可以直接进行扩增,从而缩短实验时间。
3、创新的复合物构建方式,可进行真核生物蛋白与原核生物蛋白的体外紧密结合。
发明内容
1、本发明基于生物素:亲合素结合对构建不同二抗与Tn5/MNase的复合物,该复合物的构建可以有效降低游离的Tn5和MNase数量,在CUT&Tag和CUT&Run实验中,将原一抗孵育,二抗孵育,蛋白A(PA)/蛋白G(PG)-Tn5/MNase孵育优化为一抗孵育,二抗-T5/MNase复合物孵育。此步骤减少了实验操作流程,并通过二抗-T5/MNase复合物将Tn5/MNase牢牢束缚,减少了原CUT&Tag和CUT&Run实验中的游离Tn5/MNase暴露,显著降低了阴性对照TSS区域的假阳性信号。
2、由于二抗一般来源于小鼠,家兔,山羊等物种,而Tn5酶等却来自大肠杆菌,这使得在构建融合蛋白时难度很大,在大肠杆菌中表达二抗-Tn5/MNase融合蛋白后,发现二抗结合效率很低,可能与真核生物与原核生物转录后修饰差异有关。为克服二抗与Tn5宿主来源不同导致无法获得具有功能活性的二抗-Tn5/MNase融合蛋白,我们创新的将二抗进行生物素(biotin,BT)修饰,然后与亲合素,特别是链霉亲合素(streptavidin,SA)-Tn5/MNase融合蛋白在体外进行孵育(或将二抗进行BT修饰后偶连SA,同时对Tn5/MNase进行BT修饰,然后对二者在体外进行孵育)。SA:BT的结合能力达到了Kd=10^-15,比结合程度最高的抗原-抗体结合能力还要高出至少一万倍。且一个SA蛋白能够结合4个BT分子,我们通过SA:BT的比例控制,保证Tn5/MNase酶大部分被二抗捕获,从而达到了具有功能的“二抗-BT:SA-Tn5/Mnase复合物”和“二抗-BT:SA:BT-Tn5/Mnase复合物”。
3、CUT&Tag酶切反应后加入终止液与带特殊序列接头的扩增Mix,不需要纯化可直接进行文库构建。此步骤可减少实验操作流程,并在一个EP管中完成全部的建库实验,有利于后续自动化平台的搭建。
有益效果
1、具有功能的“二抗-BT:SA:BT-Tn5/Mnase和二抗-BT:SA-Tn5/Mnase复合物”使Tn5/Mnase被二抗牢固抓取,Tn5/Mnase酶与二抗的结合程度远强于PA/PG-Tn5与二抗的结合,这使得CUT&Tag和CUT&Run实验过程中阴性对照的背景显著降低,极大的降低了CUT&Tag和CUT&Run技术中的假阳性。
2、整体有效数据占比多,实验数据信号富集程度显著增加(图5)。
3、整体CUT&Tag和CUT&Run实验时间减少了6小时,提升了实验的整体效率。
4、使用SA修饰与BT修饰的方式构建复合物,这使得整合真核生物蛋白和原核生物蛋白并保留其原有活性成为可能。尤其是免疫相关蛋白涉及VDJ重排和丰富的转录后修饰,在原核生物体系中直接表达相关蛋白并没有免疫活性。通过SA修饰可以让免疫相关蛋白在原宿主中表达后再与其他原核生物蛋白结合,从而得到具有生物学活性的真核生物蛋白-原核生物蛋白复合物,其在CUT&Tag实验中展示出准确的靶点定位,在目的基因TSS区域具有明显的信号富集(图5)。
实施方案
本发明涉及下述实施方案。
1.一种基于生物素:亲合素的二抗:转座酶复合物,其以下式表示:
转座酶-亲合素:(生物素-二抗)n,其中n为1、2、3或4。
2.实施方案1的复合物,其中亲合素为链霉亲合素。
3.实施方案1的复合物,其中转座酶为Tn5转座酶。
4.实施方案1-3任一项的复合物的混合物,其以下式表示:
转座酶-亲合素:(生物素-二抗)p,其中p大于1且小于4。
5.一种基于生物素:亲合素的二抗:转座酶复合物,其以下式表示:
(转座酶-生物素)m:亲合素:(生物素-二抗)n,其中m和n各自独立为1、2或3,
且m+n小于或等于4。
6.实施方案5的复合物,其中亲合素为链霉亲合素。
7.实施方案5的复合物,其中转座酶为Tn5转座酶。
8.实施方案5的复合物,其中生物素附着于转座体的寡核苷酸,任选地,寡核苷酸是部分配对的5'-phos-CTGTCTCTTATACACATBTCTBT-3'(SEQ ID NO:
12)和5′-TCGTBTCGGCAGCGTCAGATGTGTATBTAAGAGACAG-3′(SEQ ID NO:195)和/或部分配对的5'-phos-CTGTCTCTTATACACATBTCTBT-3'(SEQID NO:12)和5′-GTCTCGTGGGCTBTCGGAGATGTGTATBTAAGAGACAG-3
(SEQ ID NO:297),或者,寡核苷酸是部分配对的5'-phos-CTBTGTCTCTTATBTACACATCT-3'(SEQ ID NO:29)和5′-TCGTCGGCAGCGTCAGATGTGTBTATBTAAGAGACAG-3′(SEQ ID NO:
215)和/或部分配对的5'-phos-CTBTGTCTCTTATBTACACATCT-3'(SEQ ID NO:29)和5′-GTCTBTCGTGGGCTBTCGGAGATGTGTATAAGAGACAG-3′
(SEQ ID NO:317),TBT表示生物素化的T。
9.实施方案5-8任一项的复合物的混合物,其以下式表示:
(转座酶-生物素)q:亲合素:(生物素-二抗)p,其中p和q各自大于或等于1且小于或等于3,且q+p小于或等于4。
10.一种检测细胞在靶蛋白与DNA相互作用的方法,其包括:
(1)将细胞与针对靶蛋白的一抗一起温育;
(2)将细胞与实施方案1-3和5-8任一项的复合物或实施方案4或9的混合物一起温育,其中所述复合物中的二抗是对所述一抗特异性的二抗,并容许转座酶作用于细胞基因组;
(3)扩增细胞DNA;
(4)纯化扩增的DNA;并
(5)对扩增的DNA测序。
附图说明
图1:Chip-Seq技术原理图;
图2:CUT&Tag实验原理图;
图3:常规CUT&Tag实验IgG阴性对照在TSS区域具有明显的信号富集;
图4:实施例2中三组阴性对照文库(IgG+二抗+pG-Tn5、IgG+二抗:pG-Tn5和IgG+二抗-BT:SA-Tn5)信号对比;
图5:实施例2中六组样本真实信号对比;
图6:实施例2中参比文库(一抗+二抗+pG-Tn5、一抗+二抗:pG-Tn5)和测试文库(一抗+二抗-BT:SA-Tn5)分布对比;
图7:实施例3中阴性对照(IgG+二抗-BT:SA:BT-Tn5-1、IgG+二抗-BT:SA:BT-Tn5-2)信号图;
图8:实施例3中八组样本真实信号对比;
图9:实施例3中四组文库分布图。
发明详述
在一个方面,本发明提供一种基于生物素:亲合素的二抗:转座酶复合物,其以下式表示:
转座酶-亲合素:(生物素-二抗)n,其中n为1、2、3或4。n为转座酶分子加载的二抗分子数。
在一个实施方案中,所述亲合素为链霉亲合素或中性亲合素。在一个实施方案中,所述转座酶为Tn5转座酶。
在一个方面,本发明提供上述复合物的混合物,其以下式表示:
转座酶-亲合素:(生物素-二抗)p,其中p为转座酶-亲合素的平均生物素-二抗载荷,大于1且小于4。p为混合物中转座酶分子加载的平均二抗分子数。
在一个方面,本发明提供一种制备上述复合物或混合物的方法,其包括:在大肠杆菌中表达转座酶-亲合素融合蛋白;
生物素化二抗;及以1:n’的摩尔比混合转座酶-亲合素融合蛋白与生物素化的二抗。
在一个实施方案中,n’=n。在一个实施方案中,n’>n。在一个实施方案中,n’=p。在一个实施方案中,n’>p。在一个实施方案中,n’>4,例如n’=5。在一个实施方案中,转座酶-亲合素融合蛋白与生物素化的二抗的摩尔比使得不存在游离的(即未结合生物素化的二抗的)转座酶-亲合素。在一个实施方案中,转座酶-亲合素融合蛋白与生物素化的二抗的摩尔比使得转座酶-亲合素中的亲合素被生物素-二抗中的生物素饱和结合。
在一个方面,本发明提供一种基于生物素:亲合素的二抗:转座酶复合物,其以下式表示:
(转座酶-生物素)m:亲合素:(生物素-二抗)n,其中m和n各自独立为1、2或3,且m+n小于或等于4,例如m+n=2、3或4。例如,m=1,n=1、2或3;m=2,n=1或2;m=3,n=1。又例如,n=1,m=1、2或3;n=2,m=1或2;n=3,m=1。m为亲合素分子加载的转座酶分子数,n为亲合素分子加载的二抗分子数。
在一个实施方案中,所述亲合素为链霉亲合素或中性亲合素。在一个实施方案中,所述转座酶为Tn5转座酶。在一个实施方案中,所述生物素附着于转座体的寡核苷酸。在一个实施方案中,转座酶分子(或相关寡核苷酸)加载一个或多个生物素分子,例如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16或更多个。在一个实施方案中,所述寡核苷酸如CTGTCTCTTATACACATCT(SEQ ID NO:1)所示。在一个实施方案中,所述寡核苷酸如SEQ IDNO:4-182任一所示。在一个实施方案中,所述寡核苷酸如TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG(SEQ ID NO:2)所示。在一个实施方案中,所述寡核苷酸如SEQ ID NO:183-284任一所示。在一个实施方案中,所述寡核苷酸如GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG(SEQ IDNO:3)所示。在一个实施方案中,所述寡核苷酸如SEQ ID NO:285-464任一所示。在一个实施方案中,SEQ ID NO:1与SEQ ID NO:2部分配对。在一个实施方案中,SEQ ID NO:4182任一与SEQ ID NO:183-284任一部分配对。在一个实施方案中,SEQ ID NO:1与SEQ ID NO:3部分配对。在一个实施方案中,SEQ ID NO:4-182任一与SEQ ID NO:285-464任一部分配对。在一个实施方案中,转座酶与部分配对的SEQ ID NO:1和SEQ ID NO:2复合。在一个实施方案中,转座酶与部分配对的SEQ ID NO:4-182任一和SEQ ID NO:183-284任一复合。在一个实施方案中,转座酶与部分配对的SEQ ID NO:1和SEQ ID NO:3复合。在一个实施方案中,转座酶与部分配对的SEQ ID NO:4-182任一和SEQ ID NO:285-464任一复合。在一个实施方案中,转座酶与部分配对的SEQ ID NO:1和SEQ ID NO:2且与部分配对的SEQ ID NO:1和SEQ ID NO:3复合。在一个实施方案中,转座酶与部分配对的SEQ ID NO:4-182任一和SEQ ID NO:183-284任一且与部分配对的SEQ ID NO:4-182任一和SEQ ID NO:285-464任一复合。在一个实施方案中,SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3各自独立附着一个或多个生物素分子,例如1、2、3、4、5、6、7、8或更多个。在一个实施方案中,所述寡核苷酸是部分配对的5'-phos-CTGTCTCTTATACACATBTCTBT-3'(SEQ ID NO:12)和5′-TCGTBTCGGCAGCGTCAGATGTGTATBTAAGAGACAG-3′(SEQ ID NO:195)和/或部分配对的5'-phos-CTGTCTCTTATACACATBTCTBT-3'(SEQ IDNO:12)和5′-GTCTCGTGGGCTBTCGGAGATGTGTATBTAAGAGACAG-3(SEQ ID NO:297),或者,寡核苷酸是部分配对的5'-phos-CTBTGTCTCTTATBTACACATCT-3'(SEQ ID NO:29)和5′-TCGTCGGCAGCGTCAGATGTGTBTATBTAAGAGACAG-3′(SEQ ID NO:215)和/或部分配对的5'-phos-CTBTGTCTCTTATBTACACATCT-3'(SEQ ID NO:29)和5′-GTCTBTCGTGGGCTBTCGGAGATGTGTATAAGAGACAG-3′(SEQ ID NO:317),TBT表示生物素化的T。
在一个方面,本发明提供上述复合物的混合物,其以下式表示:
(转座酶-生物素)q:亲合素:(生物素-二抗)p,其中p和q各自大于或等于1且小于或等于3,且q+p小于或等于4,例如q+p≈2、3或4。例如,q≈1,p≈1、2或3;q≈2,p≈1或2;q≈3,p≈1。又例如,p≈1,q≈1、2或3;p≈2,q≈1或2;p≈3,q≈1。q为混合物中亲合素分子加载的平均转座酶分子数,p为混合物中亲合素分子加载的平均二抗分子数。
在一个方面,本发明提供一种制备上述复合物或混合物的方法,其包括:生物素化二抗;
生物素化转座酶;
以1:n’的摩尔比混合亲合素与生物素化的二抗,得到中间体;
及
以1:m’的摩尔比混合中间体与生物素化的转座酶。
在一个方面,本发明提供一种制备上述复合物或混合物的方法,其包括:生物素化二抗;
生物素化转座酶;
以1:m’的摩尔比混合亲合素与生物素化的转座酶,得到中间体;
及
以1:n’的摩尔比混合中间体与生物素化的二抗。
在一个方面,本发明提供一种制备上述复合物或混合物的方法,其包括:生物素化二抗;
生物素化转座酶;
及以m’:1:n’的摩尔比混合生物素化的转座酶、亲合素和生物素化的二抗。
在一个实施方案中,n’=n。在一个实施方案中,n’>n。在一个实施方案中,n’=p。在一个实施方案中,n’>p。在一个实施方案中,n’<4,例如n’<3,例如n’<2,例如n’=1、2或3。在一个实施方案中,n’>4。在一个实施方案中,m’=m。在一个实施方案中,m’>m。在一个实施方案中,m’=q。在一个实施方案中,m’>q。在一个实施方案中,m’<4,例如m’<3,例如m’<2,例如m’=1、2或3。在一个实施方案中,m’>4。在一个实施方案中,在n’=1的情况下,m’≤3,例如m’<3。在一个实施方案中,在n’=2的情况下,m’≤2,例如m’<2。在一个实施方案中,在n’=3的情况下,m’≤1,例如m’<1。在一个实施方案中,在m’=1的情况下,n’≤3,例如n’<3。在一个实施方案中,在m’=2的情况下,n’≤2,例如n’<2。在一个实施方案中,在m’=3的情况下,n’≤1,例如n’<1。在一个实施方案中,n’>4且m’>4。在一个实施方案中,上述摩尔比使得不存在游离的(即未结合生物素化的二抗的)生物素化的转座酶。
在一个实施方案中,本发明的数值包括±20%、±10%或±5%的误差。
在一个方面,本发明提供一种检测细胞在靶蛋白与DNA相互作用的方法,其包括:
(1)将细胞与针对靶蛋白的一抗一起温育;
(2)将细胞与上述复合物或混合物一起温育,其中所述复合物中的二抗是对所述一抗特异性的二抗,并容许转座酶作用于细胞基因组;
(3)扩增细胞DNA;
(4)纯化扩增的DNA;并
(5)对扩增的DNA测序。
在一个实施方案中,本发明的方法无需提取和纯化细胞DNA,直接扩增细胞DNA。
在一个实施方案中,所述靶蛋白是AAF、ab1、ADA2、ADA-NF1、AF-1、AFP1、AhR、AIIN3、ALL-1、α-CBF、α-CP1、α-CP2a、α-CP2b、αHo、αH2-αFB、Alx-4、aMEF-2、AML1、AML1a、AML1b、AML1c、AML1δN、AML2、AML3、AML3a、AML3b、AMY-1L、A-Myb、ANF、AP-1、AP-2αA、AP-2αB、AP-2β、AP-2γ、AP-3(1)、AP-3(2)、AP-4、AP-5、APC、AR、AREB6、Arnt、Arnt(774M形式)、ARP-1、ATBF1-A、ATBF1-B、ATF、ATF-1、ATF-2、ATF-3、ATF-3δZIP、ATF-a、ATF-aδ、ATPF1、Barhl1、Barhl2、Barx1、Barx2、Bcl-3、BCL-6、BD73、β-连环蛋白、Bin1、B-Myb、BP1、BP2、brahma、BRCA1、Brn-3a、Brn-3b、Brn-4、BTEB、BTEB2、B-TFIID、C/EBPα、C/EBPβ、C/EBPδ、CACC结合因子、Cart-1、CBF(4)、CBF(5)、CBP、CCAAT-结合因子、CCMT-结合因子、CCF、CCG1、CCK-1a、CCK-1b、CD28RC、cdk2、cdk9、Cdx-1、CDX2、Cdx-4、CFF、ChxlO、CLIM1、CLIM2、CNBP、CoS、COUP、CP1、CP1A、CP1C、CP2、CPBP、CPE结合蛋白、CREB、CREB-2、CRE-BP1、CRE-BPa、CREMα、CRF、Crx、CSBP-1、CTCF、CTF、CTF-1、CTF-2、CTF-3、CTF-5、CTF-7、CUP、CUTL1、Cx、细胞周期蛋白A、细胞周期蛋白T1、细胞周期蛋白T2、细胞周期蛋白T2a、细胞周期蛋白T2b、DAP、DAX1、DB1、DBF4、DBP、DbpA、DbpAv、DbpB、DDB、DDB-1、DDB-2、DEF、δCREB、δMax、DF-1、DF-2、DF-3、Dlx-1、Dlx-2、Dlx-3、Dlx4(长同种型)、Dlx-4(短同种型、Dlx-5、Dlx-6、DP-1、DP-2、DSIF、DSIF-p14、DSIF-p160、DTF、DUX1、DUX2、DUX3、DUX4、E、E12、E2F、E2F+E4、E2F+p107、E2F-1、E2F-2、E2F-3、E2F-4、E2F-5、E2F-6、E47、E4BP4、E4F、E4F1、E4TF2、EAR2、EBP-80、EC2、EF1、EF-C、EGR1、EGR2、EGR3、EIIaE-A、EIIaE-B、EIIaE-Cα、EIIaE-Cβ、EivF、EIf-1、EIk-1、Emx-1、Emx-2、Emx-2、En-1、En-2、ENH-bind.prot.、ENKTF-1、EPAS1、εF1、ER、Erg-1、Erg-2、ERR1、ERR2、ETF、Ets-1、Ets-1δVil、Ets-2、Evx-1、F2F、因子2、Factorname、FBP、f-EBP、FKBP59、FKHL18、FKHRL1P2、Fli-1、Fos、FOXB1、FOXC1、FOXC2、FOXD1、FOXD2、FOXD3、FOXD4、FOXE1、FOXE3、FOXF1、FOXF2、FOXG1a、FOXG1b、FOXG1c、FOXH1、FOXI1、FOXJ1a、FOXJ1b、FOXJ2(长同种型)、FOXJ2(短同种型)、FOXJ3、FOXK1a、FOXK1b、FOXK1c、FOXL1、FOXM1a、FOXM1b、FOXM1c、FOXN1、FOXN2、FOXN3、FOXO1a、FOXO1b、FOX02、FOX03a、FOX03b、FOX04、FOXP1、FOXP3、Fra-1、Fra-2、FTF、FTS、G因子、G6因子、GABP、GABP-α、GABP-β1、GABP-β2、GADD153、GAF、γCMT、γCAC1、γCAC2、GATA-1、GATA-2、GATA-3、GATA-4、GATA-5、GATA-6、Gbx-1、Gbx-2、GCF、GCMa、GCNS、GF1、GLI、GLI3、GRα、GRβ、GRF-1、Gsc、Gsc1、GT-IC、GT-IIA、GT-IIBα、GT-IIBβ、H1TF1、H1TF2、H2RIIBP、H4TF-1、H4TF-2、HAND1、HAND2、HB9、HDAC1、HDAC2、HDAC3、hDaxx、热诱导的因子、HEB、HEB1-p67、HEB1-p94、HEF-1B、HEF-1T、HEF-4C、HEN1、HEN2、Hesx1、Hex、HIF-1、HIF-1α、HIF-1β、HiNF-A、HiNF-B、HINF-C、HINF-D、HiNF-D3、HiNF-E、HiNF-P、HIP1、HIV-EP2、Hlf、HLTF、HLTF(Met123)、HLX、HMBP、HMG I、HMG I(Y)、HMGY、HMGI-C、HNF-1A、HNF-IB、HNF-1C、HNF-3、HNF-3α、HNF-3β、HNF-3γ、HNF4、HNF-4α、HNF4α1、HNF-4α2、HNF-4α3、HNF-4α4、HNF4γ、HNF-6α、hnRNPK、HOX11、HOXA1、HOXA10、HOXA10PL2、HOXA11、HOXA13、HOXA2、HOXA3、HOXA4、HOXA5、HOXA6、HOXA7、HOXA9A、HOXA9B、HOXB-1、HOXB13、HOXB2、HOXB3、HOXB4、HOXBS、HOXB6、HOXA5、HOXB7、HOXB8、HOXB9、HOXC10、HOXC11、HOXC12、HOXC13、HOXC4、HOXC5、HOXC6、HOXC8、HOXC9、HOXD10、HOXD11、HOXD12、HOXD13、HOXD3、HOXD4、HOXD8、HOXD9、Hp55、Hp65、HPX42B、HrpF、HSF、HSF1(长)、HSF1(短)、HSF2、hsp56、Hsp90、IBP-1、ICER-II、ICER-liγ、ICSBP、Id1、Id1H'、Id2、Id3、Id3/Heir-1、IF1、IgPE-1、IgPE-2、IgPE-3、IκB、IκB-α、IκB-β、IκBR、II-1RF、IL-6RE-BP、11-6RF、INSAF、IPF1、IRF-1、IRF-2、B、IRX2a、Irx-3、Irx-4、ISGF-1、ISGF-3、ISGF3α、ISGF-3γ、lst-1、ITF、ITF-1、ITF-2、JRF、Jun、JunB、JunD、κy因子、KBP-1、KER1、KER-1、Kox1、KRF-1、Ku自身抗原、KUP、LBP-1、LBP-1a、LBX1、LCR-F1、LEF-1、LEF-1B、LF-A1、LHX1、LHX2、LHX3a、LHX3b、LHXS、LHX6.1a、LHX6.1b、LIT-1、Lmo1、Lmo2、LMX1A、LMX1B、L-My1(长形式)、L-My1(短形式)、L-My2、LSF、LXRα、LyF-1、Lyl-1、M因子、Mad1、MASH-1、Max1、Max2、MAZ、MAZ1、MB67、MBF1、MBF2、MBF3、MBP-1(1)、MBP-1(2)、MBP-2、MDBP、MEF-2、MEF-2B、MEF-2C(433AA形式)、MEF-2C(465AA形式)、MEF-2C(473M形式)、MEF-2C/δ32(441AA形式)、MEF-2D00、MEF-2D0B、MEF-2DA0、MEF-2DAO、MEF-2DAB、MEF-2DA'B、Meis-1、Meis-2a、Meis-2b、Meis-2c、Meis-2d、Meis-2e、Meis3、Meox1、Meox1a、Meox2、MHox(K-2)、Mi、MIF-1、Miz-1、MM-1、MOP3、MR、Msx-1、Msx-2、MTB-Zf、MTF-1、mtTF1、Mxil、Myb、Myc、Myc1、Myf-3、Myf-4、Myf-5、Myf-6、MyoD、MZF-1、NCI、NC2、NCX、NELF、NER1、Net、NF Ill-a、NF NF-1、NF-1A、NF-1B、NF-1X、NF-4FA、NF-4FB、NF-4FC、NF-A、NF-AB、NFAT-1、NF-AT3、NF-Atc、NF-Atp、NF-Atx、NfβA、NF-CLEOa、NF-CLEOb、NFδE3A、NFδE3B、NFδE3C、NFδE4A、NFδE4B、NFδE4C、Nfe、NF-E、NFE2、NF-E2p45、NF-E3、NFE-6、NF-Gma、NF-GMb、NF-IL-2A、NF-IL-2B、NF-jun、NF-κB、NF-κB(样)、NF-κB1、NF-κB1前体、NF-κB2、NF-κB2(p49)、NF-κB2前体、NF-κE1、NF-κE2、NF-κE3、NF-MHCIIA、NF-MHCIIB、NF-muE1、NF-muE2、NF-muE3、NF-S、NF-X、NF-X1、NF-X2、NF-X3、NFXc、NF-YA、NF-Zc、NF-Zz、NHP-1、NHP-2、NHP3、NHP4、NKX2-5、NKX2B、NKX2C、NKX2G、NKX3A、NKX3Av1、NKX3Av2、NKX3Av3、NKX3Av4、NKX3B、NKX6A、Nmi、N-Myc、N-Oct-2α、N-Oct-2β、N-Oct-3、N-Oct-4、N-Oct-5a、N-Oct-5b、NP-TCII、NR2E3、NR4A2、Nrf1、Nrf-1、Nrf2、NRF-2β1、NRF-2γ1、NRL、NRSF形式1、NRSF形式2、NTF、02、OCA-B、Oct-1、Oct-2、Oct-2.1、Oct-2B、Oct-2C、Oct-4A、Oct4B、Oct-5、Oct-6、Octa-因子、八聚体-结合因子、oct-B2、oct-B3、Otx1、Otx2、OZF、p107、p130、p28调节剂、p300、p38erg、p45、p49erg、p53、p55、p55erg、p65δ、p67、Pax-1、Pax-2、Pax-3、Pax-3A、Pax-3B、Pax-4、Pax-5、Pax-6、Pax-6/Pd-5a、Pax-7、Pax-8、Pax-8a、Pax-8b、Pax-8c、Pax-8d、Pax-8e、Pax-8f、Pax-9、Pbx-1a、Pbx-1b、Pbx-2、Pbx-3a、Pbx-3b、PC2、PC4、PC5、PEA3、PEBP2α、PEBP2β、Pit-1、PITX1、PITX2、PITX3、PKNOX1、PLZF、POB、Pontin52、PPARα、PPARβ、PPARγ1、PPARγ2、PPUR、PR、PRA、pRb、PRD1-BF1、PRDI-BFc、Prop-1、PSE1、P-TEFb、PTF、PTFα、PTFβ、PTFδ、PTFγ、Pubox结合因子、Pubox结合因子(BJA-B)、PU.1、PuF、Pur因子、R1、R2、RAR-α1、RAR-β、RAR-β2、RAR-γ、RAR-γ1、RBP60、RBP-Jκ、Rel、RelA、RelB、RFX、RFX1、RFX2、RFX3、RFXS、RF-Y、RORα1、RORα2、RORα3、RORβ、RORγ、Rox、RPF1、RPGα、RREB-1、RSRFC4、RSRFC9、RVF、RXR-α、RXR-β、SAP-1a、SAP-1b、SF-1、SHOX2a、SHOX2b、SHOXa、SHOXb、SHP、SIII-p110、SIII-p15、SIII-p18、SIM'、Six-1、Six-2、Six-3、Six-4、Six-5、Six-6、SMAD-1、SMAD-2、SMAD-3、SMAD-4、SMAD-5、SOX-11、SOX-12、Sox-4、Sox-5、SOX-9、Sp1、Sp2、Sp3、Sp4、Sph因子、Spi-B、SPIN、SRCAP、SREBP-1a、SREBP-1b、SREBP-1c、SREBP-2、SRE-ZBP、SRF、SRY、SRP1、Staf-50、STAT1α、STAT1β、STAT2、STAT3、STAT4、STAT6、T3R、T3R-α1、T3R-α2、T3R-β、TAF(I)110、TAF(I)48、TAF(I)63、TAF(II)100、TAF(II)125、TAF(II)135、TAF(II)170、TAF(II)18、TAF(II)20、TAF(II)250、TAF(II)250Δ、TAF(II)28、TAF(II)30、TAF(II)31、TAF(II)55、TAF(II)70-α、TAF(II)70-β、TAF(II)70-γ、TAF-I、TAF-II、TAF-L、Tal-1、Tal-1β、Tal-2、TAR因子、TBP、TBXIA、TBXIB、TBX2、TBX4、TBXS(长同种型)、TBXS(短同种型)、TCF、TCF-1、TCF-1A、TCF-1B、TCF-1C、TCF-1D、TCF-1E、TCF-1F、TCF-1G、TCF-2α、TCF-3、TCF-4、TCF-4(K)、TCF-4B、TCF-4E、TCFβ1、TEF-1、TEF-2、tel、TFE3、TFEB、TFIIA、TFIIA-αβ前体、TFIIA-α/β前体、TFIIA-γ、TFIIB、TFIID、TFIIE、TFIIE-α、TFIIE-β、TFIIF、TFIIF-α、TFIIF-β、TFIIH、TFIIH*、TFIIH-CAK、TFIIH-细胞周期蛋白H、TFIIH-ERCC2/CAK、TFIIH-MAT1、TFIIH-M015、TFIIH-p34、TFIIH-p44、TFIIH-p62、TFIIH-p80、TFIIH-p90、TFII-I、Tf-LF1、Tf-LF2、TGIF、TGIF2、TGT3、THRA1、TIF2、TLE1、TLX3、TMF、TR2、TR2-11、TR2-9、TR3、TR4、TRAP、TREB-1、TREB-2、TREB-3、TREF1、TREF2、TRF(2)、TTF-1、TXRE BP、TxREF、UBF、UBP-1、UEF-1、UEF-2、UEF-3、UEF-4、USF1、USF2、USF2b、Vav、Vax-2、VDR、vHNF-1A、vHNF1B、vHNF-1C、VITF、WSTF、WT1、WT1I、WT1I-KTS、WT1I-del2、WT1-KTS、WT1-del2、X2BP、XBP-1、XW-V、XX、YAF2、YB-1、YEBP、YY1、ZEB、ZF1、ZF2、ZFX、ZHX1、ZIC2、ZID、ZNF174、ASH1L、ASH2、ATF2、ASXL1、BAP1、bc110、Bmil、BRG1、CARM1、KAT3A/CBP、CDC73、CHD1、CHD2、CTCF、DNMT1、DOTL1、EHMT1、ESET、EZH1、EZH2、FBXL10、FRP(Plu-1)、HDAC1、HDAC2、HMGA1、hnRNPA1、HP1γ、Hset1b、Jarid1A、Jarid1C、KIAA1718JHDM1D、KAT5、KMT4、LSD1、NFKB P100、NSD2、MBD2、MBD3、MLL2、MLL4、P300、pRB、RbAP46/48、RBP1、RbBP5、RINGIB、RNApolII PS2、RNApolII PS5、ROC1、sap30、setDB1、Sf3b1、SIRT1、Sirt6、SMYD1、SP1、SUV39H1、SUZ12、TCF4、TET1、TRRAP、TRX2、WDR5、H3K27me3、H3k4me3、WDR77和/或YY1。
在一个实施方案中,所述二抗是山羊抗小鼠、山羊抗大鼠、山羊抗兔、山羊抗驴、山羊抗鸡、山羊抗人、山羊抗豚鼠、兔抗小鼠、兔抗大鼠、兔抗山羊、兔抗驴、兔抗人、兔抗豚鼠、兔抗鸡、小鼠抗兔、小鼠抗大鼠、小鼠抗驴、小鼠抗鸡、小鼠抗人、小鼠抗豚鼠、小鼠抗山羊、大鼠抗兔、大鼠抗小鼠、大鼠抗驴、大鼠抗鸡、大鼠抗人、大鼠抗豚鼠、小鼠抗山羊、驴抗兔、驴抗大鼠、驴抗小鼠、驴抗鸡、驴抗人、驴抗豚鼠、驴抗山羊、鸡抗兔、鸡抗大鼠、鸡抗驴、鸡抗小鼠、鸡抗人、鸡抗豚鼠、鸡抗山羊、豚鼠抗兔、豚鼠抗大鼠、豚鼠抗驴、豚鼠抗鸡、豚鼠抗人、豚鼠抗小鼠、豚鼠抗山羊。
最佳实施方式(实施例)
实施例1:
转座酶-链霉亲合素的制备
可以使用本领域任何已知方法来生成转座酶-链霉亲合素融合蛋白。例如,可以在大肠杆菌中重组表达Tn5-链霉亲合素融合蛋白。
转座酶-生物素的制备
可以使用本领域任何已知方法来生物素化转座酶。例如,可以用NHS活化的生物素(Invitrogen,货号2174564)标记Tn5(Vazyme,货号S601)。见例如Muraoka et al.,Analytical Biochemistry,557,46-58,2018。
也可以使用本领域任何已知方法来合成带生物素修饰的寡核苷酸,与转座酶形成复合物(见下文“转座酶-生物素:亲合素:生物素-二抗的组装,方案2”)。
二抗-生物素的制备
可以使用本领域任何已知方法来生物素化二抗。例如,可以用NHS活化的生物素(Invitrogen,货号2174564)标记山羊抗兔IgG H&L二抗(Abcam,货号ab6702)。见例如Muraoka et al.,Analytical Biochemistry,557,46-58,2018。
转座酶-亲合素:生物素-二抗的组装
以1:5的摩尔比在PBS中混合转座酶-链霉亲合素融合蛋白与生物素化的二抗并于室温温育1小时。
转座酶-生物素:亲合素:生物素-二抗的组装,方案1
以1:1的摩尔比在PBS中混合链霉亲合素(Invitrogen,货号S888)与生物素化的二抗并于室温温育1小时,得到中间体亲合素:生物素-二抗。以5:7的摩尔比在PBS中混合中间体与生物素化的转座酶并于室温温育1小时。
转座酶-生物素:亲合素:生物素-二抗的组装,方案2
在生工生物公司合成带生物素修饰的引物:PrimerA、Primer B、Primer C(见附录),使用PBS溶解PrimerA、PrimerB、Primer C至10μM,取10μl Primer A+10μl PrimerB组成反应1,取10μl PrimerA+10μl Primer C组成反应2,分别将反应1和反应2涡旋震荡充分混匀,并短暂离心使溶液回到管底,置于PCR仪内,进行如下反应程序:
反应结束后,将反应1产物和反应2产物等体积混合,命名为AdapterMix。
随后在灭菌PCR管中依次添加如下各反应组分:
组分 | 2μg制备体系 |
Tn5(500ng/μl) | 4μl |
AdapterMix | 7μl |
PBS | 39μl |
使用移液器轻轻吹打20次充分混匀,并置于30℃反应1h。反应产物命名为BT-Tn5,-30~-15℃保存。
将生物素标记的二抗,即二抗-BT,与SA蛋白、BT-Tn5,按摩尔比5:7:7在PBS中室温孵育1h备用,并命名为:二抗-BT:SA:BT-Tn5复合物。
其中,二抗-BT:SA:BT-Tn5-1是primer A(5'-phos-CTGTCTCTTATACACATBTCTBT-3')+primerB(5′-TCGTBTCGGCAGCGTCAGATGTGTATBTAAGAGACAG-3′)+primer C(5′-GTCTCGTGGGCTBTCGGAGATGTGTATBTAAGAGACAG-3);二抗-BT:SA:BT-Tn5-2是primer A(5'-phos-CTBTGTCTCTTATBTACACATCT-3')+primer B(5′-TCGTCGGCAGCGTCAGATGTGTBTATBTAAGAGACAG-3′)+primer C(5′-GTCTBTCGTGGGCTBTCGGAGATGTGTATAAGAGACAG-3′)。
二抗:pG-Tn5的组装
使用100μL洋地黄皂苷清洗缓冲液(Dig-Wash buffer,来自诺唯赞产品TD901)对二抗进行1:100稀释,加入0.58μL 6.88μM pG-Tn5(来自诺唯赞产品TD901),室温下旋转200rpm孵育60分钟。获得预孵育的二抗:pG-TN5复合物。
实施例2:
本实施例以H3k4me3组蛋白修饰为例示性分析靶标,使用“二抗-BT:SA-Tn5复合物”和常规“pG-Tn5复合物”进行实验对比,用以说明本发明的方法步骤和有益效果。
其中,本实施例中的文库构建试剂均来自南京诺唯赞生物科技股份有限公司Hyperactive In-Situ ChIP Library Prep Kit for Illumina(pG-Tn5)(货号TD901)试剂盒,具体实验步骤如下:
按照TD901试剂盒说明书第七部分、第九部分进行缓冲液的配制和ConA珠的使用前处理。按照实施例1所述构建二抗-BT:SA-Tn5复合物。
1、收集细胞
取相同的6份Hela细胞样本,每份100μl(约100000个Hela细胞),室温下2500rpm低速离心3min,弃尽上清。在室温条件下向每份样本中加入500μl清洗缓冲液(Wash Buffer)重悬细胞,2500rpm低速离心3min,弃尽上清,用于后续实验。
其中,第一份Hela细胞样本使用Anti-Histone H3(mono methyl K4)antibody(abcam公司,货号ab8895)作为一抗和pG-Tn5复合物进行第一参比文库构建(样本1);第二份Hela细胞样本使用Rabbit(DA1E)mAb IgG(Cell Signaling Technology公司,货号66362S)作为一抗和pG-Tn5复合物进行第一阴性对照文库构建(样本2)。
第三份Hela细胞样本使用Anti-Histone H3(mono methyl K4)antibody作为一抗和二抗:pG-Tn5复合物进行第二参比文库构建(样本3);第四份Hela细胞样本使用Rabbit(DA1E)mAb IgG作为一抗和二抗:pG-Tn5复合物进行第二阴性对照文库构建(样本4)。
第五份Hela细胞样本使用Anti-Histone H3(mono methyl K4)antibody作为一抗和二抗-BT:SA-Tn5复合物进行测试文库构建(样本5);第六份Hela细胞样本使用Rabbit(DA1E)mAb IgG作为一抗和二抗-BT:SA-Tn5复合物进行第三阴性对照文库构建(样本6)。
2、细胞与ConA珠孵育
按照TD901产品说明书第九部分,将4份细胞样本分别与ConA珠进行孵育。
3、一抗孵育
按照单个样本加入50μl预冷的抗体缓冲液(Antibody Buffer),重悬细胞并轻轻涡旋混匀并置于冰上。
向第一份样本、第三份、第五份样本的EP管中加入1μl Anti-Histone H3(monomethyl K4)antibody,轻轻涡旋混匀,室温下旋转孵育2h。
向第二份样本、第四份、第六份样本的EP管中加入1μl Rabbit(DA1E)mAb IgG,轻轻涡旋混匀,室温下旋转孵育2h。
4、二抗和/或Tn5孵育
按照TD901产品说明书第九部分,将一抗孵育后的六份样本进行二抗孵育。
其中,第一份、第二份样本与山羊抗兔IgG H&L二抗进行孵育;第三份、第四份样本与二抗:pG-Tn5复合物进行孵育;第五份、第六份样本与二抗-BT:SA-Tn5复合物进行孵育。
二抗孵育后的第一份、第二份样本需要与pG-Tn5进行孵育,按照TD901产品说明书第九部分操作。
因第五份、第六份样本二抗孵育时的复合物含有Tn5转座体,因此,相对于常规的pG-Tn5复合物建库方式,二抗-BT:SA-Tn5复合物建库方式缩短1小时转座体孵育时间,并且二抗-BT:SA-Tn5复合物建库方式不需要额外的漂洗多余的二抗,将整体实验时间缩短1小时。
5、片段化反应
将与二抗-BT:SA-Tn5复合物进行孵育的第五份、第六份样本和与pG-Tn5进行孵育的第一份、第二份样本以及与二抗:pG-Tn5复合物进行孵育的第三份、第四份样本进行片段化反应,37℃孵育1小时。
6、DNA提取纯化
按照TD901产品说明书第九部分,对片段化后的第一份、第二份样本进行DNA提取纯化;片段化后的第三份、第四份、第五份、第六份样本无需DNA提取纯化,直接加入30μl 1×TE,将样本直接进行PCR扩增反应。
第五份、第六份样本无需DNA提取纯化,因此,相对于需要提取纯化的第一份、第二份样本,在时间上缩短了近3小时。
7、文库扩增
按照TD901产品说明书第九部分,进行文库扩增,其中,P5/P7引物使用南京诺唯赞生物科技股份有限公司TruePrep Index KitV2 for Illumina试剂盒(货号TD202)。
8、PCR产物纯化
按照TD901产品说明书第九部分对PCR扩增后的产物进行磁珠纯化,其中,使用的磁珠为南京诺唯赞生物科技股份有限公司VAHTS DNA CleanBeads试剂盒(货号N411)。
9、文库质量检测
将制备好的文库在Agilent 2100Bioanalyzer上进行文库长度分布检测(图6),可发现pG-Tn5组(即样本1)与二抗-BT:SA-Tn5组(即样本5)均有明显ladder状文库分布,表明二抗-BT:SA-Tn5技术方案也能正常建库,但二抗:PG-Tn5组(即样本3)ladder峰并不明显,表明其可能影响了Tn5酶对染色质的切割效率。
13、二代测序
将文库稀释至5ng/ul,取15μl送至南京世和基因生物技术有限公司进行测序,测序仪器为HiseqX。
测序结果
通过下机数据显示,在阴性对照IgG实验组中,与pG-Tn5实验方案(样本2)、二抗:PG-Tn5实验方案(样本4)相比,二抗-BT:SA-Tn5实验方案(样本6)IgG信号随机分布,降低了TSS区域的假阳性信号(图4);IGV视图展示,二抗-BT:SA-Tn5组(样本5)阳性信号更加富集,且IgG对照(样本6)假阳性信号降低(图5);文库峰型显示,二抗-BT:SA-Tn5组(样本5)与pG-Tn5组(样本1)相比,均有核小体ladder状分布,二抗:PG-Tn5组(样本3)则无明显核小体ladder状分布,暗示其可能影响了Tn5酶对染色质的切割效率(图6)。
针对CTCF转录因子、H3K27me3和H3k4me3组蛋白修饰作为靶标,得到了相似的实验结果。
实施例3:
本实施例以H3k4me3组蛋白修饰为例示性分析靶标,使用“二抗-BT:SA:BT-Tn5-1复合物”、“二抗-BT:SA:BT-Tn5-2复合物”、“二抗-BT:SA-Tn5复合物”和常规“pG-Tn5复合物”进行实验对比,用以说明本发明的方法步骤和有益效果。
其中,“二抗-BT:SA-Tn5复合物”和常规“pG-Tn5复合物”实验组步骤同实施例2;“二抗-BT:SA:BT-Tn5-1复合物”和“二抗-BT:SA:BT-Tn5-2复合物”实验步骤同实施例2中的“二抗-BT:SA-Tn5复合物”组,区别仅在于将“二抗-BT:SA-Tn5复合物”替换为“二抗-BT:SA:BT-Tn5-1复合物”或“二抗-BT:SA:BT-Tn5-2复合物”。
测序结果
通过下机数据显示,在阴性对照IgG实验组中,“二抗-BT:SA:BT-Tn5-1复合物”和“二抗-BT:SA:BT-Tn5-2复合物”的IgG无明显TSS富集(图7);IGV视图展示,与常规“pG-Tn5复合物”(一抗+二抗+pG-Tn5)相比,一抗+二抗-BT:SA:BT-Tn5-1复合物与一抗+二抗-BT:SA:BT-Tn5-2复合物以及一抗+二抗-BT:SA-Tn5均能有效降低IgG背景并提高抗体的特异性信号(图8);文库峰型显示,文库均有明显的核小体ladder分布,表明不同的修饰方式构建的复合物均能成功建库(图9)。
针对CTCF转录因子、H3K27me3和H3k4me3组蛋白修饰作为靶标,得到了相似的实验结果。
附录:转座体寡核苷酸序列(其中加粗、斜体、下划线碱基为生物素修饰的碱基)
PrimerA包括SEQ ID NO:1、SEQ ID NO:4至SEQ ID NO:182
PrimerB包括SEQ ID NO:2、SEQ ID NO:183至SEQ ID NO:284Primer C包括SEQ IDNO:3、SEQ ID NO:285至SEQ ID NO:464SEQ ID NO:1
CTGTCTCTTATACACATCT
SEQ ID NO:2
TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
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GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG
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Claims (10)
1.一种基于生物素:亲合素的二抗:转座酶复合物,其以下式表示:
转座酶-亲合素:(生物素-二抗)n,其中n为1、2、3或4。
2.权利要求1的复合物,其中亲合素为链霉亲合素。
3.权利要求1的复合物,其中转座酶为Tn5转座酶。
4.权利要求1-3任一项的复合物的混合物,其以下式表示:
转座酶-亲合素:(生物素-二抗)p,其中p大于1且小于4。
5.一种基于生物素:亲合素的二抗:转座酶复合物,其以下式表示:
(转座酶-生物素)m:亲合素:(生物素-二抗)n,其中m和n各自独立为1、2或3,且m+n小于或等于4。
6.权利要求5的复合物,其中亲合素为链霉亲合素。
7.权利要求5的复合物,其中转座酶为Tn5转座酶。
8.权利要求5的复合物,其中生物素附着于转座体的寡核苷酸,任选地,寡核苷酸是部分配对的5'-phos-CTGTCTCTTATACACATBTCTBT-3'(SEQIDNO:12)和5′-TCGTBTCGGCAGCGTCAGATGTGTATBTAAGAGACAG-3′(SEQ ID NO:195)和/或部分配对的5'-phos-CTGTCTCTTATACACATBTCTBT-3'(SEQ ID NO:12)和5′-GTCTCGTGGGCTBTCGGAGATGTGTATBTAAGAGACAG-3(SEQ ID NO:297),或者,寡核苷酸是部分配对的5'-phos-CTBTGTCTCTTATBTACACATCT-3'(SEQ ID NO:29)和5′-TCGTCGGCAGCGTCAGATGTGTBTATBTAAGAGACAG-3′(SEQ ID NO:215)和/或部分配对的5'-phos-CTBTGTCTCTTATBTACACATCT-3'(SEQIDNO:29)和5′-GTCTBTCGTGGGCTBTCGGAGATGTGTATAAGAGACAG-3′(SEQ ID NO:317),TBT表示生物素化的T。
9.权利要求5-8任一项的复合物的混合物,其以下式表示:
(转座酶-生物素)q:亲合素:(生物素-二抗)p,其中p和q各自大于或等于1且小于或等于3,且q+p小于或等于4。
10.一种检测细胞在靶蛋白与DNA相互作用的方法,其包括:
(1)将细胞与针对靶蛋白的一抗一起温育;
(2)将细胞与权利要求1-3和5-8任一项的复合物或权利要求4或9的混合物一起温育,其中所述复合物中的二抗是对所述一抗特异性的二抗,并容许转座酶作用于细胞基因组;
(3)扩增细胞DNA;
(4)纯化扩增的DNA;并
(5)对扩增的DNA测序。
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