CN113811623B - 定量检测人cdkn2a基因拷贝缺失的方法、引物及其用途 - Google Patents
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Abstract
本发明公开了一种引物对及探针组,其具有SEQ ID NO.1,SEQ ID NO.2,SEQ ID NO.3所示的寡核苷酸序列,也公开了参考序列的引物对及探针组,其具有SEQ ID NO.4,SEQ ID NO.5,SEQ ID NO.6所示的寡核苷酸序列。本发明也公开了一种检测CDKN2A共同缺失区“chr9:21970277‑21985225,hg19”的引物对及探针组在制备用于定量检测待测DNA样品中人CDKN2A基因拷贝缺失的试剂盒中的用途。本发明新方法以及特异性引物对及探针可简易、便利、特异地检测出样品中的CDKN2A基因拷贝缺失,灵敏度高于常规拷贝缺失测定方法,首次使得缺失坐标未知单样品CDKN2A基因拷贝缺失的检测成为可能,具有应用前景。
Description
技术领域
本发明涉及生物技术检测领域,更具体地涉及一种定量检测人CDKN之A基因拷贝缺失的方法,其所用的引物以及该引构的用途。
背景技术
人类第9号染色体短臂上的抑癌基因CDKN2A编码P16和P14蛋白,通过P16-CDK4/6-RB和P14-MDM2-P53-P21-RB通路,控制细胞周期G1→S期转换。该基因存在遗传性失活者易发生恶性黑色素瘤和胰腺癌等。在肿瘤发生过程中,该基因主要通过体细胞拷贝缺失或CpG岛异常甲基化两种途径失活,在多种癌组织和癌前病变组织中均可检出,是肿瘤基因组中大片段拷贝缺失频率最高的基因(平均8%,在神经组织肿瘤中>40%;参见图1)。CDKN2A基因失活与癌前病变癌变风险、肿瘤细胞对CDK4/6抑制剂敏感性和化疗药物疗效、肿瘤患者总生存时间密切相关,是重要的肿瘤候选标志物。
目前常用的体细胞基因拷贝变异(SNV)测定技术包括荧光探针原位杂交(FISH)、SNP芯片杂交、以PCR为基础的单链构象多态性(SSCP)/高效液相色谱(HPLC)测定微卫星稳定性、第一代和第二代DNA测序等技术。FISH技术可用于检测基因扩增,不能灵敏检测体细胞性(somatic)基因拷贝缺失;SNP芯片和微卫星PCR-SSCP/HPLC测定技术可用于检测成对组织(如癌组织及其切缘正常组织)的基因拷贝数深度缺失和扩增,但是灵敏度较差(最低检出限>33%),不适合非配对的单样品分析;PCR-测序法可用于测定缺失位点(断裂/融合位点)已知的基因拷贝缺失,但是无法用于检侧缺失位点未知的长片段拷贝缺失。第三代DNA单分子实时测序技术虽然可用于测定各种长片段的DNA结构变化,由于测序成本高昂,目前仅限于科学研究用途。
虽然基因拷贝扩增测定已经在临床上展示出良好的用途,目前体细胞性基因拷贝缺失的应用尚为空白。灵敏度不高的基因拷贝缺失检测技术可用于单样品的遗传性/胚系性(familial/germline)基因拷贝缺失缺失测定和成对样品体细胞性基因拷贝缺失测定,但是无法用于单样品的体细胞性基因拷贝缺失分析。由于基因大片段缺失位置多种多样,即使在肿瘤组织中缺失频率最高的CDKN2A基因拷贝缺失,至今仍然没有灵敏、特异、便利的单样品(如活检组织、血液游离DNA)测定方法,限制了其临床应用。
发明的公开
我们在对既往大量的研究文献进行综合分析时首次发现,在84份经测序确定CDKN2A基因拷贝缺失精确坐标的肿瘤细胞系或组织样品中,CDKN2A基因的拷贝大片段缺失具体坐标(断裂/融合位点)虽然多种多样,但是在CDKN2A/P16基因启动子上游至第二内含子之间存在一个15kb的共同缺失序列[chr9:21,970,277-21,985,225,hg19],参见图2。
对人类肿瘤基因组计划(TCGA)中SNV公共数据库进行挖掘,可观察到SNP芯片测定出的CDKN2A基因拷贝缺失的大致区域(SNP芯片测定法不能确定缺失区的具体坐标)分布亦存在类似的特征,参见图3。
据此,我们以这个共同缺失序列相关的微卫星为检测对象,建立了适用于成对样品比较分析的CDKN2A共同缺失区杂合性缺失(CDKN2A-LOH)测定方法;对140余例胃癌及其手术切缘正常对照组织进行分析,发现CDKN2A-LOH频率与胃癌pTNM分期呈显著正相关,而CDKN2A共同缺失区附近区域的LOH(nearCDKN2A-LOH)频率与胃癌pTNM分期相关性不显著,提示CDKN2A-LOH测定有良好的临床应用价值。
为了建立单样品的CDKN2A拷贝缺失测定方法,我们进一步以这个共同缺失序列中保守的DNA序列为PCR扩增模板,以肿瘤组织中不存在拷贝变异的GAPDH基因保守的DNA序列为内参照,设计、筛选、优化出了一组双重荧光定量PCR扩增引物;对这两个DNA片段进行双重定量PCR扩增,发现CDKN2A基因拷贝数在正常人的基因组DNA中高度稳定,不仅适用于成对样品的CDKN2A拷贝缺失测定,也适用于非成对的单样品分析,以前未见报道。
本发明提供了一种引物对及探针组,其具有SEQ ID NO.1,SEQ ID NO.2,SEQ IDNO.3所示的寡核苷酸序列。
本发明也提供了一种参考序列的引物对及探针组,其具有SEQ ID NO.4,SEQ IDNO.5,SEQ ID NO.6所示的寡核苷酸序列。
进一步,本发明提供了一种与人CDKN2A共同缺失区“chr9:21970277-21985225,hg19”的保守区序列特异性互补的引物对及探针组在制备用于定量检测待测DNA样品中人CDKN2A基因拷贝缺失的试剂或试剂盒中的用途。具体地,该引物对及探针组具有SEQ IDNO.1,SEQ ID NO.2,SEQ ID NO.3所示的寡核苷酸序列。
本发明的试剂或试剂盒还包括与GAPDH基因保守区参考序列互补的引物对及探针组,具有SEQ ID NO.4,SEQ ID NO.5,SEQ ID NO.6所示的寡核苷酸序列。
进一步,本发明中的该待测DNA样品为单样品。
本发明也提供了一种定量检测人CDKN2A基因拷贝缺失的方法,该方法包括下列步骤:
a.提取待测组织或细胞的基因组DNA样品;
b.提取和制备经过测序鉴定的CDKN2A纯合性拷贝缺失细胞和CDKN2A基因不缺失细胞的DNA标准品,按梯度级差,连续稀释成比例为0%到100%之间梯度级的CDKN2A拷贝缺失细胞DNA的PCR扩增模板,制备标准曲线;
c.根据CDKN2A共同缺失区“chr9:21970277-21985225,hg19”的无重复序列,无微卫星,不含频率>1%SNP的保守序列,设计并合成双重PCR扩增用的样品引物对及序列特异性探针;同时设计并合成针对GAPDH基因保守区参考序列的双重PCR扩增用参考的引物对和序列特异性探针;
d.双重引物荧光定量PCR扩增,测定待测组织或细胞的基因组DNA序列以及GAPDH基因保守区参考序列的各个扩增片段的PCR Ct值,计算CDKN2A共同缺失区PCR产物与参考序列PCR产物的ΔCt值[CtCDKN2A-CtGAPDH],根据标准曲线计算CDKN2A拷贝数;
e.分析正常人体细胞基因组DNA的CDKN2A基因拷贝数,根据其平均数±1.96个标准差(Mean±1.96×SD)制定参考值范围;
f.当待测组织或细胞的CDKN2A基因拷贝数低于参考值低限时,判断该待测组织或细胞的存在CDKN2A基因拷贝缺失。
进一步,本发明的方法是用于定量检测单样品的人CDKN2A基因拷贝缺失的方法,其中样品引物对及序列特异性探针组具有SEQ ID NO.1,SEQ ID NO.2,SEQ ID NO.3所示的寡核苷酸序列,参考的引物对和序列特异性探针组具有SEQ ID NO.4,SEQ ID NO.5,SEQ IDNO.6所示的寡核苷酸序列。
本发明新方法以及特异性引物对及探针可简易、便利、特异地检测出样品中的CDKN2A基因拷贝缺失,灵敏度高于常规拷贝缺失测定方法,首次使得缺失坐标未知单样品CDKN2A基因拷贝缺失的检测成为可能,具有应用前景。
为了更好地理解本发明的内容,下面结合附图及具体实施方式,对本发明的内容做进一步说明,该具体实施方式仅仅是示例目的,不意指对本发明做任何限制。
附图的简要说明
图1是参考人类肿瘤基因组计划公共数据网站www.cbioportal.org数据制作的各种肿瘤组织/细胞中CDKN2A基因拷贝深度缺失和扩增情况,其中各种肿瘤中的灰色数字表示存在CDKN2A基因拷贝深度缺失样品数/分析总样品数。
图2是测序确定的肿瘤组织/细胞样品(n=84)中CDKN2A基因拷贝缺失区的具体位置[上图]及共同缺失序列坐标[chr9:21970277-21985225,hg19;下图](MTAP、P16、P14、ANRIL、P15基因转录区坐标显示在图左侧)。
图3是SNP6.0芯片测定存在CDKN2A基因拷贝深度缺失肿瘤组织样品(n=1187)的CDKN2A基因缺失的区域[白线标示位子;上图]及共同缺失区域[chr9:21970277-21985225,hg19;下图](仅显示部分样品标识)。
图4是胃癌基因组中9p21位点上6个微卫星不稳定的代表性DHPLC色谱图,其中黑线表示胃癌组织,灰线表示正常组织。
图5是荧光定量双重引物PCR法测定P16基因拷贝缺失标准曲线。
图6是荧光定量双重引物PCR法测定存在不同P16无缺失RKO细胞比值条件下P16基因拷贝缺失检出的重现性(日间重复实验)。
实现本发明的最佳方式
实施例1:传统微卫星法确定成对胃癌组织CDKN2A基因杂合性缺失(LOH)
1.常规酚氯仿法提取149例患者的手术切除胃癌及其切缘正常组织(或外周血白细胞)DNA;
2.PCR引物设计:根据现有文献报道,合成距离CDKN2A共同缺失区最近的3个微卫星(D9S974、D9S942、D9S1748,均位于CDKN2A第一外显子α与第一外显子β之间)PCR扩增引物,同时合成CDKN2A基因周围的3个对照微卫星(D9S1604、D9S1749、D9S171,分别位于MTAP、Anril、FAM186XC3基因内)PCR扩增引物;
3.PCR扩增
PCR体系:
模板DNA(20-25ng) | 1.0μL |
10×Buffer | 2.5μL |
dNTP Mix(10mM each) | 0.5μL |
引物 | 0.5μL |
HotStar Taq DNA聚合酶 | 0.2μL |
H2O | 20.3μL |
总体积 | 25μL |
热循环条件:95℃ 15min→[95℃ 30s→55/60/61/62℃ 30s→72℃ 30s]34个循环→72℃ 10min
4.用DHPLC对PCR产物按产物的分子大小进行分离测定,结果见图4。
5.样品微卫星不稳定性判断:
(1)判断标准:与正常组织的色谱图相比,当肿瘤组织中存在部分色谱峰完全消失或峰高比值下降>50%时,或者色谱峰的形状或数目存在明显变化时,判断存在该微卫星不稳定性(MSI);
(2)当CDKN2A共同缺失区相关的3个微卫星(D9S974、D9S942、D9S1748)之中的任何一个存在MSI时,判断存在CDKN2A共同缺失区杂合性缺失(CDKN2A-LOH);
(3)当CDKN2A基因外围的3个微卫星(D9S1604、D9S1749、D9S171)之中的任何一个存在MSI时,判断存在CDKN2A基因外围区域存在杂合性缺失(nearCDKN2A-LOH);
6.CDKN2A-LOH与nearCDKN2A-LOH的临床意义比较:
(1)52个胃癌组织检出了CDKN2A-LOH,41个胃癌组织检出了nearCDKN2A-LOH;31个胃癌组织同时检出了CDKN2A-LOH和nearCDKN2A-LOH;
(2)CDKN2A-LOH的检出与胃癌pTNM分期高度相关[I期为24.0%(12/50),III期为33.3%(14/42),IV期为45.6%(26/57)],差异存在统计学显著性(趋势检验,p<0.02,双侧);nearCDKN2A-LOH的检出虽然也与胃癌pTNM分期存在一定的相关性[I期为22.0%(11/50),III期为28.6%(12/42),IV期为31.6%(18/57)],但是差异没有统计学显著性(趋势检验,p=0.55);
7.结论:虽然上述6个微卫星均常用于检测染色体9p21位点LOH,与CDKN2A共同缺失区外围的3个微卫星对照(D9S1604、D9S1749、D9S171)的MSI相比,用CDKN2A共同缺失区附近的3个微卫星(D9S974、D9S942、D9S1748)的MSI来检测9p21位点LOH更有胃癌预后判断价值。也进一步确认了检测CDKN2A共同缺失区的良好应用前景。
实施例2:荧光定量双重引物PCR法测定胃癌组织中CDKN2A基因拷贝缺失
1.常规酚氯仿法提取140例患者的手术切除胃癌及其切缘正常组织DNA;
2.配制CDKN2A共同缺失区存在纯合性缺失的A549细胞和不存在缺失的RKO细胞混合液,使A549细胞DNA的比例分别为100%、90%、80%、70%、60%、50%、40%、30%、20%、10%和0;
3.合成针对CDKN2A共同缺失区第二内含子保守序列的多重PCR扩增用引物和荧光标记探针,其寡核苷酸序列如SEQ ID NO.1、SEQ ID NO.2和SEQ ID NO.3所示;合成针对GAPDH基因保守序列的多重PCR扩增用引物和荧光标记探针,其寡核苷酸序列如SEQ IDNO.4、SEQ ID NO.5和SEQ ID NO.6所示;
4.配制PCR扩增体系:
样品DNA(20-25ng) | 2.0μL |
TaqMan Universal Master Mix II | 10.0μL |
多重引物混合物(各50μM) | 2.0μL |
探针(50μM) | 2.0μL |
H2O | 7.2μL |
总体积 | 20μL(/PCR管) |
5.每个样品设3个平行的PCR管,在多通道实时定量PCR仪上进行扩增;热循环条件:95℃变性10分钟→[95℃变性15秒→60℃退火1分钟]×40个循环);在退火环节分别读取参考序列和待测序列扩增片段的Ct值;计算各PCR管中目的序列与参考序列Ct值的差值(ΔCt值)和CDKN2A基因的相对拷贝数;
6.用P16基因拷贝缺失A549细胞DNA稀释等浓度的无P16基因缺失RKO细胞DNA,根据不同稀释浓度的CDKN2A基因缺失A549细胞DNA的相对拷贝数制定标准曲线,计算得出本方法测定CDKN2A基因拷贝缺失的线性范围为100%-10%,参见图5;
7.最低检出限设定:10%A549细胞(含90%RKO细胞)CDKN2A基因平均拷贝数虽然总是低于RKO细胞对照,但是10次重复实验中4次p>0.05;20%及更高浓度的A549细胞CDKN2A基因平均拷贝数总是低于RKO细胞对照,并且p值总是<0.05。因此该方法的最低检出限应为20%,参见图6;
8.成对样品比较分析CDKN2A基因拷贝缺失阳性结果判断:当待测样品的CDKN2A基因拷贝数比正常对照样品的拷贝数低20%,并且差异存在统计学显著性(P<0.05)时,判断该样品CDKN2A基因拷贝缺失阳性;
9.结果
(1)在140位胃癌患者胃癌组织中,37例(26.4%)P16基因拷贝缺失阳性;在脉管癌栓阳性和远处转移阳性的胃癌组织中,CDKN2A基因拷贝缺失阳性率显著高于无脉管癌栓或无远处转移者;
(2)CDKN2A基因拷贝缺失的阳性检出率与胃癌pTNM分期高度相关:I期为17.0%(8/47)、III期为23.1%(9/39)、IV期为37.0%(20/54),差异存在统计学显著性(趋势检验,p<0.02,双侧;表1)。
表1.胃癌组织中P16基因拷贝缺失与临床病理特征相关关系分析
10.单样品分析CDKN2A基因拷贝缺失阳性结果判断:140例胃癌患者的胃黏膜非癌正常配对组织CDKN2A基因相对拷贝数的平均值为3.27(100%),标准差为0.26。上下浮动1.96个标准差的参考值则为2.75~3.79。当待测样品的CDKN2A基因相对拷贝数低于参考值下限2.75时,判断为CDKN2A基因拷贝缺失阳性。
11.结果:
(1)以参考值下限2.75为标准,对胃癌组织中CDKN2A基因拷贝缺失情况进行判断,41个胃癌样品为CDKN2A基因拷贝缺失阳性。其中,32个样品是成对比较中判断为CDKN2A基因拷贝缺失阳性的样品,10个为成对比较中判断为CDKN2A基因拷贝缺失阴性的样品。如果以成对比较的结果为金标准,单样品方法的灵敏度为86.5%(32/37),特异性为91.3%(94/103);(2)临床病理特征分析表明,脉管癌栓阳性和远处转移阳性的胃癌组织中,单样分析测定的CDKN2A基因拷贝缺失阳性率仍然高于脉管癌栓阴性和无远处转移的胃癌组织,参见表1;
10.结论:本方法既适用于成对样品,也特别地适合单样品的CDKN2A/P16基因拷贝缺失分析。
工业应用性
本发明的发明人在于发现了人CDKN2A共同缺失区“chr9:21970277-21985225,hg19”,由此设计出与该共同缺失区的保守区互补的特异性引物对及探针组,以高的灵敏度为86.5%(32/37),特异性为91.3%(94/103)对单样品的CDKN2A/P16基因拷贝缺失进行分析。
由于在现有研究中已经证实了人CDKN2A基因拷贝缺失与许多癌症的发病、转移以及预后具有显著相关性,因此本发明检测人CDKN2A共同缺失区“chr9:21970277-21985225,hg19”的引物对及控针组可以制备用于定量检测待测DNA样品中人CDKN2A基因拷贝缺失的试剂或试剂盒,用于估测癌症的发病、转移以及预后。
本发明的实施例2仅仅为示例性的实施方式,其中提到的平均值和标准差等,本领域技术人员可以根据本发明给出的方法及癌症类型进行最后确定。
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Claims (4)
1.一种试剂盒,用于定量检测待测DNA样品中人CDKN2A基因拷贝缺失,其包括至少SEQID NO.1,SEQ ID NO.2,SEQ ID NO.3所示的寡核苷酸序列的引物对及探针组,并且包括与GAPDH基因保守区参考序列互补的SEQ ID NO.4,SEQ ID NO.5,SEQ ID NO.6所示的寡核苷酸序列的引物对及探针组。
2.根据权利要求1所述的试剂盒,其特征在于,该待测DNA样品为单样品。
3.根据权利要求1所述的试剂盒,其中所述定量检测待测DNA样品中人CDKN2A基因拷贝缺失,通过包括下列步骤的方法实施:
a.提取待测组织或细胞的基因组DNA样品;
b.提取和制备经过测序鉴定的CDKN2A纯合性拷贝缺失细胞和CDKN2A基因不缺失细胞的DNA标准品,按梯度级差,连续稀释成比例为0%到100%之间梯度级的CDKN2A拷贝缺失细胞DNA的PCR扩增模板,制备标准曲线;
c.根据CDKN2A共同缺失区“chr9:21970277-21985225,hg19”的无重复序列,无微卫星,不含频率>1%SNP的保守序列,设计并合成双重PCR扩增用的SEQ ID NO.1,SEQ ID NO.2,SEQ ID NO.3所示的寡核苷酸序列的样品引物对及序列特异性探针;同时设计并合成针对GAPDH基因保守区参考序列的双重PCR扩增用参考的SEQ ID NO.
4,SEQ ID NO.5,SEQ ID NO.6所示的寡核苷酸序列的引物对和序列特异性探针;
d.双重引物荧光定量PCR扩增,测定待测组织或细胞的基因组DNA序列以及GAPDH基因保守区参考序列的各个扩增片段的PCR Ct值,计算CDKN2A共同缺失区PCR产物与参考序列PCR产物的ΔCt值[CtCDKN2A-CtGAPDH],根据标准曲线计算CDKN2A拷贝数;
e.分析正常人体细胞基因组DNA的CDKN2A基因拷贝数,根据其平均数±1.96×SD个标准差制定参考值范围;
f.当待测组织或细胞的CDKN2A基因拷贝数低于参考值低限时,判断该待测组织或细胞的存在CDKN2A基因拷贝缺失。
4.根据权利要求3所述的试剂盒,其特征在于,该方法是用于定量检测单样品的人CDKN2A基因拷贝缺失的方法。
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