CN116732181A - 基因BRCA2位点g.32332272G>A突变在制备乳腺癌早期筛查试剂盒中应用 - Google Patents
基因BRCA2位点g.32332272G>A突变在制备乳腺癌早期筛查试剂盒中应用 Download PDFInfo
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Abstract
本发明公开了基因BRCA2位点g.32332272G>A突变在制备乳腺癌早期筛查试剂盒中应用以及检测基因BRCA2位点g.32332272G>A突变体的引物、试剂盒,该突变基因与BRCA2正常基因序列相比具有g.32332272G>A位点突变,所述BRCA2基因g.32332272G>A位点突变是指突变位点在SEQ ID NO:1序列的第202位碱基G突变为A。本发明获得了乳腺癌发病相关突变位点谱和特异性标志物。通过突变位点序列的改变进行相关诊断试剂盒的研制和应用,可使得乳腺癌的诊断更加方便易行。
Description
技术领域
本发明涉及于分子生物学技术领域,更具体的说是涉及基因BRCA2位点g.32332272G>A突变在制备乳腺癌早期筛查试剂盒中应用。
背景技术
近年来我国癌症的发病率和死亡率不断攀升,恶性肿瘤已成为我国重大的公共卫生问题。据我国肿瘤登记中心2016年发布的结果显示,2012年我国肿瘤新发病例数358.6万例,死亡例数218.7万例,粗发病率265/10万,死亡率161.5/10万。在恶性肿瘤发病现状中,乳腺癌在女性恶性肿瘤中处于第一位,每年新发约27.3万,且发病年龄不断呈现年轻化趋势,是严重威胁女性生命的第一杀手。
乳腺癌是具有很强遗传背景的恶性肿瘤,现有研究显示5-10%的乳腺癌是由遗传因素导致。目前BRCA1、BRCA2、TP53、PALB2、PTEN、CHEK2、ATM以及RAD等基因被认定为乳腺癌易感基因,其中BRCA1和BRCA2基因为遗传性乳腺癌的主要易感基因。研究表明,携带有BRCA1和BRCA2基因致病突变的人群在70岁之前发生乳腺癌的概率分别可达40-80%和20-85%,同时,已被诊断为遗传性乳腺癌的患者,发生复发转移的可能性也高于普通人群。
BRCA1和BRCA2属于抑癌基因,均呈常染色体显性遗传,且突变容易引发早年发病。BRCA2基因位于人类染色体13q12,包含27个外显子,编码BRCA2蛋白含3418个氨基酸,对细胞生长的调节有重要作用。BRCA2基因发生突变后表达的蛋白质失去了修复DNA损伤的功能,导致染色体不稳定,促进肿瘤发生。男性BRCA2突变携带者终生患前列腺癌,乳腺癌,胰腺癌的概率分别为20%、6%和3%,而女性BRCA2突变携带者终生患乳腺癌为26%-84%,卵巢癌的概率为20%。BRCA2基因突变相关的肿瘤往往会表达ER(雌激素受体)与PR(孕激素受体),与散发性乳腺癌表现出相似的特征。现已经报道的BRCA2突变主要是移码突变,也会发生大量的错义突变,但错义突变大多属于意义未明变异。因此,发现BRCA2新的致病突变,对开展遗传性乳腺癌的分子诊断具有重要意义。
发明内容
有鉴于此,本发明提供了基因BRCA2位点g.32332272G>A突变在制备乳腺癌早期筛查试剂盒中应用,本发明提供了乳腺癌致病的新的突变位点,可对乳腺癌筛查辅助应用。
为了实现上述目的,本发明采用如下技术方案:
基因BRCA2位点g.32332272G>A突变在制备乳腺癌早期筛查试剂盒中应用,其中,BRCA2基因序列如SEQ ID NO:1所示;突变型BRCA2基因序列如SEQ ID NO:2所示。
一种检测基因BRCA2位点g.39711928G>A突变体的引物,其特征在于,所述引物的核苷酸序列如SEQ ID NO.3~SEQ ID NO.4所示;
正向引物:5’-ATGTGCTTCTGTTTTATACTTTAACAGA-3’,如SEQ ID NO:3所示;
反向引物:5’-ACTTTTTGTAGATTTTTTGTTCTAC-3’,如SEQ ID NO:4所示。
一种检测基因BRCA2位点g.32332272G>A突变体的试剂盒,其特征在于,所述试剂盒包括上述引物。
一种基因BRCA2位点g.32332272G>A突变体的检测方法,其特征在于,包括以下步骤:
(1)提取待检样本中DNA;
(2)以该DNA为模板,利用上述引物进行PCR扩增反应,获得PCR扩增产物;
(3)检测BRCA2基因是否存在g.32332272G>A突变基因;
作为本发明优选的技术方案,所述步骤(3)中,检测BRCA2位点g.32332272G>A突变体的具体方法如下所示:
(1)利用琼脂糖凝胶将所述PCR扩增产物进行电泳;
(2)将电泳后的扩增产物进行纯化回收,以便于测序;
(3)纯化后的扩增产物在DNA测序仪上进行测序,将测序结果与野生型BRCA2参考序列进行比对,确定是否有BRCA2位点g.32332272G>A突变体。
作为本发明优选的技术方案,所述突变型BRCA2基因序列与野生型BRCA2基因序列具有g.39711928G>A位点突变。
作为本发明优选的技术方案,所述BRCA2基因g.32332272G>A位点突变是指突变位点在SEQ ID NO:1序列的第202位碱基G突变为A。
本发明的有益效果:本发明通过研究乳腺癌患者的基因序列,发现其BRCA2基因组第13号染色体的32332272位置发生碱基G到A碱基的突变,该突变位点为本研究首次在中国汉族女性乳腺癌患者中发现,且在正常人群中不存在该突变,数据库显示欧美人群该区域的扫描也未发现该突变位点。所以,本发明通过研究突变位点在乳腺癌辅助诊断的应用前景,阐述突变位点对于乳腺癌进展的影响,揭示其诊断价值。
本发明的引物是通过针对人类BRCA2基因不同基因型的特异性序列位点的改变,所设计出针对突变位点的引物序列;利用该引物以及包含该引物的试剂盒可通过分离和研究乳腺癌患者及与其年龄匹配的健康对照外周血DNA中的突变,寻找与乳腺癌相关的高特异性的突变位点,并研制出可临床或人群应用的乳腺癌辅助诊断试剂盒,为乳腺癌的筛查和诊断提供支持。
综上所述,本发明获得了乳腺癌发病相关突变位点谱和特异性标志物。通过突变位点序列的改变进行相关诊断试剂盒的研制和应用,可使得乳腺癌的诊断更加方便易行,为临床医生快速准确掌握患者病情,为临床治疗效果评价奠定基础,并为发现具有潜在治疗价值的新型小分子药物靶标提供帮助。因此,本研究丰富了BRCA2基因的致病突变谱,对开展遗传性乳腺癌的分子诊断与高危人群筛查具有重要意义。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据提供的附图获得其他的附图。
图1附图为BRCA2基因g.39711928G>A突变检测的凝胶电泳图;
图2附图为BRCA2基因g.39711928G>A突变测序图。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例中采用的仪器以及试剂
仪器:Veriti96型PCR仪、BIO-RAD Gel Doc XR+型凝胶成像仪(美国伯乐公司)、凝胶电泳仪(北京六一公司)。
试剂:QIAamp DNA提取试剂盒(德国Qiagen公司);DNA Isolation Kit提取试剂盒(北京PELFREEZ公司);PCR缓冲液、dNTP、Taq酶(美国ABI公司);引物由上海生工生物有限公司合成。
实施例1:特异性引物的设计
根据美国国家生物信息中心(NCBI)GenBank记录的BRCA2基因(序列号:NC_000013.11),通过Oligo6.0引物软件设计引物,最终确定1对特异性寡核苷酸引物序列,正向引物序列如表1中SEQ ID NO:3所示;反向引物序列如SEQ ID NO:4所示,扩增产物片段长度为202bp。
表1.相关突变位点寡核苷酸引物序列
实施例2:样本DNA的提取
本发明所用阳性样本,经过医院确诊为乳腺癌,经知情同意,取得其血样,并采用购置的试剂盒提取受试者全血基因组DNA,具体步骤如下
(1)取无菌2.0mL离心管一只,加入1mL细胞裂解液。
(2)轻轻震荡然经EDTA抗凝的全血样本,直到彻底混匀;然后吸取500μL血样加入上述含有细胞裂解液的离心管中,轻轻倾倒离心管5~6次混匀。
(3)室温孵育10分钟(期间颠倒离心管2~3次混匀)。
(4)12000rpm室温离心5分钟。
(5)用移液器缓慢的尽可能将上清液移弃干净,注意勿将两相交界处的白色物质吸出。
(6)使用涡旋振荡器(Votex)剧烈混匀,直至白细胞重悬(10~15秒)。
(7)向重悬细胞溶液中加入核裂解液300μL。用移液枪头吸放溶液5~6次裂解白细胞。此时溶液应变得很粘稠。若混合后可见细胞团块,则将溶液置于37℃孵育直至团块消散。若孵育1小时后仍可见细胞团块,则另加核裂解液100μL并重复置于37℃孵育。
(8)向核裂解物中加入蛋白沉淀液100μL,用涡旋振荡器剧烈震荡10~20秒。
(9)12000rpm室温离心5分钟。
(10)将其上清液转至对应编号的已加入300μL室温异丙醇的2.0mL离心管中。
(11)轻轻颠倒以混匀溶液,直至白色线状DNA形成沉淀。
(12)12000rpm室温离心1分钟。
(13)弃去上清液,加入与样本量等体积的室温70%乙醇,轻轻颠倒离心管数次。
(14)用移液器缓慢的尽可能将乙醇液移弃干净。将离心管置于50℃烘烤5~10分钟,尽量让残余的乙醇液挥发干净。
(15)向离心管中加入50~100μL的DNA溶解液,轻轻混匀。
(16)用1%琼脂糖凝胶电泳来评估DNA提取效果,Nanodrop核酸仪检测含量,定量到20~50ng/μL,-20℃保存。
实施例3:基因BRCA2位点g.32332272G>A突变体的检测
(1)PCR扩增:利用实施例1中设计的PCR扩增引物扩增实施例2中提取的DNA样品,并获得扩增产物。
扩增反应体系为50μL,其含PCR 5×缓冲液10μL,DNA模板5.0μL,Taq聚合酶(1U/μL)1.0μL,MgCl2终浓度2.0mmol/L,dNTP终浓度200nmol/L,以及特异性上、下引物终浓度200nmol/L,并加消毒双蒸水至总体积50μL。
扩增反应条件为95℃预变性5分钟,然后依次95℃变性30秒,接着62℃退火30秒,72℃延伸30秒至1分钟,共35个循环。
(2)凝胶电泳检测:用1.5%琼脂糖凝胶对将步骤(1)所得的扩增产物进行电泳,以检测其是否有目的片段。通过凝胶成像仪观察结果并拍照,PCR产物经电泳后显示为单一条带,无杂带,则提示PCR产物单一,无非特异扩增。若条带位置位于适当大小的位置,则为目的片段。如图1所示,M:2000DNA Marker,1:空白对照,2:野生型对照,3:BRCA2基因g.32332272G>A突变样本。
(3)PCR产物纯化:采用Takara公司的Agarose Gel DNA Purification Kit试剂盒将琼脂糖凝胶电泳后的PCR产物进行纯化回收,准备测序。
(4)Sanger测序与结果判断:将纯化后PCR产物在ABI3730型全自动DNA测序仪上进行测序。将测序结果与BRCA2野生型参考序列(NCBI Reference Sequence:NC_000013.11)进行比对,根据实际突变情况对结果进行报告。检测所得基因突变图如图2所示,图中箭头所示为BRCA2基因位点g.32332272G>A突变。
根据比对结果,我们在69例具有乳腺癌家族史的中国汉族女性乳腺癌患者中,发现有1例患者存在BRCA2基因组第13号染色体的32332272位置发生碱基G到A碱基的突变,该基因在NCBI参考数据库GRCh38.p13中的编号为NC_000013.11(32315480-32399672)。且随后进行了无家族史乳腺癌样本验证,在600例无家族史中国汉族乳腺癌女性乳腺癌患者中,存在1例患者携带该突变;在600例无肿瘤家族史,年龄匹配的正常女性对照人群中,未发现突变个体。其中,列出了在该数据库中包含有该位点野生型的部分碱基序列供参考,如SEQID NO:1所示;BRCA2基因突变相对应的序列如SEQ ID NO:2所示。
SEQ ID NO:1
AATCAGGCTTTACTAGAAGAACAGGAGAAGGGGTGACTGACCGAAAAATAAAATGCCAAGTACTCAGAATAACCCTTTAAATACTGATATGTAATATTTAGCACATTCTACATAAACTGTTTCTATGAGAAAGGTTGTGAGAATAATATAAATTATATGGCTTATAAAATATTAATGTGCTTCTGTTTTATACTTTAACAGGATTTGGAAAAACATCAGGGAATTCATTTAAAGTAAATAGCTGCAAAGACCACATTGGAAAGTCAATGCCAAATGTCCTAGAAGATGAAGTATATGAAACAGTTGTAGATACCTCTGAAGAAGATAGTTTTTCATTATGTTTTTCTAA
SEQ ID NO:2
AATCAGGCTTTACTAGAAGAACAGGAGAAGGGGTGACTGACCGAAAAATAAAATGCCAAGTACTCAGAATAACCCTTTAAATACTGATATGTAATATTTAGCACATTCTACATAAACTGTTTCTATGAGAAAGGTTGTGAGAATAATATAAATTATATGGCTTATAAAATATTAATGTGCTTCTGTTTTATACTTTAACAGAATTTGGAAAAACATCAGGGAATTCATTTAAAGTAAATAGCTGCAAAGACCACATTGGAAAGTCAATGCCAAATGTCCTAGAAGATGAAGTATATGAAACAGTTGTAGATACCTCTGAAGAAGATAGTTTTTCATTATGTTTTTCTAA
本说明书中各个实施例采用递进的方式描述,每个实施例重点说明的都是与其他实施例的不同之处,各个实施例之间相同相似部分互相参见即可。
对所公开的实施例的上述说明,使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所示的这些实施例,而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (5)
1.基因BRCA2位点g.32332272G>A突变在制备乳腺癌早期筛查试剂盒中应用,其中,BRCA2基因序列如SEQ ID NO:1所示;突变型BRCA2基因序列如SEQ ID NO:2所示。
2.一种检测基因BRCA2位点g.32332272G>A突变体的引物,其特征在于,所述引物的核苷酸序列如SEQ ID NO.3~SEQ ID NO.4所示;
正向引物:5,-ATGTGCTTCTGTTTTATACTTTAACAGA-3,,如SEQ ID NO:3所示;
反向引物:5,-ACTTTTTGTAGATTTTTTGTTCTAC-3,,如SEQ ID NO:4所示。
3.一种检测基因BRCA2位点g.32332272G>A突变体的试剂盒,其特征在于,所述试剂盒包括权利要求1所述的引物。
4.一种基因BRCA2位点g.32332272G>A突变体的检测方法,其特征在于,包括以下步骤:
(1)提取待检样本中DNA;
(2)以该DNA为模板,利用权利要求2所述的引物进行PCR扩增反应,获得PCR扩增产物;
(3)检测BRCA2基因是否存在g.32332272G>A突变基因。
5.根据权利要求4所述的一种基因BRCA2位点g.32332272G>A突变体的检测方法,其特征在于,步骤(3)中,所述检测BRCA2位点g.32332272G>A突变体的具体方法如下所示:
(1)利用琼脂糖凝胶将所述PCR扩增产物进行电泳;
(2)将电泳后的扩增产物进行纯化回收,以便于测序;
(3)纯化后的扩增产物在DNA测序仪上进行测序,将测序结果与野生型BRCA2参考序列进行比对,确定是否有BRCA2位点g.32332272G>A突变体。
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