CN103695558A - 多核苷酸引物 - Google Patents
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Abstract
含有以下引物序列之一或其互补序列的至少最后6个核苷酸的多核苷酸:SEQ.ID NO:3-16,18,20-33,35或37-39。
Description
本申请是国际申请PCT/GB2008/003306,国际申请日2008年9月29日,中国国家阶段申请号200880110038.7的发明专利申请的分案申请。
技术领域
本发明涉及多核苷酸、含有多核苷酸的试剂盒,以及在基因中检测突变存在与否的方法。
背景技术
磷酯酰肌醇3-激酶(PI3K)是参与细胞信号传递的一大类脂质激酶。PI3K-AKT途径在许多类肿瘤中被激活,导致细胞生长、增殖和存活异常(补充的1篇近期综述)。近来,在人癌症中鉴定出了1A型PI3K(PI3KCA)催化亚基中的突变[1]。这些突变在致癌作用中的确切作用仍然并没有被明确确定,但随着许多靶向PI3K抑制剂的开发,突变检测对于患者筛选来说变得日益重要。这些突变的检测中的技术难题来自于可能只包含少量突变序列的肿瘤活组织切片的限制。另外,从石蜡包埋组织提取的DNA常常已被降解且质量很差。测序检测所需要的突变DNA的最低水平是15-25%,因此对于开发能在异源样品中检测少量突变的等位基因的灵敏的检测法以及进行所述检测法所需的产品具有迫切的需求。
本发明试图满足该需求。
发明内容
本发明提供了对肿瘤引起的PIK3CA突变的灵敏耐用的检测法。
根据本发明的一个方面,提供了一种多核苷酸,其含有下列引物序列之一的至少最后6个核苷酸:SEQ.ID NO.3-16,18,20-33,35或37-39,或其互补序列。也就是说,所述多核苷酸含有下列引物序列之一3’末端的至少6个核苷酸:SEQ.IDNO.3-16,18,20-33,35或37-39,或其互补序列。
优选地,所述多核苷酸包含下列引物序列之一、或其互补序列、或与其具有80%、90%、95%或99%序列一致性的序列3’末端8、10、12、14、17、18或20个核苷酸或整个序列的至少75%的核苷酸:SEQ ID NO.3-16、18、20-33、35或37-39。
在本发明的一些实施方式中,提供了一种多核苷酸,其包含以下引物序列之一3’末端10个核苷酸的至少75%的核苷酸:SEQ.ID NO.3-16、18、20-33、35或37-39,或其互补序列。
通常,所述多核苷酸短于100个核苷酸,优选短于80个核苷酸,更优选短于60个核苷酸,更优选短于40个核苷酸,更优选短于30个核苷酸。
优选地,所述多核苷酸还含有淬灭基团和荧光团。
通常,所述淬灭基团和荧光团由含有第一和第二区域的核苷酸尾序列隔开,其中所述第一区域的核苷酸与第二区域的核苷酸互补但呈反向,从而所述第一区域与第二区域的杂交导致所述淬灭基团与所述荧光团足够靠近,以淬灭所述荧光团。
优选地,所述尾序列还含有第三区域,其具有与PIK3CA基因的一个区域互补的序列。
优选地,所述多核苷酸含有SEQ ID NO:18的3’末端的至少6个核苷酸,并且所述尾序列含有SEQ.ID NO.17。
或者,所述多核苷酸含有SEQ ID NO:35的3’末端至少最后的核苷酸,并且所述尾序列含有SEQ.ID NO.34。
或者,所述多核苷酸含有SEQ ID NO:39的3’末端至少最后的核苷酸,并且所述尾序列含有SEQ.ID NO.38。
通常,所述淬灭基团含有Dabcyl。
优选地,所述荧光团含有Hex,Fam或Rox。
根据本发明的另一个方面,提供了一种含有本发明的至少两种多核苷酸的试剂盒。
优选地,所述试剂盒含有包含SEQ ID NO.18的多核苷酸和包含SEQ ID NO.3-16之一的多核苷酸;或含有包含SEQ ID NO.35的多核苷酸和包含SEQ ID NO.20-33之一的多核苷酸;或含有包含SEQ ID NO.39的多核苷酸和包含SEQ ID NO.37的多核苷酸。
通常,所述试剂盒还含有核苷三磷酸,聚合酶和/或缓冲液。
根据本发明的另一个方面,提供了将本发明所述的多核苷酸或试剂盒,或含有SEQ.ID NO.3-16,18,20-33或35或其互补序列3’末端6个核苷酸中的4或5个的多核苷酸用于检测含有PIK3CA基因的至少一个片段的核酸样品中的突变的用途。
优选地,所述核酸样品中的PIK3CA基因所述片段长至少10个核苷酸,优选长20个核苷酸,更优选长30个核苷酸,和更优选长40个核苷酸。
根据本发明的另一个方面,提供了一种检测PIK3CA基因中突变存在与否的方法,该方法包括以下步骤:
a)混合含有PIK3CA基因的至少一个片段的核酸样品和一条多核苷酸,其含有下列引物序列之一或其互补序列3’末端至少6个核苷酸:SEQ ID NO.3-16或20-33;和
b)检测所述多核苷酸与所述核酸样品的杂交,其中杂交表示突变的存在。
通常,所述多核苷酸含有下列引物序列之一:SEQ ID NO:3-16或20-33。
优选地,该方法还在步骤a)前包括以下步骤:用热循环核酸扩增,优选PCR扩增PIK3CA基因所述片段的拷贝数。
优选地,步骤b)包括用所述多核苷酸作为第一引物进行DNA聚合,并检测聚合的延伸产物。
通常,步骤b)包括以下步骤:将所述核酸样品和多核苷酸与对应于所述多核苷酸互补区域下游的PIK3CA序列所述片段的一个区域的第二引物混合,并对该混合物进行PCR。
优选地,所述第二引物包含:SEQ.ID NO.18,并且所述多核苷酸包含SEQ.IDNOS.3-16的3’末端6个核苷酸中的至少4或5个核苷酸;或所述第二引物包含SEQ.ID NO.35,并且所述多核苷酸含有SEQ.ID NOS.20-33的3’末端6个核苷酸中的至少4或5个核苷酸。
另外,该方法还包含以下步骤:用对照引物对所述样品进行PCR,并比较PIK3CA基因的扩增和用所述多核苷酸和第二引物的扩增。
优选地,所述对照引物含有SEQ ID NO.37和39。
通常,所述多核苷酸含有淬灭基团和荧光团,并且其中步骤b)包括使所述混合物暴露于在不存在淬灭基团时荧光团对其响应的波长的光下,并检测在不存在淬灭基团的情况下荧光团发射波长处的光。
优选PIK3CA基因是GenBank登录号NM_006218版本号NM_006218.2GI:54792081可获得的序列,该序列通过参考整合在本发明中。
当说明书中提到一个参照序列“3’末端6个核苷酸中至少4或5个”时,指所述参照序列的所述6个核苷酸中的任意一个或两个核苷酸可能缺失或被不同的核苷酸取代。当然,在一些实施方式中,该序列含有参照序列的全部6个核苷酸。
在本说明书中,“ARMS”是例如EP-A-0332435中所公开的的扩增阻滞突变***。
当在本说明书中提到一种多核苷酸相对于参照多核苷酸的百分比时,这可以通过本领域已知的算法确定。
例如,两条序列之间的相同性百分数可以采用默认参数用BLASTP算法2.2.2版(Altschul,Stephen F.,Thomas L.Madden,Alejandro A.,JinghuiZhang,Zheng Zhang,Webb Miller,和David J.Lipman(1997),“缺口BLAST和PSI-BLAST:新一代蛋白质数据库检索程序”,核酸研究.25:3389-3402)加以确定。
附图说明
图1显示了对含有突变PIK3CA基因的样品进行Scorpions检测和测序的结果。
发明详述
本发明的实施方式提供了可用于在含有核酸的样品中检测PIK3CA基因突变的检测法的多核苷酸引物。
在具体实施方式中,所述多核苷酸引物是正向和反向引物,它们与PIK3CA基因杂交,从而发生PCR扩增。因此,所述正向引物与所述反向引物相反链的上游杂交,并且正向和反向引物共同确定了在PCR中扩增出的扩增子序列。选择正向引物序列,使其不和野生型序列互补,但能与突变PIK3CA序列杂交。
为了检测样品中突变PIK3CA基因的存在,将引物与样品混合。然后在样品中加入PCR必需的试剂(合适的核苷酸三磷酸、DNA聚合酶和缓冲液),进行PCR。如果样品含有正向引物能与之杂交的突变序列,那么在PCR过程中扩增出扩增子,从而表明样品中突变序列的存在。如果样品不含突变序列,那么正向引物与PIK3CA序列结合的效率低,因此几乎没有,或不存在扩增子序列的扩增。
为了检测突变E542K,所述正向引物序列可以是SEQ ID NO.3-9之一,优选为SEQ ID NO.5。为了检测突变E545K,所述正向引物序列可以是SEQ ID NO.10-16之一,优选为SEQ ID NO.14。为了检测突变H1047R,所述正向引物序列可以是SEQID NO.20-26之一,优选为SEQ ID NO.21。为了检测突变H1047L,所述正向引物序列可以是SEQ ID NO.27-33之一,优选为SEQ ID NO.28。然而,应理解正向引物的精确序列不需要与这些序列相同,只要正向引物与突变序列比与野生序列的杂交更容易。在上述序列中,引物的最后6个核苷酸(即3'末端的核苷酸)提供了结合特异性,因此这些核苷酸必需与给定序列相同。
为了检测样品中形成的扩增子的存在,反向引物在本发明实施方式中被称作“Scorpions”引物。Scorpions引物在WO-A-99/066071中被详细描述,在此通过参考整合在本发明中。Scorpions引物含有与一个基因(在本发明中为PIK3CA)的第一靶序列互补的引物序列和一个尾序列,其包含与两个互补序列侧接的探针序列。在引物序列和尾序列之间提供了DNA聚合酶封闭部分(例如六甘醇(HEG)单体)。在尾序列的一端提供了荧光团,在尾序列的另一端提供了淬灭基团。使用时,Scorpions引物的引物序列在正常方式的PCR中作为反向引物,并且因此整个Scorpions引物(包括尾序列)被掺入各扩增子。DNA聚合酶封闭部分防止尾序列复制。因此,尾序列中互相互补的序列倾向互相杂交,使荧光团和淬灭基团靠近并防止荧光团发光。然而,如果扩增子含有与所述探针序列互补的第二靶序列,所述探针序列优先与第二靶序列结合,使互补序列分开。这导致荧光团和淬灭基团在空间上分开,从而使荧光团响应不同波长的入射光而发出一定波长的光。因此,Scorpions引物能够轻易检测扩增子,并且避免假阳性结果(例如由引物二聚体引起的),因为只有扩增子含有第二靶序列时才会产生信号。
所述荧光团可以是Hex(4,7,2',4',5',7'-六氯-(3',6'-二特戊酰基荧光素基)-6-羧酰胺基己基]-1-O-(2-氰基乙基)-(N,N-二异丙基)-亚磷酰胺),Fam([(3',6'-二特戊酰基荧光素基)-6-羧酰氨基己基]-1-O-(2-氰基乙基)-(N,N-二异丙基)-亚磷酰胺)或Rox(5,6,-羧基-X-罗丹明)。所述淬灭基团可以是Dabcyl(5’-二甲氧基三苯甲氧-5-[(N-4'-羧基-4-(二甲基氨基)-偶氮苯)-氨基己基-3-丙烯亚酰胺]-2'-脱氧尿苷-3'-[(2-氰基乙基)-(N,N-二异丙基)]-亚磷酰胺)。
在本发明的实施方式中,提供了用于检测E542K和E545K突变的Scorpions引物,其中引物序列是SEQ ID NO.18,探针序列是SEQ ID NO.17。提供了用于检测H1047R和H1047L突变的Scorpions引物,其中引物序列是SEQ ID NO.35,探针序列是SEQ ID NO.34。
然而应理解,使用Scorpions引物不是本发明必需的,也可使用其它检测扩增子合成的方法,例如TaqManTM产物检测,如专利号US-A-5487972和US-A-5210015中所述。
在一些实施方式中,还进行对照试验,以检测PIK3CA基因在样品中的总浓度。这是通过用确定PIK3CA基因中另一个区域内的扩增子的对照正向和反向引物进行单独PCR反应实现的。优选正向引物是SEQ ID NO.37,反向引物是Scorpions引物,其中引物序列是SEQ ID NO.39,探针序列是SEQ ID NO.38。然后将产生阈值数量的对照扩增子所需的PCR循环数与产生含有突变序列的扩增子阈值数的PCR循环数比较,以评估样品中PIK3CA基因的突变拷贝的比例。该对照试验通常与所述检测法分开进行。
所述PCR检测优选以多重实时PCR检测的方式进行。
核酸测试样品通常是血液、粪便、痰、结肠洗出液、支气管洗出液或其它体液样品,或从个体获得的组织。所述个体通常是人,优选智人(Homo sapiens)。应理解,测试样品同样可以是对应于所述测试样品中的序列的一个核酸序列。即,首先可以用任何常规技术,例如热循环核酸扩增,尤其是PCR,或全基因组扩增(WGA)扩增样品核酸中的全部或部分区域,然后用于本发明的方法。
可使用任何常用的用于聚合的酶,只要它不在任何显著的程度上影响DNA聚合酶区分正常和突变模板序列的能力。常用的酶的例子包括没有显著3’-5'外切核酸酶活性的热稳定酶,例如Taq DNA聚合酶,尤其是“Ampli Taq Gold”TMDNA聚合酶(PE Applied Biosystems),Stoffel片段,或其它合适的Taq或Tth(嗜热栖热菌Thermus thermophilus)DNA聚合酶的N-末端缺失修饰形式。
在本发明的其它实施方式中,提供了试剂盒,其含有本发明的一种或多种多核苷酸和进行PCR反应所需的核苷酸三磷酸、DNA聚合酶和缓冲液。优选试剂盒含有用于检测特定突变的正向和反向引物,以及正向和反向对照引物。
实施例
材料和方法
根据PIK3CA基因(登录号:NM_006218)中4种最常见的突变设计引物。设计ARMS引物以检测外显子20中的两个突变:H1047R和H1047L;以及外显子9中的两个突变:E452K和E454K。对PIK3CA基因中的cDNA2450位设计对照引物。
还设计了Scorpions。为了实现在每个反应中进行多重试验,用不同荧光团标记三个Scorpion引物。
引物设计
针对每个靶区域设计多个特异性的ARMS引物。E542K和E545K突变的靶区域分别如下如SEQ ID NO.1和2所示(突变碱基在括弧中显示,其中正常形式在前)。针对所述突变的正向引物也如下所示(SEQ ID NO.3-16)。为了增强对这些反应的特异性,在接近3'-末端使用了额外的引物错配(在引物序列中用下划线表示)。针对所述实验使用了优化引物(E542K-2和E545K-4)。可与所述引物序列使用的Scorpions引物如SEQ ID NO.17和18所示。Scorpions引物和靶区域之间的对应区域用一致的高亮或下划线标出。
外显子9区域
AACAGAGAATCTCCATTTTAGCACTTACCTGTGACTCCATAGAAAATCTTTCTCC
TGCTCAGTGATTT(C/T)AGAGAGAGGATCTCGTGTAGAAATTGCTTTGAGCTGTT
CTTTGTCATTTTCCCTTAATTCATTGTCTCTAGCTAGTCTGTTACTCTGTAAAATA
AAATAATATCTTATATA(SEQ ID NO.1)
AACAGAGAATCTCCATTTTAGCACTTACCTGTGACTCCATAGAAAATCTTTCTCC
TGCT(C/T)AGTGATTTCAGAGAGAGGATCTCGTGTAGAAATTGCTTTGAGCTGTT
CTTTGTCATTTTCCCTTAATTCATTGTCTCTAGCTAGTCTGTTACTCTGTAAAATA
AAATAATATCTTATATA(SEQ ID NO.2)
外显子20区域
H1047R和H1047L突变的靶区域如下如SEQ ID NO:19所示(突变碱基如括弧中所示,其中正常形式在前)。针对突变的正向引物也如下所示(SEQ ID NO.20-33)。为了增强对这些反应的特异性,在接近3'-末端使用了额外的引物错配(如引物序列中下划线的所示)。针对所述实验使用了优化引物(H1047R-1和H1047L-1)。可与所述引物序列使用的Scorpions引物如SEQ ID NO.34和35所示。Scorpions引物和靶区域之间的对应区域用一致的高亮或下划线标出。
AGTGCAGTGTGGAATCCAGAGTGAGCTTTCATTTTCTCAGTTATCTTTTCAGTTC
AATGCATGCTGTTTAATTGTGTGGAAGATCCAATCCATTTTTGTTGTCCAGCCAC
CATGA(T/C/A)GTGCATCATTCATTTGTTTCATGAAATACTCCAAAGCCTCTTGCTC
AGTTTTATCTAAGGCTAGGGTCTTTCGAATGTATGCAATGTCATCAAAAGATTGT
AGTTCTGGCATTCCAGAGCCAAGCATCATTGAGAAAAGATTTATGAAGAGATTG
GCATGCTGTCGAATAGCTAGATAAGCCTT(SEQ ID NO.19)
对照引物
对照引物如下所示。Scorpions引物和靶区域之间的对应区域用一致的高亮或下划线标出。
AGGCTTGAAGAGTGTCGAATTATGTCCTCTGCAAAAAGGCCACTGTGGTTGAAT
TGGGAGAACCCAGACATCATGTCAGAGTTACTGTTTCAGAACAATGAGATCATC
TTTAAAAATGGGGATGG(SEQ ID NO.36)
所有引物由Invitrogen合成和提供。PCR缓冲液、Taq和镁由Eurogentec提供,dNTP购自Abgene Ltd。Scorpions由ATDBio合成和提供。
试验在包含一个对照检测和2个ARMS检测(1x外显子9和1x外显子20)的2个反应中多重进行。在含有1xPCR缓冲液、4.0mM MgCl2、200μM dNTP混合物、0.25μM的各引物(对照引物和2个ARMS引物)以及0.25μM的各Scorpion(对照Scorpion(SEQ ID NO.38和39)、外显子20Scorpion(SEQ ID NO.34和35)、以及外显子9Scorpion(SEQ ID NO.17和18))的25微升反应体系中进行试验。在各反应中加入2.5微升DNA模板。每次反应用2.5单位Taq聚合酶多重使用(multiplex)H1047R和E542K引物。每次反应用3.0单位Taq聚合酶多重使用H1047L和E545K引物。所用的E542K引物是E542K-2(SEQ ID NO.5)。所用的E545K引物是E545K-4(SEQ ID NO.14)。所用的H1047R引物是H1047R-1(SEQ ID NO.21)。所用的H1047L引物是H1047L-1(SEQ ID NO.28)。
在所有的情况下,反应在下列条件下在Stratagene Mx3000P上扩增:95℃10分钟,然后是45轮的90℃30秒和60℃1分钟循环。
根据Higuchi等[2]所述的方法构建携带点突变的DNA盒作为阳性对照。简单说,使用相应的外部和变构(mutamer)引物产生具有互补端的半个盒,每个半盒含有一个突变碱基。混合这些PCR产物,并用内部嵌套(nested)引物扩增。互补的半盒的自发引导和随后的扩增产生了具有突变碱基的终产物。对产物测序,以确定已建立了正确的序列。对每个感兴趣的突变重复该过程。将DNA盒与等量的基因组DNA混合,以产生100%阳性对照。
实施例1
为了确定反应和引物的特异性,每个反应使用5-50ng基因组DNA进行各试验,以评估错配引物延伸导致的临界信号。对于每个反应,测定△Ct值(对照Ct-突变Ct)(Ct=阈值循环)。对每个DNA浓度进行6次反应,并对各种情况重复三次,以确定截留△Ct值,在该值以下,任何扩增可被认为是由于存在突变序列而不是由于临界信号。截留△Ct值被确定为比每个试验的所有反应中的最小△Ct值小1Ct。对于H1047R和H1047L试验,截留△Ct被定为12,对于E542K试验,截留△Ct是9,对于E545K试验,截留△Ct是8。
实施例2
为了评估试验的灵敏度,将5个突变DNA拷贝稀释于不同浓度的基因组DNA中,使得最终浓度为突变DNA相对于野生型DNA的5,2,1,0.5和0.1%。表1显示了4个ARMS试验的灵敏度。该表格显示了在野生型DNA背景下递减浓度的突变DNA的△Ct值。预定的截留△Ct如最后一栏所示。外显子20试验能够在仅占0.1%总DNA的情况下检测5个突变DNA拷贝(在先前预定的截留△Ct下)。外显子9试验能够在仅占1%总DNA的情况下检测5个突变DNA拷贝,其中△Ct位于先前预定的截留值范围内)(表1)。
表1:
实施例3
用含有突变H1047R(HCT-116)和E545K(MCF-7)的细胞系的混合物比较ARMS试验与测序相比的相对灵敏度。两个细胞系对于突变都是杂合的。用Samuels等[1]所述的引物和PCR循环条件进行测序。ARMS试验和测序在突变基因占总混合物100%,50%,30%,10%和1%的浓度下进行。结果如图1所示,其中“Scorpions”标题下所示的结果显示了在持续的PCR循环后扩增子拷贝数的增加(使用对照引物和突变引物的结果用单独的线条表示)。在标题“DNA测序”下所示的是对混合物中基因反向链测序的结果。当H1047R突变体少于50%的总混合物时,测序不能检测其存在,而当存在的E545K突变体少于30%的总混合物时,也不能检测其存在。相反,使用本发明引物的试验能检测以1%浓度存在的突变体。
实施例4
对从通过ARMS/Scorpion检测对存在PIK3CA突变进行了评估的不同肿瘤类型的新鲜冷冻组织中提取的DNA进行该试验。总共从279个肿瘤样品中获得了DNA。该试验在49个结肠癌样品中的5个(10.2%),49个乳腺癌样品中的19个(38.7%),51个肺癌样品中的1个(1.9%),和34个黑素瘤样品中的1个(2.9%)中报告了突变。在50个***或46个卵巢癌样品中未检测到突变。在对于PIK3CA突变阳性的结肠样品中,3个是H1047R,1个是H1047L,而1个是E542K;在对于PIK3CA阳性的乳腺癌样品中,15个是H1047R,1个是H1047L,而3个是E545K;对于PIK3CA突变阳性的肺癌样品和黑素瘤样品中的两个突变都是H1047R。测序仅仅鉴定出被检测出的26个突变中的14个(53%)。测序检测出一乳腺癌标本中的突变,它是ARMS试验并没有设计用于检测的(c1634A>G;p E545G)。这不是新的突变,且先前已经在乳腺癌和结肠癌中进行过描述[3-5]。
除了卵巢癌,所分析的样品中PIK3CA突变的出现率与先前研究一致[1,3-9]。在卵巢癌中先前已经描述过PIK3CA突变,但被认为可能与子宫内膜样和透明细胞癌有关[8,10]。所有在本研究中测试的卵巢癌都是严重腺癌,这可能解释了为何不存在任何PIK3CA突变。
ARMS试验明显在临床样品中鉴定了比直接测序所见更多的突变。细胞系混合物确定该试验比测序在检测感兴趣的PI3KCA突变中更灵敏。可能同时含有肿瘤和正常组织的临床样品的异源性意味着在一些情况下,突变发生率将会比测序法可检测的低,因此ARMS试验更适合临床应用。缺点是,仅有某些ARMS特异突变可被检测。然而在该系列的279个样品中,在PI3KCA基因的外显子9或20中仅检测出一个ARMS试验没有设计检测的突变。
总的说,实施例显示了本发明提供了一种用于检测PI3KCA基因中4个最常见突变的灵敏的高通量的检测法。该检测法可用于少量DNA,并可检测样品中低水平的突变PIK3CA。
Claims (6)
1.一种检测PIK3CA基因中突变存在与否的方法,所述方法包括以下步骤:
a)混合含有PIK3CA基因的至少一个片段的核酸样品和第一引物与第二引物,并对该混合物进行PCR;和
b)检测所述第一引物与所述核酸样品的杂交,其中杂交表示突变的存在;
所述第一引物能够与PIK3CA基因的H1047L突变序列杂交,但与野生型序列不互补,所述第一引物的3’末端最后6个核苷酸与SEQ ID NO.28的3’末端最后6个核苷酸相同;
所述第二引物在PIK3CA序列片段内的对应区域位于第一序列互补区的下游。
2.如权利要求1所述的方法,第二引物的序列如SEQ ID NO.35所示。
3.一种正向引物,能够与PIK3CA基因的H1047L突变序列杂交,但与野生型序列不互补,所述第一引物的3’末端最后6个核苷酸与SEQ ID NO.28的3’末端最后6个核苷酸相同。
4.一种试剂盒,含有至少两条引物,其中,第一引物是正向引物,第二引物是反向引物,所述第一引物能够与PIK3CA基因的H1047L突变序列杂交,但与野生型序列不互补,所述第一引物的3’末端最后6个核苷酸与SEQ ID NO.28的3’末端最后6个核苷酸相同;
所述第二引物在PIK3CA序列片段内的对应区域位于第一序列互补区的下游。
5.如权利要求4所述的试剂盒,所述第二引物的序列如SEQ ID NO.35所示。
6.权利要求3所述正向引物或权利要求4所述试剂盒用于检测核酸样品中突变的用途,所述核酸样品至少含有PIK3CA基因的片段。
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