CN1782096B - 利用zip编码的pna芯片及其制备方法 - Google Patents
利用zip编码的pna芯片及其制备方法 Download PDFInfo
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- CN1782096B CN1782096B CN2005100713955A CN200510071395A CN1782096B CN 1782096 B CN1782096 B CN 1782096B CN 2005100713955 A CN2005100713955 A CN 2005100713955A CN 200510071395 A CN200510071395 A CN 200510071395A CN 1782096 B CN1782096 B CN 1782096B
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Abstract
本发明涉及利用zip编码的PNA芯片及其制备方法,尤其是,本发明涉及PNA zip编码芯片,其采用环氧化合物作为连接剂将PNA zip编码探针高密度地固定,本发明还涉及其制备方法。与使用DNA芯片相比,使用本发明的PNA芯片对先天性疾病或者碱基突变进行诊断的精确性更高,并且具有更高的灵敏性。并且,使用PNA zip编码探针仅通过杂交反应就能诊断出基因突变,而无需在每次靶基因改变时都要直接将探针固定在基底上的复杂步骤。
Description
技术领域
本发明涉及利用zip编码的PNA芯片及其制备方法,更特异地,本发明所涉及的PNA zip编码芯片,其采用环氧化合物作为连接剂将PNA zip编码探针高密度地固定,本发明还涉及该芯片的制备方法。
背景技术
由于对包括人类基因组计划的多种生物体的基因组计划最终得到了许多的遗传信息,因此目前正积极进行的研究包括对这些基因信息进行翻译以及对其中的关系进行分析。分子生物学和生物工程学的研究方向正逐渐从对DNA进行结构分析转移到对基因的功能进行分析,以及对基因之间的关系进行鉴别。顺应该种研究趋势,目前开发了多种分析遗传信息的方法,其中DNA芯片技术是一种能够对基因组计划得到的巨大量的遗传信息样品进行分析的方法,因此尤其吸引了人们的注意力。这是由于DNA芯片能够在短时间内对大量的遗传信息进行分析,并且易于自动化操作。
在DNA芯片中,是将1,000-1,000,000个寡聚核苷酸排列于固态表面上并与表面相连接,其中每一寡聚核苷酸具有8-25个碱基,所述固态表面包括例如硅、表面修饰的玻璃、聚丙稀或者活化的聚丙烯酰胺等。根据靶基因的DNA碱基序列来确定待固定的DNA。DNA芯片可以与重组基因技术以及PCR一样具有多种应用,并且还有许多优于现有技术的特点。根据不同的使用方式可将DNA芯片应用于广泛的样本中,因此其应用领域广泛。利用DNA芯片甚至可能分析出非常少量的样品,并能同时鉴别出不同位点的靶基因的碱基序列。
预计采用DNA芯片的DNA分析系统能够将DNA分析的速度加快几十倍至几百倍,因此能够显著缩短目前正在进行的多物种基因组计划的完成时间,并能显著降低分析每一碱基的单位成本。目前已经将DNA分析系统广泛应用于各领域,包括对先天性疾病的诊断、突变的研究、癌症诊断、致病细菌的检测、基因表达的分析、以及新药的开发等。
在具有zip编码的DNA芯片(Gerry,N.P.et al.,J.Mol.Biol.,292:251,1999;Hirschhom,J.N.,Proc.Natl.Acad.Sci.,97:12164,2000)中,将探针固定至固态基底上,其中所述探针为具有给定长度且彼此之间没有同源性的碱基序列,并且将靶DNA的碱基序列设计为以靶基因5’-末端具有与探针互补的碱基序列、3’-末端具有与所述基因互补的碱基序列的方式进行杂交。由此使得DNA芯片具有下述优点:其能通过简单的方式进行构建,并且可不经过构建探针的分离步骤就能得到诊断结果,这是通过将靶DNA的碱基序列位点重新构建而与基因相匹配来实现的。
PNA是DNA的类似物,其以肽键作为框架,包括与DNA相同的四种碱基。与DNA不同,PNA的框架中没有负电荷(Nielsen,P.E.et al.,Sci.,254:1497,1991)。图1显示了具有糖-磷酸盐框架的DNA的结构以及具有肽键框架的PNA的结构。与DNA相比,PNA对核酸酶和多种化学物质具有更高的稳定性,并且在很大的温度和pH值范围内也表现得比DNA更稳定。PNA与DNA和RNA形成强键,并且比DNA具有更高的特异性和选择性(Nielsen,P.E Acc.Chem.Res.,32:624,1999),因此其可用于多种用途,包括抗原-抗体反应、杂交技术以及药物输送等(Dean,D.A.,Adv.Drug Delivery Rev.,44:81,2000)。
迄今为止,所开发的DNA芯片都是通过很麻烦的过程而进行制备的,在该过程中,将探针的碱基序列固定在芯片上,根据碱基突变或者待诊断的SNPs的类型而与靶DNA相匹配。换言之,构成DNA芯片的探针需要根据待诊断的靶DNA的类型进行重新构建,而这非常麻烦,并且增加了探针的制作成本。
因此,本发明人为解决上述问题进行了大量研究,最终发现可使用PNA芯片,该PNA芯片是利用PNA通过zip编码技术进行制备的,所述PNA具有优于DNA的特性,利用所述PNA芯片能够高灵敏性地对多碱基以及碱基突变进行精确分析,因而完善了本发明。
发明概述
本发明涉及一种制备PNA zip编码芯片的方法,该方法利用环氧化合物作为连接剂,将PNA探针固定在胺化基底(以胺类修饰的基底)上。
本发明还涉及PNAzip编码芯片,在该芯片中利用环氧化合物作为连接剂将zip编码的探针固定在基底上。
本发明还涉及检测DNA、分析多碱基突变以及单核苷酸多态性(SNPs)的方法,其包括使用PNA芯片。
下述公开内容以及所附权利要求能够更加全面地对本发明的其它方面、特征、以及优点进行描述。
附图简要说明
图1为PNA(肽核酸)和DNA的结构式;
图2为采用本发明方法利用环氧化合物将DNA以及PNA等固定在胺化玻璃基底上的示意图;
图3为PNA zip编码芯片在6×SSPET杂交溶液中与荧光物质(Cy3)标记的靶DNA杂交的荧光图像;
图4为SSPET杂交溶液浓度发生变化时的杂交水平;
图5为根据杂交溶液浓度的变化,本发明的PNA zip编码芯片和DNA zip编码芯片对于单核苷酸多态性的检测能力。图5(A)为通过改变杂交溶液浓度,PNA zip编码芯片对于单核苷酸多态性的检测能力,图5(B)为通过改变杂交溶液浓度,DNA zip编码芯片对于单核苷酸多态性的检测能力;
图6为利用TAMRA-ddCTP对PNA zip编码芯片的多碱基突变进行分析;
在图7中,按照本发明实施方案制备PNA zip编码芯片,对其突变位点的几个碱基同时进行分析,获得荧光图像示于图7。图7(A)为PNAzip编码芯片的排列图片(数字代表基因的位置,AA,GG,CC和TT代表基因的正常表型),图7(B)为同时检测4个PNA芯片中的10个碱基突变的荧光图像结果;
图8为利用本发明实施例6的PNAzip编码来分析碱基突变的示意图。
具体实施方式
本发明一方面提供了一种利用zip编码制备PNA芯片的方法,所述方法包括下述步骤:(a)将二甲基亚砜(DMSO)、PNA zip编码探针和环氧化合物的混合物在胺化基底上进行点样从而使PNA探针固定在基底上;以及(b)利用SDS溶液洗涤固定了PNA zip编码探针的基底,然后干燥。
在本发明方法中,步骤(a)中二甲基亚砜(DMSO)、PNA探针和环氧化合物的体积比优选为1∶1∶1,并且所述环氧化合物优选以下式1所表示:
式1
在本发明方法中,PNA zip编码探针的浓度优选为10μM至1mM。还优选所述PNA zip编码探针为多个,并且所述多个PNA zip编码探针所包括的碱基序列彼此之间的同源性小于30%。所述PNAzip编码探针优选包括SEQ IDNO:1至SEQ ID NO:10中的任一序列。此外,所述PNA zip编码探针还包括SEQ ID NO:1至SEQ ID NO:10和SEQ ID NO:16至SEQ ID NO:45中的任一序列。
本发明另一方面提供了一种按照所述方法制备的PNA zip编码的芯片,其中PNA zip编码探针用环氧化合物作为连接剂进行固定。
本发明又一方面提供了检测靶DNA的方法,所述方法包括使靶DNA与所述PNAzip编码芯片杂交的步骤。在该方法中,所述检测利用荧光信号进行。
本发明再一方面提供了一种分析多碱基突变的方法,所述方法包括下述步骤:(a)选择靶基因的碱基突变位点;(b)设计SBE引物;(c)利用PCR扩增步骤(a)的靶基因;(d)采用步骤(b)的SBE引物以及荧光物质标记的ddATP,ddTTP,ddCTP或ddGTP对扩增的靶基因进行SBE反应;(e)使SBE反应产物与PNA zip编码芯片相杂交;以及(f)检测荧光物质的信号。
在本发明的该方法中,所述SBE引物优选包括5’-末端与固定于PNA zip编码芯片上的PNA zip编码相互补的序列,以及3’-末端与靶基因相互补的序列。另外,所述SBE引物为多个。更进一步,所述靶基因优选为糖尿病相关基因HNF1-a。
本发明又一方面提供了一种分析多碱基突变的方法,所述方法包括下述步骤:(a)选择靶基因的碱基突变位点;(b)设计SBE引物;(c)利用PCR扩增步骤(a)的靶基因;(d)在分别包含ddATP、ddTTP、ddCTP和ddGTP的四个管中采用步骤(b)的SBE引物以及特定化合物标记的ddNTP对扩增的靶基因进行SBE反应;(e)使SBE反应产物与PNA zip编码芯片相杂交;以及(f)检测标记化合物的信号。
在该方法中,所述SBE引物优选包括5’-末端与固定于PNA zip编码芯片的PNA zip编码相互补的序列,以及3’-末端与靶基因相互补的序列。另外,所述SBE引物优选为多个。
在本发明的该方法中,所述特定化合物优选为生物素。对信号的检测步骤通过采用偶联了链霉抗生物素蛋白的藻红蛋白来染色,其中所述链霉抗生物素蛋白为与生物素具有强亲和力的蛋白。
本发明另一方面提供了分析SNP的方法,所述方法使用PNA芯片。
下面将对本发明进行详细描述。
zip编码芯片中,将包括给定长度碱基序列的探针(zip编码)固定于固态基底上,其中所述碱基序列之间没有同源性。特异地,所述zip编码芯片可与一寡聚核苷酸相杂交从而检测信号,其中该寡聚核苷酸在5’端具有与探针的碱基序列同源的序列,在3’端具有与靶DNA的碱基序列相互补的序列。
也就是说,在本发明中,选择彼此之间同源性最低的碱基序列作为探针,将具有这种碱基序列的探针称为zip编码。
在本发明中采用PNA作为zip编码芯片的探针。
在本发明中,可以不受限制地使用任意的靶物质进行杂交,只要其可通过反应进行检测或者可与PNA探针相连接即可。该类靶物质的优选实施例包括来源于活体的分子,例如DNA、RNA等。
在本发明中,将PNA zip编码探针固定在胺化的固态基底上,所述固定采用环氧化合物作为连接剂通过化学连接将PNA与胺化固态基底相连接。
本发明中所用的环氧化合物具有下面式1所示结构。如图2所示,环氧化合物的环氧基团与胺化玻璃基底化学连接,PNA探针的氨基与连接至玻璃基底的环氧化合物的其它环氧基团化学连接。籍此将PNA探针固定于玻璃基底上,并且PNA zip编码探针和玻璃基底之间还可生成一些由于物理吸附而造成的连接。
实施例
下面将通过实施例对本发明进行详细描述。但是,很显然,对于本领域技术人员来说,可对本发明进行多种形式的修饰,并且本发明并不限于所述实施例。所述实施例仅是对本发明进行进一步的阐述。
实施例1:PNA zip编码芯片的制备
在本实施例中,采用胺化的玻璃基底作为PNA芯片的基底。在基底上固定10个PNA zip编码探针。这些探针分别包括SEQ ID NO:1至SEQ ID NO:10的碱基序列,这些碱基序列之间的同源性很低。每一碱基序列具有12个碱基。由于PNA探针的N末端具有氨基(-NH2),因此利用氨基和环氧基团之间的连接来将探针固定在基底上。
N-TGCGGGTAATCG-C(SEQ ID NO:1)
N-TGCGACCTATCG-C(SEQ ID NO:2)
N-ATCGTGCGACCT-C(SEQ ID NO:3)
N-ATCGGGTATGCG-C(SEQ ID NO:4)
N-CAGCATCGTGCG-C(SEQ ID NO:5)
N-CAGCACCTTGCG-C(SEQ ID NO:6)
N-GGTAATCGACCT-C(SEQ ID NO:7)
N-GACCATCGACCT-C(SEQ ID NO:8)
N-GACCCAGCATCG-C(SEQ ID NO:9)
N-ACCTGACCATCG-C(SEQ ID NO:10)
采用下述方法将PNA zip编码探针固定在玻璃基底上,从而制备PNA zip编码探针。
将二甲基亚砜(DMSO)、PNA zip编码探针和环氧化合物以1∶1∶1的体积比进行混合,得到PNA的终浓度为100μM的混合物。利用点样仪将混合物点样至玻璃基底上。然后,用0.2%SDS溶液洗涤点样后的基底,室温干燥,从而制备了PNA zip编码芯片。
实施例2:DNA与PNA zip编码芯片的杂交
为了检验PNA zip编码芯片与DNA的杂交能力,采用实施例1方法得到的PNA zip编码芯片来进行杂交实验,其中固定了四个具有SEQ ID NO:3,SEQID NO:6,SEQ ID NO:7和SEQ ID NO:9碱基序列的PNA zip编码探针。
首先,制备由荧光物质(Cy3)标记的具有25个碱基的靶DNA(SEQ ID NO:11)。将含有1nM靶DNA的6X SSPET(生理盐水磷酸钠EDTA)杂交溶液30μl置于PNA zip编码芯片上,将其置于杂交室中,在37℃使靶DNA和PNA芯片杂交12小时。在固定于芯片的4个PNA探针中,所述靶DNA具有与SEQID NO:7探针互补的碱基序列。
5’-AAGAAGAAGGTCGATTACCAAAGGA-3’(SEQ ID NO:11)(靶DNA)
因此,当与PNA zip编码芯片杂交时,在4个探针中,仅有一个探针能够检测到荧光信号。如图3所示,仅在与靶DNA有互补序列的第三个PNA探针(SEQ ID NO:7)检测到荧光信号,显示仅在第三个探针发生了杂交。
实施例3:按照杂交溶液浓度的改变所得到的PNA zip编码芯片敏感性的变化
为了寻找本发明PNA zip编码芯片可表现最有效选择性的条件,在不同杂交溶液浓度下进行杂交。利用0.1X,0.3X,0.5X,1X,3X和6X SSPET杂交溶液对实施例2所用的PNA和靶DNA(SEQ ID NO:11)进行杂交。结果发现,在0.1X SSPET杂交溶液观察到最强的荧光信号,而在最高浓度的6X SSPET杂交溶液中观察到最弱的荧光信号(图4)。由此发现,杂交溶液的浓度越低,PNAzip编码芯片的杂交效率越高。
实施例4:PNA zip编码芯片和DNA zip编码芯片对单核苷酸多态性检测能力的实验
为了证实PNA zip编码芯片对单核苷酸多态性的检测能力要远远优于DNA zip编码芯片,分别将具有同样序列的PNA和DNA探针固定在胺化玻璃基底上,对检测结果进行比较。
用于检测单核苷酸多态性能力的探针的序列如下所示,对于DNA探针,为了将探针与环氧化合物相连接以进行固定,将氨基连于5’末端。
DNA(PM)-5’-NH2-GGTAATCGACCT-3’(具有SEQ ID NO:7碱基序列的DNA探针)
DNA(MM)-5’-NH2-GGTAATTGACCT-3’(具有SEQ ID NO:7单突变序列的DNA探针)
PNA(PM)-N-GGTAATCGACCT-C(具有SEQ ID NO:7碱基序列的PNA探针)
PNA(MM)-N-GGTAATTGACCT-C(具有SEQ ID NO:7单突变碱基序列的PNA探针)
为了检验对单核苷酸多态性的检测能力,利用多个浓度的杂交溶液(0.1XSSPET,0.3X SSPET,1X SSPET,3X SSPET,和6X SSPET)来进行杂交。
如图5所示,可以发现随着杂交溶液浓度的增加,PNA zip编码芯片在全部荧光信号值的范围内都有降低,但是检测单核苷酸多态性的能力却增加。相反,对DNA zip编码芯片来说,随着杂交溶液浓度的增加,其对单核苷酸多态性的检测能力以及荧光信号值都有所增加,但是其荧光信号的绝对值要远远低于PNA zip编码芯片的荧光信号绝对值(参照图5)。
实施例5:利用PNA zip编码芯片对多碱基突变的分析(1)
在本实施例中,采用与MODY-3(为一种类型的糖尿病)相关的HNF1-a基因(SEQ ID NO:12)作为靶基因,如下面表1所示,选择在基因外显子2上包括3个碱基突变位点。
Zip编码 | 位置 | 野生型 | 突变 | 突变结果 |
PNA 6P | 1845 | TCC | TTC | S142F |
PNA 18P | 1847 | CCA | CTA | H143Y |
PNA 31P | 1791 | TCCT | TCT | S121fsdelC |
突变位点为黑体。
设计与PNA zip编码探针相结合的SBE引物,所述引物的5’端具有与固定在PNA zip编码芯片上的每一PNA探针相互补的序列、3’端具有与包含碱基突变的靶基因互补的序列。将本实施例中所用的SBE引物设计为具有不同的长度,从而使得在约60℃的均一温度下其能保持Tm值。将所有引物构建成在靶基因的碱基突变位点前一个碱基。
为了分析PNA zip编码芯片上的多碱基突变,利用PCR扩增与糖尿病相关的HNF1-a基因的外显子2。利用荧光物质(TAMRA)标记的ddCTP对扩增的DNA进行SBE反应。同时在同一试管中用SBE01-6P(SEQ ID NO:13),SBE01-18P(SEQ ID NO:14)和SBE01-31P(SEQ ID NO:15)三种引物来进行SBE反应。SBE反应完成后,使反应产物与上面固定了40个zip编码的PNA zip编码芯片进行杂交(表2)。
SBE01-6P:5’-TACCAGGTCGCAGATACCACTGGCCTCAACCAG-3’(SEQ ID NO:13)
SBE01-18P:5’-CGCAAGGTGCTGCACTGGCCTCAACCAGTCC-3’(SEQ ID NO:14)
SBE01-31P:5’-TGCTGGGTCAGATGGTCAAGT-3’(SEQ ID NO:15)
表2:实施例5所用的40个zip编码
PNA | N末端→C末端 | PNA | N末端→C末端 |
1 | TGCGGGTAATCG(SEQ ID NO:1) | 21 | GGTAATCGTGCG(SEQ ID NO:30) |
2 | TGCGGGTACAGC(SEQ ID NO:16) | 22 | GGTAATCGACCT(SEQ ID NO:7) |
3 | TGCGGACCATCG(SEQ ID NO:17) | 23 | GGTACAGCATCG(SEQ ID NO:31) |
4 | TGCGGACCACCT(SEQ ID NO:18) | 24 | GGTACAGCGACC(SEQ ID NO:32) |
5 | TGCGACCTATCG(SEQ ID NO:2) | 25 | GGTAGACCTGCG(SEQ ID NO:33) |
6 | TGCGACCTGGTA(SEQ ID NO:19) | 26 | GGTAGACCACCT(SEQ ID NO:34) |
7 | ATCGTGCGACCT(SEQ ID NO:3) | 27 | GGTAACCTTGCG(SEQ ID NO:35) |
8 | ATCGGGTATGCG(SEQ ID NO:4) | 28 | GGTAACCTCAGC(SEQ ID NO:36) |
9 | ATCGGGTAGACC(SEQ ID NO:20) | 29 | GACTATCGTGCG(SEQ ID NO:37) |
10 | ATCGGACCTGCG(SEQ ID NO:21) | 30 | GACCATCGACCT(SEQ ID NO:8) |
11 | ATCGACCTTGCG(SEQ ID NO:22) | 31 | GACCCAGCATCG(SEQ ID NO:9) |
12 | ATCGACCTCAGC(SEQ ID NO:23) | 32 | GACCCAGCACCT(SEQ ID NO:38) |
13 | CAGCATCGTGCG(SEQ ID NO:5) | 33 | GACCACCTTGCG(SEQ ID NO:39) |
14 | CAGCATCGACCT(SEQ ID NO:24) | 34 | GACCACCTATCG(SEQ ID NO:40) |
15 | CAGCGGTAATCG(SEQ ID NO:25) | 35 | ACCTATCGTGCG(SEQ ID NO:41) |
16 | CAGCGGTAGACC(SEQ ID NO:26) | 36 | ACCTATCGCAGC(SEQ ID NO:42) |
17 | CAGCGACCTGCG(SEQ ID NO:27) | 37 | ACCTCAGCGACC(SEQ ID NO:43) |
18 | CAGCACCTTGCG(SEQ ID NO:6) | 38 | ACCTGGTAATCG(SEQ ID NO:44) |
19 | CAGCACCTGACC(SEQ ID NO:28) | 39 | ACCTGACCTGCG(SEQ ID NO:45) |
20 | GGTATGCGGACC(SEQ ID NO:29) | 40 | ACCTGACCATCG(SEQ ID NO:10) |
结果发现,在PNA zip编码探针6(SEQ ID NO:19)、PNA zip编码探针18(SEQ ID NO:6)、以及PNA zip编码探针31(SEQ ID NO:9)中检测到了荧光信号(参照图6)。由此可以发现,使用本发明的PNA zip编码探针能够检测多碱基突变。
实施例6:利用PNA zip编码芯片对多碱基突变的分析(2)
在本实施例中,利用实施例5的靶基因采用生物素标记而不是TAMRA标记的ddNTP来进行SBE反应,使反应产物与实施例1制备的芯片进行杂交,所述芯片上固定了10个PNA zip编码探针。为观察荧光信号,将杂交的芯片以偶联了链霉抗生物素蛋白的藻红蛋白进行染色,其中所述链霉抗生物素蛋白是一种与生物素具有强亲和力的蛋白质。分别使用包括生物素-ddATP、生物素-ddTTP、生物素-ddCTP和生物素-ddGTP的四种反应溶液中的每一种进行SBE反应(表3)。
表3:多SBE反应中所用反应溶液的组分
对于四种反应溶液,都加入包括10个碱基突变位点的10个SBE引物(SEQID NO:46至SEQ ID NO:55)。碱基突变分析中所用的SBE引物序列如下所示,其中与PNA zip编码序列互补的序列被加了下划线。
(SEQ ID NO:46)5’-CGATTACCCGCAGCAGCACAACATCCCACAGC-3’(SBE02-P1)
(SEQ ID NO:47)5’-CGATAGGTCGCATACCTGCAGCAGCACAACATC-3’(SBE02-P2)
(SEQ ID NO:48)5’-AGGTCGCACGATCCGTGGCGTGTGGCG-3’(SBE02-P3)
(SEQ ID NO:49)5′CGCATACCCGATAGCAGCACAACATCCCACAG-3’(SBE02-P4)
(SEQ ID NO:50)5′-CGCACGATGCTGACAGCGGGAGGTGGTCG-3’(SBE02-P5)
(SEQ ID NO:51)5′-CGCAAGGTGCTGCACTGGCCTCAACCAGTCC-3’(SBE02-P6)
(SEQ ID NO:52)5’-AGGTCGATTACCGACGCAGAAGCGGGCC-3’(SBE02-P7)
(SEQ ID NO:53)5’-AGGTCGATGGTCAACCAGTCCCACCTGTCCCAAC-3’(SBE02-P8)
(SEQ ID NO:54)5’-CGATGCTGGGTCTCCCATGAAGACGCAGAAGC-3’(SBE02-P9)
(SEQ ID NO:46)5’-CGATGGTCAGGTCCTACCTGCAGCAGCACAACA-3’(SBE02-P10)
完成SBE反应后,将每一反应产物与PNA zip编码芯片杂交。
图7所示为用荧光扫描仪观察PNA zip编码芯片的结果,所述芯片已经与每一组合下的SBE反应产物相杂交并用偶联了链霉抗生物素蛋白的藻红蛋白进行染色。如图7(B)所示,预计为AA表现型的芯片(引入生物素-ddATP)仅在AA表现型显示出荧光信号。并且,其余三种情况(GG,CC和TT)也与预期结果相吻合。这一结果显示,在同一试管内,采用一个SBE反应就可精确判断出10个碱基突变位点。采用本方法能够同时大量分析与几种疾病相关的碱基突变位点或者SNPs。图8为按照实施例6的方法分析碱基突变的示意图。
如上所述,本发明提供了一种利用zip编码制备PNA zip编码芯片的方法,其包括采用环氧化合物作为连接剂将PNA探针与胺化基底相连接。本发明还提供了按照该方法制备的PNA zip编码芯片,其中所述基底和PNA zip编码探针采用环氧化合物作为连接剂彼此相连接。此外,本发明还提供了检测DNA的方法以及分析多碱基突变和SNPs的方法,其包括使用所述PNA芯片。
根据本发明,与使用DNA相比,采用PNA能更精确地诊断先天性疾病或者碱基突变,并且采用PNA芯片比采用DNA芯片具有更高的灵敏性。并且,采用PNA zip编码芯片可仅通过杂交反应即能简单地判断基因突变,而无需在每次靶基因改变时就要将探针固定于基底上的复杂步骤。
尽管已经对本发明进行了详细描述,但是对本领域技术人员来说,说明书中的内容仅是本发明的优选技术方案,而并非对本发明进行限制。因此,本发明的实质保护范围将由所附权利要求书及其等同物来确定。本领域技术人员可以在不偏离本发明精神的情况下根据权利要求书的内容对本发明进行简单修饰、变换以及增加。
SEQUENCE LISTING
<110> 韩国科学技术院
<120> 利用ZIP编码的PNA芯片及其制备方法
<130> PIF050792C
<140> KR10-2004-0099514
<141> 2004-11-30
<150> KR10-2004-0099514
<151> 2004-11-30
<160> 55
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Claims (19)
1.一种利用zip编码制备PNA芯片的方法,所述方法包括下列步骤:
(a)将二甲基亚砜、包括SEQ ID NO:7的PNA zip编码探针和环氧化合物的混合物点样至胺化基底上,从而将所述PNA zip编码探针固定在基底上;以及
(b)用SDS溶液洗涤固定了PNA zip编码探针的基底,然后进行干燥。
2.根据权利要求1的方法,其中步骤(a)中二甲基亚砜、PNA探针和环氧化合物的体积比为1∶1∶1。
4.根据权利要求2的方法,其中所述PNA zip编码探针的浓度为10μM至1mM。
5.根据权利要求1的方法,其中进一步将包括SEQ ID NO:1至SEQ IDNO:6、SEQ ID NO:8至SEQ ID NO:10以及SEQ ID NO:16至SEQ ID NO:45的PNA zip编码探针固定在基底上。
6.根据权利要求1的方法,其中进一步将包括SEQ ID NO:1至SEQ IDNO:6和SEQ ID NO:8至SEQ ID NO:10的PNA zip编码探针固定在基底上。
7.根据权利要求1的方法,其中进一步将包括SEQ ID NO:3、SEQ ID NO:6和SEQ ID NO:9的PNAzip编码探针固定在基底上。
8.按照权利要求1-7中任一方法制备的PNA zip编码芯片,其中采用环氧化合物作为连接剂来固定PNA zip编码探针。
9.一种检测靶DNA的方法,所述方法包括使靶DNA与权利要求8的PNAzip编码芯片相杂交的步骤,其中所述方法不用于疾病诊断。
10.根据权利要求9的方法,其中所述检测利用荧光信号进行。
11.一种分析多碱基突变的方法,所述方法包括下述步骤:
(a)选择靶基因的碱基突变位点;
(b)设计SBE引物;
(c)利用PCR扩增步骤(a)的靶基因;
(d)采用步骤(b)的SBE引物以及荧光物质标记的ddATP、ddTTP、ddCTP或ddGTP对扩增的靶基因进行SBE反应;
(e)使SBE反应产物与权利要求8的PNA zip编码芯片相杂交;以及
(f)检测荧光物质的信号;
其中,所述方法不用于疾病诊断。
12.根据权利要求11的方法,其中所述SBE引物包括5’-末端与固定在权利要求8的PNA zip编码芯片上的PNA zip编码相互补的序列,以及3’-末端与靶基因相互补的序列。
13.根据权利要求11的方法,其中所述SBE引物为多个。
14.根据权利要11的方法,其中所述靶基因为糖尿病相关基因HNF 1-a。
15.一种分析多碱基突变的方法,所述方法包括下述步骤:
(a)选择靶基因的碱基突变位点;
(b)设计SBE引物;
(c)用PCR扩增步骤(a)的靶基因;
(d)在四个管中采用步骤(b)的SBE引物以及分别为ddATP、ddTTP、ddCTP和ddGTP的生物素标记的ddNTP对扩增的靶基因进行SBE反应;
(e)使SBE反应产物与权利要求8的PNA zip编码芯片相杂交;以及
(f)检测标记化合物的信号;
其中所述方法不用于疾病诊断。
16.根据权利要求15的方法,其中所述SBE引物包括5’-末端与固定在权利要求8的PNA zip编码芯片上的PNA zip编码相互补的序列,以及3’-末端与靶基因相互补的序列。
17.根据权利要求15的方法,其中所述SBE引物为多个。
18.根据权利要求15的方法,其中对信号的检测步骤通过用偶联了链霉抗生物素蛋白的藻红蛋白来染色,其中所述链霉抗生物素蛋白是与生物素具有强亲和力的蛋白。
19.一种分析SNP的方法,其中所述方法使用权利要求8的PNA zip编码芯片,并且所述方法不用于疾病诊断。
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US7659064B2 (en) | 2010-02-09 |
KR20060060443A (ko) | 2006-06-05 |
US20060115825A1 (en) | 2006-06-01 |
KR100653975B1 (ko) | 2006-12-05 |
JP4189929B2 (ja) | 2008-12-03 |
JP2006153839A (ja) | 2006-06-15 |
CN1782096A (zh) | 2006-06-07 |
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