CN107573331B - 能够提高寡核苷酸链Tm值的标记化合物及其应用 - Google Patents
能够提高寡核苷酸链Tm值的标记化合物及其应用 Download PDFInfo
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Abstract
本发明涉及能够提高寡核苷酸链Tm值的标记化合物(I)及其应用。本发明的有益效果主要体现在:本发明提供了一种能够提高寡核苷酸链Tm值的标记化合物(HT琥珀酸酯)及其应用,采用HT标记的探针能够有效的甄别DPYD*9A位点野生型和突变型模板,可用于检测消化系统肿瘤,具有重要应用前景。
Description
(一)技术领域
本发明涉及一种能够提高寡核苷酸链Tm值的标记化合物及其应用。
(二)背景技术
实时荧光定量PCR技术通过检测PCR产物中荧光讯号强度来达到定量的目的,该技术不仅实现了PCR从定性到定量的飞跃,而且与常规PCR相比,它具有特异性更强、有效解决PCR污染问题、自动化程度高等特点,目前已在动植物基因工程,微生物和医学领域中得到广泛应用。
目前有下面几种常用的荧光定量方法:TaqMan探针、Beacon探针、FRET技术等。MGB探针是在TaqMan探针基础上研发的一种探针修饰方法,该探针是指带有一个小沟结合物基团的DNA探针,它可与单链的靶DNA形成极为稳定的异源双链,同时这种探针相对于通常的探针序列更短一些。
研究表明,一般情况下,同MGB-寡核苷酸形成的错配异源双链其熔点温度比没有MGB的寡核苷酸形成的双链熔点温度高,而且由于ΔTm(完全匹配的Tm值与错配的Tm值之差)由错配的位置和类型决定,在小沟结合区内形成的错配特别不稳定。比起通常的寡核苷酸探针,MGB寡核苷酸探针更容易辨认在MGB结合区形成的错配。同时分子分析表明,与寡核苷酸探针3′连接的MGB可向探针的5′-末端滑行1-2bp,以便有更好的小沟结合位。MGB结构连接到12-18mer的寡核苷酸时,能显著增加异源双链的稳定性。据报道,Tm值在66-70℃之间,而没有MGB的相同序列Tm值在44~56℃。探针越短,整体异源双链的稳定性越大。有研究表明,一个没有MGB的27mer寡核苷酸探针形成的异源双链的稳定性(Tm=65℃)与有MGB的12mer寡核苷酸探针(Tm=66℃)相同,从这12mer的寡核苷酸探针的序列中可以看出,富含A/T的3′-末端附近的MGB配体对稳定性的作用等于在序列的5′-末端另加15个mer。
目前的MGB探针是MGB结构连接在3’端的淬灭基团上,而且结构单一的使用了DPI3,因次,目前的MGB探针不适合做多重荧光定量检测,另外针对不同基因设计的灵活性不够高,造成很多地方应用的局限性。
但是MGB探针虽然可以提高探针的Tm值,但是大部分情况下会抑制PCR的扩增,因此,需要寻找一种新的方法,对PCR扩增不产生影响且可以提高探针的Tm值。
(三)发明内容
本发明目的即是提供一种提高寡核苷酸链Tm值的标记化合物,及其在提高寡核苷酸链Tm值中的应用。
本发明采用的技术方案是:
一种能够提高寡核苷酸链Tm值的标记化合物(Hoechst 33258-琥珀酸酯,以下简称HT琥珀酸酯),所述化合物结构如式(I)所示:
该标记化合物能直接嵌入DNA双螺旋结构或者能够与DNA双螺旋结构中的小沟结合从而形成稳定DNA的双螺旋,从而提高被修饰的寡核苷酸链的Tm值。
本发明还涉及所述标记化合物在提高寡核苷酸链Tm值中的应用。
具体的,所述应用方法如下:
(1)将所述标记化合物与带有氨基的寡核苷酸按照摩尔比1~30:1溶于pH 7~10的碳酸氢钠水溶液中,在20~60℃下进行反应1~48小时,得到带有标记化合物的寡核苷酸粗产物;
(2)将步骤(1)得到的粗产物采用沉淀、PAGE或者高效液相方法进行纯化,得到纯化后的带有标记化合物的寡核苷酸,即Tm值提高后的寡核苷酸链。
进一步,所述Tm值提高后的寡核苷酸链可用于检测消化系统肿瘤,采用该Tm值提高后的寡核苷酸链,能有效的甄别野生型和突变型模板。
本发明的有益效果主要体现在:本发明提供了一种能够提高寡核苷酸链Tm值的标记化合物(HT琥珀酸酯)及其应用,采用HT琥珀酸酯标记的探针能够有效的甄别DPYD*9A位点野生型和突变型模板,可用于检测消化系统肿瘤,具有重要应用前景。
(四)附图说明
图1为采用HT琥珀酸酯标记的荧光定量PCR探针检测DPYD*9A位点(T85C)的检测图;其中,A:野生型模板检测结果;B:突变型模板检测结果;
图2为采用MGB标记的荧光定量PCR探针检测DPYD*9A位点(T85C)的检测图;其中,A:野生型模板检测结果;B:突变型模板检测结果;
图3为采用TaqMan标记的荧光定量PCR探针检测DPYD*9A位点(T85C)的检测图;其中,A:野生型模板检测结果;B:突变型模板检测结果;
图4为采用LNA标记的荧光定量PCR探针检测DPYD*9A位点(T85C)的检测图;其中,A:野生型模板检测结果;B:突变型模板检测结果。
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并不仅限于此:
实施例1:合成标记化合物
一种提高寡核苷酸链Tm值的标记化合物,该标记化合物为一种琥珀酸酯,简称HT琥珀酸酯,HT琥珀酸酯的制备步骤为:
将1.8g Hoechst 33258与1.48g碳酸钾溶解在20ml DMF中,加入0.8ml 4-溴丁酸乙酯,将反应混合物在60℃加热12小时,冷却,过滤,旋蒸,剩余物质溶解在甲醇中,用硅胶柱纯化,甲醇和二氯甲烷最为洗脱剂,得到的产品用乙醇重结晶得到hoechst 33258-O-丁酸乙酯1.4g。1H NMR(DMSO-d6)δ1.19(3H),2.03(2H,m),2.45(2H,m),2.90(3H),3.08 and3.16(4H,m),3.90(4H,m),4.08-4.12(4H,m),7.21(3H,m),7.34(1H,m),7.73(1H,d),7.94(1H,d),8.14(1H,m),8.24(2H,m),8.57(1H,s).
将1.3g hoechst 33258-O-丁酸乙酯放入5ml 1M NaOH和5ml乙醇中,加热到60℃1h,反应混合物冷却然后用10%的盐酸酸化到pH为4,产物析出,过滤,滤饼用水,丙酮和乙醚洗涤,干燥的到hoechst 33258-O-丁酸。1H NMR(DMSOd6)δ1.99(2H,m),2.42(2H,t),2.45(3H,s),2.84(4H,m),3.44(4H,s),4.10(2H,t),6.99(1H,d),7.11(3H,m),7.50(1H,d),7.66(1H,m),8.06(1H,d),8.24(2H,d),8.40(1H,s).
琥珀酸酯的合成:
将0.43g hoechst 33258-O-丁酸溶解在5ml DMF中,加入0.68ml三乙胺,然后加入0.31g TSTU,室温反应1h后倒入石油醚中,过滤,得到产物hoechst 33258-琥珀酸酯0.3g。
1H NMR(DMSOd6)δ1.99(2H,m),2.42(2H,t),2.45(3H,s),2.64(4H,m),2.84(4H,m),3.44(4H,s),4.10(2H,t),6.99(1H,d),7.11(3H,m),7.50(1H,d),7.66(1H,m),8.06(1H,d),8.24(2H,d),8.40(1H,s).
实施例2:带有标记化合物的寡核苷酸的合成与纯化
提高寡核苷酸链Tm值的标记化合物的标记方法,主要包括以下步骤:
(1)将Hoechst 33258-琥珀酸酯(化合物结构式I)5mg溶解在3mlDMF(二甲基甲酰胺)与3ml 0.1M NaHCO3中,加入3.2mg amino修饰的寡核苷酸(C*TGCTCCCCGCGT*G和C*TGCTCCCCCCGT*G,其中*为氨基修饰)25℃反应2小时,可得到带有标记化合物的寡核苷酸粗产物;
(2)将步骤(1)得到带有标记化合物的寡核苷酸粗产物采用HPLC(高效液相色谱法)(C18柱,TEAAPH=7.0体系分离)方法对寡核苷酸进行纯化,即可得到纯的带有标记化合物的寡核苷酸(野生型:C#TGCTCCCCGCGT#G,tm:67;突变型:C#TGCTCCCCCCGT#G,tm:67,其中,带#为HT琥珀酸酯修饰的核苷酸)。
实施例3:HT琥珀酸酯在荧光定量PCR检测的探针特异性评价(在荧光定量PCR探针检TP53位点中的应用)
1、材料:
质粒基因组DNA提取试剂、PCR反应体系和Taq DNA聚合酶,pGEM-T-Easy克隆系统购自Promage公司、CFX96定量PCR仪为美国Biorad公司产品。
2、质粒标准品检测:
野生型和突变型TP53位点质粒为人工合成,由山东维真生物科技有限公司提供,采用质粒基因组DNA提取试剂提取基因组DNA,分别取1.0μL做模板,用检测用上下游引物在Biorad公司CFX96型定量PCR仪上进行PCR扩增。
PCR反应液组成如下:
1×PCR buffer 2μL
野生型探针(10μM) 0.2μL
突变型探针(10μM) 0.2μL
上游引物:CAATGGTTCACTGAAGACCCA (10μM)1μL
下游引物:CGGTGTAGGAGCTGCTGGT (10μM)1μL
DNA聚合酶(5U/μL) 0.2μL
dNTPs(各250mM) 1.60μL
(dATP、dTTP、dCTP、dGTP物质的量比1:1:1:1)
模板DNA(50ng/μL) 1μL
水补足至20μL。
PCR反应条件为:95℃预变性5分钟,95℃15秒、60℃45秒进行40个循环扩增。
3、四种修饰探针的特异性比较:
上游引物:CAATGGTTCACTGAAGACCCA tm:58
下游引物:CGGTGTAGGAGCTGCTGGT tm:58
TaqMan标记:
TaqMan-C(野生型):CAGAGGCTGCTCCCCGCGT,tm:66
TaqMan-T(突变型):CAGAGGCTGCTCCCCCCGT,tm:66
以ABI primer express3.0软件设计。
MGB标记:
TaqMan-MGB-C(野生型):CTGCTCCCCGCGTG,tm:66
TaqMan-MGB-T(突变型):CTGCTCCCCCCGTG,tm:66
以ABI primer express3.0软件设计。
LNA标记:
LNA-TaqMan-C(野生型):CTGC+TCCCCG+CGTG,tm:70
LNA-TaqMan-T(突变型):CTGC+TCCCCC+CGTG,tm:70
其中+号代表后面一个碱基为LNA修饰碱基,tm值符合EXIQON公司设计原则。
HT琥珀酸酯标记:
HT-C(野生型):C#TGCTCCCCGCGT#G,tm:67
HT-T(突变型):C#TGCTCCCCCCGT#G,tm:67
其中,带#为HT琥珀酸酯修饰的核苷酸,其他组探针的MGB修饰、TaqMan修饰和LNA修饰的核苷酸按本领域常规。
将野生型和突变型检测探针分别采用HT琥珀酸酯标记、MGB标记、TaqMan标记和LNA标记,采用上述反应体系分别对野生型和突变型TP53位点质粒进行检测,图1为HT琥珀酸酯标记探针分别检测野生型(A)和突变型(B)模板的结果;图2为MGB标记探针分别检测野生型(A)和突变型(B)模板的结果;图3为TaqMan标记探针分别检测野生型(A)和突变型(B)模板的结果;图4为LNA标记探针分别检测野生型(A)和突变型(B)模板的结果。
结果显示HT琥珀酸酯标记的探针和MGB标记的探针均能有效的甄别野生型和突变型模板(见图1和图2),而TaqMan标记和LNA标记的探针无法准确的检测与探针序列完全匹配的质粒标准品模板(见图3和图4)。
Claims (3)
1.一种能够提高寡核苷酸链Tm值的标记化合物,所述化合物结构如式(I)所示:
2.如权利要求1所述标记化合物在提高寡核苷酸链Tm值中的应用。
3.如权利要求2所述的应用,其特征在于所述应用方法如下:
(1)将所述标记化合物与带有氨基的寡核苷酸按照摩尔比1~30:1溶于pH7~10的碳酸氢钠水溶液中,在20~60℃下进行反应1~48小时,得到带有标记化合物的寡核苷酸粗产物;
(2)将步骤(1)得到的粗产物采用沉淀、PAGE或者高效液相方法进行纯化,得到纯化后的带有标记化合物的寡核苷酸,即Tm值提高后的寡核苷酸链。
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