利用无标记的分子信标及核酸染料Hoechst 33258 检测特定序列核酸

向东山等
摘要利用无标记的分子信标及核酸染料Hoechst 33258建立了一种高灵敏、高选择性的特定序列核酸检测方法,并以野生型乙型肝炎病毒的一段寡核苷酸序列为目标DNA,对这种方法进行了验证。
1引言
特定序列单链核酸(DNA)的特异性检测在临床诊断、基因治疗、环境调查、食品安全及生物医学研究等领域都具有十分重要的意义\[1~5\]。分子信标(Molecular beacons)是一种呈发夹结构的茎环双标记的寡核苷酸探针,具有操作简单、选择性好以及不必与未反应的探针分离即可实时检测等特点,在特定序列单链DNA的检测中扮演着越来越重要的角色\[6~13\]。近年来,有关分子信标对特定序列单链DNA检测的报道逐渐增多\[14~16\]。尽管分子信标在DNA的定性及定量分析中显示出广阔的应用前景,但经典分子信标在实际定量分析中还存在一些不足: (1) 经典分子信标具有较强的背景信号,影响定量检测的检出限\[17\];(2) 相对于核酸染料而言,分子信标对核酸检测的灵敏度较低;(3) 经典分子信标需要在两端分别标记荧光基团及猝灭基团,制备时间长且成本高。
核酸染料Hoechst 33258是一种可以穿透细胞膜的蓝色荧光染料,具有很高的灵敏度\[18,19\]。它在水溶液中荧光很弱,与单链DNA几乎不发生作用,但与双链DNA具有很强的亲和性,能嵌入双链DNA的小沟中,与双链中的A/T碱基对发生特异性结合,使其结构发生变化,导致荧光强度大大增加\[20,21\]。根据此原理,Hoechst 33258在双链 DNA 含量测定中,已得到广泛应用\[22,23\]。
Hoechst 33258 与分子信标联合应用的报道较少。James等\[24\]利用Hoechst 33258 与分子信标研究了发夹聚酰胺对双链DNA的解链温度的影响,并获得了较好的结果。到目前为止,利用Hoechst 33258 结合分子信标对特定序列核酸的检测还未见报道。
本研究利用无标记的分子信标(无有机荧光基团和荧灭基团)及Hoechst 33258建立一种高灵敏、高选择性的特定序列核酸检测方法。在这种分析方法中,将无标记分子信标的茎完全设计成C/G碱基对,分子信标的环设计为目标DNA的互补序列。利用分子信标与目标DNA反应之前,Hoechst 33258荧光信号很弱,而与目标DNA反应之后,其荧光信号显著增强的基本原理,实现对特定序列DNA的定量检测。
相对于经典分子信标对核酸的检测而言,本方法具有以下特点:(1)使用无标记分子信标,省去了标记步骤, 降低了分析成本; (2)将分子信标的茎完全设计成C/G碱基对,而Hoechst 33258不与C/G碱基对发生作用,因此背景荧光很低,可显著降低分析方法的检出限; (3)利用Hoechst 33258的荧光代替经典分子信标中的荧光基团对目标核酸进行检测,可显著提高检测的灵敏度。这主要是因为利用核酸染料检测核酸时,一个核酸分子能与多个核酸染料相结合,而每个分子信标只有一个荧光基团。同时无标记的分子信标又保留了经典分子信标中茎环的结构,仍然具有很高的选择性。
3结果与讨论
3.1检测原理
利用无标记的分子信标及Hoechst 33258对特定序列核酸检测的原理如图1所示。在没有目标DNA时,尽管分子信标处于茎环结构状态,但分子信标的茎全部由C/G碱基对组成,不与Hoechst 33258作用,此时Hoechst 33258的荧光信号很弱;在有目标DNA时,其与分子信标发生杂交反应,形成双链DNA后再与Hoechst 33258结合,Hoechst 33258的荧光强度显著增强。根据荧光增强的程度,实现对特定单链DNA的检测。
3.3.6核酸染料Hoechst 33258与双链DNA作用的时间的影响核酸染料Hoechst 33258只有与双链DNA结合之后才会发出较强的荧光,因此它与双链DNA的结合时间是影响其荧光强度的重要因素。本实验对Hoechst 33258与双链DNA的结合时间进行了考察。结果表明,在9 min内,Hoechst 33258的荧光强度随着时间的增加而增大;当结合时间超过9 min后,Hoechst 33258的荧光强度不再发生变化。
3.4工作曲线及复杂样品中核酸的检测
在3.5碱基错配分析
对不同碱基错配序列DNA进行了分析,以考察方法的特异性,结果如图4所示。对于不同碱基错配序列的DNA,Hoechst 33258的荧光强度具有显著区别。对于目标DNA序列、单碱基错配DNA序列、双碱基错配DNA序列及三碱基错配DNA序列,所对应的荧光强度之比
4结论
利用无标记的分子信标与单链DNA特异性反应生成双链,再与Hoechst 33258结合后,其荧光显著增强的基本原理,建立了检测特定序列核酸的新方法。本方法操作简单、检测速度快、灵敏度高、重现性好、检出限低。
References
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AbstractA highly sensitive and selective method for specific DNA sequence detection is developed using a nonlabeled molecular beacon (MB) and a nucleic acid dye Hoechst 33258. It is demonstrated by a specific DNA sequence of wildtype HBV as a model system. In this strategy, the stem of MB is completely designed as C/G base pairs. In the absence of target DNA, the interaction between Hoechst 33258 and the MBs is very weak,and the fluorescence signals of Hoechst 33258 is very low. In the presence of target DNA, the MBs hybridize with the target DNA and form doublestranded structure. Hoechst 33258 binds to dsDNA, and the fluorescence intensity is significantly enhanced. Under the optimum conditions, the fluorescence intensity of Hoechst 33258 exhibits good linear dependence on target DNA concentration in the range of 2×10
The proposed method has good precision, simple operation, fast detection speed, low detection limit, high accuracy and high sensitivity.
KeywordsNonlabeled molecular beacon; Hoechst 33258 nucleic acid dye; Singlestranded nucleic acid; Fluorescence
5Song S P, Liang Z Q, Zhang J, Wang L H, Li G X, Fan C H. Angew. Chem. Int. Et., 2009, 48(46): 8670-8674
6Nesterova I V, Erdem S S, Pakhomov S, Hammer R P, Soper S A. J. Am. Chem. Soc., 2009, 131(7): 2432-2433
7Radi A E, Sanchez J L A, Baldrich E, O′Sullivan C K. J. Am. Chem. Soc., 2006, 128(1): 117-124
8ZHENG AiHua, ZHU Qing, XIANG DongShan, HE ZhiKe. Chinese J. Anal. Chem., 2013, 41(3): 325-329
郑爱华, 朱 庆, 向东山, 何治柯. 分析化学, 2013, 41(3): 325-329
9Haner R, Biner S M, Langenegger S M, Meng T, Malinovskii V L. Angew. Chem. Int. Et., 2010, 49(7): 1227-1230
10Tang Z W, Liu P, Ma C B, Yang X H, Wang K M, Tan W H, Lv X Y. Anal. Chem., 2011, 83(7): 2505-2510
11LIU Bin, YANG XiaoHai, WANG KeMin,TAN WeiHong. Chem. J. Chinese Universities, 2012, 32(3): 486-491
刘 斌, 羊小海, 王柯敏, 谭蔚泓. 高等学校化学学报, 2012, 32(3): 486-491
12Sheng P P, Yang Z Y, Kim Y M, Wu Y R, Tan W H, Benner S A. Chem. Commun., 2008, 44(41): 5128-5130
13Qiao G M, Zhuo L H, Gao Y, Yu L J, Li N, Tang B. Chem. Commun., 2011, 47(26): 7458-7460
14Wu C S, Oo M K K, Cupps J M, Fan X D. Biosens. Bioelectron., 2011, 26(9): 3870-3875
15Li F, Huang Y, Yang Q, Zhong Z T, Li D, Wang L H, Song S P, Fan C H. Nanoscale, 2010, 2(6): 1021-1026
16Wu J K, Huang C H, Cheng G F, Zhang F, He P G, Fang Y Z. Electrochem. Commun., 2009, 11(1): 177-180
17Zhang P, Beck T, Tan W H. Angew. Chem. Int. Ed., 2001, 40(2): 402-405
18Buurma N J, Haq I. J. Mol. Biol., 2008, 381(3): 607-621
19Furse K E, Corcelli S A. J. Am. Chem. Soc., 2008, 130(39): 13103-13109
20Anuradha, Alam M S, Chaudhury N K. Chem. Pharm. Bull., 2010, 58(11): 1447-1454
21Ojha H, Murari B M, Anand S, Hassan M I, Ahmad F, Chaudhury N K. Chem. Pharm. Bull., 2009, 57(5): 481-486
22Kobayashi M, Takashi K B, Saito M, Kaji S, Oomura M, Iwabuchi S, Morita Y, Hasan Q, Tamiya E. Electrochem. Commun., 2004, 6(4): 323-343
23Zhou Y L, Mao S N, Li Y Z, Chang W B. Microchim. Acta, 2004, 144(1): 191-197
24James P L, Le S L, Ellervik U, Bratwall C, Norden B, Brown T, Fox K R. Biophys. Chem., 2004, 111(3): 205-212
AbstractA highly sensitive and selective method for specific DNA sequence detection is developed using a nonlabeled molecular beacon (MB) and a nucleic acid dye Hoechst 33258. It is demonstrated by a specific DNA sequence of wildtype HBV as a model system. In this strategy, the stem of MB is completely designed as C/G base pairs. In the absence of target DNA, the interaction between Hoechst 33258 and the MBs is very weak,and the fluorescence signals of Hoechst 33258 is very low. In the presence of target DNA, the MBs hybridize with the target DNA and form doublestranded structure. Hoechst 33258 binds to dsDNA, and the fluorescence intensity is significantly enhanced. Under the optimum conditions, the fluorescence intensity of Hoechst 33258 exhibits good linear dependence on target DNA concentration in the range of 2×10
The proposed method has good precision, simple operation, fast detection speed, low detection limit, high accuracy and high sensitivity.
KeywordsNonlabeled molecular beacon; Hoechst 33258 nucleic acid dye; Singlestranded nucleic acid; Fluorescence
5Song S P, Liang Z Q, Zhang J, Wang L H, Li G X, Fan C H. Angew. Chem. Int. Et., 2009, 48(46): 8670-8674
6Nesterova I V, Erdem S S, Pakhomov S, Hammer R P, Soper S A. J. Am. Chem. Soc., 2009, 131(7): 2432-2433
7Radi A E, Sanchez J L A, Baldrich E, O′Sullivan C K. J. Am. Chem. Soc., 2006, 128(1): 117-124
8ZHENG AiHua, ZHU Qing, XIANG DongShan, HE ZhiKe. Chinese J. Anal. Chem., 2013, 41(3): 325-329
郑爱华, 朱 庆, 向东山, 何治柯. 分析化学, 2013, 41(3): 325-329
9Haner R, Biner S M, Langenegger S M, Meng T, Malinovskii V L. Angew. Chem. Int. Et., 2010, 49(7): 1227-1230
10Tang Z W, Liu P, Ma C B, Yang X H, Wang K M, Tan W H, Lv X Y. Anal. Chem., 2011, 83(7): 2505-2510
11LIU Bin, YANG XiaoHai, WANG KeMin,TAN WeiHong. Chem. J. Chinese Universities, 2012, 32(3): 486-491
刘 斌, 羊小海, 王柯敏, 谭蔚泓. 高等学校化学学报, 2012, 32(3): 486-491
12Sheng P P, Yang Z Y, Kim Y M, Wu Y R, Tan W H, Benner S A. Chem. Commun., 2008, 44(41): 5128-5130
13Qiao G M, Zhuo L H, Gao Y, Yu L J, Li N, Tang B. Chem. Commun., 2011, 47(26): 7458-7460
14Wu C S, Oo M K K, Cupps J M, Fan X D. Biosens. Bioelectron., 2011, 26(9): 3870-3875
15Li F, Huang Y, Yang Q, Zhong Z T, Li D, Wang L H, Song S P, Fan C H. Nanoscale, 2010, 2(6): 1021-1026
16Wu J K, Huang C H, Cheng G F, Zhang F, He P G, Fang Y Z. Electrochem. Commun., 2009, 11(1): 177-180
17Zhang P, Beck T, Tan W H. Angew. Chem. Int. Ed., 2001, 40(2): 402-405
18Buurma N J, Haq I. J. Mol. Biol., 2008, 381(3): 607-621
19Furse K E, Corcelli S A. J. Am. Chem. Soc., 2008, 130(39): 13103-13109
20Anuradha, Alam M S, Chaudhury N K. Chem. Pharm. Bull., 2010, 58(11): 1447-1454
21Ojha H, Murari B M, Anand S, Hassan M I, Ahmad F, Chaudhury N K. Chem. Pharm. Bull., 2009, 57(5): 481-486
22Kobayashi M, Takashi K B, Saito M, Kaji S, Oomura M, Iwabuchi S, Morita Y, Hasan Q, Tamiya E. Electrochem. Commun., 2004, 6(4): 323-343
23Zhou Y L, Mao S N, Li Y Z, Chang W B. Microchim. Acta, 2004, 144(1): 191-197
24James P L, Le S L, Ellervik U, Bratwall C, Norden B, Brown T, Fox K R. Biophys. Chem., 2004, 111(3): 205-212
AbstractA highly sensitive and selective method for specific DNA sequence detection is developed using a nonlabeled molecular beacon (MB) and a nucleic acid dye Hoechst 33258. It is demonstrated by a specific DNA sequence of wildtype HBV as a model system. In this strategy, the stem of MB is completely designed as C/G base pairs. In the absence of target DNA, the interaction between Hoechst 33258 and the MBs is very weak,and the fluorescence signals of Hoechst 33258 is very low. In the presence of target DNA, the MBs hybridize with the target DNA and form doublestranded structure. Hoechst 33258 binds to dsDNA, and the fluorescence intensity is significantly enhanced. Under the optimum conditions, the fluorescence intensity of Hoechst 33258 exhibits good linear dependence on target DNA concentration in the range of 2×10
The proposed method has good precision, simple operation, fast detection speed, low detection limit, high accuracy and high sensitivity.
KeywordsNonlabeled molecular beacon; Hoechst 33258 nucleic acid dye; Singlestranded nucleic acid; Fluorescence
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