CN115060694A - 一种检测双链dna形成过程的荧光方法 - Google Patents
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Abstract
本发明一种检测双链DNA形成过程的荧光方法,属于生物传感领域。利用建立的方法设计出能够灵敏检测DNA双链结构的荧光探针。特点在于利用核酸碱基互补配对原理,形成双链结构,导致探针与硫黄素T的结合力增加,荧光检测信号增强。首先将所设计检测探针与待测单链核酸分子结合,形成分子间双链结构,再加入硫黄素T分子,通过测定混合物的荧光信号强度,能够对待测单链核酸分子浓度进行检测。
Description
技术领域
本发明专利涉及一种构建双链DNA荧光检测探针的技术,可用于碱基互补配对形成的双链DNA的检测。特点在于利用含鸟嘌呤(G)重复单元的核酸片段与硫黄素T结合,并在双链核酸存在时使荧光增强的性质,实现对核酸分子从单链到双链的检测,属于生物传感领域。
背景技术
两条特定序列的单链核酸分子由于分子间碱基互补配对作用能够形成双链核酸分子,传统方法中常使用溴化乙锭嵌入到双链核酸的碱基中,对双链核酸分子进行检测,然而溴化乙锭具有强致癌性、对细胞产生强的毒性,阻碍了其广泛应用;为解决这一问题,一些低毒性的有机荧光分子,如4S Green Plus,SYBR Green,4S Gelblue,4S GelRed等染料被用于双链DNA形成过程的检测,然而这类核酸染料存在价格高,单链和双链核酸的区分度不高的问题,制约了其应用。研究通用、低成本、生物安全、选择性高的双链DNA荧光检测方法十分必要。
目前,对单链核酸片段的灵敏检测在许多领域得到应用,如特定转基因片段的检测能够对转基因大豆和非转基因大豆进行甄别,新型冠状病毒特定DNA片段的检测有助于新冠病毒肺炎阳性病例的确定,早期癌症的筛查往往依赖于microRNA的液体活检技术。目前对这类分子的检测多依赖于PCR技术,在PCR过程中形成大量的双链DNA,结合荧光染料对双链DNA进行检测,但专门化的检测仪器和相对较长的检测时间制约了其广泛应用;另一方面,传统PCR检测方法往往需要设计特定序列的上游和下游引物,增加了实验步骤,给核酸分子的便捷检测带来不利影响。为了克服这些限制,需要建立通用型的双链DNA荧光检测技术,以克服传统方法选择性差、检测成本高、检测时间长的不足。
发明内容
针对现有技术存在的问题,本技术旨在发明一种检测双链DNA形成过程的荧光方法。
本项目通过使用含有多个鸟嘌呤(G)的核酸片段DNA-B,再将能与单链核酸分子形成碱基互补配对作用的互补单链DNA通过多个腺嘌呤(A)与DNA-B连接形成检测探针P1,用于单链核酸分子的检测。P1与目标分子:单链核酸分子作用后,产生分子间核酸双链结构,当DNA-B相邻位置存在双链DNA时,DNA-B与硫黄素T分子结合力增强,荧光信号提高,从而使用荧光法实现双链DNA形成过程的灵敏检测,并实现对单链核酸片段的灵敏检测。
技术方案:
(1)对于分子间双链DNA形成过程检测,首先将含有1-5个腺嘌呤(A)的核酸片段连接到碱基序列为XGGGXAGGGYGGG的核酸片段的5’端,形成含有n个腺嘌呤(An,n=1-5)的A1……An(n=1-5)XGGGXAGGGYGGG核酸片段,再根据待检测的单链核酸分子M,将与M反向互补配对的核酸序列M'连接到A1……An(n=1-5)XGGGXAGGGYGGG上,形成M'A1……An(n=1-5)XGGGXAGGGYGGG检测探针P1,其中X代表胸腺嘧啶(T)或腺嘌呤(A),Y代表胸腺嘧啶(T)或腺嘌呤(A)或胞嘧啶(C)。然后将一定浓度的P1与单链核酸分子M溶解于水中,加入一定pH值的含钠离子,镁离子和硫黄素T的10 mM Tris缓冲溶液中,充分混合并静置反应一段时间后,利用荧光光度计测定混合溶液的荧光光谱,激发光波长和发射光波长分别为400-450纳米和460-600纳米;
(2)优选的,步骤(1)中P1探针的设计,X代表胸腺嘧啶(T),Y代表胞嘧啶(C);
(3)优选的,步骤(1)中P1探针中腺嘌呤(A)数量为3个,P1浓度为0.2微摩尔/升,Tris缓冲溶液pH值为7.3,钠离子和镁离子浓度为40 mM,硫黄素T最终浓度为6微摩尔/升,静置反应时间为15分钟;
(4)优选的,步骤(1)中溶液荧光光谱的测定所用激发光波长为435纳米,发射光波长为495纳米。
本发明的有益效果:
(1)本发明不需要选择具有毒性和价格昂贵的核酸荧光染料,使用的检测探针不需要进行专门的荧光分子修饰,具有操作安全,节约检测成本的优点;
(2)本发明根据所检测单链核酸序列,仅需要在DNA-B核酸片段进行相应的核酸片段延伸,方法具有很好的通用性,可有效避免繁琐的核酸序列设计过程。
附图说明
图1是本发明荧光检测可行性分析中不同溶液的荧光光谱图;
图2中(A)是使用核酸探针P0对不同浓度核酸序列M1检测的溶液荧光光谱图;(B)是溶液在发射波长为495纳米处荧光强度与M1浓度之间的关系图;
图3是使用核酸探针P0对0.15微摩尔/升的核酸M1、M2、M3和M4检测时溶液在发射波长为495纳米处荧光强度。
具体实施方式
下面结合附图对本发明的实施例作详细说明:实施例在本发明的技术方案为前提下进行,给出了一般性的实施步骤和具体操作过程,但本发明的保护范围不限于下述的实施例。
本发明所使用的所有核酸分子购自生工生物工程(上海)股份有限公司。碱基序列为:P0序列为GGCCATCGTTGAAGATGCCTCTGCC-AAA-TGGGTAGGGCGGG,核酸序列M1为GGCAGAGGCATCTTCAACGATGGCC;核酸序列M2为TAGCCTCCTTCAAACCCATTTG,核酸序列M3为TCAACCCACTTCATTAGCCTTG,核酸序列M4为TCGGCTTACTTCAGCCTCAAC。
实施例1:缓冲溶液中M1检测
(1)将P0检测探针溶解于6份50微升水中,使其浓度为2微摩尔/升,再分别加入50微升溶于水的不同浓度的核酸M1,M1的浓度依次为0,20 nM,50 nM,100 nM,500 nM,1000nM,1500 nM,混合均匀,依次记为A1,A2,A3,A4,A5,A6;
(2)将硫黄素T溶解于900微升含40 mM钠离子和40 mM镁离子的10 mM Tris缓冲溶液中,使硫黄素T最终浓度为6微摩尔/升,记为B液;
(3)将100微升A1至A6溶液分别与6份900微升B液混合,使最终溶液中M1的浓度依次为0纳摩尔/升,2纳摩尔/升,5纳摩尔/升,10纳摩尔/升,50纳摩尔/升,100纳摩尔/升,150纳摩尔/升,在25摄氏度下静置15分钟,利用荧光光度计依次测定混合溶液的荧光光谱,激发光波长和发射光波长分别为435纳米和495纳米,见附图1和附图2(A)所示;
(4)根据不同浓度M1溶液对应的495纳米处荧光强度绘制浓度响应标准曲线,见附图2(B)所示,计算线性回归方程式为荧光强度=243.74+8.58CM1,CM1代表最终溶液中M1浓度。
实施例2:本发明对不同核酸序列的检测
(1)将P0检测探针溶解于3份50微升水中,使其浓度为2微摩尔/升,再分别加入50微升溶于水的1.5微摩尔/升的核酸M1、M2、M3和M4,混合均匀后,分别记为M1液、M2液、M3液和M4液;
(2)将硫黄素T溶解于900微升含40 mM钠离子和40 mM镁离子的10 mM Tris缓冲溶液中,使硫黄素T最终浓度为6微摩尔/升,记为B液;
(3)将100微升的M1液、M2液、M3液和M4液分别与4份900微升B液混合,在25摄氏度下静置15分钟,利用荧光光度计测定混合溶液的荧光光谱,激发光波长和发射光波长分别为435纳米和495纳米,记录4份溶液在495纳米处的荧光强度,见附图3所示。
实施例3:具体玉米样品检测
(1)选择转基因玉米样品作为实际样品,转基因玉米样品取自中储粮镇江公司,按照《GB/T 19495.3-2004 转基因产品检测 核酸提取纯化方法》提取玉米中的核酸片段,获得含转基因片段的检测样品。
(2)将P0检测探针溶解于50微升水中,使其浓度为2微摩尔/升,再加入50微升转基因片段检测样品,混合均匀后,记为S液;
(3)将硫黄素T溶解于900微升含40 mM钠离子和40 mM镁离子的10 mM Tris缓冲溶液,使硫黄素T最终浓度为6微摩尔/升,记为T液;
(4)将100微升S液和900微升T液混合,在25摄氏度下静置15分钟,利用荧光光度计测定混合溶液的荧光光谱,激发光波长和发射光波长分别为435纳米和495纳米。
(5)将荧光强度代入实施例1中获得的标准曲线,计算待测溶液中转基因片段的浓度,测得浓度为每千克玉米中含150个M1片段。
说明:以上实施例仅用以说明本发明而并非限制本发明所描述的技术方案;因此,尽管本说明书参照上述的各个实施例对本发明已进行了说明,但是本领域的普通技术人员应当理解,仍然可以对本发明进行修改或等同替换;而一切不脱离本发明的精神和范围的技术方案及其改进,其均应涵盖在本发明的权利要求范围内。
序列表
<110> 鲁东大学
<120> 一种检测双链DNA形成过程的荧光方法
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<170> SIPOSequenceListing 1.0
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<212> DNA
<213> 人工序列(Patent In Version 3.3)
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Claims (5)
1.一种检测双链DNA形成过程的荧光方法,其特征在于按照下述步骤进行:
首先选取含有核酸序列为XGGGXAGGGYGGG的DNA,记为DNA-B,在其5’端连接多个腺嘌呤(A),再在多个腺嘌呤序列的5’端连接一个与待测单链核酸分子反向互补的碱基序列,记为检测探针P1;
将步骤(1)设计的检测探针P1与一定量待测单链核酸分子加入到一定pH值的含钠离子和镁离子的10 mM Tris缓冲溶液中,充分混合后加入硫黄素T溶液,静置反应一段时间,得到检测单链核酸分子的混合溶液;
使用荧光分光光度计测定步骤(2)获得的混合溶液荧光光谱,绘制荧光信号最大值与待测溶液中单链核酸分子的浓度关系曲线。
2.根据权利要求1所述的一种检测双链DNA形成过程的荧光方法,其特征在于在步骤(1)中,DNA-B序列中X代表胸腺嘧啶(T)或腺嘌呤(A),Y代表胸腺嘧啶(T)或腺嘌呤(A)或胞嘧啶(C)。
3.根据权利要求1所述的一种检测双链DNA形成过程的荧光方法,其特征在于在步骤(1)中,检测探针P1中,DNA-B序列的5‘端连接腺嘌呤(A)数为1-5个,腺嘌呤(A)序列的5’端连接1个与待测单链核酸分子反向互补的碱基序列。
4.根据权利要求1所述的一种检测双链DNA形成过程的荧光方法,其特征在于在步骤(2)中,待测单链核酸分子浓度为0-0.2微摩尔/升,检测探针P1浓度为0.05-0.25微摩尔/升,Tris缓冲溶液pH值为6-8,钠离子和镁离子浓度为20-50mM,硫黄素T浓度为2-6微摩尔/升,静置反应时间为10-60分钟。
5.根据权利要求1所述的一种检测双链DNA形成过程的荧光方法,其特征在于在步骤(3)中,溶液荧光光谱测定所用激发光波长为400-450纳米,发射光波长为460-600纳米。
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