CN105039519B - 一种基于足点域杂交的dna测定新方法 - Google Patents

一种基于足点域杂交的dna测定新方法 Download PDF

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CN105039519B
CN105039519B CN201510325830.6A CN201510325830A CN105039519B CN 105039519 B CN105039519 B CN 105039519B CN 201510325830 A CN201510325830 A CN 201510325830A CN 105039519 B CN105039519 B CN 105039519B
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混旭
孟妍
王学刚
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Abstract

一种基于足点域杂交的DNA测定新方法，属于化学发光技术领域。将目标DNA加入DNA1修饰磁珠中，目标DNA的一端与DNA1的一端单链部分杂交形成足点域，在足点域作用下目标DNA未杂交的部分与DNA1继续进行杂交反应，将DNA1的发卡结构打开，同时形成双链DNA，再加入FITC标记DNA2探针，DNA2的一端与所形成的双链DNA的一端单链部分杂交形成足点域，在足点域的作用下DNA2未杂交的部分与双链DNA继续进行杂交反应，形成新的双链DNA，将目标DNA替换下来进行循环利用，并将大量的DNA2附着在磁珠上，利用光二极管诱导化学发光原理实现对目标DNA的测定。本发明方法简单、成本低、灵敏度高。

Description

一种基于足点域杂交的DNA测定新方法
技术领域
本发明属于化学发光技术领域，涉及一种基于足点域杂交的DNA测定新方法。
背景技术
DNA催化自组装技术是在DNA链式杂交反应技术的基础上发展而来的一种放大技术。它不同于DNA链式杂交反应技术中需要两条发卡DNA，而是涉及了一条或者多条发卡DNA。近年来DNA测定方法主要有化学发光法(Zhang X,Liu H,Li R,Zhang N,Xiong Y,NiuS.Chemiluminescence detection of DNA/microRNA based on cation-exchange of CuSnanoparticles and rolling circle amplification.Chem Commun.2015.DOI:10.1039/C5CC01317H)；电化学法(Liu L,Song C,Zhang Z,Yang J,Zhou L,Zhang X,XieG.Ultrasensitive electrochemical detection of microRNA-21 combining layerednanostructure of oxidized single-walled carbon nanotubes and nanodiamonds byhybridization chain reaction.Biosens Bioelectron.2015,28；70:351-357)；荧光法(Wang Q,Wang W,Lei J,Xu N,Gao F,Ju H.Anal Chem.Fluorescence quenching ofcarbon nitride nanosheet through its interaction with DNA for versatilefluorescence sensing.2013.17；85(24):12182-12188)；光度法(Thavanathan J,HuangNM,Thong KL.Colorimetric detection of DNA hybridization based on a dualplatform of gold nanoparticles and graphene oxide.BiosensBioelectron.2014.15；55:91-98)；表面等离子共振法(Szunerits S,Maalouli N,WijayaE,Vilcot JP,Boukherroub R.Recent advances in the development of graphene-based surface plasmon resonance(SPR)interfaces.Anal Bioanal Chem.2013,405(5):1435-43)等。
但是，这些方法各有其优缺点，本发明利用有DNA1标记的磁珠为载体，以异硫氰酸荧光素FITC为标记物，利用DNA取代反应，实现了目标DNA的循环利用，从而实现了信号的循环放大，并最终实现了对目标DNA的高灵敏度测定，具有方法简单、灵敏度高，成本低的优点。
发明内容
本发明目的是提供一种基于足点域杂交的DNA测定新方法，利用DNA1标记的磁珠为载体，以异硫氰酸荧光素FITC标记DNA2为探针，当目标DNA加入DNA1修饰磁珠溶液中时，目标DNA的一端与DNA1的一端单链部分杂交形成足点域，在足点域的作用下目标DNA未杂交的部分与DNA1继续进行杂交反应，将DNA1的发卡结构打开，同时形成双链DNA，然后加入FITC标记DNA2探针，DNA2的一端与所形成的双链DNA的一端单链部分杂交又形成足点域，在足点域的作用下DNA2未杂交的部分与双链DNA继续进行杂交反应，形成新的双链DNA，将DNA2附着在磁珠上，同时，将目标DNA替换下来，被替换下来的目标DNA进行下一个双链的形成，并使另外一条DNA2附着在磁珠上，如此目标DNA被循环利用，同时将大量的DNA2附着在磁珠上，利用光二极管诱导化学发光原理，利用化学发光仪检测化学发光强度，根据化学发光强度实现对目标DNA的测定。
技术方案
一种基于足点域杂交的DNA测定新方法，其特征在于根据目标DNA序列，设计好有特定序列的发卡DNA1，发卡DNA2，并利用DNA1标记的磁珠为载体，以异硫氰酸荧光素FITC标记的DNA2为探针，利用足点域杂交反应实现目标DNA的循环使用，同时实现了信号的循环放大，最终实现了对目标DNA的测定。测定步骤如下：
(1)磁珠的前处理
取10μL链霉亲和素修饰的磁珠至1.5mL的离心管中，用100μL pH 8.0的Tris-HCl缓冲溶液清洗两遍，然后将其分散到Tris-HCl缓冲溶液中。
(2)发卡DNA的前处理
发卡DNA在使用之前在95℃温度下孵化2min，并逐步降至室温。
(3)捕获DNA1在磁珠表面的固定
在含10μL链霉亲和素修饰的磁珠的1.5mL的离心管中，加入10μL 1.0×10-5M的生物素修饰的DNA1，在37℃下振荡反应30min，并用100μL pH 6.8的磷酸缓冲溶液洗涤两遍，得到DNA1修饰磁珠，并分散到1mL的磷酸缓冲溶液中。
(4)目标DNA检测
取(3)所得磁珠溶液100μL，并加入含有目标DNA的样品溶液100μL，然后再加入100μL含有1.0×10-7M DNA2的pH为6.8的磷酸缓冲溶液，并在37℃振荡反应1h。然后进行磁分离，除去上清液后，将磁珠用100μL pH 6.8的磷酸缓冲溶液洗涤两遍，并将其分散于100μLpH 6.8的磷酸缓冲溶液中。再加入200μL pH 11.3的鲁米诺溶液，打开光二极管，15秒后关闭光二极管，用化学发光仪检测化学发光信号，根据化学发光信号强度对目标物进行定量分析。
本发明的化学试剂优选分析纯试剂，所有溶液均用二次蒸馏水配置。
本发明的0.2M pH 6.8磷酸缓冲溶液的配制方法：称取0.2g KH2PO4、2.9gNa2HPO4·12H2O溶解于1L水中。
Tris-HCl缓冲溶液配制方法：称取2.429g三(羟甲基)氨基甲烷，用500mL超纯水溶解后，用0.1M盐酸调pH至8.0，最后用超纯水稀释至1000mL。
所用人工合成DNA序列(购自北京赛百盛生物工程有限公司)如下。
DNA的部分序列如下：
DNA1：5’-biotin-GCGGTAGGCCGTCCTAATCTCCTCCCCCAACACGGCC-3’
DNA2：5’-GTTGGGGGAGGAGATTAGGACGGCC-FITC-3’
目标DNA：5’-AGATTAGGACGGCCTACCGC-3’
本发明的化学发光测定选用MPI-E型化学发光分析***(西安瑞迈分析仪器有限公司)。
本发明的振荡孵育选用THZ-82A气浴恒温振荡器(全坛市医疗仪器厂)。
本发明的离心机选用Anke-TGL-16C飞翁牌高速离心机(上海市安亭科学仪器厂)。
本发明的pH测量选用PHS-3D型酸度计(上海雷磁仪器厂)。
本发明的显著效果
本发明实现了目标DNA的循环使用，同时实现了信号的循环放大，最终实现了对目标DNA的测定，得到了检测目标DNA的标准曲线，线性范围及线性方程。
当目标DNA的浓度在1.0pM-700pM之间时，随着目标DNA浓度的变化，化学发光强度有明显变化。经计算得到检测目标DNA的线性方程为ICL＝9.8136x+59.506(ICL是体系的化学发光强度；x是目标DNA的浓度，pM；n＝12，n表示同一浓度测定次数，R＝0.9997)，检测限是0.5pM。同时，考察了利用三明治夹心法测定目标DNA的检测限为500pM，基于足点域杂交的DNA测定方法检测限较三明治夹心法的低1000倍，表明该方法的灵敏度高。
该测定方法的精密度通过对浓度为30.0pM的目标DNA进行11次平行测定而计算得出，相对标准偏差为3.9％，表明本发明的测定方法有较好的重现性。
附图说明
图1.测定目标DNA的方法原理示意图。
图2.目标DNA标准曲线，横坐标是目标DNA浓度，单位是pM，纵坐标ICL是体系的化学发光强度。
具体实施方式
按照技术方案的步骤(1)至(4)得到目标DNA标准曲线见图2。DNA由赛百盛基因技术有限公司(上海，中国)合成。实验所用其他试剂均为分析纯，并且不用对其进一步纯化可以直接使用。
根据发明的方法对目标DNA含量进行了测定，并采用标准加入法对方法进行了评价，样品测定回收率为96.0–103.4％，测定结果见表1，本发明的方法在目标DNA检测中具有精密度高的特点。
表1.样品分析测定结果
编号 含量a,b 标准样品加入量 测得量 回收率(％)
1 3.2 5.0 8.1 96.0
2 27.6 30.0 58.3 102.3
3 46.9 50.0 98.6 103.4
a7次测量结果
b单位：pM。

Claims (2)

1.一种基于足点域杂交的DNA测定新方法，其特征包括以下步骤：
a.发卡DNA在使用之前在95℃温度下孵化2min，并逐步降至室温；
b.取10μL链霉亲和素修饰的磁珠至1.5mL的离心管中，用100μL pH 8.0的Tris-HCl缓冲溶液清洗两遍，然后将其分散到Tris-HCl缓冲溶液中；
c.在含10μL链霉亲和素修饰的磁珠的1.5mL的离心管中，加入10μL 1.0×10-5M的生物素修饰的DNA1，在37℃下振荡反应30min，并用100μL pH 6.8的磷酸缓冲溶液洗涤两遍，得到DNA1修饰磁珠，并分散到1mL的磷酸缓冲溶液中；
d.取c所得磁珠溶液100μL，并加入含有目标DNA的样品溶液100μL，然后再加入100μL含有1.0×10-7M DNA2的pH为6.8的磷酸缓冲溶液，并在37℃振荡反应1h；然后进行磁分离，除去上清液后，将磁珠用100μL pH 6.8的磷酸缓冲溶液洗涤两遍，并将其分散于100μL pH6.8的磷酸缓冲溶液中；再加入200μL pH 11.3的鲁米诺溶液，打开光二极管，15秒后关闭光二极管，用化学发光仪检测化学发光信号，根据化学发光信号强度对目标物进行定量分析；
其中，DNA1、DNA2和目标DNA的序列如下：
DNA1：5’-biotin-GCGGTAGGCCGTCCTAATCTCCTCCCCCAACACGGCC-3’
DNA2：5’-GTTGGGGGAGGAGATTAGGACGGCC-FITC-3’
目标DNA：5’-AGATTAGGACGGCCTACCGC-3’。
2.根据权利要求1的测定DNA的方法，其特征在于所述的磁珠粒径为0.4-0.6μm，浓度为8-12mg/mL。
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