CN1348096A - 一种均相特异性检测核酸的探针及应用方法 - Google Patents
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Abstract
本发明涉及一种新的均相特异性检测核酸的探针及应用方法。这种探针由两条分别标记荧光剂和淬灭剂并且反向互补的寡核苷酸组成,正常条件下,探针因碱基互补而成双链结构,当与靶序列杂交时可以发生荧光信号的改变。这种探针可以在室温自发地与靶序列发生置换式杂交,从而检测核酸是否存在。而单碱基突变的靶序列则不能发生杂交反应。另外,还可以利用荧光双链探针对PCR反应进行实时检测。
Description
本发明涉及一种均相特异性检测核酸的探针。
用探针特异性检测核酸一般都需要将未杂交的探针分离出来,而均相检测方法则不须分离步骤,具有反应速率快,操作简单,容易定量等突出优点。如:Molecular beacons技术。
均相核酸检测近几年在核酸扩增中应用广泛。如聚合酶链式反应(PCR),这是一种80年代后期发展起来的一种体外扩增核酸片段的技术,可在数小时内使特定的核酸片段扩增至几百万倍。该项技术自发明以来已广泛应用于生物和医学研究的各个领域。早期的PCR是通过电泳来检测结果,其操作繁琐,不能进行实时监测。实时荧光PCR是近几年发展起来的新检测技术,具有诸多优点,尤其在临床诊断中优势明显。由于采用闭管测定方式,消除了传统凝胶电泳或其它非均相方法的扩增产物污染的隐患,从而在测定步骤中杜绝了假阳性的主要来源。并且通过实时动态监测可以获得准确的原始模板定量,动态范围也较终点测定方式大大加宽。同时荧光测定在分析灵敏度、简便性等方面都具有明显优势。
目前实时荧光PCR检测技术已有多种方式,可以分为非探针型和探针型两类。非探针型有荧光嵌入剂法、Sunrise Primer(又称Amplifluor)、AmpliSensor等;探针型包括:5’—核酸外切酶技术(TaqMan探针)、分子信标(molecularbeacon)技术、应用于Lightcycler的双探针技术、蝎子引物(Scorpions primer)等。前者由于无法识别非特异扩增产物,在实际应用中受到很大限制。后一种类型,由于增加了探针识别步骤,提高了测定的可靠性,但普遍存在实验设计复杂,价格高昂等缺点,并且有些方法识别单碱基突变的能力不强。
本发明设计的荧光双链探针,可以均相特异性检测核酸,要求的实验设计简单,可操作性强。在保证特异性和灵敏度的同时,实现对核酸的测定。
荧光双链探针的构成
荧光双链探针是一对(两条)按照脱氧核糖核酸(DNA)碱基互补配对原则而反向互补的寡核苷酸,两条链长度不同。因为两条链不等长,所以从整体上看探针可以分为两部分,一是单链的触手区,一是双链区。这种探针我们称为“触手探针”。在其中的一条寡核苷酸上标记荧光供体,在另一条寡核苷酸上标记荧光受体或淬灭剂。并且探针序列与靶序列互补,可以发生杂交反应。荧光双链探针在不同的条件下可以形成多种形态,并且伴随荧光强度的改变。探针两条链在稳定的双链结构,淬灭剂靠近荧光剂,荧光剂的荧光被淬灭,此时探针没有荧光;在酸、碱、热等条件下探针分开成两条单链,淬灭剂远离荧光剂,荧光剂发出荧光;当恢复至温和条件,两条链复性为双链状态,淬灭剂靠近荧光剂,探针没有荧光;当有靶序列存在时,探针的两条链分别与各自互补的靶序列杂交,两条链因此而分开,淬灭剂远离荧光剂,荧光剂发出荧光。探针的应用主要有以下两方面:
“触手探针”与靶序列自发地发生置换式杂交反应:
“触手探针”可以与溶液中的单链靶序列发生置换式杂交反应,探针的“触手”是单链的,它会首先与溶液中的单链靶序列碰撞结合,然后靶序列将置换短链与长链形成更稳定的靶序列和长链的杂交体,此时探针的长链和短链分离,荧光剂远离淬灭剂,探针发出荧光。见图1。
用“触手探针”PCR反应的实时监测:
所述的荧光双链探针在进行PCR检测中,两条寡核苷酸的3’端必须进行封闭,使其不能作为引物延伸。
在由荧光双链探针存在的PCR反应混合体系中,进入热变性阶段,“触手探针”和模板均发生变性,荧光剂远离淬灭剂,荧光性质得以恢复。
温度降低至退火温度时,如果没有靶序列的存在,两条探针将恢复至双链结构,荧光剂与淬灭剂靠近,荧光剂被淬灭;如果有靶序列的存在,两条探针将分别与靶序列杂交,使两条探针无法恢复至双链结构,荧光剂远离淬灭剂,荧光剂发出荧光。
在PCR的延伸阶段,因为高温两条探针从靶序列上解离,使延伸反应顺利进行。然后进入下一个循环。
由以上过程可见,我们可以通过实时荧光PCR仪在PCR反应的复性阶段检测荧光的变化,从而动态监测靶序列的有无及含量。见图2。
荧光双链探针的设计方法:
1.两条链的长度:两条链一般不等长,探针的碱基互补配对使其呈双链结构,长出的“触手”长度最好在2-10核苷酸之间。
2.荧光剂和淬灭剂的标记位置:荧光剂和淬灭剂可以标记在探针的顶端也可标记在探针的中间,但最好标记在同一对互补的碱基上,这样荧光剂和淬灭剂的位置很近,可以达到最大的淬灭效率。
3.做PCR实验所用仪器和数据采集:可以使用PE公司PE-7700仪器,BIO-RAD公司iCycler仪器,ROCHE公司Lightcycler仪器,THE ROTER-GENE公司GP2000仪器等,荧光数据在退火时采集。
“触手探针”的积极效果;
1.探针设计简单,实验成功率高:对探针设计以及整个反应体系没有太多要求,整个系统非常简单,容易掌握。
2.探针制备较简单:荧光双链探针的标记是寡核苷酸的单一荧光物质修饰,可以通过固相合成直接制备,纯化一步完成,因此产率也高。其它的探针或引物,多数为双修饰(双端修饰或内部插入修饰),且须按照彼此的规则进行特殊处理,双修饰纯化繁琐,产率低,价格也较昂贵。
3.特异性高:有文献报道,能量限制型探针的特异性高于非能量限制型探针,在已有的荧光PCR技术中只有分子信标(molecular beacon)是分子内能量限制型探针,它与靶序列杂交所释放的能量必须高于分子信标本身闭和成发夹结构所释放的能量,所以它的特异性高于线性探针的特异性。而触手探针是一种分子间能量限制型探针,它与靶序列杂交所释放的能量也必须高于两条链互相复性所释放的能量。这样就保证了探针杂交的高特异性,即如果靶序列发生了突变或有非特异扩增产物,荧光标记双链探针将自身恢复至双链状态,不会形成非特异杂交体。
图1.自发杂交反应原理图
图2.荧光双链探针实时检测PCR产物原理图
图3.荧光双链探针检测靶序列结果图
图4.荧光双链探针实时检测PCR产物结果图
实施例一
与靶序列自发地发生置换式杂交反应:
在探针的两条链已稳定结合成双链结构的溶液中,加入过量的寡核苷酸靶序列,可以观测到荧光强度升高了20倍以上。图3。靶序列和荧光双链探针的置换反应是特异的,加入的核酸必须是完全与探针互补的,否则即使有一个碱基的不同也不能发生置换反应,荧光强度不会升高。图3。
自发进行置换式杂交反应:探针由上海申友技术公司合成,长链:5’-FAM-ACG AAC CTC AAA CAG ACA CCA T-3’;短链:5’-TGT CTG TTT GAG GTTGCT-DABCYL-3’。50μL反应液,内含10mM Tris-HCl pH:8.0,1.5mM MgCl2,两探针分别为0.8μ mol/L,先94℃变性2分钟,再50℃杂交5分钟,使两条链成稳定的双链结构,在25℃测定荧光强度5分钟,然后加入靶序列,野生靶序列:5’-CCA TGG TGT CTG TTT GAG GTT GCT-3’突变探针:5’-CCA TGG TGT CTGTTT CAG GTT GCT-3’,下画线碱基为突变碱基。靶序列的终浓度为探针浓度的2倍,然后每分钟测定1次。由图3可见:完全互补地靶序列(◆)荧光升高20倍以上,而点突变序列(■)荧光强度几乎没有升高。
实施例二
人β-珠蛋白荧光标记双链探针实时荧光PCR检测
一、扩增片段及引物和探针:
扩增人β-珠蛋白基因,扩增片段长268bp。
引物为:
5’-GAA GAG CCA AGG ACA GGT AC-3’
5’-CAA CTT CAT CCA CGT TCA CC-3’:
探针为不等长双链探针,两条链按照碱基互补配对原则反向互补:
5’-FAM-AGC AAC CTC AAA CAG ACA CCA TGG-3’-封闭,
5’-TGT CTG TTT GAG GTT GCT-Dabcyl-3’。
引物和探针由上海申友生物技术公司合成。
二、样品提取:
正常人外周血由实验室志愿者提供。用淋巴细胞液分离法分离血液白细胞和用常规酚氯仿抽提,乙醇沉淀法提取白细胞DNA。
三、PCR反应:
PCR反应总体积为50μL,内含10mmol/L Tris-HCl,pH8.3,50mmol/L KCl,,2.0U Taq酶,200μ mol/L dNTP,2.0mmol/L MgCl2,0.4μmol/L两引物,0.2μmol/L探针,各梯度模板5μL。经95℃,5min预变性,再95℃ 30秒,50℃30秒,72℃1分钟,40个循环后。荧光数据采集在退火阶段即50℃时采集。结果见图4。
Claims (9)
1.一种均相特异性检测核酸的探针,其特征是两条链分别标记荧光剂和淬灭剂的双链寡核苷酸探针。
2.根据权利要求1的均相特异性检测核酸的探针,其特征在于双链探针是两条根据碱基互补配对原则而反向互补的寡核苷酸。
3.根据权利要求1的均相特异性检测核酸的探针,其特征在于两条寡核苷酸链不等长。
4.根据权利要求1的均相特异性检测核酸的探针,其特征在于双链探针的两条链分别标记荧光供体和受体,使其在探针和靶序列杂交时可以发生荧光强度的改变。
5.根据权利要求1均相特异性检测核酸的探针,其特征在于特异性检测核酸时双链探针序列与待测核酸序列碱基互补。
6.根据权利要求1的均相特异性检测核酸的探针,其特征在于双链探针的两条链3’端用化学基团封闭,使其不能作为引物延伸。
7.根据权利要求1的均相特异性检测核酸的探针,其特征在于荧光剂和淬灭剂分别标记在两条链上,并且是在同一对互补的核苷上,并且是在两条链的3’端或5’端。
8.一种应用“触手探针”自发地均相检测溶液中的单链核酸的方法,其特征是荧光双链探针的溶液中,加入单链核酸靶序列,可以观测到荧光强度的升高。
9.一种应用“触手探针”实时检测PCR扩增产物的方法,其特征在于可以在PCR反应的退火阶段实时监测扩增产物,而不必分离未杂交的探针。
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EP01977535A EP1339732B1 (en) | 2000-10-10 | 2001-10-05 | Specific double-stranded probes for homogeneous detection of nucleic acid and their application methods |
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JP2002534331A JP3999653B2 (ja) | 2000-10-10 | 2001-10-05 | 核酸の同種内検出用の特異的二本鎖プローブおよびその適用方法 |
PCT/US2001/031246 WO2002030946A1 (en) | 2000-10-10 | 2001-10-05 | Specific double-stranded probes for homogeneous detection of nucleic acid and their application methods |
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-
2001
- 2001-01-13 CN CN01101446A patent/CN1348096A/zh active Pending
- 2001-10-05 BR BR0114575-4A patent/BR0114575A/pt not_active IP Right Cessation
- 2001-10-05 AU AU2001296647A patent/AU2001296647B2/en not_active Ceased
- 2001-10-05 US US10/398,832 patent/US7799522B2/en not_active Expired - Lifetime
- 2001-10-05 AU AU9664701A patent/AU9664701A/xx active Pending
- 2001-10-05 CN CN018170129A patent/CN1468251B/zh not_active Expired - Fee Related
- 2001-10-05 CA CA2424856A patent/CA2424856C/en not_active Expired - Fee Related
- 2001-10-05 WO PCT/US2001/031246 patent/WO2002030946A1/en active Application Filing
- 2001-10-05 JP JP2002534331A patent/JP3999653B2/ja not_active Expired - Fee Related
- 2001-10-05 EP EP01977535A patent/EP1339732B1/en not_active Expired - Lifetime
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Cited By (9)
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CN1294276C (zh) * | 2003-07-22 | 2007-01-10 | 厦门大学 | 检测核酸的等长双链特异性探针 |
CN101845498A (zh) * | 2010-04-29 | 2010-09-29 | 厦门艾德生物医药科技有限公司 | 一种用于核酸特异识别检测的探针 |
CN101845498B (zh) * | 2010-04-29 | 2013-05-08 | 厦门艾德生物医药科技有限公司 | 一种用于核酸特异识别检测的探针 |
CN103975061A (zh) * | 2011-10-31 | 2014-08-06 | 荣研化学株式会社 | 靶核酸的检测方法 |
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CN112996899A (zh) * | 2018-11-09 | 2021-06-18 | 横河电机株式会社 | 核酸序列检测用装置 |
CN112996899B (zh) * | 2018-11-09 | 2024-07-05 | 横河电机株式会社 | 核酸序列检测用装置 |
CN109652516A (zh) * | 2018-12-29 | 2019-04-19 | 中国人民解放军军事科学院军事医学研究院 | 一种双链寡核苷酸核酸探针的结构和用途 |
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US9157112B2 (en) | 2015-10-13 |
JP3999653B2 (ja) | 2007-10-31 |
JP2004511227A (ja) | 2004-04-15 |
CA2424856A1 (en) | 2002-04-18 |
AU2001296647B2 (en) | 2006-03-16 |
AU9664701A (en) | 2002-04-22 |
CA2424856C (en) | 2014-03-11 |
WO2002030946A1 (en) | 2002-04-18 |
EP1339732A4 (en) | 2004-04-21 |
CN1468251A (zh) | 2004-01-14 |
US20110129828A1 (en) | 2011-06-02 |
CN1468251B (zh) | 2012-11-21 |
EP1339732B1 (en) | 2012-08-01 |
US20040023269A1 (en) | 2004-02-05 |
BR0114575A (pt) | 2004-01-06 |
US7799522B2 (en) | 2010-09-21 |
EP1339732A1 (en) | 2003-09-03 |
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