CN1116859A - 检测从体液或组织提取的合成寡核苷酸的方法 - Google Patents
检测从体液或组织提取的合成寡核苷酸的方法 Download PDFInfo
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Abstract
本发明提供了一种检测存在于体液或组织样品中特异性合成寡核苷酸的方法，该体液或组织样品取自服用过寡核苷酸的动物或人类患者。
Description
本发明涉及检测特定核酸序列的方法。更具体地说，本发明涉及检测存在于体液或组织内的合成寡核苷酸的方法。
对存在于细胞内的特定核酸序列的检测方法在现有技术中通常是已知的。Southern(J.Mol.Biol.98：503-517，1975)提出了在由凝胶电泳分离的DNA片断中检测特定序列的方法，该方法使用“印迹”方法将DNA片断转移至薄膜，然后用放射性探针与变性的DNA片断杂交并放射自显影。这一方法也已经用于检测从细胞或组织中提取出的RNA分子，近些年来，已经开发了更快且定量的“点印迹”方法用于快速检测从组织或细胞中获得的DNA或RNA。
最近，人们对开发合成寡核苷酸在所谓的反义方法中作为治疗剂或基因表达调节剂方面已产生相当大的兴趣。例如，Agrawal(Trends in Biotechnology 10：152-158，1991)广泛地阐述了反义治疗方法的发展。为了使反义治疗方法有效，必须将寡核苷酸引入患者体内并达到所需治疗的特定组织处。因此，需要在体液或组织中检测寡核苷酸的存在。在动物模型中，已经将放射性标记的寡核苷酸施用给动物，它们在动物体液和组织中的分布也已经由提取这些寡核苷酸然后用放射性自显影术检测到。(参见Agrawal，Tems-amani和Tang，Proc.Natl.Acad.Sci.88：7595-7599，1991)。然而，作为一种实用的方式，这些方法都不能延用于人类患者。不幸的是，用于检测存在于体液或组织内的特定未标记核酸序列的各种技术仅适用于多核苷酸，例如大的DNA或RNA分子。由于寡核苷酸分子较小，存在与非特异性结合或背景以及无结合相关的、检测不到或假阴性的特殊问题。因此，仍需要开发一种用于检测存在于体液和组织中的特定合成寡核苷酸序列的方法。
本发明提供了一种检测从实验动物或人类患者获得的体液或组织样品中合成寡核苷酸存在的方法。在本发明的方法中，体液或组织样品是从已经施用了寡核苷酸的动物或人类中获取的，经蛋白酶消化后进行提取，然后将全部核酸转移至杂交膜。将该附着有核酸的杂交膜进行预杂交，然后与被标记的寡核苷酸杂交。该被标记的寡核苷酸与给动物或患者施用的寡核苷酸是互补的。然后用标准的方法检测杂交上的被标记的寡核苷酸。本发明的方法用于在经历反义寡核苷酸治疗的患者和用于研究寡核苷酸药物动力学性质的动物模型中检测和定位寡核苷酸。
附图的简单要说明
图1是根据本发明方法，使用如实施例1-5中详细描述的放射性标记探针的一个方案的图解表示。
图2表示从描述于实施例1-5的使用放射性标记探针的实验中获得的放射自显影结果。在A组(即对照组)中，将50、25、12.5、6.2、3.1、0ng寡核苷酸(从上到下)直接涂于杂交膜上。B组表示将相同量的寡核苷酸加至血清中，然后如实施例1-5中所述的方法处理。
图3是根据本发明的方法，使用如实施例1-5中详细描述的抗原标记探针的一个方案的图解表示。
图4表示从描述于实施例1-5的、使用放射性标记探针的实验中获得的放射自显影结果。上图表示放射自显影。下图表示放射自显影的扫描光密度测定对已知浓度的寡核苷酸的曲线图。
图5说明使用同位素或非同位素检测的本发明方法的应用。
本发明涉及检测存在于体液或组织中的特定核酸序列的方法。具体地说，本发明涉及在施用过这类寡核苷酸的动物或人类患者的体液或组织中检测合成寡核苷酸的方法。
本发明提供了一种检测从体液或组织中提取的合成寡核苷酸的方法。这里所使用的“寡核苷酸”包括(但不限于)所有5＇至3＇连接的、2＇一修饰的核苷和/或脱氧核糖核苷聚合物，其中的连接可以是天然的磷酯连接或诸如硫代磷酸酯、二硫代磷酸酯、氨基磷酸酯、烷基膦酸酯、烷基硫代膦酸酯、磺酸酯、氨基甲酸酯或磷酸三酯的人造连接。另外，这样的寡核苷酸还包括在碱基和/或糖残基上有修饰的寡核苷酸以及那些在3＇端和/或5＇端具有核酸酶抗性的大取代基的寡核苷酸。这里所用的“体液”包括(但不限于)血液、尿液、汗液、粘性分泌物、脑脊髓液和滑液。当使用血液时，最好离心分离出细胞以获得血清或血浆，并从血清或血浆中提取核酸。“组织”包括构成任一器官的那些物质，例如淋巴组织、肝、肾、肺、脑、肠、平滑肌、心肌、横纹肌、真皮和表皮等等。
在本发明的方法中，按下列方式处理体液样品或组织样品。首先，用合适的蛋白酶，例如蛋白酶K、链霉蛋白酶或另一种常规蛋白酶，将体液进行蛋白酶消化。然后，用分配剂，优选用苯酚/氯仿/异戊醇抽提该样品。然后，用异丁醇和乙醚提取核酸。下一步，将核酸重新悬浮于溶液中，使其单链化并将其涂覆且结合于合适的杂交膜上。这样的膜包括(但不限于)尼龙和Pall A膜。下一步，用标记的寡核苷酸处理该结合有核酸的膜，该标记的寡核苷酸与待检测的寡核苷酸是互补的。用该互补的寡核苷酸杂交，然后将未杂交的寡核苷酸洗掉。适宜的标记包括放射性同位素标记，如32P或35S，和任何其它常规标记，如荧光标记(如若丹明或荧光素)、化学发光标记或生物素和酶。
这样的标记可直接结合在寡核苷酸探针上。可以选择的是，在优选方案中将抗原连接在寡核苷酸上，通过将标记与特异地与抗原结合的抗体偶联使标记与探针相连接(该探针与其靶序列杂交)。最优选的方案使用连接有抗原地高辛配基(digoxigenin DIG)的探针和接合有碱性磷酸酯酶的抗DIG抗体或抗原结合抗体片断。在这一方案中，通过加入稳定的磷酸化的化合物来检测探针，这种化合物一经去磷酸化作用就产生可用放射自显影术检测的发光。为了本发明的目的，术语“放射自显影”意为：通过将底片与经探针杂交的杂交膜并置而使照相底片曝光的曝光方法，不管该光线是日光、X射线，或由放射活性化合物衰变而释放的α或β粒子或γ射线。本领域的技术人员将认识到在本发明的方法中任何其它抗原均可用来代替DIG而且在作用上与DIG相当。本领域的技术人员还将认识到其它配体一受体对能取代抗原抗体对并与其功能相当。最后，本领域的技术人员还将认识到任何其它产生化学发光的酶一底物的组合都可代替碱性磷酸酶和其试剂，而且功能相当。
杂交和洗涤的条件是特别重要的，对于具有离子型核苷酸间连结的寡核苷酸，如寡核苷酸磷酸二酯或硫代磷酸酯来说，发现在6×SSC中37℃下杂交3-16小时，然后在6×SSC中室温下洗涤5-10分钟，对于使用具有62％G＋C含量的25个碱基的探针检测具有56％G＋C含量的25个碱基的寡核苷酸来说是适宜的。当使用的条件较重时，检测不到靶寡核苷酸。但当使用的条件较轻时，背景杂交会掩盖真正的信号。对于具有非离子型修饰的核苷酸间连结或较低G＋C含量的寡核苷酸来说，使用较轻的条件(例如较低的温度、较高的盐浓度)可能是有益的。对较长的寡核苷酸或具有较高G＋T含量或RNA组分的寡核苷酸，使用较重的条件(例如较高温度、较低盐浓度和/或存在如甲酰胺的氢键竞争剂)可能是有益的。解链温度和不同经修饰的核苷酸间连接之间的关系已有详细的描述(参见例如美国专利5,149,798号的图9，其内容在此引入作为参考)。对于任何给定的经修饰的寡核苷酸，杂交是用靶寡核苷酸直接涂布的杂交膜，以下面实施例4所述的条件开始的。然后，对于不同修饰的寡核苷酸，减轻或增重所用条件，直至达到可检测3ng靶寡核苷酸的检测极限。只有在这时，背景和检测不到的问题才被解决。
洗涤后，将膜干燥，并用常规方法，如荧光检测、β-放射检测、化学发光检测或放射自显影术，来检测信号。
本发明方法在寡核苷酸药物动力学的动物研究中非常有用，该方法不需要在动物中使用大量放射性标记的寡核苷酸。此外，本发明的方法对于检测在经历反义寡核苷酸治疗的人类患者体内寡核苷酸的浓度和分布非常有用，为剂量的优化工作提供了便利。
下述实施例意在进一步说明本发明的某些优选方案，并不在本质上限制。
实施例1
体液和组织样品的制备
将已知量的寡核苷酸掺入血清或血液中。将250μl掺料的血液血清于含有2mg/ml蛋白酶K的提取缓冲液(0.5％SDS/10mM NaCl/20mM Tris-HCl，pH7.6/10mM EDTA)中，在60℃下温育1.5至2小时。加入200毫升的水至样品中，然后用500μl苯酚/氯仿/异戊醇(25∶24∶1体积/体积)抽提一次并用500μl氯仿抽提一次，然后用1ml异丁醇将水相抽提两次并用500μl乙醚抽提一次。将含有核酸的剩余溶液干燥成丸。将这些丸再悬浮于10μ1TE缓冲液中(10mMTris-HCl，pH8.0/1mM EDTA)，然后加热至95℃，保持5分钟。再加入40μl20×SSC(3M NaCl/0.3 M柠檬酸钠，pH7.0)。使用500μl尿或0.25cm3淋巴样组织也可进行相似的处理。
实施例2
将提取的核酸转移至膜
在10×SSC中将每张尼龙膜(Zeta ProbeTM，Bio Rad)和Whatman3MM纸浸湿。将浸湿的Whatman纸放置在一个点杂交装置中(Minif-old IITM，Schleicher & Schuell)，并将浸湿的尼龙膜放置在Whatman纸的上面。将具有多个加样孔的盖放在装置上并锁紧，然后将该装置与真空源相连接。用100μl 20×SSC漂洗加样孔，然后将根据实施例1制备的样品在10μl TE＋40ml 20×SSG中加至加样孔，然后用100μl 20×SSC漂洗加样孔。随后将真空关闭并将尼龙膜移走。然后在10cm距离处，膜上侧正对UV源，将尼龙膜在短波(＜300mm)UV光下照射十分钟使得核酸交联至膜上。
实施例3
被标记探针的制备
为了制备放射性标记探针，在37℃，在含有100ng寡核苷酸(5μ1)、3μl[γ-32P]ATP(3000居里/毫摩尔，在10毫居里/毫升中)、1μl 10×激酶缓冲液和1μl T4聚核苷酸激酶(8-10单位/μl)的反应混合液中，用32P，将与掺入血液、尿或组织样品的寡核苷酸互补的寡核苷酸的5＇末端标记30分钟，然后加热至65℃，保持3分钟。然后用0.4M NH4OAc和乙醇使被标记的寡核苷酸沉淀，并再悬浮于50μl H2O中。
或者，根据制造商的说明，用Genius 5TM寡核苷酸加尾仪(Boehringer Mannheim)制备非放射活性的化学发光探针。在末端转移酶的存在下，使1μg寡核苷酸在3＇与辛高地配基-11-dduTP/dATP(DIG-11-dduTP/dATP)接尾。20μl反应混合液体积包括4μl 5X反应缓冲液(1M卡可酸钾、125mM Tris-HCl、1.25mg/ml牛血清白蛋白，在25℃时pH6.6)、4μl 25mM氧化钴、100微微摩尔寡核苷酸、1μl 1mM的DIG-ll-dduTP(2＇，3＇-二脱氧尿苷-5＇-三磷酸酯通过一个11原子的间隔臂与辛高地配基偶联)和50单位的末端转移酶。在37℃温育反应混合物15分钟，然后置于冰上。向反应混合物中加入1μl 20mg/ml的糖原和1μl 200mM的EDTA(pH8.0)，然后通过加入0.1体积4M的氯化锂和2.5体积的冰乙醇，然后在-70℃混合、温育30分钟使其沉淀。将寡核苷酸制成小球并用70％的乙醇洗涤该小球，直接重新悬浮于30μl的10mM Tris-HCl(pH7.0-8.0)/1mM EDTA/1％SDS。
实施例4
探针寡核苷酸的预杂交和杂交
在37℃下，10ml杂交缓冲液(1M NaCl/1％SDS/10％葡聚糖硫酸酯和150μg/ml tRNA)中将按照实施例2制备的膜预杂交1至3小时。向杂交溶液中的膜加入用3μg/ml最终浓度的未标记互补寡核苷酸(5×105cpm/ml；250ng/ml探针最终浓度)稀释的被标记寡核苷酸探针。在37℃下温育连续进行3-16小时。在6×SSC中洗涤膜2次(每次5-10分钟)，然后在室温下干燥。
实施例5
检测与膜特异性连接的探针
检测按照实施例4制备的与膜特异性连接的探针按如下步骤进行。将膜曝光于X射线底片，然后将其冲洗并经扫描光密度测定，与直接涂于膜的已知量的寡核苷酸样品比较。示于图2的对于血清样品的结果表明，每0.25ml血清检测到约3ng寡核苷酸。用尿和组织样品获得了相似的结果。
在抗原DIG连结至探针的实验中，探针是通过碱性磷酸酯酶接合的抗-DIG抗体Fab片断的结合，并按照制造商的说明，使用Genius 3TM核酸检测试剂盒(Boehringer Mannheim)来检测的。简单地说，在100mM Tris-HCl(pH7.5)/150mM NaCl溶液中，用封闭剂将实施例4所述方法制备的、具有特异连接的探针寡核苷酸的杂交膜封闭30-60分钟，然后在相同溶液中，将与碱性磷酸酯酶接合的抗-DIG抗体Fab片断加至膜上。将抗体溶液与膜一起温育30分钟，然后将膜移走并放置于一个新的杂交袋中。然后在室温下，在预先通过0.45μM滤膜过滤的100mM Tris-HCl(pH7.5)/150mMNaCl中将膜洗涤两次，每次15分钟。洗涤后，在膜还是湿的情况下，将膜置于两层醋酸纤维素膜之间。然后将0.5ml(每100cm2膜)的0.33mM4-甲氧基-4-(3-磷酸苯基)-螺(1，2＇-二氧杂环丁烷-3，2＇-金刚烷)二钠盐/750mM2-氨基-2-甲基-1-丙醇(pH9.6)/0.88mM MgCl2/1.13mM十六烷基三甲基溴化铵/0.035mM荧光素表面活性剂涂敷至膜的上表面，并在膜和乙酸纤维素膜之间形成液封。然后使覆盖有乙酸纤维膜的膜在一片XARTM底片(Kodak)上曝光60分钟。冲洗该曝光的底片并使用光密度检测仪和RV9342.0版光密度测定软件(EC仪器公司)扫描至计算机中。然后将平均密度对寡核苷酸的已知浓度作图。结果示于图4中。
这些结果表明本发明的方法能检测出体液或组织中以低至3ng/ml浓度存在的寡核苷酸。而且，用本发明的方法可对存在于体液或组织内的寡核苷酸进行定量分析。
Claims (6)
1、一种检测存在于体液或组织中的合成寡核苷酸的方法，该方法包括下列步骤：
(a)用蛋白酶消化体液或组织样品；
(b)用分配剂抽提该蛋白酶消化的样品；
(c)用异丁醇和乙醚抽提存在于样品中的核酸，包括待检测的合成寡核苷酸；
(d)将核酸重新悬浮至溶液中；
(e)将核酸连接至杂交膜上；
(f)用与待检测的合成寡核苷酸互补的标记寡核苷酸探针处理具有连接核酸的膜；
(g)洗涤膜以除去任何不与连接在膜上的核酸杂交的标记寡核苷酸；和
(h)检测膜上探针。
2、根据权利要求1的方法，其中体液是血液。
3、根据权利要求1的方法，其中体液是尿液。
4、根据权利要求1的方法，其中组织是淋巴样组织。
5、根据权利要求1的方法，其中标记的寡核苷酸探针是放射性标记的探针。
6、根据权利要求1的方法，其中标记的寡核苷酸探针是抗原或配体，而且探针的检测包括下列步骤：
(a)用合适的封闭剂封闭膜；
(b)将膜与特异性地与抗原或配体连接的、并与能作用于底物产生化学发光的酶接合的抗原-结合性抗体片断或受体一起温育；
(c)洗涤膜以除去任何非特异性连接的抗体、抗原结合性抗体片断或受体；
(d)加入酶作用的底物；和
(e)使膜曝光至照片底片。
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US278693A | 1993-01-08 | 1993-01-08 | |
| US08/002,786 | 1993-01-08 | ||
| US5636393A | 1993-04-30 | 1993-04-30 | |
| US08/056,363 | 1993-04-30 |
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| Publication Number | Publication Date |
|---|---|
| CN1116859A true CN1116859A (zh) | 1996-02-14 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN94190976A Pending CN1116859A (zh) | 1993-01-08 | 1994-01-07 | 检测从体液或组织提取的合成寡核苷酸的方法 |
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| Country | Link |
|---|---|
| US (1) | US5693466A (zh) |
| EP (1) | EP0680518B1 (zh) |
| JP (1) | JPH08507437A (zh) |
| KR (1) | KR960700349A (zh) |
| CN (1) | CN1116859A (zh) |
| AT (1) | ATE151816T1 (zh) |
| AU (1) | AU678821B2 (zh) |
| CA (1) | CA2153504A1 (zh) |
| DE (1) | DE69402683T2 (zh) |
| FI (1) | FI953364A (zh) |
| NZ (1) | NZ262561A (zh) |
| WO (1) | WO1994016103A2 (zh) |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4483920A (en) * | 1982-05-17 | 1984-11-20 | Hahnemann University | Immobilization of message RNA directly from cells onto filter material |
| US5118605A (en) * | 1984-10-16 | 1992-06-02 | Chiron Corporation | Polynucleotide determination with selectable cleavage sites |
| WO1988008036A1 (en) * | 1987-04-08 | 1988-10-20 | Invitron Corporation | Detection and identification of dna and/or rna in protein-containing samples |
| DE3813278A1 (de) * | 1988-01-12 | 1989-07-20 | Boehringer Mannheim Gmbh | Verfahren zum nachweis von nukleinsaeuren |
| US5149798A (en) * | 1989-04-06 | 1992-09-22 | Worcester Foundation For Experimental Biology | Process for synthesizing oligonucleotides and their analogs adaptable to large scale syntheses |
| ATE172466T1 (de) * | 1990-07-13 | 1998-11-15 | Microprobe Corp | Nicht-ätzende zusammensetzung und verfahren zur gewinnung von nukleinsäuren |
| US5223402A (en) * | 1990-08-30 | 1993-06-29 | Difco Laboratories | Method of detecting microbes utilizing chemiluminescent compound |
| WO1992018649A1 (en) * | 1991-04-12 | 1992-10-29 | Microprobe Corporation | Compositions and methods for improved extraction and hybridization of nucleic acid |
-
1994
- 1994-01-07 CA CA002153504A patent/CA2153504A1/en not_active Abandoned
- 1994-01-07 AT AT94909444T patent/ATE151816T1/de not_active IP Right Cessation
- 1994-01-07 EP EP94909444A patent/EP0680518B1/en not_active Expired - Lifetime
- 1994-01-07 KR KR1019950702852A patent/KR960700349A/ko not_active Application Discontinuation
- 1994-01-07 CN CN94190976A patent/CN1116859A/zh active Pending
- 1994-01-07 DE DE69402683T patent/DE69402683T2/de not_active Expired - Fee Related
- 1994-01-07 NZ NZ262561A patent/NZ262561A/en unknown
- 1994-01-07 AU AU62288/94A patent/AU678821B2/en not_active Ceased
- 1994-01-07 JP JP6516256A patent/JPH08507437A/ja active Pending
- 1994-01-07 WO PCT/US1994/000297 patent/WO1994016103A2/en active IP Right Grant
-
1995
- 1995-01-03 US US08/368,243 patent/US5693466A/en not_active Expired - Lifetime
- 1995-07-07 FI FI953364A patent/FI953364A/fi not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| EP0680518A1 (en) | 1995-11-08 |
| AU6228894A (en) | 1994-08-15 |
| DE69402683T2 (de) | 1997-09-25 |
| NZ262561A (en) | 1997-01-29 |
| KR960700349A (ko) | 1996-01-19 |
| EP0680518B1 (en) | 1997-04-16 |
| JPH08507437A (ja) | 1996-08-13 |
| FI953364A0 (fi) | 1995-07-07 |
| FI953364A (fi) | 1995-07-07 |
| DE69402683D1 (de) | 1997-05-22 |
| CA2153504A1 (en) | 1994-07-21 |
| ATE151816T1 (de) | 1997-05-15 |
| WO1994016103A2 (en) | 1994-07-21 |
| AU678821B2 (en) | 1997-06-12 |
| US5693466A (en) | 1997-12-02 |
| WO1994016103A3 (en) | 1994-09-01 |
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