CN101360819A - 反应容器及dna的扩增反应方法 - Google Patents

反应容器及dna的扩增反应方法 Download PDF

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CN101360819A
CN101360819A CNA2006800511123A CN200680051112A CN101360819A CN 101360819 A CN101360819 A CN 101360819A CN A2006800511123 A CNA2006800511123 A CN A2006800511123A CN 200680051112 A CN200680051112 A CN 200680051112A CN 101360819 A CN101360819 A CN 101360819A
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China
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substrate
reaction
film body
protuberance
reaction chamber
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佐藤里佳
植山公助
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Independent Administrative Institution Physical Chemistry Institute
Shimadzu Corp
Toppan Inc
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Independent Administrative Institution Physical Chemistry Institute
Shimadzu Corp
Toppan Printing Co Ltd
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Publication of CN101360819A publication Critical patent/CN101360819A/zh
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Abstract

在合成树脂制的基板(1)的背面设有线状凹部，以膜体(3)塞封所述线状凹部，构成由膜体(3)和基板围成的隧道状反应室(12)，同时膜体(3)从基板(1)的背面表面伸入反应室(12)侧而形成突出部(31)。能够抑制试剂等的蒸发，并且，由于基板(1)和膜体(3)的热膨胀率的差异所引起的缝隙只不过是在反应室(12)的侧壁和膜体(3)的突出部上(31)产生而可防止其减少，从而获得足量的反应生成物。

Description

反应容器及DNA的扩增反应方法
技术领域
本发明涉及适于生物体反应的一次性容器。例如，涉及一次性反应容器，其可适用于，采集含有来自人体的微量DNA的样品，使其进行PCR扩增并检查其单核苷酸多态性。
背景技术
单核苷酸多态性(SNP)，表示在DNA序列中单碱基被其他的碱基置换，由于该单碱基的不同，产生个人的个体差异，即易患病度、所开药剂的种类和效果以及副作用等上的差异。因此，为在遗传基因水平上对其体质进行检查并且对每种体质决定治疗方针和预防方针，此SNP的检查受到关注。
作为使用于该检查的DNA，例如，利用从人体采集的血液等样品中所包含的DNA，但是为了使采集的血液等样品少量即可，并有效率地进行检查，通过使采集的样品中的DNA扩增且检查扩增后的DNA，来检知所述SNP。
对于扩增样品中所包含的微量DNA的方法来说，已知有各种方法，其中PCR法作为代表性的方法被公知。该方法，将样品中的双链DNA的变性阶段(生成单链DNA)、退火阶段(将被称为引物的寡聚核苷酸和单链DNA的一部分进行杂交)、以及延伸阶段(以所述引物作为起点使核苷酸延伸)所构成的三个阶段作为一个循环、并且反复进行该循环使样品中的DNA扩增。在理论上，循环数作为n，则可扩增至2n倍。变性阶段在80～100℃进行，退火阶段在50～60℃进行，而延伸阶段在60～80℃进行。一个循环所需要的时间最多为10分钟左右，但是为了反复进行该循环扩增必要量的DNA，则也有花费数小时的情况。
另外，扩增获得DNA被用于此DNA中所包含的SNP的检查阶段(分型阶段)。分型方法有多种，其中侵入法作为代表性的方法被例举。在该方法中，使用两种非荧光标记寡聚核苷酸(等位基因探针(Allele probe)、侵入探针(Invader probe))、一种荧光标记寡聚核苷酸(FRET探针)以及对DNA结构特异性的核酸内切酶(裂解酶)。等位基因探针为5’端具有与模版DNA的序列不相关的序列(标签(フラツプ))、3’端具有相对模版DNA特异性的互补序列的寡聚核苷酸，其互补序列的5’端末端成为SNP部分。另一方面，侵入探针被设计，以从所述SNP部分开始与模版DNA的3’端互补性地结合。另外，FRET探针为具有荧光标记的寡聚核苷酸，在其5’末端具有荧光标记(报告基团(Reporter))，在其上游结合猝灭基团(Quencher)。并且，从报告基团开始3’端的部分进行自体杂交并构成双链，从该双链开始在3’末端侧具有与等位基因探针的标签呈互补序列的单链的部分。另外，裂解酶为识别核苷酸的三层重叠的部分、切断三层重叠的核苷酸的3’端并使其游离的酶。
另外，在该侵入法中，首先，在将检查对象的模版DNA和等位基因探针进行杂交后时，侵入探针的3’末端侵入SNP部分。因此，在该SNP部分，模版DNA、等位基因探针以及侵入探针相重叠形成三层结构。裂解酶识别该SNP部分的结构，使等位基因探针的标签切断或游离。然后，来自等位基因探针的所述游离标签与FRET探针进行杂交。通过杂交，在自体杂交的双链和来自等位基因探针的所述游离标签的交点形成三层结构，裂解酶再次识别此结构并切断FRET探针的报告基团，而从猝灭基团上被放开。然后，通过利用激发光进行照射，被切断游离的报告基团的荧光标记进行荧光显色。如果SNP部分的碱基与等位基因探针不相配，则不切断或游离来自等位基因探针的标签，因而，由于荧光发光率非常低，通过检出该荧光强度的差，可以检查SNP。另外，一般利用紫外光或者可视光作为激发光。
上述的DNA的检查技术提出了以下方法(JP特开平05-317030号公报)：即，由于污染(contamination)而使得其检查精度降低，因而使用一次性反应容器，在基板上设置多个凹部以分别作为收容室、反应室及检查室，在收容室内收容必要的试剂，同时在反应室使用该试剂等进行PCR扩增反应，在检查室进行分型(typing)反应。通过该方法，由于可在一次性反应容器中完成单一的检查对象的检查，可防止污染而进行正确地检查。
专利文献1：JP特开平05-317030号公报
发明内容
然而，如前所述，PCR扩增反应需要在80～100℃的范围反复进行热循环，并且为了能够获得必要量的DNA需花费数小时。在这种进行长时间加热的情况下，DNA和试剂蒸发，反而使其减量，从而出现不能获得必要量的DNA的情况。
因此，本发明的目的在于提供一种防止这些DNA和试剂蒸发且能够获得必要量的DNA的一次性反应容器和利用该反应容器的扩增反应方法。
即，技术方案1记载的发明为，一种反应容器，所述反应容器由基板和与所述基板一体化的膜体构成，所述基板在其背面具有线状凹部，通过所述膜体塞封该线状凹部构成由所述膜体和所述基板围成的隧道状反应室，其特征在于，所述膜体从所述基板背面表面伸入反应室侧而形成突出部。
另外，技术方案2记载的发明为，根据技术方案1所述的反应容器，其特征在于，所述突出部的高度为0.1～10μm。
另外，技术方案3记载的发明为，根据技术方案1或2所述的反应容器，其特征在于，所述膜体通过固化型粘接剂与基板形成一体化。
另外，技术方案4记载的发明为，根据技术方案1或2所述的反应容器，其特征在于，所述膜体通过热密封与基板形成一体化。
另外，技术方案5记载的发明为，根据技术方案1-4中任意一项所述的反应容器，其特征在于，在所述隧道状反应室的两端处具有贯通基板的贯通孔。
另外，技术方案6记载的发明为，根据技术方案1-5中任意一项所述的反应容器，其特征在于，所述隧道状反应室为遗传基因扩增反应室。
另外，技术方案7记载的发明为，一种遗传基因扩增反应方法，其特征在于，向技术方案5所述的反应容器的反应室中注入含有遗传基因的样品、遗传基因扩增试剂以及轻比重的不挥发性液体，进行所述遗传基因的扩增反应。
另外，技术方案8记载的发明为，根据技术方案7所述的遗传基因扩增反应方法，其特征在于，所述样品为生物体样品。
另外，技术方案9记载的发明为，根据技术方案7或8所述的遗传基因扩增反应方法，其特征在于，所述遗传基因扩增试剂为PCR反应试剂。
另外，技术方案10记载的发明为，根据技术方案7-9中任意一项所述的遗传基因扩增反应方法，其特征在于，所述不挥发性液体为矿物油、植物油或硅油。
附图说明
图1A是表示本发明的反应容器的例子的分解立体图，图1B为基板的背面立体图。
图2是表示收容室的要部剖面图。
图3是表示反应室的要部剖面图。
图4是表示检查室的要部剖面图。
图5A～C是表示剥离引导凸部形状的说明用平面图。
图6A～B是表示剥离引导凸部形状的说明用平面图。
图7是表示本发明的基板的其他例子的立体图，B是其背面立体图。
具体实施方式
本发明所涉及的反应容器由作为必须要件的基板和膜体构成。基板，在其背面上具有线状凹部，所述膜体将该线状凹部塞封，构成由膜体和基板围成的隧道状的反应室，该隧道状的反应室，例如，可用于DNA和RNA等遗传基因的扩增反应中。并且，膜体比基板的背面表面，更伸入反应室侧，形成突出部。该突出部仅为微微突起即可，例如其高度为0.1μm以上即可。并且，优选为10μm以下。另外，作为扩增反应，例如，可以DNA的PCR扩增反应为代表性示例。
在基板中，除所述隧道状反应室，还可设有其他的检查室。并且，在基板中，还可设有收容室，收容在这些反应室或检查室中进行化学反应所利用的试剂。另外，检查室和收容室可由膜体等进行密闭，也可不进行密闭而曝露在外。
例如，在基板上设有多个的收容室和检查室，在收容室中收容用于扩增反应的扩增试剂，其他的收容室中可收容用于扩增反应的稀释液等。在这种情况下，反应室可作为用于PCR扩增反应的PCR扩增反应室，该PCR扩增反应室中扩增检测体DNA所获得的检查对象可向多个检查室中分别注入，并在多个检查室中可进行各自不同的分型反应。并且，在这种情况下，优选地，该多个收容室和PCR扩增反应室都由盖材进行密封。另外，分型试剂可收容于所述收容室的一部分中，还可预先分别收容于多个所述的检查室中。收容室中收容用于PCR扩增反应的PCR试剂和稀释液等的同时，由于在各检查室中预先收容了分型试剂，从而使SNP检查阶段可在该反应容器上全部完成，可防止在PCR扩增阶段和分型阶段中出现操作误差，或者使用错误的试剂等人为差错，从而使SNP的检查正确地进行。
基板可通过合成树脂注射成型而被制造。作为合成树脂，只要在能够耐受反应时的热等反应条件的同时，不阻碍正确的反应即可。此外，当反应容器用于DNA的分型反应时，优选地使用激发光(紫外光或可见光)及荧光(可见光)的透过率高的合成树脂。例如，作为荧光标记物质所知的FAM的激发光的波长为494nm，荧光的波长为518nm，RED的激发光的波长为579nm，荧光的波长为595nm。优选地，基板对这些激发光及荧光的透过率为70％以上。最优选地为85％以上。
作为这种合成树脂，例如，聚乙烯和聚丙烯等聚烯烃树脂适于使用。或者，也可使用聚丙烯酸甲酯和聚甲基丙烯酸甲酯等丙烯酸系合成树脂。另外，除此之外，也可使用聚碳酸酯、聚对苯二酸乙二醇酯等聚酯系合成树脂、聚氯乙烯树脂、聚苯乙烯树脂等。
此外，出于同样的理由，优选地基板的厚度为2mm以下。超过2mm，激发光或荧光的透过率降低。优选地为1mm以下。并且，为防止热变形，优选地最少有0.3mm的厚度。
图1A是表示本发明的反应容器的例子，该反应容器由基板1，收容室用的膜状盖材2，反应室形成用膜体3以及检查室用保护膜体4构成，基板1具有横方向延长的长方形的外形。基板1的长边为5～15cm，短边为1～5cm。并且，沿其纵向方向，依次地设有9个收容室11，单一的PCR扩增反应室12，以及以3列×8个进行排列的24个检查室13。并且，为避免弄错基板1的方向，其纵向方向的侧边中的一边上设有切槽15，为防止纵向方向的弯曲等变形，沿其背面侧的两侧边上设有肋体14。另外，图1B是表示从背面侧看基板1的立体图。
9个收容室11都收容PCR扩增反应中适用的药品。也就是说，一部分收容室11中收容了含有聚合酶等的PCR试剂。并且，其他的收容室11中收容了稀释液。另外，这些PCR试剂、稀释液都为液体，因此，收容室11由膜状盖材2进行液密性地密封(参照图2)。并且，收容室11以凹部的形态在基板1中被设置，其内容积，较后述的检查室的内容积更大地被构成。例如，收容室11的开口部直径为5～10mm，深度为5mm以下。并且，其开口部的周围设有从基板1突出的线状的凸部111。并且，该凸部111的高度都相同，膜状盖材2横跨该凸部111的全体，整体地被密接。
这些收容室11，其内壁都被锥形地构成，以使得面向底部112，其横剖面积逐渐减少，收容于这些收容室11中的试剂等即使少量时，也可在预先确定的位置，即底部112中央汇集。这样，在试剂等在预先确定的位置上一直地汇集的情况下，使用注射器状注射器(syringe)或移液管等，可容易且准确地取出这些试剂。另外，作为收容室11，也可以内侧面的上部为均一横剖面积，其底部112形成锥形形状，此时试剂等也在预先确定的位置上汇集，也可容易且准确地取出。
另外，优选地，收容室11，至少其底部112与所收容的试剂等具有良好的亲和性。当亲和性不足时，如果试剂等的量少，因其表面张力，试剂等形成微小的球状，分散于各位置，难于在预先确定的位置上汇集。试剂等为亲水性时，为增大与试剂等的亲和性，可预先对收容室的底部112实施亲水化处理。例如，大气压等离子体处理，电晕放电处理，或者由臭氧气体等的氧化性药品进行的表面处理等。
另外，通过在收容室的底部112上设置微小的凹凸体，使表面能增大，提高与亲水性试剂等的亲和性，可使所收容的试剂等汇集在规定的位置。微小的凹凸体，例如，可通过对该底面实施喷砂处理进行设置。另外，对该底部112进行激光照射，也可在该底部112上形成微小的凹凸体。另外，使用在底部112对应的位置上实施了喷砂处理的模具，通过注射成型法制造基板1，也可在收容室底部112上设置微小的凹凸体。作为该微小的凹凸体，优选地，十点平均粗糙度为1.0μm以上。更优选地为1.5μm以上。并且，优选地，该凹凸体的十点平均粗糙度为100μm以下。更优选地为30μm以下。
在理论容量96.5μl的收容室中收容58.0μl的亲水性试剂，当***注射器状的尖端或移液管等，取出该试剂时，收容室底部112为平滑(十点平均粗糙度约0.2μm)的情况下，底部112残留的试剂为17～38μl(回收率70～34％)，与此相对，在使用砂编号A220(2μm)、压力4kg/mm、放射距离15cm的条件下实施喷砂处理，通过该实施了喷砂处理的模具进行注射成型，收容室底部112上形成十点平均粗糙度为2μm的凹凸体的基板1，在使用该基板1的情况下，其底部残留的试剂为9～17μl(回收率84～70％)。
另外，如前所述，在这些收容室11中预先收容使用的试剂等，因而非常方便(其中，优选地，至少一个的收容室保持空的状态。该空的收容室用于作为收容从人体采取的血液等样品的部位)。因为PCR试剂、稀释液等为液体状态，优选地，这些液状的试剂等收容于收容室11中的一部分的收容室中，由盖材2液密性地密封。作为盖材2，可使用合成树脂的注射成型品，优选地，如该实施例，使用膜状的盖材与收容室11的开口部周围的凸部111相密接。虽然，对于各个收容室11而言，也可适用分别独立的个别的膜状盖材，由于凸部111线状地被构成且形成为相同的高度，优选地，使用1枚较大面积的膜状盖材2与收容室11的全部凸部111整体相密接。另外，例如，通过使用注射器状注射器等，从膜状盖材2刺穿***，可取出收容室内的试剂。并且，收容从人体采取的血液等样品的情况时，也可将注射器状注射器等刺穿***，收容于收容室中。因此，膜状盖材2没有必要可从基板剥离。当然，可将该膜状盖材2可剥离地密接在凸部111上，在这种情况下，可剥离膜状盖材2的一部分或全部，使收容室11露出，进行使用。
作为收容室用的膜状盖材2，可使用合成树脂膜。作为这种合成树脂膜，例如，可举例为聚乙烯和聚丙烯或者聚甲基戊烯等的聚烯烃膜、聚丙烯酸甲酯或聚甲基丙烯酸甲酯等的丙烯酸系合成树脂膜、聚苯乙烯膜、聚缩醛膜、聚酰胺膜、聚丙烯腈膜、聚碳酸酯膜、聚环烯烃系膜、硅树脂系膜、氟素系树脂膜等。另外，作为膜状盖材3，也可使用金属箔或金属箔上层积合成树脂膜的层积膜。另外，膜状盖材2可为透明的膜，也可为不透明的膜。
收容室用的膜状盖材2，例如，可使用耐热性的粘接剂与凸部111相密接。例如，固化性粘接剂。作为这种固化性粘接剂，可举例为环氧(epoxy)系粘接剂、尿烷(urethane)系粘接剂等。另外，也可使用含有丙烯酸系单体和光开始剂的光固化性粘接剂。另外，也可通过热密封进行密接。
其次，PCR扩增反应室12为进行PCR扩增反应的部位。如图3的要部剖面图所示，该PCR扩增反应室12呈隧道状结构。即，基板1的背面具有线状的凹部，反应室形成用膜体3与该线状凹部的周围相密接、塞封该线状凹部，从而形成被反应室形成用膜体3和基板1围绕的隧道状部位，该隧道状部位作为PCR扩增反应室12。如前所述，虽然PCR扩增反应有时在高温下加热1小时以上，而使其反应，但通过在机密性高的隧道状的PCR扩增反应室12中进行反应，注入比前述样品和试剂的比重更轻的不挥发性液体，由于其表面被不挥发性液体覆盖，可防止反应液的蒸发。另外，作为不挥发性液体，可使用矿物油、植物油或硅油。
另外，在该隧道状PCR扩增反应室12的两端设有贯通基板1的贯通孔121，在基板1的表面设有具有与贯通孔121连通的中心孔的筒状突出部122，筒状突出部122的中心孔与贯通孔121连通，可将PCR试剂、稀释液以及不挥发性液体向隧道状PCR扩增反应室12中注入，并且可从隧道状PCR扩增反应室12中取出扩增所获得的检查对象。另外，为防止中心孔的污染等，在筒状突出部122的上部可密接图中未示出的保护膜。
另外，优选地，隧道状PCR扩增反应室12的高度，即线状凹部的深度在0.1～5.0mm的范围内。若较之更浅，则通过PCR扩增反应，难于反应获得向各检查室分注所必须的量的检查对象。并且，若较之更深，则不能充分传递PCR扩增反应所必须的热，有可能不产生必要的扩增反应。
另外，隧道状PCR扩增反应室12可呈与所述两贯通孔121直线地连接形状，但为抑制反应液的蒸发，优选地，具有在所述两贯通孔121之间弯曲的线形状。例如，圆弧状、Z字状、コ字状，或者由它们组合而成的形状。图7A及B示出了设有コ字状的隧道状PCR扩增反应室12的基板的立体图以及背面立体图。
另外，优选地，塞封线状凹部而构成PCR扩增反应室12的反应室形成用膜体3，在该线状凹部内的位置上，一般地比基板1表面稍微更向PCR扩增反应室12侧伸入，构成突出部31。因为基板1和膜体3的热膨胀率一般不相同，在PCR扩增反应的三阶段(DNA变性阶段、退火阶段，延伸阶段)的热循环下，在基板1和膜体3之间有时会产生缝隙。由于膜体3比基板1的背面表面更向PCR扩增反应室12突出，即使产生这样的缝隙时，也只不过是在PCR扩增反应室12的侧壁和膜体3的突出部31上产生，该缝隙不会在基板1和膜体3之间产生。所述突出部31的高度x可为0.1～10μm。
作为反应室形成用膜体3，优选地使用押压下可延伸的膜体，例如，可使用热可塑性合成树脂膜。膜体4即可为透明，也可为不透明。另外，也可使用金属箔上层积合成树脂膜的层积膜。作为这种热可塑性金属箔，例如，优选地可使用铝箔。在使用含有金属箔的层积膜的情况中，因该层积膜水蒸气阻碍性高，反应时传热性良好，可使PCR扩增反应高效率地进行。
反应室形成用膜体3，例如，可使用耐热性粘接剂粘接至基板1上。例如，热固化性粘接剂。作为这种固化性粘接剂，可举例为环氧系粘接剂、尿烷系粘接剂等。另外，也可使用含有丙烯酸系单体和光开始剂的光固化性粘接剂。并且，在反应室形成用膜体3的一面上整面地涂布固化性粘接剂，将该粘接剂面叠放在基板1上，在隧道状PCR扩增反应室12的底部中央附近以具有突出部的押压型进行按压，一边使反应室形成用膜体3稍微延伸，一边使其向PCR扩增反应室12内突出，以这种状态进行加热或照射紫外线，可使其固化而粘接。此时，虽然固化后的粘接剂在PCR扩增反应室12的底面上露出，因该粘接剂固化完成，PCR扩增反应不受该粘接剂的阻害，可进行正确的PCR扩增反应。
另外，反应室形成用膜体3也可通过热密封与基板1密接。也就是说，所述热可塑性树脂膜面向基板1叠放，在隧道状PCR扩增反应室12的底部中央附近以具有突出部的押压型进行按压的同时，进行加热，该膜体3可与基板1密接。此时，膜体3在PCR扩增反应室12的底面上露出，并不阻害PCR扩增反应，可进行正确的PCR扩增反应。
下面，对检查室13进行说明。图示中以3列×8个进行排列的24个检查室13，为分型反应进行的部位。检查室13不限于24个，因为各检查对象不同调查的SNP的数量不同，各SNP使用的试剂不同，优选地，设有各种的试剂的数量的检查室13。另外，在这些多个的检查室13中，通过预先收容相互不同种类的分型试剂，各个检查室13的分型反应可特定化，从而防止检查错误。
如图4的要部剖面图所示，检查室13在基板1中以凹部的形态被设置。另外，优选地，进行分型反应的检查室13，相比所述收容室11，由较小内容量的凹部构成。例如，其开口部的直径以及深度可为5mm以下。优选地，为0.01～5mm。分型反应以PCR扩增反应室13中DNA扩增后的微量的反应生成物作为检查对象，对于这样微量的检查对象有必要使其进行精度高的反应，并且，有必要使从基板1的背面照射的激发光在该检查对象上正确地集光的同时，在该被集光的激发光作用下使正确地发生荧光，并由检查装置准确地检知该荧光。假如使微量的检查对象在较大的反应室中进行反应，由激发光发生的荧光很弱，因此，可能不被检知。
另外，优选地，检查室13，通过将其侧壁131做成锥状，在将PCR扩增反应室13中DNA扩增后的检查对象进行分注时，可防止气泡卷入，使这些检查对象准确地收容在检查室13的底部，同时，其底面132呈平面状，防止从基板背面照射的激发光的弯曲或偏向。另外，出于同样的理由，优选地，与该底面相对的基板1的背面133也呈与底面132平行的平面。
另外，优选地，为确保激发光及荧光的透过率为70％以上，底面132与相对于其的基板1的背面133的距离(基板1的厚度)为2mm以下。更优选地，为1mm以下。
另外，为防止检查对象分注时的气泡，优选地，底面132与侧壁131的构成角度在100～140度的范围内。分型试剂可以固体的形态收容或预置成与检查室13的底面132接触的状态。
为密闭检查室13、防止其污染，有必要在构成该检查室13的凹部的开口部周围设置线状凸部134，该凸部134处预先可剥离地粘接保护膜体4而与基板形成一体化。保护膜体4在检查室13使用之前被剥离除去。
保护膜体4与全部24个检查室13整体密接，因此，有必要被构成为覆盖全部24个检查室13的大小。并且，通过从其端部开始剥下，可使全部24个的检查室13同时开口。
另外，沿保护膜体4的预定剥离方向，该反应容器有必要具有剥离引导凸部。如前所述，剥离引导凸部与开口部周围的线状凸部134可独立地被设置，但图示的例子，作为其一部分包含凹部周围的前述凸部134，而由凹部周围的该凸部134和连接该凸部彼此的连接凸部135构成。在该剥离引导凸部上密接前述保护膜体4，并引导保护膜体4的剥离。也就是说，通过相对于该剥离引导凸部的全体以均匀的粘接力密接保护膜体4，从剥离开始到剥离结束，可以一定的力进行剥离使该24个检查室13同时开口。
出于上述理由，优选地，构成剥离引导凸部的凹部周围的前述凸部134和前述连接凸部135都为线状。此时，在保部膜体4上施加均匀的压力的同时，可使保护膜体4和线状凸部粘接。并且，均匀的压力下被粘接的膜体和线状凸部，其粘接力也均匀，从剥离开始到剥离结束，可以一定的力进行剥离。
另外，优选地，构成剥离引导凸部的凹部周围的前述凸部134的宽度和前述连接凸部135的宽度为基本相同。此时，从剥离开始到剥离结束，可以一定的力且不断破地进行剥离。优选地，最窄部位的宽度作为100％、最宽的部位为200％以内。
另外，优选地，凹部周围的前述凸部134的宽度和前述连接凸部135的宽度都为0.1～3mm。当这些凸部134及连接凸部135的宽度为0.1～3mm时，可容易且中途不断破地剥离保护膜体4。
另外，当剥离引导凸部与凹部周围的前述凸部134独立地被构成时，优选地，剥离引导凸部和凹部周围的前述凸部134都为线状，并且，优选地，其顶部(即，与保护膜体4密接的部位)以基本相同的宽度被构成。并且，优选地，这些剥离引导凸部的宽度与凹部周围的前述凸部134的宽度都为0.1～3mm。
另外，作为剥离引导凸部的剖面形状，可呈长方形、梯形、或外形线呈圆弧或椭圆弧的扇形等。另外，构成剥离引导凸部的凹部周围的前述凸部134的剖面形状与前述连接凸部135的剖面形状也可不同。
另外，优选地，以保护膜体4的面积作为基准，剥离引导凸部为其1～80％范围的面积。若较之更宽，则难于剥离。并且，若较之更窄，则密接强度将不足。当剥离引导凸部与凹部周围的前述凸部134独立地被构成时，凸部全体的面积在1～80％的范围内。
其次，剥离引导凸部并不必须以全体1条线状凸部构成，例如，也可由相互独立的多条线状凸部的全体构成。无论哪种情况，优选地，从剥离的开始位置到结束位置，在剥离预定方向的全体上连续。此时，通过朝剥离方向进行剥离，可将保护膜体5中途不断破地全部地剥离，露出前述多个的凹部。另外，当前述剥离预定方向为反应容器的纵向方向时，因与剥离预定方向正交的方向为短边方向，剥离时剥离力在剥离正交方向上不分散，从而可沿剥离预定方向膜体不破裂地，在所述短边方向整体上剥离膜体。
图1的例子中，沿反应容器的纵向方向排列了3列×8个的共计24个的检查室13，因其纵向方向(图示中的横向方向)作为剥离预定方向，设有连接凸部135，以连接包含在各列上的凹部周围的前述凸部134，设有相互独立地三条线状凸部，其全体作为引导凸部。另外，图5A是表示图1的连接凸部135的形状的说明用平面图。
另外，图5B是表示其他的连接凸部135的形状的说明用平面图，以×字状的连接凸部135将斜向相邻的检查室13的凸部134之间共计24个的检查室13相连，作为整体在其剥离预定方向的全体上呈连续的形状。另外，图5C的例子中，第二列的第二个检查室13与第一列及第三列的第一个检查室13、第一列及第三列的第三个检查室13连接，如此地，各列的检查室13交替地且相互不同地连接，从而，凹部周围的前述凸部134与前述连接凸部135构成Z字形线的同时，作为整体在其剥离预定方向的全体上呈连续的形状。另外，也可相互邻接的检查室13之间以连接凸部135被连接，作为整体呈连接全部检查室13的矩阵状(参考图6D)。另外，全部24个的检查室13中，也可连接位于外侧的18个检查室13，作为整体在其剥离方向的全体上呈连续形状(参考图6E)。
其次，优选地，保护膜体4在其一部分上具有作为剥离开始部41的未粘接部。并且，优选地，该剥离开始部位于保护膜体4的纵向方向的端部。图1的例子中，保护膜体5因横方向为纵向方向，图中可设在左侧端部(即，收容室11侧的端部)。此时，可不弄错剥离开始位置地、从该开始位置开始剥离，并将保护膜体4的整体正确地剥离，露出全部的前述多个的凹部13。
另外，优选地，该剥离开始部41位于连接保护膜体4被粘接的部位彼此的直线上。此时，保护膜体4因在剥离开始部的两侧被粘接或固定，由于该粘接部位间的张力，其间的剥离开始部41可不松动地被固定，当剥离开始时能够正确地把持并容易地剥离。
其次，优选地，剥离引导凸部在顶部(即，与保护膜体4的密接部位)无高低差别，其顶部全体呈平面或光滑的曲面。此时，当保护膜体4粘接时，可使该保护膜体4在剥离引导凸部的整体上均匀地可靠地粘接。另外，无论凹部周围的前述凸部134是否构成剥离引导凸部的一部分，优选地，凹部周围的前述凸部134与剥离引导凸部具有基本相同的高度。即使有高度不相同的部位，最高部位的高度也在最低部位高度的150％以下。
另外，优选地，剥离引导凸部的高度为0.05mm以上。当剥离引导凸部与凹部周围的前述凸部134独立地构成时，这些剥离引导凸部的高度与凹部周围的前述凸部134的高度都为0.05mm以上。此时，当保护膜体4粘接时，该膜体与剥离引导凸部和凹部周围的前述134粘接，且不与凸部以外的部位粘接，因而从剥离开始至剥离结束，可以一定的力且不断破地进行剥离。优选地，高度为0.05～2mm。
其次，作为保护膜体4，例如，可举例为聚乙烯和聚丙烯或者聚甲基戊烯等的聚烯烃膜、聚丙烯酸甲酯和聚甲基丙烯酸甲酯等的丙烯系合成树脂膜、聚苯乙烯膜、聚缩醛膜、聚酰胺膜、聚丙烯腈膜、聚碳酸酯膜、聚环烯烃系膜、硅树脂系膜、氟素系树脂膜等。另外，作为保护膜体5，也可使用金属箔或金属箔上层积合成树脂膜的层积膜。另外，保护膜体4可为透明的，也可为不透明的。
该保护膜体4，例如，通过热密封，可与前述凸部可剥离地粘接。为调整其粘接强度使剥离容易，优选地，保护膜体4的热密封面与基板1使用不同种类的树脂材料。例如，当基板1为聚丙烯制时，作为保护膜体4可使用聚乙烯膜和热密封面为聚乙烯的层积膜。
此外，如前所述，基板1具有长边5～15cm、短边1～5cm的细长形长方形形状，因此，在成型时的热量、反应室形成用膜体3和检查室用保护膜体4热密封时的热量、或者PCR扩增反应或分型反应时的热量作用下，有时产生弯曲等变形。假如，检查室13的存在部位的基板1上发生弯曲等变形，则检查室13的底面132及与此底面相对的基板1的背面133发生倾斜，由于该倾斜，通过检查室13背面照射的激发光发生弯曲或偏向，并且，距该激发光的光源的距离发生变化，检查对象的位置与集光的位置不一致，再加上该激发光下产生的荧光也发生弯曲或偏向，难于准确地检查。因此，优选地，多个检查室13的底面132位于同一平面上。如果即使倾斜，位于多个检查室13中的两端的检查室13的底面132处建立的法线，尽可能为4度以下的角度的相交的角度。优选地为1度以下。并且，位于两端的检查室13的底面132的高度差尽可能为4.0mm以下，优选地为1.0mm以下。
变形防止肋体14，是防止这样的变形而相对多个检查室13的全体保持其底面在同一平面上的元件，其设置于检查室13存在部位的纵向方向两侧边上。变形防止肋体14，也可超过检查室13的存在部位而在反应容器的纵向方向两侧边的全长上设置，优选地，为用于把持该反应容器，收容室11侧的端部的纵向方向两侧边y的基板1的表面及背面均为平坦。
在图1所示的例子中，虽然变形防止肋体14设置于基板1的背面，也可设置于基板1的表面，或者表面和背面两者上。并且，该变形防止肋体14可以突出于基板1的表面或背面的直线状凸部的形态进行设置。基板1的厚度为0.3～2mm时，优选地，变形防止肋体14的高度为0.1～5mm、宽度为0.5～5mm。
此处，注射成型长边为10.0cm、短边为2.5cm、厚度为0.5mm的聚丙烯制基板，对其弯曲量进行测定。并且，测定如下进行。即，准备两个平坦的支持台，固定该2个支持台以其表面形成同一平面，以该平面作为基准面后，将前述基板的两端在这2个支持台上放置，测定基板1背面的中央与基准面的距离作为弯曲量。并且，测定位于两端的检查室13的底面处建立的法线的交叉角度。
在无变形防止肋体14时，注射成型后即刻测量的弯曲量为0.9mm，前述交叉角度为约0度55分。随后，在190℃、5秒的条件下用反应室形成用膜体3及检查室用保护膜体4进行热密封后，弯曲量为4.2mm，前述交叉角度为约4度20分。
随后，以同一尺寸，注射成型长边的两侧边上具有变形防止肋体14的聚丙烯制基板。并且，变形防止肋体14被形成于基板的表侧，从检查室13的侧端部开始长度约8.0mm，收容室11的端部的两侧边y上不形成变形防止肋体14而保留平坦。并且，该变形防止肋体14高度为1.0mm、宽度为1.0mm。注射成型后即刻测量的弯曲量为0.9mm，前述交叉角度为约0度55分。并且，在前述条件下用反应室形成用膜体3及检查室用保护膜体4进行热密封后，弯曲量为3.2mm，前述交叉角度为约3度20分。
另外，以同一尺寸，注射成型长边的两侧边上具有变形防止肋体14的聚丙烯制基板。并且，变形防止肋体14被形成于基板的表侧，从检查室13的侧端部开始长度约8.0mm，收容室11端部的两侧边y上不形成变形防止肋体14而保持平坦。并且，该变形防止肋体14高度为1.0mm、宽度为1.5mm。注射成型后即刻测量的弯曲量为0.4mm，前述交叉角度为约0度15分。并且，在前述条件下用反应室形成用膜体3及检查室用保护膜体4进行热密封后，弯曲量为0.75mm，前述交叉角度为约0度45分。
如上，涉及技术方案1的本发明中，通过塞封基板背面的线状凹部，被膜体和基板围成的隧道状部位作为反应室，增大了密闭性，可抑制反应液等的蒸发，即使进行长时间的加热反应也能够抑制其减少。
另外，由于所述膜体从基板的表面伸入反应室侧而形成突出部，基板和膜体的热膨胀率的差异所引起的缝隙只不过在反应室的侧壁和膜体的突出部上产生，可降低进入该缝隙而损失的试剂等。
并且，相应地，即使通过长时间的加热反应，也可获得足够的反应生成物。
另外，涉及技术方案2的本发明中，因所述突出部的高度为0.1～10μm，进入缝隙而损失的试剂等很少，其量可减少到最小限度。
另外，涉及技术方案3的本发明中，所述膜体通过固化型粘接剂与基板形成一体化，另一方面，涉及技术方案4的本发明中，因通过热密封形成一体化，可不妨碍反应室中的反应地使反应正确地进行。
另外，涉及技术方案5的本发明中，因在所述隧道状反应室的两端处具有贯通基板的贯通孔，可从该贯通孔向隧道状反应室注入试剂，并取出反应生成物。
另外，涉及技术方案6的本发明中，因所述隧道状的反应室作为DNA等遗传基因的扩增反应室，长时间地反复进行热循环后，也可充分地获得作为反应生成物的扩增后的遗传基因。
另外，涉及技术方案7～10的本发明中，由于除含遗传基因的样品和遗传基因扩增试剂之外，还注入了比重较轻的不挥发性液体，进行遗传基因的扩增反应，尽管隧道状反应室两端处具有贯通孔，也能够防止从贯通孔中蒸发反应液，从而获得必要量的扩增后遗传基因。

Claims (10)

1.一种反应容器，该反应容器由基板和与该基板一体化的膜体构成，所述基板在其背面具有线状凹部，通过所述膜体塞封该线状凹部构成由所述膜体和所述基板围成的隧道状反应室，其特征在于，所述膜体从所述基板背面表面伸入反应室侧而形成有突出部。
2.根据权利要求1所述的反应容器，其特征在于，所述突出部的高度为0.1～10μm。
3.根据权利要求1或2所述的反应容器，其特征在于，所述膜体通过固化型粘接剂与基板形成一体化。
4.根据权利要求1或2所述的反应容器，其特征在于，所述膜体通过热密封与基板形成一体化。
5.根据权利要求1-4中任意一项所述的反应容器，其特征在于，在所述隧道状反应室的两端具有贯通基板的贯通孔。
6.根据权利要求1-5中任意一项所述的反应容器，其特征在于，所述隧道状反应室为遗传基因扩增反应室。
7.一种遗传基因扩增反应方法，其特征在于，向权利要求5所述的反应容器的反应室中注入含有遗传基因的样品、遗传基因扩增试剂以及轻比重的不挥发性液体，进行所述遗传基因的扩增反应。
8.根据权利要求7所述的遗传基因扩增反应，其特征在于，所述样品为生物体样品。
9.根据权利要求7或8所述的遗传基因扩增反应，其特征在于，所述遗传基因扩增试剂为PCR反应试剂。
10.根据权利要求7-9中任意一项所述的遗传基因扩增反应，其特征在于，所述不挥发性液体为矿物油、植物油或硅油。
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