CN1180248C - 优化电子杂交反应的方法 - Google Patents
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Abstract
本发明涉及关于在微电子片和装置中改进或最优化DNA迁移速度,DNA杂交反应的效率,和总的杂交特异性的各种参数,电解质(缓冲液),和其它条件的发现。具体地说,本发明涉及的发现是,低导电性两性离子缓冲溶液,特别是那些含有在大约50mM浓度和在接近pI(等电点大约pH7.47)下制备的氨基酸组氨酸的溶液提供用于迅速电泳DNA迁移和高效杂交反应的最佳条件。实现了相对于次佳已知缓冲液,半胱氨酸至少10倍的杂交效率。试验数据证实与半胱氨酸相比杂交效率增加大约50,000倍。
Description
本发明领域
本发明涉及适应于医学诊断,生物学和其它用途的用于电子装置的缓冲液和电解质。更具体地说,本发明涉及对于在微电子医学诊断装置上进行的DNA杂交分析有利的缓冲液和电解质。
本发明背景
最近,对于结合微电子学和分子生物学的装置的兴趣逐渐增加。在申请日为1993年11月1日的系列号08/146,504,“用于分子生物学分析和诊断学的可主动程序化的电子装置”中公开了一种这类系统,该文献现已作为美国专利号5,605,662出版,本文作为参考文献引用。其中公开的系统将称为APEX系统。APEX系统可执行许多功能,在诸如核酸杂交,抗体/抗原反应,临床诊断和生物多聚体合成的分子生物学反应中使用具有优势。
APEX型装置利用缓冲液和电解质用于其操作。缓冲液定义为在加入酸或碱时对pH改变具有抗性的化学溶液。例如,参见,生物技术词典,第二版,James Coombs,Stockton出版。如其中所述,“传统上说,以无机盐(磷酸盐,碳酸盐)和有机酸盐(乙酸盐,柠檬酸盐,琥珀酸盐,甘氨酸,马来酸盐,巴比妥盐等)为基础的缓冲液用于生物学实验。”
本发明的目的是发现了在进行杂交,反应,诊断或合成的分子生物学电子装置中使用有优势的缓冲液和电解质。
本发明概述
下述发明涉及我们关于在APEX微电子片和装置中改进或最优化DNA迁移速度,DNA杂交反应的效率,和总的杂交特异性的各种参数,电解质(缓冲液),和其它条件的发现。具体地说,本发明涉及的发现是,低导电性两性离子缓冲溶液,特别是那些含有在10-100mM,优选大约50mM浓度和在接近pI(等电点大约pH7.47)下制备的氨基酸组氨酸的溶液提供用于迅速DNA迁移和高效杂交反应的最佳条件。实现了相对于次佳已知缓冲液,半胱氨酸,至少10倍的杂交效率。试验数据证实与半胱氨酸相比杂交效率增加大约50,000倍。
附图的简要描述
图1是用于组氨酸缓冲液的棋盘排列的平面图。
本发明的详细描述
有各种物理参数涉及DNA和其它带电分析物在各种类型的电解质/缓冲液中的电泳转移。某些装置,例如,在上文引用的美国专利号5,605,662中所述的申请人的APEX装置,基本上是DC(直流)电装置,它在装置的表面产生电场。这些电场接着又引起带电分子在该装置表面相对(+/-)有偏压的微区域之间出现电泳迁移。相反,所谓的Genosensor(阻抗传感器),参见,例如,Hollis等,“用于分子检测的光学和电学方法及装置”,WO93/22678,和双向电泳装置,参见,例如,Washizu 25静电学杂志,109-123,1990涉及使用AC电场。涉及这些装置的重要区别是,当应用AC电场时,在任何这类系统中基本上没有净电流,即,没有使带电分子迁移的电泳推进力。当应用电压时APEX型装置产生明显的净直流(DC)电,它被承认为“电泳的特征”。在电泳中,离子和带电颗粒的迁移由沿电场梯度方向的电作用力产生,且电流和电压的关系对于该技术是重要的。电泳迁移本身在宏观上表现为在所用电压影响下溶液中的电流传导且遵从欧姆定律:
V=R×I
V是电势
R是电解质的电阻[V×A-1=R(Ω)]
I是电流[A]。
溶液的电阻是导电性的倒数,导电性可用电导计测量。导电性主要取决于缓冲液/电解质中的离子种类和其浓度;因此这些参数对于涉及电场的分子生物学技术非常重要。基础电流/电压关系对于APEX技术与对于任何其它电泳系统基本上是相同的,尽管产生的电场事实上是微观环境。
APEX系统关于产生电流和电压的各种方式具有独特的特征,且已发现电流和电压方案如何改进该系统的效果。具体地说,各种DC脉冲方案(线性和对数梯度)似乎提高了杂交严格性。
电泳迁移与离子强度
在电泳领域已充分认识到带电分析物种类(蛋白质,DNA等)的迁移率对数减少,它与电解质溶液离子强度的平方根成反比(参见“毛细电泳:原理和实践”,R.Kuhn和S.Hoffstetter,Springer-Verlag,1993第83页和图3.16)。在任何给定的恒定电场强度下,随着电解质浓度相对于分析物种类(蛋白质,DNA等)的减少,分析物以更快的速率迁移。证实丹磺酰化氨基酸的该效应的相似的结果由J.J.Issaq等,色谱,第32卷,#3/4,1991年8月,155页至161页(特别是参见第157页图3)显示。在不同的电解质溶液中证实DNA的该效应的结果在P.D.Ross和R.L.Scruggs,生物多聚体,第2卷,第231至236页,1964(特别是参见232页图1)中显示。
离子强度/导电性关系
对于那些涉及在溶液中完全解离的阴离子和阳离子种类(Na+← →CL-,K+← →CL-,等)的非缓冲电解质,离子强度与导电性是相当的,即,导电性通常与离子强度成比例。对于那些解离状态(例如,2Na+← →PO4 -2)的缓冲电解质(磷酸盐,乙酸盐,柠檬酸盐,琥珀酸盐等),离子强度与导电性通常是相当的,即,导电性与离子强度成比例。对于那些具有两性离子种类(在其pI下无净电荷)的缓冲电解质[Good缓冲液(MOPS,HEPES,TAPS,Tricine,Bicine),氨基酸缓冲液,两性电解质等],导电性在等电点(pI)和(pKa)之间每pH单位差异将减少大约10倍。例如,氨基酸在其两性离子状态(-OOC-CH(R)-NH3 +)导电性值比“氨基酸基团”具有全部净正电荷(HOOC-CH(R)-NH2 +← →X-),或全部负电荷(Y+← →-OOC-CH(R)-NH2)时低大约1000倍。因此,外部负或正电荷随着其离开pI在氨基酸基团上形成,且导电性和离子强度开始相关。然而,处于或接近pI时,对于给定离子强度或浓度导电性比预期低得多。当在处于或接近其pI处使用时,电泳试验涉及Good缓冲液和氨基酸缓冲液,因为“在高离子强度或浓度时具有低导电性”(参见“毛细电泳:原理和实践”第88页,R.Kuhn和S.Hoffstetter,Springer-Verlag,1993)。常用的电泳缓冲液“Tris-硼酸盐”实际上比从其离子强度和浓度所预期的具有明显更低的导电性。这可能是由于“Tris阳离子”和“硼酸阴离子”在溶液中形成相对稳定的两性离子复合物。测定了100mM Tris-硼酸盐溶液的导电性是694μS/cm,比从其离子强度所预期的大约低20倍,且大约相当于5mM磷酸钠或氯化钠溶液。表1显示了许多迁移缓冲液的导电性测量值。
表1
溶液/缓冲液 | 测量值 | 测量值 | 测量值 | 平均/标准偏差 |
10mMMgCl2 | 1.95mS/cm | 2.02mS/cm | 2.13mS/cm | 2.03+/-0.09mS/cm |
1mM MgCl2 | 174μS/cm | 208μS/cm | 177μS/cm | 186+/-18.8μS/cm |
0.1mMMgCl2 | 16.9μS/cm | 16.7μS/cm | 18.3μS/cm | 17.3+/-0.87μS/cm |
10mM NaCl | 1.07mS/cm | 1.10mS/cm | 1.18mS/cm | 1.12+/-0.057mS/cm |
1mM NaCl | 112μS/cm | 115μS/cm | 111μS/cm | 112.7+/-2.08μS/cm |
0.1mMNaCl | 8.80μS/cm | 8.98μS/cm | 10.5μS/cm | 9.43+/-0.93μS/cm |
20mMNaPO4 | 2.90mS/cm | 2.79mS/cm | 3.00mS/cm | 2.90+/-0.11mS/cm |
10mMNaPO4 | 1.40mS/cm | 1.44mS/cm | 1.48mS/cm | 1.44+/-0.04mS/cm |
1mM NaPO4 | 122μS/cm | 128μS/cm | 136μS/cm | 128.7+/-7.0μS/cm |
50mM TRIS | 3.50mS/cm | 3.14mS/cm | 3.40mS/cm | 3.35+/-0.19mS/cm |
10mM TRIS | 572μS/cm | 562μS/cm | 583μS/cm | 572+/-10.5μS/cm |
250mMHEPES | 141μS/cm | 144μS/cm | 158μS/cm | 147.6+/-9.07μS/cm |
25mMHEPES | 9.16μS/cm | 9.44μS/cm | 10.5μS/cm | 9.7+/-0.71μS/cm |
3.3mM柠檬酸钠 | 964μS/cm | 964μS/cm | 1.03mS/cm | 986+/-38.1μS/cm |
5mM琥珀酸钠 | 1.05mS/cm | 960μS/cm | 1.01mS/cm | 1.01+/-0.045mS/cm |
5mM草酸钠 | 1.02mS/cm | 1.03mS/cm | 1.12mS/cm | 1.06+/-0.055mS/cm |
10mM乙酸钠 | 901μS/cm | 917μS/cm | 983μS/cm | 934+/-43.5μS/cm |
250mM半胱氨酸 | 27.4μS/cm | 17.3μS/cm | 23.5μS/cm | 22.7+/-5.09μS/cm |
Milli-Q水 | <0.5μS/cm | 检测极限 | ||
0.1单元太低 |
两性离子缓冲液/导电性/迁移率
当使用处于或靠近其pI的两性离子缓冲液(Good缓冲液,氨基酸缓冲液)或Tris-硼酸盐缓冲液时对于DNA电泳迁移的速率或速度具有优势。这些优势有:1)这些缓冲液可以相当高的浓度使用以提高缓冲能力,2)其导电性在相同浓度下比其它类型的缓冲液明显更低,3)对于感兴趣的分析物(DNA)可获得更高电泳迁移速率的优势。
两性离子在等电点(pI)的缓冲能力
氨基酸缓冲液在其pI处具有缓冲特性。尽管给定氨基酸在其pI处具有或不具有其“最高缓冲能力”,但它具有一定程度的缓冲能力。在pI和pK之间每个pH单位的差异使缓冲能力下降10倍;那些具有三个可离子化基团的氨基酸(组氨酸,半胱氨酸,赖氨酸,谷氨酸,天冬氨酸等)一般比那些仅具有两个解离基团的氨基酸(甘氨酸,丙氨酸,亮氨酸等)在其pI处具有更高的缓冲能力。例如,组氨酸pI=7.47,赖氨酸pI=9.74和谷氨酸pI=3.22在其pI处相对于丙氨酸或甘氨酸均具有相对较好的缓冲能力,而后者在其pI处具有相对较低的缓冲能力(参见,A.L.Lehninger,生物化学,第二版,Worth Publishers,纽约,1975;特别是第79页图4-8和第80页图4-9页)。组氨酸已被建议作为缓冲液用于凝胶电泳,参见,例如,美国专利4,936,963,但在该系统中未进行杂交。半胱氨酸的缓冲能力位于更中间的位置。半胱氨酸的pI是5.02,α羧基的pKa是1.71,巯基的pKa是8.33,α氨基的pKa是10.78。250mM半胱氨酸的酸/碱滴定曲线表明半胱氨酸在~pH5比20mM磷酸钠具有更好的“缓冲能力”。在pH4至6的范围内,半胱氨酸的缓冲能力比20mM磷酸钠明显更好,特别是在更高的pH.然而,在这些pH范围中,250mM半胱氨酸溶液的导电性与具有~2.9mS/cm值的20mM磷酸钠相比极低,为~23μS/cm,低100倍。图1显示了各种迁移缓冲液的导电性测量。
超过20年以前形成的一些电泳技术以在“其pI”的两性离子缓冲液中分离蛋白质的能力为基础,这些技术称为等电点电泳,等速电泳和等电聚焦(参见由B.D.Hames&D.Rickwood编辑的“蛋白质凝胶电泳:实践方法”IRL Press 1981中的第3和4章)。在所有这些应用中使用了各种氨基酸缓冲液和Good缓冲液,均在其pI点(参见上述参考文献第168页表2)。
在低离子强度和低导电性缓冲液中的DNA迁移
使用2.5%琼脂糖覆盖的5580小片和ByTr-RCA5荧光探针进行一系列荧光棋盘实验。我们可在所有下列系统中实现迅速(6秒)的棋盘装载:(1)250mM HEPES(低导电性),(2)10μM琥珀酸钠,(3)10μM柠檬酸钠和(4)蒸馏水。柠檬酸钠的结果在图1中显示。尽管某些类型的低导电性或低离子强度溶液具有某些更好的特性,但使用所有这些系统均实现了棋盘装载和迅速的DNA迁移(6至12秒内DNA积累到80μm的条上)。另外,DNA可在蒸馏水中装载APEX片,因为DNA(本身是一种多聚阴离子)是存在于混合溶液中的提供导电性的电解质。图1显示了使用组氨酸的APEX小片的平面图。
电泳迁移率和阳离子/阴离子种类的关系
除了带电分析物种类(DNA,蛋白质等)的迁移率与电解质溶液的离子强度相关的事实外,迁移率也极大地受电解质溶液中阳离子和阴离子种类的特性的影响(参见“毛细电泳:原理和实践”参考文献的第89页)。在上文生物多聚体,第2卷,231-236页,1964的参考文献中证实了DNA迁移的这一具体点。该参考文献第232页图1显示了当在相同离子强度下使用具有不同单价阴离子(Li+>Na+>K+>TMA+)的电解质时DNA迁移率的变化。基本上,不同的阳离子与DNA磷酸基团具有不同的缔合常数,和/或改变DNA分子周围的水化球,导致其迁移率改变。
本发明涉及我们关于在电场分子生物学装置,特别是APEX微电子片和装置中改进或最优化DNA迁移速度,DNA杂交反应的效率,和总的杂交特异性的各种参数,电解质(缓冲液),和其它条件的发现。具体地说,本发明涉及的发现是,含有在10-100mM,特别是大约50mM浓度和在处于或接近pI(等电点大约pH7.47)下制备的氨基酸组氨酸的低导电性两性离子缓冲溶液提供用于迅速电泳DNA迁移和高效杂交反应的最佳条件。组氨酸缓冲液的该优势对于APEX小片类型的装置特别重要。这些具体装置(与微机械型装置相反)对于可应用的电流和电压量有限制。这些限制使得难以使用相同缓冲系统同时实现迅速迁移和高效杂交。在这些情况下,以低导电性缓冲液(半胱氨酸或丙氨酸)进行DNA迁移,其中限制的电流/电压仍产生迅速迁移。在这些条件下,DNA在试验位点积累,但不能有效杂交。在这些低导电性缓冲液中迁移后,将该溶液变成高盐缓冲液(>100mM氯化钠或磷酸钠),然后在试验位点产生有效杂交。
表2显示了使用APEX小片装置确定缓冲能力,pH,和导电性参数与DNA积累和杂交灵敏性(效率)关系的一系列实验的结果。
表2
溶液 | 缓冲能力pH 4-10 | pH在PI | 导电性(μS) | 相对DNA迁移率 | SA-生物素T12灵敏性 | DNA的杂交灵敏性 | |
β-丙氨酸 | pK1-3.6pK2-10.2 | + | 7.3 | 10.0 | 最快+++++ | 3×106 | |
牛磺酸 | pK1-1.5pK2-8.7 | +/- | 4.6 | 4.5 | ++++ | >7.5×1010 | |
半胱氨酸 | pK1-1.7pK2-8.3pK3-10.8 | +/- | 5.2 | 25.0 | ++++ | 3×107 | 7.5×1010 |
组氨酸 | PK1-1.8pK1-6.0pK3-9.0 | +++ | 7.6 | 212.0(172.0)高纯度 | +++ | 3×106 | 3×106 |
赖氨酸 | pK1-2.2pK2-8.9pK3-10.3 | ++ | 9.6 | 477.0 | ++ | >7.5×1010 | |
NaPO4 | 复合体 | + | 7.4 1/ | 1,400.0 | +最慢 |
具体地说,表2显示了各种两性离子氨基酸缓冲液[β-丙氨酸,牛磺酸,半胱氨酸,组氨酸,赖氨酸和磷酸钠(不是两性离子缓冲液)]对迁移的靶DNA与特异性捕捉DNA在试验位点的杂交能力的影响。对于迁移,在相同电场条件下导电性一般与迁移相关。β-丙氨酸,牛磺酸,半胱氨酸显示出极好的迁移,组氨酸显示出较好的迁移,赖氨酸和磷酸钠显示出一般性迁移。报道了具有带负电的多聚阴离子磷酸骨架的“正常DNA”的DNA杂交灵敏性。除了杂交灵敏性外,表2还报道了链霉抗生物素蛋白/生物
素DNA探针捕捉亲和性的灵敏度。
表2清楚地显示了DNA迁移(积累)与低导电性(β-丙氨酸,牛磺酸,半胱氨酸,组氨酸)的关系。表中显示了使用β-丙氨酸,牛磺酸,半胱氨酸和组氨酸对于链霉抗生物素蛋白/生物素探针亲和性较好的灵敏性。正如表2中灵敏性数据所反应的,组氨酸比半胱氨酸或其它缓冲液,如20mNaPO4杂交效率好4个数量级。相对于半胱氨酸至少提高10倍,更具体地说为102倍,最特别的是至少104倍。最重要的是,表2显示了对于组氨酸缓冲液DNA杂交敏感性(效率)极好。因此,目前所试验的所有两性离子氨基酸缓冲液中,组氨酸是唯一一个既有较好的迁移,又有较好的DNA/DNA杂交效率的缓冲液。
据信组氨酸缓冲系统的低导电性是快速DNA迁移(积累)的原因。对于组氨酸缓冲液为什么能产生相当高效的DNA/DNA杂交有一些可能的解释。一个优势可能是组氨酸较好的缓冲能力。由于其pI为7.47,因此组氨酸在酸性或碱性条件下都能较好地缓冲(参见A.L.Lehninger,生物化学,第二版,Worth Publisher,纽约,1975,第80页图4-9)。APEX小片在积累DNA用于杂交的正电极上产生酸,组氨酸可有效缓冲这些条件。更重要的是,在这些酸性条件(pH<5)下,组氨酸上的咪唑基质子化开始将分子转变成双阳离子种类。可能这种带有阳性电荷的α氨基和带有阳性电荷的咪唑基的双阳离子种类可帮助促进杂交和稳定在APEX小片阳极形成的DNA/DNA杂交体。阳离子,双阳离子和多阳离子已知经过减小双链DNA结构上带负电的磷酸骨架的互斥来帮助稳定DNA/DNA杂交体。也可能DNA/DNA/组氨酸从在阳极产生的其它电化学产物(过氧化氢等)也形成一些稳定加合物类型。
尽管本实施方案利用了天然存在的组氨酸,但本发明完全可应用于其它天然或合成的化合物,该化合物具有较好的缓冲能力,较低的导电性(或两性离子特征)且具有允许DNA杂交被电荷稳定或加合物形成所稳定的特性。
尽管为了清楚和容易理解以说明和实施例的方式在某些细节上描述了上述发明,但是对本领域的普通技术人员而言显而易见的是在本发明的教导下可对其作出某些改变和修饰而不偏离所附权利要求的实质或范围。
Claims (13)
1.一种用于在微电子杂交装置中增强靶核酸杂交效率的方法,该装置包括一种具有捕捉核酸的微区域试验位点,该微区域适合于至少被置于不产生电场的第一状态和在该微区域产生吸引靶核酸的电场的第二状态,包括如下步骤:
给该装置使用一种低导电性的两性离子缓冲液,其中的缓冲液包括10-100mM的浓度的在等电点处制备的组氨酸,
给该装置提供靶核酸,
通过给该装置施加电能将该微区域置于所述的第二状态以便在该装置微区域试验位点引起靶核酸的积累,和
使靶核酸与捕捉核酸杂交,其在所述第二状态中具有比在所述第一状态中更高的效率。
2.权利要求1的方法,其中等电点是pH7.47。
3.权利要求1的方法,其中缓冲液稳定所述第二状态中靶核酸和捕捉核酸之间的杂交。
4.权利要求3的方法,其中缓冲液是具有低导电性的天然化合物。
5.权利要求3的方法,其中缓冲液是天然两性离子化合物。
6.权利要求3的方法,其中缓冲液是具有低导电性的合成化合物。
7.权利要求3的方法,其中缓冲液是合成的两性离子化合物。
8.权利要求1的方法,其中杂交效率比在相同条件下用半胱氨酸至少高100倍。
9.权利要求8的方法,其中杂交效率比在相同条件下用半胱氨酸至少高1,000倍。
10.权利要求9的方法,其中杂交效率比在相同条件下用半胱氨酸至少高50,000倍。
11.权利要求1的方法,其中缓冲液减少了在所述第二状态中捕捉核酸与靶核酸间的排斥。
12.权利要求1的方法,其中缓冲液减少了捕捉核酸与靶核酸间加合物的形成。
13.权利要求1的方法,其中缓冲液是低导电性缓冲液。
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US70826296A | 1996-09-06 | 1996-09-06 | |
US08/708,262 | 1996-09-06 |
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JP (1) | JP4213216B2 (zh) |
KR (1) | KR100591626B1 (zh) |
CN (1) | CN1180248C (zh) |
AU (1) | AU723564B2 (zh) |
BR (1) | BR9712800A (zh) |
CA (1) | CA2264780C (zh) |
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WO (1) | WO1998010273A1 (zh) |
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US6051380A (en) * | 1993-11-01 | 2000-04-18 | Nanogen, Inc. | Methods and procedures for molecular biological analysis and diagnostics |
US6468742B2 (en) | 1993-11-01 | 2002-10-22 | Nanogen, Inc. | Methods for determination of single nucleic acid polymorphisms using bioelectronic microchip |
US6379897B1 (en) * | 2000-11-09 | 2002-04-30 | Nanogen, Inc. | Methods for gene expression monitoring on electronic microarrays |
US5964995A (en) * | 1997-04-04 | 1999-10-12 | Caliper Technologies Corp. | Methods and systems for enhanced fluid transport |
US6238909B1 (en) * | 1999-05-04 | 2001-05-29 | Motorola, Inc. | Method and apparatus for obtaining electric field-enhanced bioconjugation |
MXPA02003063A (es) * | 1999-09-27 | 2003-10-14 | Monsanto Technology Llc | Metodos para determinar aceites en semillas. |
US7309581B2 (en) * | 2000-11-01 | 2007-12-18 | Sysmex Corporation | Method of staining, detection and counting bacteria, and a diluent for bacterial stain |
GB0205455D0 (en) | 2002-03-07 | 2002-04-24 | Molecular Sensing Plc | Nucleic acid probes, their synthesis and use |
US7153687B2 (en) | 2002-08-13 | 2006-12-26 | Hong Kong Dna Chips Limited | Apparatus and methods for detecting DNA in biological samples |
JP4464664B2 (ja) | 2003-06-13 | 2010-05-19 | 独立行政法人理化学研究所 | 生体分子マイクロアレイ用基板、生体分子マイクロアレイ、相互作用促進用装置および方法、ならびに、相互作用の検出方法 |
US7314542B2 (en) * | 2004-09-23 | 2008-01-01 | Nanogen, Inc. | Methods and materials for optimization of electronic transportation and hybridization reactions |
KR100785011B1 (ko) * | 2006-04-07 | 2007-12-11 | 삼성전자주식회사 | 쌍이온 화합물을 이용한 핵산 혼성화 특이성을 증가시키는방법 |
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US4936963A (en) * | 1987-05-27 | 1990-06-26 | Abbott Laboratories | Polycationic buffers and method for gel electrophoresis of nucleic acids |
US5188963A (en) * | 1989-11-17 | 1993-02-23 | Gene Tec Corporation | Device for processing biological specimens for analysis of nucleic acids |
US6017696A (en) * | 1993-11-01 | 2000-01-25 | Nanogen, Inc. | Methods for electronic stringency control for molecular biological analysis and diagnostics |
CA2204912C (en) * | 1994-11-10 | 2005-01-04 | David Sarnoff Research Center, Inc. | Liquid distribution system |
US5585069A (en) * | 1994-11-10 | 1996-12-17 | David Sarnoff Research Center, Inc. | Partitioned microelectronic and fluidic device array for clinical diagnostics and chemical synthesis |
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EP1019711A1 (en) | 2000-07-19 |
CA2264780A1 (en) | 1998-03-12 |
JP4213216B2 (ja) | 2009-01-21 |
WO1998010273A1 (en) | 1998-03-12 |
CA2264780C (en) | 2006-08-01 |
KR20010029477A (ko) | 2001-04-06 |
JP2001501301A (ja) | 2001-01-30 |
BR9712800A (pt) | 1999-11-23 |
KR100591626B1 (ko) | 2006-06-20 |
AU4071997A (en) | 1998-03-26 |
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