CN1249214C - 用于固定化和扩增dna的基体,在基体上固定了dna的dna－固定化芯片,以及扩增dna的方法 - Google Patents
用于固定化和扩增dna的基体,在基体上固定了dna的dna－固定化芯片,以及扩增dna的方法 Download PDFInfo
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- CN1249214C CN1249214C CNB99804668XA CN99804668A CN1249214C CN 1249214 C CN1249214 C CN 1249214C CN B99804668X A CNB99804668X A CN B99804668XA CN 99804668 A CN99804668 A CN 99804668A CN 1249214 C CN1249214 C CN 1249214C
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Classifications
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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Abstract
用于固定化DNA等以产生DNA文库的基体和适于通过PCR扩增用于扩增DNA的芯片。PCR扩增方法是使用在卓越传导率基体固定DNA的固相基体进行的。这些基体是用末端有极性基团的化学基团表面修饰的。在PCR方法中，使用DNA固定化在基体上的DNA-固定化芯片，可以在短时间内扩增DNA。
Description
本发明领域
本发明涉及用于分子生物学领域和生物化学领域的用于固定化DNA，肽等等的基体，特别地涉及具有卓越热传导率的基体，更特别地涉及在基体的末端用羟基、羧基、环氧基、氨基及其它基团化学修饰的基体以及在基体上固定了DNA的DNA-固定化芯片。
本发明背景
现有技术中，为了获得特定量的DNA，在DNA扩增步骤中需要重复包括下列步骤(1)至(3)的热循环：
1)将样品的温度增加至95℃，以解开DNA双链的氢键；
2)将样品的温度降低至45℃，为了复制DNA使其与引物杂交；
以及
3)将样品的温度增加至74℃，通过使用耐聚合酶延伸引物以复制DNA。
在通过这种称为PCR的方法进行的DNA扩增反应中，样品和反应溶液是包含在管形塑料反应容器中的。将该容器置入铝块中并重复上述热循环。
但是，上述的扩增反应重复加热和冷却DNA及反应溶液，因而要获得一定量的DNA需要很长的时间。此外，控制反应溶液的温度精度低，亦复制了靶DNA之外的其它DNA。
为了解决上述缺陷，本发明的一个目标是提供能容易地固定化DNA，最适合通过DNA扩增反应复制DNA的固相基体，在基体上固定化了DNA的DNA-固定化芯片，以及用于扩增DNA的方法。
本发明的公开
本发明的基体特征是具有适于固定化和扩增DNA的卓越的热传导率。这些基体优选的是金刚石和化学修饰的金刚石。这些基体优选的在其末端由极性基团，羧基、环氧基或氨基化学修饰。
优选地，羧基通过酯键或肽(酰胺)键连接到基体表面，或羧基通过硅烷偶联试剂连接到基体的表面，或者环氧基或氨基通过硅烷偶联试剂连接到基体的表面。
本发明的DNA-固定化芯片特征是在基体的表面固定化了DNA。
本发明的扩增DNA的方法特征是用这些基体或芯片扩增DNA。
实施本发明的最佳方式
本发明的基体是用于固定化DNA和扩增DNA的固态基体，优选的具有卓越的热传导率。例如，在所有的材料中，金刚石是具有最佳热传导率的材料之一，因此金刚石可以快速的加热/冷却。通过提供本发明的基体，可以缩短重复加热/冷却诸如DNA扩增反应的热循环周期。
在本发明的基体中，羟基、羧基、环氧基、氨基等化学修饰到基体表面。DNA等可以容易的固定化，因而本发明的基体是通过DNA扩增反应复制DNA的最适合的芯片。在基体表面污染时，可通过水解将本发明的芯片重新化学修饰。
在本发明的基体中，固态基体的热传导率优选的相当于或大于0.1W/cm·K。更优选的，热传导率相当于或大于0.5W/cm·K。进一步，更优选的，热传导率相当于或大于1W/cm·K。如果基体的热传导率相当于或大于0.1W/cm·K，在本发明的DNA-固定化基体通过PCR(聚合酶链式反应)进行反应时，加热和冷却的追随性优良。
作为良好的热传导率材料的例子，可以考虑金刚石，金属诸如银、铜、铝、钨、钼等等。亦可考虑陶瓷诸如氧化铝，氮化铝、碳化钛、碳化硅、二氧化硅及其它等等。与上述材料和陶瓷混合的材料也是适用的。另外，塑料材料诸如聚碳酸酯和氟树脂等也是适用的。如果材料是化学稳定的，除了上述金属、陶瓷和塑料之外其它材料也可能是合适的。例如，金刚石和类金刚石可以是合适的。混合了塑料和上述金属、陶瓷和金刚石的基体也是合适的。
作为金刚石基体的原料，可以使用合成金刚石、高压合成金刚石或天然金刚石等的材料。这些金刚石可以为单晶体或多晶体。考虑到生产率，通过汽相复合方法诸如微波等离子CVD方法生产金刚石是优选的。
制成本发明的基体的的方法是可选的。例如，考虑了微波等离子CVD方法，ECRCVD方法，高频等离子CVD方法，IPC方法，DC溅射法，ECR溅射法，离子镀法(ion plating method)，槽离子镀法(ark ion platingmethod)，EB沉积法，耐热沉积法。亦考虑了将金属颗粒、陶瓷颗粒或其它材料与粘合剂诸如树脂混合的方法。亦考虑了使用压力成型机器将金属颗粒、陶瓷颗粒或其它材料加压并在高温烧结加压材料的方法。
优选地，有意使本发明的基体的表面粗糙。这样的表面适于固定大量的DNA，因为与光滑表面相比粗糙表面的面积扩大了。基体的形状没有限制。板形、线形、球形、多角形、粉末状及其它等等均可考虑。另外，亦可考虑这些基体和其它材料的复合形式诸如双层形式。
在本发明的基体中，基体的表面是化学修饰的。优选的，在化学修饰的末端含有极性基团，羟基或羧基。也就是说，将特定基团加到了(化学修饰)基体的表面。通过提供这种化学修饰，很容易将DNA固定化到基体的表面。对于化学修饰到基体表面的特定基团和在末端含有的极性基团，可以考虑羟基、羧基、硫酸基、氰基、硝基、硫羟基、氨基、环氧基和其它基团等等。此外，可以考虑有机羧酸。在上述基团中，羧基可以直接加到诸如金刚石等基体或通过其它末端的羟基基团间接加到基体。
在这种情况下，为了固定化DNA，优选有1到10个碳原子的烃基基团。对于要改变为烃基的酸，可以考虑一元羧酸诸如甲酸、乙酸和丙酸，二元羧酸诸如草酸、丙二酸、琥珀酸、马来酸、延胡索酸、苯二甲酸和多元羧酸诸如偏苯三酸等。
在本发明的基体用于通过PCR方法的DNA扩增时，有两种情况。第一种情况需要抗水解特征，另一种情况通过水解再生化学修饰。
在需要抗水解特征时，优选使上述在烃基末端结合了羧基的基团，通过肽(酰胺)键与基体表面连接以提供耐碱特征。
另一方面，在必须通过水解除去产生的化学修饰并再生的情况下，优选使上述在烃基末端结合了羧基的基团，通过酯键与基体表面连接，从而提供在碱溶液中的水解特征。
作为将与烃基末端连接的羟基基团连接到基体表面的一种方法，可以考虑使用氧等离子体氧化基体表面然后用水蒸汽处理的方法，或在氯气中照射紫外光将基体表面氯化然后在碱性溶液中水解羟化的方法，以及用氧等离子体氧化基体表面、然后氯化之后在碱性溶液中水解羟化的方法。
在将羟基连接到基体表面时，通过用硅烷偶联剂、钛偶联剂和铝偶联剂处理，可以使羧基等的化学修饰更牢固。
作为将在烃基末端结合了羧基的基团通过肽(酰胺)键与基体表面连接的方法，可以考虑在氯气中照射紫外光将基体表面氯化，然后在氨气中照射紫外光将基团氨化，在非水溶剂中与酰氯反应然后在弱碱性溶液中中和的方法。
作为将在烃基末端结合了羧基的基团通过酯键与基体表面连接的方法，可以考虑在氯气中照射紫外光将基体表面氯化，之后在非水溶剂中与羧酸钠反应然后在弱酸性溶液中中和的方法，或者用氧等离子体氧化基体表面，再氯化，在碱性溶液中水解羟基化，在非水溶剂中与酰氯反应然后在弱碱性溶液中中和的方法。以下将详细描述本发明的实施方案。
(实施例1)
通过微波等离子体CVD方法制造了直径64mm，厚度0.3mm的气相合成金刚石。然后，将金刚石抛光形成为0.25mm的均匀厚度。对于10mm×10mm的抛光面，通过FTIR方法(傅立叶变换红外光谱检测方法)测量了由于碳和氢的拉伸振动产生的在2879cm-1的吸收强度，各检测值几乎是恒定的。使用激光束从金刚石切下若干10mm×10mm大小的小块。各小块用作实施方案1到6的样品。在实施方案1中，使用由微波激发的氧等离子体氧化金刚石表面。然后，将样品放入可分烧瓶(separableflask)并将可分烧瓶内的气体置换成水蒸气。当水蒸气充入可分烧瓶后，将烧瓶加热至400℃维持30分钟然后冷却。
从烧瓶中取出样品并干燥以获得在末端有羟基基团的金刚石。通过SIMS方法(二次离子质谱方法)测定抛光金刚石表面后，用氧等离子体和水蒸气处理金刚石后的羟基基团的峰强度。以氢的峰强度为1，羟基基团的峰强度示于表1。
| 表1 | |
| 步骤 | 羟基基团的峰强度 |
| 抛光表面后 | 0.14 |
| 氧等离子体处理后 | 0.70 |
| 水蒸气处理后 | 1.14 |
如表1所示，羟基基团的峰强度在氧等离子体处理和水蒸气处理后增加。结论是，可以确认金刚石的表面已被羟基基团化学修饰了。
(实施例2)
在抛光了实施例1中获得的气相合成金刚石的表面后，使用激光束切下10mm×10mm大小的样品。将样品放入可分烧瓶并将可分烧瓶内的气体置换成氩气。以流量1SCCM通入氯气，通过主波长为3600的Hg-Xe灯的紫外光束照射60分钟氯化金刚石表面。用氩气取代了烧瓶中的气体后，取出样品。将样品在10wt％氢氧化钠水溶液中沸腾15分钟后，水洗清洁样品并干燥，以获得在末端有羟基基团的金刚石。通过SIMS方法，分别测定了抛光金刚石表面、氯化金刚石和氢氧化钠处理后氢、羟基基团和氯基团的峰强度。以氢的峰强度为1，羟基基团和氯基团的峰强度比示于表2。
| 表2 | ||
| 步骤 | 峰强度比 | |
| 羟基基团 | 氯基团 | |
| 抛光表面后 | 0.14 | - |
| 氯化后 | 0.20 | 0.50 |
| 氢氧化钠处理后 | 0.47 | 0.35 |
如表2所示，羟基基团的峰强度在氯化处理和氢氧化钠处理后增加。结论是，确认金刚石的表面用羟基基团化学修饰了。由氯基团的降低判断，确认氯基团被羟基取代了。
(实施例3)
将实施例1中获得的气相合成金刚石的表面抛光。使用激光束切下10mm×10mm大小的样品。用微波激发的氧等离子体氧化金刚石表面后，类似实施例2氯化金刚石的表面。用氩气置换烧瓶内的气体后取出样品。将样品在(10wt％)氢氧化钠溶液中沸腾15分钟后，清洁样品并干燥。结果是获得了在末端有羟基基团的金刚石。按照SIMS方法，分别测定了抛光，用氧等离子体处理，氯化和用氢氧化钠处理金刚石表面后氢、羟基基团和氯基团的峰强度。以氢的峰强度为1，羟基基团和氯基团的每个测定的峰强度示于表3。
| 表3 | ||
| 步骤 | 峰强度比率 | |
| 羟基基团 | 氯基团 | |
| 抛光表面后 | 0.14 | - |
| 氧等离子体处理后 | 0.70 | - |
| 氯化后 | 0.21 | 0.47 |
| 氢氧化钠处理后 | 0.54 | 0.33 |
如表3所示，羟基基团的峰强度在氧等离子体处理、氯化处理和氢氧化钠处理后增加。结论是，金刚石的表面用羟基基团化学修饰了。由氯基团的降低判断，可以认识到氯基团被羟基取代了。
(实施例4)
在抛光了实施方案1中获得的气相合成金刚石的表面后，使用激光束切下10mm×10mm大小的样品。将样品放入可分烧瓶并将可分烧瓶内的气体置换成氩气。以1SCCM的流量通入氯气，通过主波长为3600的Hg-Xe灯的照射紫外光束60分钟氯化金刚石表面。用氩气再次取代烧瓶中的气体后，加入100m1(癸二酸钠)的N，N-二甲基甲酰胺溶液。将冷凝器置入可分烧瓶回流2小时。取出样品后，将其在乙酸溶液中(1wt％)洗涤，用丙酮清洗然后干燥。结果是得到了癸二酸连接到酯键末端连接有羧基的金刚石。按照SIMS方法，分别测定了抛光金刚石表面，氯化金刚石，用癸二酸钠处理后氢、羟基基团和氯基团的峰强度。以氢的峰强度为1，羟基基团和氯基团的每一测定的峰强度示于表4。
| 表4 | ||
| 步骤 | 峰强度比率 | |
| 羟基基团 | 氯基团 | |
| 抛光表面后 | 0.14 | - |
| 氯化后 | 0.20 | 0.50 |
| 癸二酸钠处理后 | 0.40 | 0.34 |
如表4所示，羟基基团的峰强度在氯化处理和癸二酸钠处理后增加。按照FTIR方法，通过FTIR方法测定了由碳和氢的拉伸振动引起的吸收强度和由碳和氧的拉伸振动引起的吸收强度。结果是，各吸收强度都增加了(与金刚石空白相比，吸收强度比率增加大约30％)。
因而，可以认识到金刚石的表面用基团化学修饰了，其中羧基连接到癸二酸的烃基的末端。
可以认识到氯基团被羟基基团取代了，因为氯基团的数量减少了。
(实施例5)
抛光实施例1中获得的气相合成金刚石的表面。使用激光束切下10mm×10mm大小的样品。用微波激发氧等离子体氧化金刚石表面后，类似实施例2将金刚石表面氯化。将烧瓶内的气体置换成氩气后，取出样品。将样品在(10wt％)氢氧化钾溶液中沸腾15分钟以在金刚石表面修饰羟基。干燥后，将样品置入可分烧瓶，在其上方安置了有氯化钙干燥管的冷凝器。加入50ml氯仿和1g三乙胺，用氩气置换烧瓶中的气体。用冰冷却可分烧瓶，逐渐加入50ml氯仿和10g琥珀酰氯。回流4小时后，取出样品，用10wt％的碳酸钾溶液洗涤，丙酮清洗然后干燥。结果是获得连接了丙二酸的金刚石，它是通过酯键连接的，其末端连接到羧基基团。按照SIMS方法，分别测定了抛光金刚石表面，用氧等离子体处理，氯化，羟化并用琥珀酰氯处理后氢、羟基基团的峰强度。以氢的峰强度为1，测定的羟基基团的峰强度示于表5。
| 表5 | |
| 步骤 | 羟基基团的峰强度 |
| 抛光表面后 | 0.14 |
| 氧等离子体处理后 | 0.70 |
| 氯化后 | 0.21 |
| 羟化后 | 0.70 |
| 琥珀酰氯处理后 | 0.50 |
如表5所示，羟基基团的峰强度在氧等离子体处理、氯化、羟化和琥珀酰氯处理后增加。按照FTIR方法，测定了由碳和氢的拉伸振动引起的吸收强度和由碳和氧的拉伸振动引起的吸收强度。结果是，各吸收强度都增加了(吸收强度比率与金刚石空白相比增加大约为25％)。
因而，可以认识到金刚石的表面用基团化学修饰了，其中羧基连接到丙二酸的烃基的末端。
(实施例6)
抛光实施方案1中获得的气相合成金刚石的表面。使用激光束切下10mm×10mm大小的样品。用微波激发的氧等离子体氧化金刚石表面后，类似实施方案2将金刚石表面氯化。
将放置样品的可分烧瓶内的气体置换成氩气。当将流速是1SCCM的氨气充入烧瓶时，通过主波长为3600的Hg-Xe灯产生的紫外光束照射60分钟加入将金刚石表面氨化。用氩气取代烧瓶中的气体后，在可分烧瓶上方安装有氯化钙干燥管的冷凝器。加入50m1氯仿到烧瓶中，然后用氩气置换烧瓶中的气体。
随后，当可分烧瓶在冰块中冰冷时，逐渐加入10g琥珀酰氯的50ml氯仿溶液。回流4小时后，取出样品。用10wt％的碳酸钾溶液洗涤，丙酮清洗然后干燥。结果是获得连接了丙二酸的金刚石，它是通过肽键连接的，其末端具有羧基基团。按照SIMS方法，在每次处理前分别测定了氢、羟基基团和氯基团的峰强度。以氢的峰强度为1，每个测定的羟基基团和氯基团的峰强度示于表6。
| 表6 | ||
| 步骤 | 峰强度比率 | |
| 羟基基团 | 氯基团 | |
| 氢等离子体处理后 | 0.06 | - |
| 氯化后 | 0.21 | 0.47 |
| 氨基处理后 | 0.18 | 0.10 |
| 琥珀酰氯处理后 | 0.58 | 0.10 |
如表6所示，羟基基团的峰强度在氢等离子体处理、氯化、羟化和琥珀酰氯处理后增加。按照FTIR方法，测定了由碳和氢的拉伸振动引起的吸收强度和由碳和氧的拉伸振动引起的吸收强度。结果是，各吸收强度都增加了(吸收强度的增加比率与金刚石空白相比是大约25％)。因而，可以认识到金刚石的表面用基团化学修饰了，其中羧基连接到丙二酸的烃基的末端。
采用实施例1至6中获得的含有末端用羧基化学修饰了的基团的金刚石，进行扩增DNA的反应。1小时后，可以获得预定量的DNA。
关于本发明的化学修饰的金刚石芯片，可将寡核苷酸的末端碱基通过氢键固定在末端羟基基团或末端羧基基团上，然后可将具有与该寡核苷酸互补的碱基序列的DNA固定化，从而用作DNA文库芯片。或者，也可将核苷酸、寡核苷酸、DNA片段等固定化到金刚石表面用作文库。
(实施例7)
通过高频等离子体CVD方法气相合成了直径64mm、厚度0.1mm、表面糙度Ra＝0.1mm的碳化钛圆板。使用激光束从圆板切下3mm×5mm的样品。测定的热传导率结果是0.44W/cm·K。
在实施例7中，用高频激发的氧等离子体将样品的表面氧化。然后，将样品置入可分烧瓶。水蒸气置换烧瓶内的气体后，在水蒸气流入烧瓶的情况下，将烧瓶加热至400℃保持30分钟，将烧瓶放置冷却。取出样品并干燥。结果是，可以获得末端有羟基基团的基体。进一步，将基体表面浸入硅烷偶联溶液以获得表面覆盖硅烷偶联剂的基体。将基体直接与珀耳帖元件接触，它做为加热/冷却装置控制基体温度。然后操作下面的PCR方法。
(DNA固定化)
在mRNA(信使RNA)时，将寡-dT16-20固定在基体表面。另一方面，在gDNA(基因组DNA)时，通过化学酯键反应固定含有靶限制酶位点的寡核苷酸。随后，在固定化cDNA(互补DNA)时，在0℃至4℃低温下加入从组织和细胞提取的总RNA溶液，使mRNA和固定化寡-dT16-20杂交。然后，控制基体温度在37℃至60℃并通过RT酶(逆转录酶)合成cDNA。这种情况下，使用通过向寡-dT16-20的5’延长反应而固定化的cDNA。
将合成的固定化的cDNA和mRNA的在杂交条件下溶液加热到90℃将mRNA去杂交，将反应溶液替换为TE缓冲液(Tris-EDTA)，再将基体冷却到低温0℃至4℃并用乙醇清洗。结果是，产生了精制的单链固定化cDNA芯片。
固定化gDNA时，类似上述的寡-dT16-20，将含有靶限制酶位点的固定侧寡核苷酸固定在本发明基体的表面。随后，在低温0℃至4℃下用含有杂交侧寡核苷酸和靶限制酶的反应溶液取代用于化学固定的反应溶液。各寡核苷酸间杂交后，将基体加热到37℃以对半固化寡核苷酸进行限制酶切断。
用限制酶切断后，基体的温度转为0℃至4℃的范围。使用包括靶限制酶切断的gDNA片段和连接酶的反应溶液取代上述溶液。再将基体的温度增加到37℃以进行连接酶反应。这样产生了单链的gDNA固定化芯片。
对于上述产生的在基体表面的cDNA固定化芯片或上述产生的在基体表面的gDNA固定化芯片，依据下面描述的各自目的可将这些芯片分到不同的容器。
(1)比较多个测试样品的同种组织和细胞中的基因变异；
(2)比较同一测试样品的各组织和细胞的基因的表达和变化。
(3)依据治疗和手术(sergeant)后的时间，同一实验样品中的基因的表达和变化。
例如，在(1)的情况下，为了比较同种组织和细胞的基因变异，将多种实验样品(多种DNA固定化芯片)分别置入不同容器。这些多个连接的容器作为一个盒。将这些盒放在反应器主体。如果系统地产生至少两个盒，可以有效的进行实验样品的比较。连接了多个容器的上述一个盒或上述多个盒称为一个集合并且将DNA固定化芯片置入集合的各容器。这样，它即可用作盒型DNA文库。
使用这些盒型DNA文库，可以系统地进行上述(1)和(2)的比较，从而有效的搜寻基因的变化。
在TE缓冲液和70～75％的乙醇溶液中充分清洗本发明的DNA固定化芯片后，将芯片在100％乙醇中浸湿并冰冻条件保存，按照比较数据的要求，可以半永久的使用该文库。
(用DNA-固定化芯片扩增DNA)
使用多种上述cDNA固定化芯片制成了反应容器。反应容器可控地与加热/冷却装置接触。用TE缓冲液充分清洗反应容器的内表面后，置入扩增靶DNA的引物并加入包含四种核苷酸及DNA聚合酶的PCR反应溶液。将反应容器瞬时升温至变性温度95℃，使双链DNA分离为单链DNA。使容器保持在95℃大约1.5分钟。然后，将容器瞬时冷却至退火温度45℃使单链DNA与DNA引物连接。使容器保持在45℃大约1分钟。然后，将容器升温至DNA扩增温度74℃，通过耐热DNA聚合延伸DNA链。将容器保持在此温度大约2分钟。将上述热循环重复30次进行PCR。PCR的总时间是保持时间的总和，大约135分钟，因为几乎不需要加热/冷却时间。
(实施例8)
用激光束切实施方案1中获得的气相合成金刚石，提供10mm×10mm大小的样品。用微波等离子体激发的氢等离子体处理样品表面后，类似实施方案2，将金刚石表面氯化。用氩气取代气体后，取出小块并在10wt％的氢氧化钾溶液中沸腾15分钟进行羟化。另一方面，通过加入乙酸将100cc 95％乙醇的5％溶液PH控制为PH5，搅拌下加入2cc 3-环氧丙氧丙基三甲氧基硅烷。将羟基金刚石浸入溶液。取出羟基金刚石后，用乙醇轻轻清洗金刚石并通过110℃处理5分钟将环氧基引入金刚石表面。
(实施例9)
对于环氧基基团，固定5’端氨基化寡核苷酸(引物)。与mRNA退火后，通过RT酶产生cDNA复制物并固定在芯片上。
(实施例10)
用激光束切实施方案1中获得的气相合成金刚石，提供10mm×10mm大小的样品。用微波等离子体激发的氢等离子体处理样品表面后，类似实施方案2，将金刚石表面氯化。用氩气取代气体后，取出小块并在10wt％的氢氧化钾溶液(hydroxyl potassium)中沸腾15分钟对金刚石表面进行羟化。另一方面，将2cc 3-氨基丙基三甲氧基硅烷搅拌下添加到100cc的95％乙醇的5％溶液中。取出羟基金刚石后，用乙醇轻轻清洗金刚石并通过110℃处理5分钟将氨基引入金刚石表面。
(实施例11)
用肽键将在5端修饰了羧基的寡核苷酸固定在实施例10获得的引入了氨基的金刚石的表面，以mRNA用作模板通过RT酶将cDNA固定在金刚石上。
(实施例12)
通过电弧离子镀方法气相合成了直径64mm、厚度0.1mm、表面糙度Ra为0.3mm的氮化铝圆板。使用激光束从圆板切下3mm×5mm的样品。可以获得热传导率的数值是1.70W/cm·K。将样品置入可分烧瓶并用氩气置换烧瓶中的气体。以1SCCM流量通入氯气时，通过用He-Xe灯照射主波长为3600的紫外线60分钟氯化样品的表面。用氩气取代气体后，取出样品并在10wt％的氢氧化钠溶液中沸腾15分钟，然后用水清洗并干燥。结果是获得了末端具有羟基基团的基体。然后，类似实施方案1，用该基体进行了利用PCR方法的DNA固定化和扩增。
(实施例13)
通过粉末烧结法获得直径64mm、厚度为0.5mm，平均表面糙度的钨圆板。从圆板用激光切出3mm×5mm的样品。测定的热传导率的数值是1.67W/cm·K。通过微波激发的氧等离子体氧化样品的表面，然后氯化试样表面。用氩气取代气体后，取出样品并在10wt％氢氧化钠溶液中沸腾15分钟。然后用水清洗并干燥，获得了末端具有羟基基团的基体。之后，类似实施方案1，用该基体进行了利用PCR方法的DNA固定化和扩增。
(实施方案14)
通过粉末烧结方法产生了氧化铝圆板。使用激光束从圆板切下3mm×5mm的样品。可以获得热传导率的数值是0.3W/cm·K。将样品置入可分烧瓶并用氩气置换烧瓶中的气体。以1SCCM通入氯气，通过用Hg-Xe灯照射主波长为3600的紫外线60分钟氯化样品的表面。再用氩气取代气体后，加入含1wt％癸二酸钠的100ml N，N-二甲基甲酰胺溶液。在可分烧瓶上安置冷凝器，回流2小时。然后取出样品并用1wt％的乙酸溶液清洗。用丙酮清洗并干燥，获得了末端具有通过酯键连接的癸二酸羧基基团的基体。然后，类似实施方案1，用该基体进行了利用PCR方法的DNA固定化和扩增。
除了将容器与上述的加热/冷却装置诸如热敏电阻(thermister)接触，本发明的基体和DNA芯片还可插入反应溶液，为类似常规PCR方法中DNA扩增的Eppen dorf管。这种情况下，与上述实施方案相比，虽不能在很短时间内完成PCR扩增方法。但是，这种时间仍比将DNA加入反应溶液的常规方法短。
工业应用可能性
本发明中，使用了具有卓越热传导率的基体，从而与PCR方法相比，可在很短的时间内完成DNA扩增反应。
通过直接将基体与加热/冷却装置接触，可以改进上述PCR反应的温度控制的精确度，从而几乎不扩增靶DNA之外的DNA。这是一个优点。
在本发明的基体中，DNA直接固定化到具有卓越热传导率的固相基体，从而通过将基体与加热/冷却装置直接接触改进了PCR等之中热循环的追随性，在短时间内获得目的量的DNA。
在本发明的基体中，表面被羟基基团、羧基基团、环氧基基团和氨基基团化学修饰。因此，DNA固定变得很稳定并且对于将芯片用于通过使用PCR方法等通过DNA扩增反应来复制DNA是最合适的。
当本发明的基体表面受到污染时，通过水解再生化学修饰。因此，可以节省昂贵的DNA芯片。
Claims (7)
1.用于DNA固定化的固态基体，其由选自以下的物质构成：合成的金刚石，天然金刚石，金刚石样碳，包括银、铜、铝、钨或钼的金属，包括氧化铝、氮化铝、碳化钛、碳化硅或硅的陶瓷，包括聚碳酸酯或氟树脂的塑料材料或者塑料与所述金属、陶瓷和金刚石的混合材料，并且对于固定化和扩增DNA该基体具有至少0.1w/cm·k的热传导率，其中所述基体表面被修饰有极性基团，通过在氯气氛下用紫外线照射基体表面使其结合氯，并通过将基体浸入沸腾的碱溶液或水蒸汽中而以羟基取代氯，或者通过在氨气氛中用紫外线照射基体而将氨基基团结合到基体上或者通过将基体浸入带羧基的烃或带环氧基的烃的溶液而将羧基结合到基体上。
2.权利要求1的基体，其中所述极性基团是羧基且所述羧基通过肽键连接到上述基体的表面。
3.权利要求1的基体，其中所述极性基团是羧基且所述羧基通过硅烷偶联剂、钛偶联剂和铝偶联剂引入到所述基体的表面。
4.权利要求1的基体，其中所述极性基团是环氧基且所述环氧基通过硅烷偶联剂、钛偶联剂和铝偶联剂引入到所述基体的表面。
5.权利要求1的基体，其中所述极性基团是氨基且所述氨基通过硅烷偶联剂、钛偶联剂和铝偶联剂引入到所述基体的表面。
6.由在权利要求1至5的任一项所述的基体上固定DNA得到的芯片。
7.扩增DNA的方法，其利用权利要求1至5任一项的基体或权利要求6的芯片，并包括以下步骤：
(a)在所述基体表面固定DNA或直接利用固定了DNA的芯片；(b)用Tris-EDTA缓冲溶液清洗基体或芯片；(c)将清洗过的基体或芯片浸入含以下成份的溶液：扩增目标DNA的引物、四种核苷酸及DNA聚合酶；(d)保持所述溶液温度于95℃1.5分钟；(e)在45℃保持所述溶液温度1分钟；(f)在74℃保持所述溶液温度2分钟；和(g)重复步骤(d)-(f)。
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- 1999-02-08 ID IDW20001744A patent/ID28181A/id unknown
- 1999-02-08 CN CNB99804668XA patent/CN1249214C/zh not_active Expired - Fee Related
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| EP1063286A1 (en) | 2000-12-27 |
| AU2187199A (en) | 1999-08-23 |
| ID28181A (id) | 2001-05-10 |
| WO1999040173A1 (fr) | 1999-08-12 |
| US7022473B1 (en) | 2006-04-04 |
| KR20010040747A (ko) | 2001-05-15 |
| JP3607199B2 (ja) | 2005-01-05 |
| EP1063286A4 (en) | 2004-03-24 |
| CA2320462A1 (en) | 1999-08-12 |
| AU765387B2 (en) | 2003-09-18 |
| CN1295611A (zh) | 2001-05-16 |
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