CN1484764A - Dna-固定化基质的再使用方法 - Google Patents
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Abstract
本发明提供DNA-固定化基质的再使用方法,由此使得昂贵的DNA-固定化基质可有效被利用,且提供可再使用的DNA-固定化基质,该基质具有与新产品相同的性能且在实际应用中无任何问题。即,一种DNA-固定化基质的再使用方法,其特征在于从携带由酰胺键通过寡核苷酸固定的DNA的DNA-固定化基质上去除DNA而由此使得可以固定新的DNA,在基质和DNA之间的酰胺键用酸或碱来水解。
Description
技术领域
本发明涉及DNA-固定化基质的再使用方法。
背景领域
迄今,一种DNA诊断的基质已用作基因分析等的方法。DNA-固定化基质接受大量的DNA片段和寡核苷酸排列在其固相表面,且在生物化学、分子生物学和基因工程或医学检查中得到广泛的注意,这使得它可能以迅速且简化的方式来处理大量的小的样品并通过光谱学来分析反应数据。
尽管这样的DNA-固定化基质极其昂贵,但再使用用过的基质以在其上再次固定任何其他DNA的方法尚未发展出来。因此,用过的基质通常被扔掉,且即使再使用,之前已经结合其上的DNA可能仍然保留在其上,且大多数用过的基质不再可实际利用。
本发明要解决上述问题,并提供用过一次的DNA-固定化基质的再使用方法以使得有效利用昂贵的DNA-固定化基质,其中再使用的DNA-固定化基质与新基质具有相同的性能且不具有实际应用中的问题,并且本发明减少DNA-固定化基质使用者的经济和技术负担。
发明公开
我们本发明者经刻苦研究已达到上述的目标,并作为结果,发现了从已重复用于PCR扩增的DNA-固定化基质上完全去除DNA的方法和将DNA再固定在更新了的基质上的可能性。
具体地,权利要求1的发明提供DNA-固定化基质的再使用方法,它包括从携带通过酰胺键经寡核苷酸在其上固定了DNA的DNA-固定化基质上将固定的DNA去除以此更新基质,而使得它可接受新的DNA固定其上,且其特征在于基质-DNA之间的酰胺键是用酸或碱来水解的。
权利要求2的发明提供了DNA-固定化基质的再使用方法,它包括处理其上带有通过酰胺键固定的DNA的DNA-固定化基质,由此用酸水解基质-DNA之间的酰胺键,然后浸入碱中以去除阴离子,从而将基质表面转变为氨基基团修饰的表面。
权利要求3的发明提供DNA-固定化基质的再使用方法,它包括处理其上带有通过酰胺键固定的DNA的DNA-固定化基质,由此用碱水解基质-DNA之间的酰胺键,从而将基质表面转变为氨基基团修饰的表面。
本发明中用于水解的酸优选为一或多种如在权利要求4中所述的无机酸和有机酸。
同样优选,用于水解的碱为如在权利要求5中所述的任何碱金属氢氧化物和/或碱金属盐。
附图简述
图1为迁移图,显示固定化和再固定化的证实结果。
实施本发明的最佳方式
本发明再使用DNA-固定化基质的方法包含从其上携带通过酰胺键经寡核苷酸固定的DNA的DNA-固定化基质上将固定的DNA去除由此更新基质,而使得它可接受新的DNA固定其上,且其特征在于基质-DNA之间的酰胺键用酸或碱来水解。
在本发明的再使用方法中要处理的DNA-固定化基质具有经酰胺键固定在其氨基基团修饰的基质表面上的DNA。
优选,该氨基基团修饰的基质通过氨化以下基质的表面而制备,如金刚石、硅、玻璃,或金属如金、银、铜、铝、钨、钼,或者覆盖于金属上的陶瓷层,或者塑料如聚碳酸酯、氟树脂(fluororesin)。
除上述之外,任何其他化学稳定的材料均可用作基质,如石墨和金刚石样的碳。同样可用塑料与上述金属、陶瓷、金刚石等的混合物。同样优选用于此处的是硅、玻璃、金属、石墨、塑料和其他用金刚石或金刚石样的碳包被的材料。
在那些材料中,金刚石由于其导热性而优选。金刚石具有良好的导热性并可接受快速地加热和冷却。因此,加热或冷却基质以在其上结合DNA的热循环周期可有效地缩短。
具体地,基质材料的导热性可为至少0.1W/cm·K,优选至少0.5W/cm·K,更优选至少1.0W/cm·K。
对于金刚石基质材料,可用任何合成的金刚石、高压塑造的金刚石、天然的金刚石等。其结构可为单晶体或多晶体的任何形式。以其生产的观点看,金刚石优选通过气相合成法生产,如通过微波等离子体辅助的CVD(microwave plasma-assisted CVD)。
该基质可以任何已知的方法形成。例如,该方法包括微波等离子体辅助的CVD、ECRCVD、IPC、DC反应溅射法、ECR反应溅射法、离子电镀、电弧离子电镀、EB蒸汽沉积作用、电阻加热蒸汽沉积作用等。可将金属粉末、陶瓷粉末等与树脂粘合剂混合,并可塑造为基质以用于此处。除此之外,可将金属粉末、陶瓷粉末等起始材料通过用压力机挤压成未淬火的压块(green compact),且该压块可在高温进行烧结。
优选,基质的表面故意制成粗糙样。粗糙的表面由于其表面积增加而有利于固定大量的DNA等。基质的形状没有特定限定,且可为扁平的、线状的、球形的、多角形的、粉状的等。此外,基质的大小也没有特定地限定。
在本发明中所用的金刚石基质可包括包含金刚石和任何其他物质的复合材料(如金刚石或金刚石样的碳的两相复合材料)。
基质表面的氨基基团修饰可通过如在氯气中用UV射线照射该固体基质而氯化基质表面并随后在氨气中用UV射线进一步地照射以氨化而获得。
氨基基团修饰的基质表面可通过用适当的酸性氯化物羧化并随后将末端羧基基团与碳二亚胺或二环己基碳二亚胺和N-羟基琥珀酰亚胺脱水和缩合而用羧酸来修饰。
描述了根据本方法的脱水缩合的一个实例。将其表面已被修饰而具有羧基基团的固体基质浸入到1,4-二氧六烷溶液中,在其中溶解有碳二亚胺或二环己基碳二亚胺和N-羟基琥珀酰亚胺或对硝基酚,然后洗涤并干燥。这样处理后,该基质即在其末端具有结合了N-琥珀酰亚胺酯基或对硝基酚酯的烃基基团。
上述方法中更优选,预先在金刚石基质上形成了伯氨基基团,且这是用活化的二酯处理而使得该二酯的一个酯基通过脱水缩合结合到该伯氨基基团上。
该活化的二酯具有上文所述的两个活化的酯基。优选,该酯基位于该活化二酯的两个末端,且各具有0~12,更优选0~6个碳原子。同样优选,除酯基之外的主链部分为线性饱和脂肪酸。
在使用这样的活化酯的方法中,可以用与上述相同的方式将基质氯化然后氨化,其后其中的氨基基团可与一个活化酯反应以获得预期的基质,该活化酯预先通过用琥珀酸(二羧酸)对N-羟基琥珀酰亚胺的活化酯化反应而制备。
因而,具有羧酸修饰的表面并具有经寡核苷酸固定于其表面的DNA的基质将根据本发明来处理以供再次使用。
将要固定于基质上的DNA没有特定的限定,且可为任何单链的cDNA(互补DNA)或双链的gDNA(基因组DNA、染色体DNA)。
DNA的链长度没有特定的限定。
该寡核苷酸应该依赖于要结合到基质上的DNA的类型而适当地选择。在单链cDNA固定到基质上的情况下,可利用以mRNA为模板的反转录,且可将作为反转录引物的寡脱氧胸苷酸等用作寡核苷酸。
另一方面,双链gDNA的固定可通过使用限制性内切酶切割位点来实施。在这种情况下,所用的寡核苷酸应该在DNA结合一侧具有限制性内切酶切割位点。
在本发明中,将用过的无用的DNA-固定化基质浸入酸或碱溶液中,由此基质-DNA之间的酰胺键被水解和切割,然后其表面由此更新为具有氨基基团修饰的表面。
用于水解的酸优选为无机酸如盐酸,但可为任何其他的能够水解酰胺键的无机酸或有机酸。
在碱用于水解的情况下,优选碱金属氢氧化物,但可为任何其他的碱性盐如碱金属碳酸盐。
描述了水解的条件。对于酸水解,将DNA-固定化的基质置于6M盐酸中并在80~100℃反应几个小时至24小时以由此完全水解基质-DNA之间的酰胺键。
对于碱水解,将DNA-固定化的基质置于2M NaOH中并在室温反应几个小时至24小时以借此水解基质-DNA之间的酰胺键。
在酸水解后,基质表面上有阴离子。因此,优选将其浸入碱中以除去阴离子然后用水洗涤。
另一方面,碱水解的基质可用水洗涤而不需任何额外的处理。
因此,DNA-固定化的基质中基质-DNA之间的酰胺键即被水解,且因此基质的氨基基团修饰的表面暴露出来。
在这样处理后,该基质可用上述的方式再用于羧酸修饰、寡核苷酸固定化和DNA固定化。
实施例
本发明参考下面的实施例而详细描述。
实施例1
使用金刚石基质。下述的基质是金刚石基质。
将在其上固定了DNA的基质置于6N的盐酸中并在95℃反应5小时以使其水解。然后将其洗涤(约3次)。在该条件下,该基质具有氯化氢。因此,将其浸入0.1N的氢氧化钾溶液中以从中去除氯离子。随即将基质用水洗涤(约3~5次)以,由此使其具有氨基基团修饰的表面。
将0.1mmols的琥珀酸氯化物加入到1ml氯仿中,并将可再使用的氨基基团修饰的基质插入其中且在室温反应30分钟。下一步,将基质用氯仿洗涤,干燥,然后浸入到纯水中放置1小时,最后用纯水洗涤(约3~5次)以使其具有琥珀酸修饰的表面。
将0.1mmols水溶性的碳二亚胺和0.1mmols的羟基琥珀酰亚胺溶解于1ml 90%的1,4-二氧六烷中,并将琥珀酸修饰的基质浸入其中并在高的温度下振荡反应15分钟。然后将基质取出并用1,4-二氧六烷(约2~4次)和纯水(约3~5次)洗涤。
将基质浸入到寡核苷酸的水溶液中并于室温反应1小时,该寡核苷酸具有限制性内切酶(EcoR I)位点和5’末端的(dA)3(浓度:500fmol/μl,每一基质50μl)。
然后,将其于4℃浸入到与固定的寡核苷酸具有互补序列的寡核苷酸水溶液中(浓度=500fmol/μl,每一基质50μl)30分钟以进行杂交。
将基质浸入到EcoR I反应液体中并于37℃反应1小时。然后,将反应混合物冷却到4℃,并将基质从中取出。将其用于4℃冷却的无菌水洗涤(约3~5次),然后用于4℃冷却的60%的乙醇水溶液洗涤,其后稍微干燥(使乙醇蒸发)。
将基质浸入到含有通过用EcoR I处理λDNA所获得的21-kbp的片段的连接酶溶液中,并于4℃反应过夜以借此将DNA固定在基质上。
水解和DNA在基质上的再固定可证实如下:将基质用能够扩增21-kbp的λDNA片段中500-bp的片段的引物进行PCR,并在其上检查扩增的DNA。图1显示了结果。
图1是迁移图,显示固定化和再固定化的证实结果。在图1中,在迁移图下描述的标记M显示100bp的滞后标记(lag marker);泳道0显示水解前的基质;泳道1和2显示水解后的基质;以及泳道3和4显示再固定化后的基质。
如图1,证实了已固定在基质上的DNA被从水解的基质上去除了,并且新的DNA再固定在基质上。
工业应用
根据本发明的结果,可从无用的DNA-固定化基质上将DNA完全去除,且新的DNA可结合到该更新的基质上。
此外,由于本发明的再使用方法不需要任何特殊的试剂和仪器,所以其简单且便宜。
进一步地,由于昂贵的DNA-固定化基质可根据本发明而再使用,所以本发明促进了使用DNA-固定化基质进行基因诊断等的经济的诊断和研究。
因此,本发明的再使用方法可用于医学领域和其他各种分子生物学、生物化学和基因工程技术领域。
Claims (5)
1.DNA-固定化基质的再使用方法,它包括从其上携带有通过酰胺键经寡核苷酸固定的DNA的DNA-固定化基质上将固定的DNA去除而由此更新基质,以使得它可接受新的DNA固定于其上,且其特征在于基质-DNA之间的酰胺键用酸或碱来水解。
2.DNA-固定化基质的再使用方法,它包括处理其上携带有通过酰胺键固定的DNA的DNA-固定化基质,由此用酸水解基质-DNA之间的酰胺键,然后将其浸入碱中以去除阴离子从而将基质表面转变为氨基基团修饰的表面。
3.DNA-固定化基质的再使用方法,它包括处理其上携带有通过酰胺键固定的DNA的DNA-固定化基质,由此用碱水解基质-DNA之间的酰胺键从而将基质表面转变为氨基基团修饰的表面。
4.根据权利要求1或2的DNA-固定化基质的再使用方法,其中用于水解的酸为一种或多种无机酸和有机酸。
5.根据权利要求1或3的DNA-固定化基质的再使用方法,其中用于水解的碱为任何碱金属氢氧化物和/或碱金属盐。
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