建立一种简便、快速、特异的制备基因芯片探针的方法.以K 562细胞和正常人淋巴细胞作为消减对象,利用自行建立的消减方法进行消减杂交,结合限制性显示技术,分组扩增差异cD N A,回收K 562细胞特异基因片段,制作基因芯片探针.结果显示,分...建立一种简便、快速、特异的制备基因芯片探针的方法.以K 562细胞和正常人淋巴细胞作为消减对象,利用自行建立的消减方法进行消减杂交,结合限制性显示技术,分组扩增差异cD N A,回收K 562细胞特异基因片段,制作基因芯片探针.结果显示,分离到400个K 562特异的基因,片段大小均一,适于制作cD N A芯片.消减杂交技术结合限制性显示技术制备基因芯片探针,具有快速、简便、特异的特点,降低了芯片制作成本,可加速芯片的推广应用.展开更多
In order to isolate expeditiously the HIV1U26942 DNA fragments for preparation of DNA microarrays, the multiple gene fragments with sizes suitable for DNA microarrays, produced by digesting the dissociated HIV gene wi...In order to isolate expeditiously the HIV1U26942 DNA fragments for preparation of DNA microarrays, the multiple gene fragments with sizes suitable for DNA microarrays, produced by digesting the dissociated HIV gene with Sau 3AⅠ, were ligated with universal adapters. PCR primers were designed to match the universal adapters (including the restriction site sequence) but with one "nesting" base overhanging at the 3’ end. The PCR reactions that were performed with various single primers or primer combinations were divided into ten subgroups. PCR products were purified and then cloned into the T vectors. The positive clones were propagated and the plasmids were extracted. The target HIV gene fragments were isolated and sequenced, which were correlated precisely with the prediction of restriction analysis. Eighteen gene fragments ranging from 0.1kb to 1kb were prepared for DNA microarray. Restriction display is an effective and rapid method for the isolation of gene展开更多
目的采用双重限制性荧光标记技术(doub le restriction fluorescent labeling,DRFL)标记微量核酸样品,提高基因芯片用于病原体检测的灵敏度。方法以限制性显示技术处理SARS-CoV核酸样品,分别采用传统限制性荧光标记技术(直接采用荧光标...目的采用双重限制性荧光标记技术(doub le restriction fluorescent labeling,DRFL)标记微量核酸样品,提高基因芯片用于病原体检测的灵敏度。方法以限制性显示技术处理SARS-CoV核酸样品,分别采用传统限制性荧光标记技术(直接采用荧光标记的通用引物标记)和双重限制性荧光标记技术(荧光标记的通用引物和荧光标记的dNTP的双重荧光标记)标记,进一步在同等条件下与基因芯片进行杂交、清洗和芯片扫描检测。通过对杂交结果的分析,比较2种标记方法的标记效果。结果以DRFL方法标记SARS基因片段,其荧光强度均值比传统荧光标记方法提高了3.5835倍。结论DRFL技术有效地提高单位分子标记片段的荧光强度值,提高了检测的灵敏度,可用于微量病原体核酸样品的基因芯片检测。展开更多
文摘建立一种简便、快速、特异的制备基因芯片探针的方法.以K 562细胞和正常人淋巴细胞作为消减对象,利用自行建立的消减方法进行消减杂交,结合限制性显示技术,分组扩增差异cD N A,回收K 562细胞特异基因片段,制作基因芯片探针.结果显示,分离到400个K 562特异的基因,片段大小均一,适于制作cD N A芯片.消减杂交技术结合限制性显示技术制备基因芯片探针,具有快速、简便、特异的特点,降低了芯片制作成本,可加速芯片的推广应用.
文摘In order to isolate expeditiously the HIV1U26942 DNA fragments for preparation of DNA microarrays, the multiple gene fragments with sizes suitable for DNA microarrays, produced by digesting the dissociated HIV gene with Sau 3AⅠ, were ligated with universal adapters. PCR primers were designed to match the universal adapters (including the restriction site sequence) but with one "nesting" base overhanging at the 3’ end. The PCR reactions that were performed with various single primers or primer combinations were divided into ten subgroups. PCR products were purified and then cloned into the T vectors. The positive clones were propagated and the plasmids were extracted. The target HIV gene fragments were isolated and sequenced, which were correlated precisely with the prediction of restriction analysis. Eighteen gene fragments ranging from 0.1kb to 1kb were prepared for DNA microarray. Restriction display is an effective and rapid method for the isolation of gene
文摘目的采用双重限制性荧光标记技术(doub le restriction fluorescent labeling,DRFL)标记微量核酸样品,提高基因芯片用于病原体检测的灵敏度。方法以限制性显示技术处理SARS-CoV核酸样品,分别采用传统限制性荧光标记技术(直接采用荧光标记的通用引物标记)和双重限制性荧光标记技术(荧光标记的通用引物和荧光标记的dNTP的双重荧光标记)标记,进一步在同等条件下与基因芯片进行杂交、清洗和芯片扫描检测。通过对杂交结果的分析,比较2种标记方法的标记效果。结果以DRFL方法标记SARS基因片段,其荧光强度均值比传统荧光标记方法提高了3.5835倍。结论DRFL技术有效地提高单位分子标记片段的荧光强度值,提高了检测的灵敏度,可用于微量病原体核酸样品的基因芯片检测。
