PRIMER EXTENSION ANALYSIS PROVIDES A SENSITIVE TOOL FOR THE IDENTIFICATION OF PCR-AMPLIFIED DNA FROM HIV-1
PRIMER EXTENSION ANALYSIS PROVIDES A SENSITIVE TOOL FOR THE IDENTIFICATION OF PCR-AMPLIFIED DNA FROM HIV-1
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DOI：
10.1016/0166-0934(93)90042-p
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发表时间：
1993-05-01
影响因子：
3.1
通讯作者：
SCHUPBACH, J
中科院分区：
文献类型：
--
作者：
BONI, J;SCHUPBACH, J
评价引物延伸分析作为鉴定HIV-1 PCR扩增DNA的方法。与放射性标记的寡核苷酸的溶液杂交，然后与大肠杆菌DNA聚合酶I的Klenow片段进行延伸反应，随后在变性聚丙烯酰胺凝胶上分离，揭示了预测大小的单链DNA产物。通过特异性内切酶消化进一步证明反应的特异性。当双链DNA探针用于杂交时，该分析比Southern印迹法更灵敏，并且与Slot印迹法同样灵敏。与末端标记的寡核苷酸探针，引物延伸分析证明了一个数量级更敏感的膜杂交。该分析还允许从1 pg至约1 ng的DNA产物定量扩增的DNA。在所述扩增条件下，引物延伸分析能够在1 μ g总DNA存在下检测单个HIV-1质粒DNA分子。寡核苷酸探针的3 '端错配不会导致检测限显著改变。引物延伸分析也可以在同一反应管中用至少三种不同的PCR扩增的DNA进行。
Primer extension analysis was evaluated as a means to identify PCR-amplified DNA from HIV-1. Solution hybridization with radioactive labeled oligonucleotides followed by an extension reaction with Klenow fragment of Escherichia coli DNA polymerase I and subsequent separation on denaturing polyacrylamide gels reveals single stranded DNA products of the predicted size. The specificity of the reaction is further demonstrated by specific endonuclease digestion. The analysis is more sensitive than Southern blotting and about equally sensitive as Slot blot analysis when double-stranded DNA probes are used for hybridization. With end-labeled oligonucleotide probes, primer extension analysis proved an order of magnitude more sensitive than membrane hybridization. The analysis also allows quantitation of amplified DNA from 1 pg to about 1 ng of DNA product. Under the conditions described for amplification, primer extension analysis is capable of detecting a single HIV-1 plasmid DNA molecule in the presence of 1 mug of total DNA. 3'-end mismatching of the oligonucleotide probe does not result in a significantly altered detection limit. Primer extension analysis can also be carried out with at least three different PCR-amplified DNAs in the same reaction tube.