A novel real-time quantitative PCR method using attached universal template probe
A novel real-time quantitative PCR method using attached universal template probe
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DOI：
10.1093/nar/gng123
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发表时间：
2003-10-15
影响因子：
14.9
通讯作者：
Cao, W
中科院分区：
文献类型：
--
作者：
Zhang, YL;Zhang, DB;Cao, W
描述了一种新的实时定量聚合酶链反应（PCR）方法，该方法使用附加通用模板（UT）探针。UT是附着在PCR引物5'端的大约20个碱基，它可以与互补的TaqMan探针杂交。该方法的优点之一是可以使用相同的UT探针检测不同的目标DNA序列，这大大降低了实时PCR设置的成本。此外，该方法还可以在多重反应中用6-羧基荧光素标记的UT探针检测靶基因，用5-六氯荧光素标记的UT探针检测参比基因。此外，UT-PCR分析的靶DNA长度要求相对灵活，在本报告中可能短至56 bp，这表明从部分降解的样品中检测靶DNA是可能的。采用简并引物的UT-PCR系统也可用于筛选同源基因。综上所述，我们的研究结果表明，UT-PCR技术是一种高效、可靠、廉价、劳动强度低的定量PCR分析方法。
A novel real-time quantitative polymerase chain reaction (PCR) method using an attached universal template (UT) probe is described. The UT is an approximately 20 base attachment to the 5' end of a PCR primer, and it can hybridize with a complementary TaqMan probe. One of the advantages of this method is that different target DNA sequences can be detected employing the same UT probe, which substantially reduces the cost of real-time PCR set-up. In addition, this method could be used for simultaneous detection using a 6-carboxy-fluorescein-labeled UT probe for the target gene and a 5-hexachloro-fluorescein-labeled UT probe for the reference gene in a multiplex reaction. Moreover, the requirement of target DNA length for UT-PCR analysis is relatively flexible, and it could be as short as 56 bp in this report, suggesting the possibility of detecting target DNA from partially degraded samples. The UT-PCR system with degenerate primers could also be designed to screen homologous genes. Taken together, our results suggest that the UT-PCR technique is efficient, reliable, inexpensive and less labor-intensive for quantitative PCR analysis.