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Nucleic Acids Research, 2003, Vol. 31, No. 20 e123
© 2003 Oxford University Press

A novel real-time quantitative PCR method using attached universal template probe

Yuanli Zhang1,2, Dabing Zhang*,2, Wenquan Li3, Jianqun Chen1, Yufa Peng4 and Wei Cao3

1 Department of Biological Science and Technology, Nanjing University, 22 Hankou Road, Nanjing 210093, PR China, 2 Shanghai Key Laboratory of Agricultural Genetics and Breeding, Agro-biotech Research Center, Shanghai Academy of Agricultural Sciences, 2901 Beidi Road, Shanghai 201106, PR China, 3 The Department of Technology, GenoMultix Co. Ltd, 1289 Yishan Road, Shanghai 200233, PR China and 4 Institute of Crop Protection, Chinese Academy of Agricultural Sciences, 2 Yuanmingyuan Road, Beijing 100094, PR China

*To whom correspondence should be addressed. Tel/Fax: +86 21 6220 8750; Email: zdb30{at}hotmail.com

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.


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