A novel real-time quantitative PCR method using attached universal template probe.
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.
Zhang
Yuanli
Y
Department of Biological Science and Technology, Nanjing University, 22 Hankou Road, Nanjing 210093, PR China.
Zhang
Dabing
D
Li
Wenquan
W
Chen
Jianqun
J
Peng
Yufa
Y
Cao
Wei
W
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics; D001427:Bacterial Toxins; D052160:Benzothiazoles; D018744:DNA, Plant / Q000235:genetics; D003959:Diamines; D004731:Endotoxins / Q000235:genetics; D006460:Hemolysin Proteins; D015345:Oligonucleotide Probes / Q000737:chemistry / Q000235:genetics*; D009930:Organic Chemicals / Q000737:chemistry; D030821:Plants, Genetically Modified; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013698:Templates, Genetic; D003313:Zea mays / Q000235:genetics; D043324:beta-Fructofuranosidase / Q000235:genetics
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D052160:Benzothiazoles; D018744:DNA, Plant; D003959:Diamines; D004731:Endotoxins; D006460:Hemolysin Proteins; D015345:Oligonucleotide Probes; D009930:Organic Chemicals; D011804:Quinolines; C030956:insecticidal crystal protein, Bacillus Thuringiensis; C098022:SYBR Green I; D043324:beta-Fructofuranosidase