A novel real-time quantitative PCR method using attached universal template probe - PubMed (original) (raw)

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

Yuanli Zhang et al. Nucleic Acids Res. 2003.

Abstract

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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Figures

Figure 1

Figure 1

Schematic drawings of signal generation of the UT–PCR amplification. (A) The UT–PCR primer is composed of the 5′ end-attached UT sequence hybridized with the UT probe, and the 3′ end specifically hybridizes to the target sequence. (B) During the first cycle of the PCR amplification, the 3′ end of the UT–PCR primer is extended, generating a chimeric DNA fragment with the UT sequence on the 5′ end and newly synthetic target DNA on the 3′ end. (C) During the second cycle of the PCR amplification, the 3′ end of a free UT–PCR primer and the other primer anneal to available chimeric target DNA. The UT probe specifically anneals to the UT in the chimeric DNA fragments. Then, the 5′ exonuclease activity of DNA polymerase begins to hydrolyze the hybridized UT probe, and sets the reporter moiety free, thus generating a fluorescent signal. This amplification generates more chimeric DNA fragments.

Figure 2

Figure 2

Comparison of the amplification efficiency between the target DNA-specific primers and the same primer pairs with one attached with UT using SYBR Green I fluorogenic dye (Lectin gene, four replicates per reaction).

Figure 3

Figure 3

Sensitivity, precision and dynamic range of fluorogenic real-time PCR. Serial dilutions (10-fold) of transgenic maize Event 176 ranging from 0.01 to 100 ng were detected using a FAM-labeled fluorogenic primer (primer set 2, Table 2). (A) Amplification plot. (B) Initial DNA concentration versus Ct standard curve (_R_2 = 0.994, reaction efficiency = 0.99, three replicates per dilution).

Figure 3

Figure 3

Sensitivity, precision and dynamic range of fluorogenic real-time PCR. Serial dilutions (10-fold) of transgenic maize Event 176 ranging from 0.01 to 100 ng were detected using a FAM-labeled fluorogenic primer (primer set 2, Table 2). (A) Amplification plot. (B) Initial DNA concentration versus Ct standard curve (_R_2 = 0.994, reaction efficiency = 0.99, three replicates per dilution).

Figure 4

Figure 4

Multiplex fluorogenic PCR to detect the Invertase 1 and CryIA(b) gene using serial dilutions (10-fold) of transgenic maize Event 176. (A) Amplification plot of endogenous Invertase 1 gene. Each dilution contains 100 ng of total maize DNA. (B) Amplification plot of the transgenic CryIA(b) gene, serial dilutions of transgenic maize Event 176 ranging from 0.01 to 100 ng. (C) Initial Event 176 DNA concentration versus Ct standard curve. (D) Standard curve, plotting log (GMO amount) versus ΔCt (_R_2 = 0.993, reaction efficiency = 0.98, three replicates per dilution).

Figure 4

Figure 4

Multiplex fluorogenic PCR to detect the Invertase 1 and CryIA(b) gene using serial dilutions (10-fold) of transgenic maize Event 176. (A) Amplification plot of endogenous Invertase 1 gene. Each dilution contains 100 ng of total maize DNA. (B) Amplification plot of the transgenic CryIA(b) gene, serial dilutions of transgenic maize Event 176 ranging from 0.01 to 100 ng. (C) Initial Event 176 DNA concentration versus Ct standard curve. (D) Standard curve, plotting log (GMO amount) versus ΔCt (_R_2 = 0.993, reaction efficiency = 0.98, three replicates per dilution).

Figure 4

Figure 4

Multiplex fluorogenic PCR to detect the Invertase 1 and CryIA(b) gene using serial dilutions (10-fold) of transgenic maize Event 176. (A) Amplification plot of endogenous Invertase 1 gene. Each dilution contains 100 ng of total maize DNA. (B) Amplification plot of the transgenic CryIA(b) gene, serial dilutions of transgenic maize Event 176 ranging from 0.01 to 100 ng. (C) Initial Event 176 DNA concentration versus Ct standard curve. (D) Standard curve, plotting log (GMO amount) versus ΔCt (_R_2 = 0.993, reaction efficiency = 0.98, three replicates per dilution).

Figure 4

Figure 4

Multiplex fluorogenic PCR to detect the Invertase 1 and CryIA(b) gene using serial dilutions (10-fold) of transgenic maize Event 176. (A) Amplification plot of endogenous Invertase 1 gene. Each dilution contains 100 ng of total maize DNA. (B) Amplification plot of the transgenic CryIA(b) gene, serial dilutions of transgenic maize Event 176 ranging from 0.01 to 100 ng. (C) Initial Event 176 DNA concentration versus Ct standard curve. (D) Standard curve, plotting log (GMO amount) versus ΔCt (_R_2 = 0.993, reaction efficiency = 0.98, three replicates per dilution).

Figure 5

Figure 5

Screening of the CryIA(b) fragment in three lines of insect- resistant maizes (Bt11, Event 176, MON810) using degenerate UT–PCR primers (three replicates per sample).

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