While the T-DNA flanking sequence analysis is technically unstable and complicated owing to possible rearrangement or broken-end structure of integrated T-DNA [6]. Recently, quantitative real-time PCR (qPCR) has also been applied to estimate transgene copy number [7?6]. Unlike the traditional hybridization-based methods, qPCR assay allows more Title Loaded From File samples to be analyzed in a Title Loaded From File shorter time and requires much less plant tissue. However, despite its increasing application, there are controversies concerning its accuracy. In some studies, the results of transgene copy number determination have shown mismatches between qPCR and Southern blot analysis [8,12,13,15,17]. Some researchers have suggested that qPCR can only be viewed as complementary to Southern blot analysis for determination of transgene copy number, and that the technique is not sufficiently accurate and reproducible to discriminate twofold differences in transgene copy number [10,17]. Thus, the accurate determination of transgene copy number by 18325633 qPCR is not always possible by these approaches. In a survey of previous literature on qPCR-based transgene copy number determination, it was found that some studies assumed that the PCR efficiency was 100 , or took for granted that efficiencies for the integrated target gene (t) and the internalA qPCR Approach for Transgene Copy Number Analysisreference gene (r) were equal, while most 26001275 other studies estimated PCR efficiency either through the establishment of a standard curve by serial dilution [8,13], or a mathematical calculation from the amplification curve itself [18?1]. However, none of these strategies is perfect. The true PCR efficiency cannot be 100 , and the efficiency for t and r can sometimes be quite different. PCR efficiency estimation based on a serial dilution standard curve can be erroneous because only one PCR efficiency value is generated, but this cannot hold true for serial samples where the concentrations of template DNA and potential inhibitors varies over several orders of magnitude [20,22?5]. Also, estimation from the amplification curve itself can sometimes be quite difficult and complicated, and the results can vary between different approaches. Furthermore, even using the identical approach, the results can still be inconsistent when different analysis parameter settings for calculation were selected. In summary, it is often difficult to obtain accurate PCR efficiency, which results in possible erroneous estimation of transgene copy number from qPCR. In this study, we present a novel qPCR approach, named standard addition qPCR (SAQPCR), to accurately determine transgene copy number. The strategy is to add known amounts of standard DNA to test samples to change fluorescence intensity and Ct values, which is similar to standard addition in quantitative chemical analysis [26]. In this assay, the estimation of PCR efficiency can be bypassed, which is not the case in the previously mentioned approaches.transgene copy number chromosome ploidy| chromosome ploidy| N0t N0r Fnt |(1zEr ) Fnr |(1zEt )CntCnr??For selected values of Cnt and Cnr, Fr and Ft data can be obtained, and therefore, in the above equation E is the only unknown parameter required for copy number determination. However, as described above in Introduction Section, PCR efficiency cannot be accurately determined either based on a serial dilution standard curve or from the amplification curve itself. Similar situations also occur in quantitative chemical anal.While the T-DNA flanking sequence analysis is technically unstable and complicated owing to possible rearrangement or broken-end structure of integrated T-DNA [6]. Recently, quantitative real-time PCR (qPCR) has also been applied to estimate transgene copy number [7?6]. Unlike the traditional hybridization-based methods, qPCR assay allows more samples to be analyzed in a shorter time and requires much less plant tissue. However, despite its increasing application, there are controversies concerning its accuracy. In some studies, the results of transgene copy number determination have shown mismatches between qPCR and Southern blot analysis [8,12,13,15,17]. Some researchers have suggested that qPCR can only be viewed as complementary to Southern blot analysis for determination of transgene copy number, and that the technique is not sufficiently accurate and reproducible to discriminate twofold differences in transgene copy number [10,17]. Thus, the accurate determination of transgene copy number by 18325633 qPCR is not always possible by these approaches. In a survey of previous literature on qPCR-based transgene copy number determination, it was found that some studies assumed that the PCR efficiency was 100 , or took for granted that efficiencies for the integrated target gene (t) and the internalA qPCR Approach for Transgene Copy Number Analysisreference gene (r) were equal, while most 26001275 other studies estimated PCR efficiency either through the establishment of a standard curve by serial dilution [8,13], or a mathematical calculation from the amplification curve itself [18?1]. However, none of these strategies is perfect. The true PCR efficiency cannot be 100 , and the efficiency for t and r can sometimes be quite different. PCR efficiency estimation based on a serial dilution standard curve can be erroneous because only one PCR efficiency value is generated, but this cannot hold true for serial samples where the concentrations of template DNA and potential inhibitors varies over several orders of magnitude [20,22?5]. Also, estimation from the amplification curve itself can sometimes be quite difficult and complicated, and the results can vary between different approaches. Furthermore, even using the identical approach, the results can still be inconsistent when different analysis parameter settings for calculation were selected. In summary, it is often difficult to obtain accurate PCR efficiency, which results in possible erroneous estimation of transgene copy number from qPCR. In this study, we present a novel qPCR approach, named standard addition qPCR (SAQPCR), to accurately determine transgene copy number. The strategy is to add known amounts of standard DNA to test samples to change fluorescence intensity and Ct values, which is similar to standard addition in quantitative chemical analysis [26]. In this assay, the estimation of PCR efficiency can be bypassed, which is not the case in the previously mentioned approaches.transgene copy number chromosome ploidy| chromosome ploidy| N0t N0r Fnt |(1zEr ) Fnr |(1zEt )CntCnr??For selected values of Cnt and Cnr, Fr and Ft data can be obtained, and therefore, in the above equation E is the only unknown parameter required for copy number determination. However, as described above in Introduction Section, PCR efficiency cannot be accurately determined either based on a serial dilution standard curve or from the amplification curve itself. Similar situations also occur in quantitative chemical anal.
