Dual PCR-capillary electrophoresis for detection of soybean transgenes can be applied for rapid detection of glyphosate-resistant transgenic soybeans.
Operation method
Dual PCR capillary electrophoresis
Principle
Two pairs of primers were designed to simultaneously amplify the above genes by dual PCR using 8g/L hydroxypropyl methylcellulose as the sieving medium, and the products were detected by laser-induced fluorescence-capillary electrophoresis under a voltage of 10 kV in a 50 cm×100 μm i.d. coated capillary using 8g/L hydroxypropyl methylcellulose as the sieving medium. The PCR amplification products of transgenic soybean were detected by laser-induced fluorescence-capillary electrophoresis at a voltage of 10 kV.
Materials and Instruments
Positive Glyphosate Resistant GM Soybean Standard Imported GM Soybean Samples Non-GM Soybean Oligonucleotide Primers Move 1. Total DNA extraction Total plant DNA was extracted by CTAB method. 2.PCR system and reaction conditions Dual PCR system: 1×buffer, 2.5 mmol/L MgC12, 0.25 mmol/L dNTP, primers (0.4 μmol/L ZY1-35S, ZY2-EPSPS and 0.4 μmol/L ZY3-NOS, ZY4-NOS), 1.5units of Taq DNA PCR cycle parameters: 95℃ pre-denaturation for 3min; 95℃ for 45s, 55℃ for 60s, 72℃ for 45s, total 35 cycles; 72℃ extension for 5min. PCR reaction system for Lectin: 1×buffer,2.0mmol/L MgC12,0.2mmol/L dNTP,0.2μmol/L primers.1.0 units of Taq DNA Polymerase(MBI),template DNA1O0--200ng,total PCR system: 1×buffer,2.0mmol/L MgC12,0.2mmol/L dNTP,1.0 units of Taq DNA Polymerase(MBI),template DNA1O0--200ng. 200 ng, total reaction volume 25 μ1.PCR cycling parameters were: 95 ℃ pre-denaturation 3min; 95 ℃ 45s, 58 ℃ 60s, 72 ℃ 45s, a total of 35 cycles; 72 ℃ extension 5min. 3.High efficiency capillary electrophoresis of PCR products Before electrophoretic analysis, the capillary column was filled with 1g/L HPMC, then 8g/L HPMC containing 3.75μmol/L ethidium bromide, and the buffer solution for electrophoretic separation was TBE solution (45mmol/L Tris, 45mmol/L boric acid, 1 mmol/L EDTA, pH 8.5). The PCR amplification product was taken as 1O μl and added with 0.5 μl of the internal standard 2×0.0001 mol/L Sulforhdamine B solution, and electrophoresis was performed at -1OkV for 20 s with electrodynamic injection at -1OkV under constant pressure, and the fluorescence of the DNA fragments was generated by the conjugation of DNA fragments and ethidium bromide using a laser excitation at a wavelength of 543.5 nm, which was detected at a wavelength of 590 nm. The electrophoresis temperature was 20℃. After each electrophoresis, the capillary was rinsed with 0.01 mol/L H3PO4 solution for 5 min, and then proceeded to the next detection. pUC19DNA/Msp I (Hpa II) Marker was used as a DNA marker, and was diluted with appropriate amount of sterile water by Maker when used. The electrophoretic profiles and experimental data were collected and analyzed by windows software compiled in C++ language. 4. Agarose gel electrophoresis of PCR products 5 μl of PCR products were mixed with 1 μl of sampling buffer and sampled on a 30 g/L agarose gel (containing 0.1 μg/ml ethidium bromide). In 0.5×TBE electrophoresis buffer at 100V, electrophoresis was performed for 50min, and pUC19DNA/Msp I (Hpa II) DNA Marker was used for simultaneous electrophoresis, and then the results were observed by UVI UV imaging analyzer. Caveat 1. The capillary with an inner diameter of 100 μm was able to separate 110 (111)/147 bp DNA fragments in the DNA fragment standard pUC19DNA/Msp I (Hpa II) Marker with a separation of up to 2.91, and the capillary with an inner diameter of 100 μm has a larger detection range, which results in a higher detection sensitivity than those of the capillary with an inner diameter of 50 μm and 75 μm. The detection sensitivity was higher than that of capillary columns of 50 μm and 75 μm. By using a capillary column with an i.d. of 100 μm and reducing its length to 40 cm, the separation of the 110(111)/147 bp standard DNA fragment was reduced to 2. 12. Therefore, a capillary of 50 cm × 100 μm i.d. was used for electrophoretic separation in this experiment. 2. As the mass concentration of cellulose increased, the ability to separate the DNA fragments became better, and when the gel mass concentration reached 8g/L, the separation of 110/147bp DNA fragments was maximum (R=2.91). Continuing to increase the mass concentration of the gum, the separation degree did not change much, but the viscosity of the sieving medium increased and the analysis time was prolonged. Therefore, HPMC of 8g/L was chosen for this experiment. 3. The migration times of DNA standard fragments of different sizes all decreased with increasing field strength. For 147bp DNA fragments, the results of column efficiency showed that the highest theoretical plate number of 2.3×1O0000 was obtained when the field strength was 150V/cm-200V/cm. When the electric field strength was higher than 200V/cm, the migration time became shorter, but the Joule-heating effect was also more significant, which led to the band broadening and to a certain extent reduced the band length of the spectrum. The broadening of the spectral band, to a certain extent, reduces the column efficiency, so that the separation degree is reduced. In order to carry out rapid separation, we chose to carry out electrophoretic analysis at a higher field strength of 200V/cm. Common Problems 1. Reproducibility of migration time The reproducibility of DNA fragment migration time is one of the important indicators in genetic analysis. We examined the reproducibility of the migration time of DAN fragments. Under optimized experimental conditions, the DNA relative molecular mass marker and PCR products used were analyzed six times consecutively, and Sulforhdamine B fluorescent reagent was added to the samples to be tested as an internal standard, and the relative migration time was used in order to eliminate the effects of fluctuations in the injection time and voltage on the migration time. Under the optimized experimental conditions Under the optimized experimental conditions, the analysis of the DNA marker used was very reproducible, and the relative standard deviation of the relative migration time was 0.90-1.5. The relative migration times of the dual PCR products of imported soybean were 0.504 and 0.526 for the relative migration times of 125bp and 142bp DNA fragments, respectively, with the relative standard deviations of 0.504 and 0.526. The relative standard deviation was 2.O--3.2 . 2. Comparison of capillary electrophoresis and agarose gel electrophoresis The double PCR products of positive glyphosate-resistant transgenic soybean standard, blank control and imported soybean, as well as the endogenous gene PCR products of negative non-transgenic soybean were compared with capillary electrophoresis and agarose gel electrophoresis at the same time. The results of traditional agarose gel electrophoresis showed that the imported soybean and the transgenic soybean standard of positive control amplified two bands, respectively 125 bp and 142 bp; the non-transgenic soybean as negative control only amplified 118 bp bands of the endogenous gene; the blank control using sterile water as template could not amplify any bands. Comparing the results of agarose gel electrophoresis and capillary electrophoresis, they were basically the same. Capillary electrophoresis uses a high voltage of tens of thousands of volts, which can more effectively separate fragments with smaller differences. And it can complete the analysis in a shorter time. The target DNA fragments of this experiment can be separated in 15 min, which shortens the analysis time. While agarose gel electrophoresis requires 50 min to separate the above two fragments. It can be seen that compared with agarose gel electrophoresis, capillary electrophoresis is faster to analyze Capillary electrophoresis requires less sample volume. This experiment uses electric injection, the injection volume is about 5nl, while agarose gel electrophoresis needs 5--10μl of sample volume, so the analysis with capillary can greatly save the sample volume, suitable for trace analysis. The bands of gel electrophoresis are generally wide, which is not favorable for the determination of the size of DNA fragments. And capillary electrophoresis column efficiency is high. The peak shape is sharp and the identification ability is strong. In this experiment, the simultaneous electrophoresis of the internal standard and the sample can reduce the experimental errors caused by the fluctuation of the injection time and voltage, etc., and improve the accuracy of the results. According to the capillary electrophoresis profiles of DNA Marker and samples, the sizes of DNA fragments were 124 bp and 145 bp respectively, which were compared with the sequencing results. The difference was 1 bp and 3 hp, respectively; the DNA fragment size of the peak in the figure was 121 bp, which was only 3 bp difference compared with the fragment size reported in the literature.It can be seen. It is more accurate to determine the DNA fragment size by capillary electrophoresis than by gel electrophoresis. In summary, this study used dual PCR to simultaneously amplify the three outer bath target genes of glyphosate-resistant transgenic soybeans, and the glyphosate-resistant soybeans could be accurately identified with only one PCR reaction The use of laser-induced fluorescence-capillary electrophoresis to analyze the PCR products shortened the time of analysis and significantly increased the analytical sensitivity compared to the traditional agarose gel electropenetration: and the amount of samples used was small The method established in this study is a good solution for the analysis of transgenic ingredients in food. The method established in this study provides a reliable analytical tool for the rapid detection of genetically modified (GM) ingredients in food. For more product details, please visit Aladdin Scientific website.
Ethidium bromide Hydroxypropylmethylcellulose Trihydroxymethylaminomethane Sulforhdamine B Tris(hydroxymethyl)aminomethane Ethylenediaminetetraacetic acid Boric acid Taq DNA polymerase dNTPs pUC19DNA Msp I(Hpa II) Marker
Capillary Electrophoresis-Laser Induced Fluorescence Detection Unit Quartz Capillary UNO Model PCR Instrument PAC3000 Electrophoresis Instrument UVI Ultraviolet Imaging Analyzer