The Establishment Of Capillary Gel Electrophoresis-ultraviolet Detection System For Highly Sensitive Analyzing Nucleic Acids With Its Application In Molecular Identification Of Polygonum Multiflorum

Low sensitivity is the main restriction for the analysis of nucleic acids using capillary electrophoresis, and for the establishment of capillary electrophoresis system based on nucleic acid detection. To address the problem of low sensitivity, field-amplified stacking techniques were used to enhance the sensitivity of CGE-UV for analyzing DNA. And after the methods were established, CGE-UV was used to identify Polygonum multiflorum with its adulterants at gene level; the achievements in our research were as follows:1. By means of studying literatures extensively, the key problem for establishing capillary electrophoresis system based on nucleic acid detection was deeply investigated, the feasible solutions for solving the sensitivity dilemma, and the technical procedures for CE was used to analyze nucleic acid after capillary electrophoresis system was set up, were put forward.(1) Field-amplified stacking techniques were come up to settle the low sensitivity dilemma in CE. Moreover, two modes, termed as M-FASI and C-FASI, were firstly proposed and the possible highly-sensitive mechanism for C-FASI was firstly put forward and illustrated, which offered the solutions for solving low sensitivity in CE.(2) After highy-sensitive capillary electrophoresis system based on nucleic acid detection was set up, we proposed that capillary electrophoresis methods should couple with the optimized nucleic acid detection strategies to analyze nucleic acid better, which provided technical process for the subsequently molecular identification of Polygonum multiflorum with its adulterants.2. Field-amplified sample stacking(FASS) was used to improve the sensitivity of CGE-UV for DNA. The optimal conditions were that TE was as the dilution solution, water-plug was injected(0.5 psi, 40 s), and then sample was injected(25 psi, 40 s), the voltage for separation was-250 V/cm. Under the optimal conditions, compared with normal pressure injection(0.5 psi, 10 s), the sensitivity for FASS was 665-fold higher and the LOD for DNA was reduced to 20 ng/m L. The experiment also proved the stacking effect was far stronger than dilution effect, which offered the strategy that samples of high ionic strength could be enriched and detected using FASS with large amounts of dilution because PCR products or clinical samples are often high in ionic strength and low in concentration. To validate the stategy, samples of ?X174-Hae III digest DNA produced by enzyme digestion of bacteriophage ?X174am3 DNA without purification and high ionic strength was tested in FASS, the 11 DNA bands were successfully detected confirmed the usefulness of the strategy. The linear range for FASS is 20-400 ng/m L, regression equation is Y=92.10X-736.5, and correlation coefficient is 0.999. The RSDs for migration time and peak area under FASS are(1.07-5.36) and(0.04-0.91), respectively. These results demonstrate that FASS has good qualitative and quantitative abilities for the analysis of DNA, is one of field-amplified modes that effectively enhance the sensitivity of CGE-UV for DNA.3. Although FASS enhanced the volume of injection, the prolongation of injection time together with the extended sample plug existing in capillary that interrupted the subsequent separation increased the total analysis time. Moreover, the sensitivity effect using pressure injection was restrained by the volume of capillary. Thus, M-FASI using electrokinetic injection was used to further enhance the sensitivity of CGE-UV for DNA. The optimized conditions were that(50 μM tris-Hcl and 5 μM EDTA)/L TE solution was as the dilution solution, sample was injected(-10 k V, 50 s) and the voltage for separation was-250 V/cm. Under the optimized conditions, LOD(S/N=3) for DNA under M-FASI was reduced to 4 ng/m L, the sensitivity was 100-fold higher compared to normal electrokinetic injection(-10 k V,10 s) and 4-fold higher compared to FASS. The experiment also proved the stacking effect was far stronger than dilution effect and the successful detection of ?X174-Hae III digest DNA without purification validated the effectiveness of M-FASI. The linear range for M-FASI is 20-134 ng/m L, RSDs for migration time and peak areas are(0.04-0.91) and(0.66-6.21), respectively. These results demonstrate that M-FASI has good qualitative and quantitative abilities for the analysis of DNA, and is the effective method for analyzing DNA in low concentration.4. Although M-FASI using electrokinetic injection acquired better sensitivity than FASS, the stacking interface between sample solution(high field-strength) and BGE(low field-strength) was easily disrupted, electrokinetic injection time was limited, together with overproduced Joule heat interrupted the efficiency of CE separation,etc. These troubles restricted the improvement of CE sensitivity. Therefore, to further stabilize the stacking interface and develop a highly sensitive method for analyzing nucleic acids using capillary gel electrophoresis with ultraviolet detection(CGE-UV), we combined matrix field-amplified with head-column field-amplified stacking injection(C-FASI) to employ the advantages of two methods and eliminate the shortness of FASS with M-FASI. Without diminishing the resolution, a limit of detection of 0.13 ng/ml(signal/noise=3) in a 300,000-fold diluted sample was obtained, the sensitivity is 102,308 times higher than that achieved with normal pressure injection, 3,077 times that with normal electrokinetic injection, 154 times that with pressure field-amplified sample stacking injection, and 31 times that with matrix field-amplified stacking injection. After establishing the method, we tested the detection of a ?X174-Hae III digest DNA product without purification and with a high ionic strength. At the lowest dilution of 5000-fold, sample at a concentration of 10 ng/ml was enriched and detected. The relative standard deviations for migration time and peak area(n=3) were 0.03–1.15 and 0.72–6.42, respectively. To further validate C-FASI was applicable for real sample, a 400 bp PCR product without purification was directly detected with a limit of detection at the concentration of 6,000-fold dilution(signal/noise=3), The relative standard deviations for migration time and peak area(n=6) were 0.44 and 4.8, respectively. These results indicated that C-FASI had good qualitative and quantitative detection abilities and CGE-UV based on C-FASI is easy to perform, practical, highly-sensitive and robust for nucleic acid detection, which makes it a highly valuable tool for nucleic acid analysis.5. After capillary gel electrophoresis-ultraviolet detection system for highly-sensitive analyzing nucleic acid was set up, to validate the usefulness of this system, CGE-UV based on field-amplified stacking was used to identify Polygonum multiflorum with its adulterants by means of PCR-RFLP technique, and compared with traditional slab gel electrophoresis(SGE). Both were able to differentiate Polygonum multiflorum with its adulterants. However, CGE possesses the advantages that a 6000-fold higher sensitivity over SGE, more than million plates in analytical efficiency, clear electrophoregrams, automation with parallelly analyzing data, clean without pollution, no harm for the health of operators, cost-effective, is able to quantify with good quantitative precision. These results indicate that CGE-UV based on field-amplified stacking has a great potential for replacing SGE in DNA analysis. However, because PCR-RFLP procedure spent too much time so that PCR-RFLP-CGE-UV was unable to quickly identify Polygonum multiflorum, therefore, the experiment tried to optimize the PCR strategy.6. SNP-PCR instead of PCR-RFLP was used to integrate with CGE-UV to rapidly identify Polygonum multiflorum with its adulterants(SNP-PCR-CGE-UV), completing the process from PCR to DNA detection with data analysis in 23 mins demonstrated SNP-PCR-CGE-UV was the rapidest identification method among all approaches for molecularly identifying Polygonum multiflorum. Together with SNP-PCR-CGE-UV costed less than PCR-RFLP-CGE-UV, these results proved it was a true high-speed, high-throughput, high cost-effective method for identifying Polygonum multiflorum. Moveover, optimizing the nucleic acid analytical system from two factors that were PCR strategy and the detection mode for DNA set an example for using CGE-UV to apply in other nucleic acid analysis. At the same time, the experiment also showed CGE-UV based on field-amplified stacking had a great potential for replacing SGE in DNA analysis.Based on the above results in our researches, we concluded that the established field-amplified stacking methods successfully settled the sensitivity problem for using CGE-UV to analyze DNA, and CGE-UV was able to detect DNA sensitively and rapidly, and was applicable to analyze the real nucleic acid samples that were often high in ionic strength with low in concentration, which laid the foundation for using CGE-UV to analyze DNA. In the molecular identification of Polygonum multiflorum with its adulterants, CGE-UV coupled with SNP-PCR offered an example for applying CGE-UV in other nucleic acid analysis. These results indicat that highly sensitive CGE-UV based on nucleic acid detection has agreat potential in future nucleic acid analysis.