| Emulsion PCR is single-molecule PCR technology. Separated by the oil phase,the water droplets become independent PCR sections in which PCR can be carried out without interference each other. Under the most ideal scenario, there is only one DNA template within each water droplet and the PCR within a droplet will not be interfered by other DNA templates.Emulsion PCR has many advantages. (1) Strong anti-interference ability. (2)Elimination of the competitive inhibition. (3) High amplification efficiency and high-throughput. (4) High sensitivity. (5) Absolute quantitative.Here, we established the emulsion asymmetric PCR and emulsion single primer PCR method to build the ssDNA library. Furthermore, we also setup the two-step emulsion PCR method for detection of P16 gene methylation in serum form colorectal cancer patients and Hepatitis B virus DNA in serum from patients.Part I Construction and optimization of the efficient amplification method of a random ssDNA library with emulsion asymmetric PCRObjective: To construct a random ssDNA library using an emulsion asymmetric PCRMethods: In emulsion asymmetric PCR, the single molecule PCR is performed within single template who is separated by emulsion particles from different templates and the non-specific hybridization was avoided. The total volum of emulsion asymmetric PCR system was 100 ?l and it was composed of 50 mmol/L KC1, 10 mmol/L Tris-HCl (pH 8.6), 2.5 mmol/L MgCl2, 0.5 mmol/L dNTP, 0.4 ?mol/L upstream primer, 0.02 ?mol/L downstream primer, 0.04 ?g/mL template, 0.1 U/?L Pfu DNA polymerase and the original random ssDNA library was used as the template. To optimizate the PCR reaction condition, the concentration of template was used from 0.0004 ?g/mL to 40 ?g/mL, the concentration of upstream primer ranged from 0.2 ?mol/L to 1 ?mol/L and the concentration of downstream primer performed between 0.0025 ?mol/L and 0.02 ?mol/L. The emulsion asymmetric PCR condition was 30 sec denaturation at 94 ?, followed by 35 cycles of 40 sec denaturation at 94?, 30 sec annealing at 60 ?, 30 sec extension at 72 ?, and 3 min final extension at 72 ?. The cycle number to be optimized ranged from 10 to 50. Products,recovered by breaking emulsion, were analyzed by PAGE and silver stain. The finally ssDNA library was purified by Streptavidin system and magnetic beads isolation technique.Moreover, we compared conventional asymmetric PCR and emulsion asymmetric PCR.Results: In conventional asymmetric PCR, by-products were found in the 20th cycle.In the 25th cycle, the bands of target products were mixed with those of by-products.After 25 cycle,the number of dsDNA product was decreased with the increasing of cycle number. In the end, there was no template could be used and specific dsDNA were disappeared and only nonspecific product was generated. There were many by-products at 25th amplication cycle, ssDNA products were isolated by magnetic bead and unpurified. Therefore,it is suitable to separate and detect the concentration of the products at 10th, 15th and 20th cycle and the concentrations of products were 0.76,1.43 and 2.02 ?g/mL respectively. In emulsion asymmetric PCR, after 50 cycles, no by-products were formed, and only the ssDNA and dsDNA belts were found, and the amounts of dsDNA products were not decreased. At the 10th, 15th, 20th, 25th, 30th, 35th,40tth, 45th and 50th cycle, the products were isolated and detected, and the concentrations of products were 0.58?1.22?1.78?2.25?3.21?4.32?6.43?6.71 and 6.55 ?g/mL respectively and higher than those of products from conventional asymmetric PCR.During the optimization of emulsion asymmetric PCR, we firstly optimized template concentration. When the template concentration was increased from 0.0004?g/mL to 0.04 ?g/mL, the amount of products were increased. When template concentration was increased above 0.04 ?g/mL, the amount of products were not increased. When the template concentration was 4 ?g/mL, a large amount of by-products were generated and smear bands were eviden.When the template concentration was from 0.04 to 0.4 ?/mL, by-products could not be found and the amount of products were enough. When the template concentration was 0.4 ?g/mL,the concentrations of dsDNA were decreased obviously. So the appropriate template concentration ranged from 0.04 to 0.4 ?g/mL.During the optimization of primer concentration, we optimized both the ratio of upstream primer to downstream primer and the concentrations of upstream primer and downstream primer. Since primers were prone to react with ssDNA to form non-specific hybridization. The higher primer concentration was easier to form the non-specific amplification. Therefore, we firstly optimized the upstream primer with the high concentration. When the upstream primer concentration varied from 0.2 ?mol/L to 1 ?mol/L, the amount of products showed no significant change. While the upstream primer concentration was 0.6 ?mol/L, a small amount of by-products were generated. When the upstream primer concentration was 1 ?mol/L, a large amount of by-products were generated. During the optimization of downstream primer, the upstream primer concentration was 0.4 ?mol/L and downstream primer concentrations ranged from 0.0025 ?mol/L, to 0.02 ?mol/L, the amount of products was increased gradually and no by-product was generated. So the sutiable upstream primer concentration ranged from 0.2 to 0.4 ?mol/L, and the best downstream primer concentration was 0.02 ?mol/L.Conclusion: In the emulsion asymmetric PCR, the optimization of cycle number was not needed, and the by-products formation dut to overamplification in conventional PCR was avoided. The high quality ssDNA library could be gernerated easy via optimizating of the concentration of template and primers. The method,described here, could be used to construct an ssDNA sub-library easily, conveniently,efficiently, and improved the screening efficiency of aptamers.Part II Emulsion single primer PCR : a high efficient method for generating random ssDNA libraryObjective: To develop a high efficient method to generate random ssDNA library using emulsion single primer PCR.Methods: The library was constructed with a single-stranded 90-oligonucleotide.The random dsDNA library was amplified by emulsion PCR. After that, a emulsion single primer PCR was performed to construct the ssDNA library with the templates from dsDNA library and compared with the conventional single primer PCR. The total volume of 100-?L emulsion single primer PCR system was composed of 50 mmol/L KCl, 10 mmol/L Tris-HCl (pH 8.6), 2.5 mmol/L MgCl2, 0.5 mmol/L dNTP,0.75 ?mol/L upstream primer, 9 pmol/mL dsDNA template, and 0.1 U/?L pfu DNA polymerase. In the optimization experiments, the concentrations of template were ranged from 1 pmol/mL to 18 pmol/mL, the concentrations of primer were ranged from 0.5 ?mol/L to 3 ?mol/L, the concentrations of pfu DNA polymerase were ranged from 0.075 U/?L to 0.175 U/?L and the concentrations of dNTP were ranged from 0.25 mmol/L to 1.25 mmol/L. Emulsion single primer PCR was performed with 30 sec denaturation at 94 ?, followed by 35 cycles of 30 sec denaturation at 94 ?, 30 sec annealing at 60 ?, and 30 sec extension at 72 ?. and 3 min extension at 72 ?.The optimization cycle numbers were ranged from 10 to 35.Results: Emulsion PCR can convert ssDNA to high quality of dsDNA. In single primer PCR, we found that by-products were generated after 15 cycles which could not be separated from target products. In emulsion single primer PCR system, after 35 cycles, the bands of by-products were not observed and the amounts of target products were significantly higher than the conventional single primer PCR. During the optimization of emulsion single primer PCR, it was found that the amount of template affected the amount of products significantly. When the template concentration was decreased from 18 pmol/mL to 1 pmol/mL, the amount of product was almost decreased to zero. The primer concentration affected by-products significantly.Annealing temperature affected the amounts of specific products and by-products.When the DNA polymerase concentration was greater than 0.1 U/?L, by-products were appeared. The influence of dNTP concentration on the amplification products was relatively small.Conclusions : In the emulsion single primer PCR, the optimization of cycle number was not needed and the formation of by-product was avoided. The template was enough for the emulsion single primer PCR templates were dsDNA from the products of PCR amplification. The emulsion single primer PCR were more advantages and more powerful than the emulsion asymmetric PCR for ssDNA library construction.Part ? Establishment a two-step emulsion PCR and aplicated in detection of P16 gene methylation in serum from patients with colorectal cancerObjective : To establishe a two-step emulsion PCR for detecting P16 DNA methylation in serum from patients with colorectal cancer.Methods : Including methylation and non-methylation of P16 gene sequence plasmids were constructed and transfered with e.coli DH5a. A two-step Emulsion PCR was be established : (1) Water-in-oil emulsion: PCR emulsion was contained 0.1 mL PCR mixture that were composed with 10 mM KCl, 20 mM Tris-HCl (pH 8.8),0.2 mM MgSO4,16 mM (NH4)2SO4, 0.1% TritonX-100, 0.5 mM dNTP,0.2?M primers, 4 ?L template, and 0.1 U/?L Pfu DNA polymerase. Aliquots of 100?L/tube of the prepared emulsion was used in PCR amplification under the following conditions: denaturation at 94 ? for 5 min, followed by 15 cycles of denaturation at 94 ? for 30 sec, 60 sec for annealing at either 69? for methylated or 65? for non-methylated DNA, 60 sec for extension at 72?, and 3 min for final extension. (2) Oil-in-water emulsion PCR: After the first amplification was completed, the samples were centrifuged at 14,000 rpm for 10 min to remove the top oil phase and mixed with 33?L PCR solution containinglO mM KCl, 20 mM Tris-HCl (pH 8.8), 0.2 mM MgSO4,16 mM (NH4)2SO4, 0.1% TritonX-100, 0.5 mM dNTP,0.4 ?M primers, and 0.1 U/?L Pfu DNA polymerase. Oil-in-water emulsion was generated by a thorough mixing and PCR was performed following the above conditions for 35 cycles of amplification.MSP and two-step emulsion PCR was used to detect different concentrations of methylation of P16 gene sequence of bacteria respectively,and the characteristics of the two methods was compared. The P16 gene methylation were detected in serum from 36 patients with colorectal cancer and 25 healthy donors.Results: The results of sensitivity and interference assay were shown that the two-step emulsion PCR has higher sensitivity and stronger anti-interference ability than those of MSP. The result showed the detection sensitivity of two-step emulsion PCR is 10 times of MSP. With the interference of non-methylation gene, the methylation could be detected in methylation and non-methylation ratio of 1:100 by two-step emulsion PCR, while the methylation could be detected in methylation and non-methylation ratio of 1:10 by MSP. In the detection of p16 gene methylation in serum from colorectal cancer patients, two-step emulsion PCR detection rate was 55.5%,while MSP detection rate was only 44.4%.Conclusion : The two-step emulsion PCR system has high sensitivity and stronganti-interference ability for detecting P16 gene methylation. detection rate of which was higher than that of MSP and can be used to the early diagnosis of clinical oncology.Part ? Establishment of a two-step emulsion PCR and aplicated in detection of hepatitis B virus DNAObjective : To establish a two-step method emulsion PCR to detect hepatitis B virus DNA.Methods: 78 samples, with the results of < 500 IU/mL in hepatitis B virus DNA were detected by qPCR and without amplification curve, were selected. We use two-step emulsion PCR and conventional PCR to detect the these samples. And the serum marker of hepatitis B virus was detected by ELISA.Results: 14 positive samples (35%) among the 40 samples, without amplification curve, were detected by two-step emulsion PCR, while 0 positive samples were detected by conventional PCR. Among the 40 samples, 27 (67.5%) were HBsAg positive and HBeAg negative (67.5%), which were inactive HBSAg positive carriers.However, HBV DNA was founded using the two-step emulsion PCR method in 10 of the 27 patients.Conclusion: The two-step emulsion PCR has higher sensitivity than that of conventional PCR, and could further detect whether there are hepatitis B virus DNA in serum from patients with <500 IU/mL HBV DNA evaluated by conventional PCR.In conclusion, the two-step emulsion PCR can be used as a supplement method for the fluorescence quantitative PCR and provid the basis for the clinical diagnosis and evaluation of treatment in anti-hepatitis B virous. |
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