4/27/2023 0 Comments Emulsion pcr![]() The low specificity of DNA hybridization is apt to generate the false positive result. Similarly, the assays of gene variation based on DNA hybridization technique like southern blot cannot accurately discriminate the rare mutations among the predominant wild-type DNA alleles. The predominant wild-type DNA tends to be amplified prior to the mutant alleles in PCR, which leads to the dilution of mutation. Attempts to increase the percentage of rare mutation by polymerase chain reaction (PCR) often fail owing to the interference of wild-type DNA (11,12). The mutation with the percentage less than 20% is hardly to be identified by gene sequencing. Take the gene sequencing technique, the golden standard for detecting gene variation first. Thus, the rare mutations among large excess wild-type alleles are hard to be detected by conventional assays for gene variation. And there is only one nucleotide variation in DNA sequence between wild-type DNA and point mutation. There are predominant wild-type alleles derived from the apoptosis of normal cells in the blood. The percentage of mutation is usually less than 10% in the total cfDNA. However, the detection of rare mutations in cfDNA has been proved very difficult (8-10). Detection of such mutations in the blood can provide wealth of information for tumor diagnosis, prognosis, and therapy. The mutations in the blood are contribute to the DNA release of apoptosis and necrosis of tumor cells because they bear the uncommon variations that consistent with that of DNA isolated from tumor cells. In the last decades, the detection of rare mutation of cancer genes in the circulating free DNA (cfDNA) of patient’s blood has attracted increasing attention for its potential in tumor diagnosis and personalized treatment (4-7). 5 and 10: PCR mixture include all the masteries.The detection of point mutation or single nucleotide polymorphism (SNP) is essential in many fields of biomedical research such as molecular diagnosis, personalized therapy, and drug development (1-3). 4 and 9: PCR mixture include all except DNA polymerase. 3 and 8: PCR mixture include all except dNTP. 2 and 7: PCR mixture include all except primer. 1 and 6: PCR mixture only include template. Influence of DNA polymerase concentrations.Įmulsion PCR amplification was carried out for 35 cycles the number of templates was 0.01 pmol/ml the primer concentration was 0.4 µmol/L and the annealing temperature was 65☌.Įmulsion PCR amplification was carried out for 35 cycles the number of templates was 0.01 pmol/ml the concentration of Taq DNA polymerase was 0.125 U/µl and the annealing temperature was 65☌.Ĭonventional PCR was carried out for 1 and 30 cycles with 5 µg/ml template. Emulsion PCR amplification was carried out for 35 cycles the number of template was 0.01 pmol/ml the primer concentration was 0.4 µmol/L and the concentration of Taq DNA polymerase was 0.125 U/µl. The concentration of the dsDNA products and by-products was obtained from microfluidic chip electrophoresis. Emulsion PCR amplification was carried out for 35 cycles the annealing temperature was 65☌ the primer concentration was 0.4 µmol/L and the concentration of Taq DNA polymerase was 0.125 U/µl. Panel B indicates the concentration of dsDNA products and by-products obtained from microfluidic chip electrophoresis. Panel A indicates the PAGE electropherograms of PCR reaction mixtures for amplification of a random DNA library with different amounts of template molecules. Influence of the number of template molecules. Emulsion PCR amplification was performed with cycles ranging from 10 to 40 the template concentration was 0.01 pmol/ml the primer concentration was 0.4 µmol/L and the concentration of Taq DNA polymerase was 0.125 U/µl. Conventional PCR amplification was performed with cycles ranging from 10 to 32 the template concentration was 0.01 pmol/ml the primer concentration was 0.4 µmol/L and the concentration of Taq DNA polymerase was 0.05 U/µl. Dynamics of product and by-product concentrations in conventional PCR and emusion PCR (E). Microfluidic chip electrophoresis electropherograms of PCR reaction mixtures for amplification of a random DNA library with conventional PCR (B) and emulsion PCR (D). PAGE electropherograms of PCR reaction mixtures for amplification of a random DNA library with conventional PCR (A) and emulsion PCR (C). Simulation diagrams of emulsion PCR amplification of a random DNA library.ĭifferent compartment contained different templates marked with different colors, and some compartment are empty.Ĭonventional PCR amplification of a DNA random library versus emulsion PCR.
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