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Application of bioluminescence to detect DNA polymerase activity


LED working fluid processing to remove one of the pyrophosphate, as described in experimental approach by 4.1 light work of fluid, not detected inorganic pyrophosphate. Determination of the reaction in two steps into the 26 * line: First use of APS and ATP S acylase to quantitatively pyrophosphate into ATP: PPi + APSATP + SO then add the processed light working fluid to determine the amount of ATP: ATP + luciferin + O Zhu AMP + PPi + oxyluciferin + CO2 + h7ATP sulfur acylase has been identified and purified commercial,[link widoczny dla zalogowanych], ATP produced by the reaction can be more easily Chemiluminescence determination. Under this approach, exogenous ATP and the reaction of produced PPi Ⅱ small amounts, does not affect the determination. 2APS and the amount of ATP S acylase choose the presence of different concentrations of APS, adding a certain amount (10pmo1) pyrophosphate, was determined by 4.2 points 6s luminous intensity, when the APS concentration greater than 0.6 # mol / L, the maximum luminous intensity of the reaction in the determination of this Act continue to increase within the APS concentration, light intensity remain basically unchanged. The best selection of the experiment the concentration of APS 5 # mol / L, APS solution should be prepared using the new in order to prevent decomposition. Pyrophosphate in the presence of 10pmol, the addition of different concentrations of ATP S acylase (per unit of this enzyme in pH8.0, 30C conditions,[link widoczny dla zalogowanych], the catalyst generated per minute 10. PmolATP), determined by 4.2 points light 6s strength, when using 0.2UATP sulfur acylase, they can obtain a stable maximum luminous intensity. 3 acidity select all reactions were carried out under conditions in pH7.75, pH7.75 are sulfur acylation ATP insect luciferase enzyme and acidity of [3]. In this study 0.10mol/LpH7.75 of Tris-Ac buffer. 4 pyrophosphate calibration curve,[link widoczny dla zalogowanych], detection limit and repeatability were obtained with different concentrations of pyrophosphate standard solution (volume 101) by 4.2 points determined by the method 6s luminous intensity,[link widoczny dla zalogowanych], drawing the calibration curve. Experimental results show that the amount of pyrophosphate in the (1 × 1O called ~ 5 × 10) mol / I a good linear relationship. Calculated signal to noise ratio of 3 times the detection limit of 2.8 × 10. mol / L. Pyrophosphate standards take 0.10pmol (volume 101), 8 were determined by experiment, the relative standard deviation was 4.6%. 5 determination of the different preservation time TaqDNA polymerase activity LClinTransfusLabMed, Jan. 2005, Vol7, No. 1PCR is an efficient in vitro nucleic acid amplification technology, accompanied by the amplified nucleic acid pyrophosphate generated quantitative: (DNA) + dNTPDNApolymerase (DNA) l1 + + PPi different preservation time were taken for the PCR reaction TaqDNA polymerase , take the PCR amplification products (diluted 10 times) 10l added to the light tube,[link widoczny dla zalogowanych], which was detected by the determination of pyrophosphate content. Measured results shown in Table 1 (4 average). Pyrophosphorylase activity than a volume (using different storage time of TaqDNA polymerase) / pyrophosphate content (using a known standard TaqDNA polymerase activity) × 100. Table 1 save time TaqDNA polymerase activity of this method is a simple, sensitive detection methods TaqDNA polymerase activity, saving time by measuring the different TaqDNA polymerase activity, the results were satisfactory. 【
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