Bisulfite conversion of DNA
a technology of dna and bisulfite, which is applied in the field of cytosine methylation detection in dna, can solve the problems of high degradation and problems within subsequent purification and amplification, and achieve the effect of improving the efficacy of conversion and increasing the water solubility
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example 1
Automation of the Bisulfite Reaction
[0061] The application of the method for detecting the methylation state of cytosines in the factor VIII gene of a genomic DNA sample, which is treated with a restriction endonuclease according to the instructions of the manufacturer, is described in the present example. The method is based on the use of an automatic pipetting system (MWG RoboSeq 4204) with four separate vertically movable adapters for exchangeable pipetting tips, so as to exclude cross contaminations. The pipetting system makes possible the pipetting of 100 μl [aliquots] with an error of less than ±2 μl. The operating plate of the automatic pipetting system is equipped with six racks for pipetting tips and eight pipetting positions, two of which can be cooled, a reagent rack that can be cooled, a stacking system for 10 microtiter plates, a pipette tip washing station and a device for separating the pipette tips from the adapter.
[0062] The automatic pipetting system is connected...
example 2
Optimized Bisulfite Conversion by Addition of Dioxane for the Detection of DNA in Plasma Samples
[0066] The inventive optimized bisulfite method makes possible a sensitive methylation analysis of DNA obtained from body fluids. For example, 1 ml of human plasma was mixed with a specific quantity of human DNA. The DNA was isolated from the plasma samples via the Magna Pure method (Roche) according to the manufacturer's instructions. The 100 μl of eluate resulting from the purification were utilized completely in the following bisulfite reaction. The conversion according to a standard method (Frommer et al., loc. cit.) was conducted as a control. The procedure for the method according to the invention was as follows: The eluate was mixed with 354 μl of bisulfite solution (5.89 mol / l) and 146 μl of dioxane containing a radical scavenger(6-hydroxy-2,5,7,8-tetramethylchromane 2-carboxylic acid, 98.6 mg in 2.5 ml of dioxane). The reaction mixture was denatured for 3 min at 99° C. and subse...
example 3
Optimized Bisulfite Conversion by Addition of DME for the Detection of DNA in Plasma Samples
[0068] It will be shown that the optimized bisulfite method makes possible a sensitive methylation analysis of DNA obtained from body fluids. For this purpose, 1 ml of human plasma was mixed with a specific quantity of human DNA. The DNA was isolated from the plasma samples via the Magna Pure method (Roche) according to the manufacturer's instructions. The 100 μl of eluate resulting from the purification were utilized completely in the following bisulfite reaction. Conversion according to a standard method (Frommer et al., loc. cit.) was conducted as a control. The procedure for the method according to the invention was as follows: The eluate was mixed with 354 μl of bisulfite solution (5.89 mol / l) and 46 μl of DME containing a radical scavenger(6-hydroxy-2,5,7,8-tetramethylchromane 2-carboxylic acid, 98.6 mg in 787 μl of DME). The reaction mixture was denatured for 3 min at 99° C. and subse...
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