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Method of rapidly quantifying global DNA methylation

Inactive Publication Date: 2008-08-14
LI WEIWEI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0015]Thus the invention allows a rapid quantification of global DNA methylation to be achieved. The invention is based on the finding that nucleic acid binding substances coated on the plastic surface can more rapidly and conveniently enhance DNA immobilization and retention on the plastic surface in a simple dry-capture manner at appropriate temperature. The invention is also based on the finding that the immunodetection of 5-methylcytosine can be quantitatively achieved through specific antibody recognition followed by color or fluorescence development and measurement. Therefore the method presented in this invention significantly overcomes the weaknesses existing in the prior technologies and enables global DNA methylation to be quantified rapidly and efficiently.

Problems solved by technology

However these methods are labor intensive, time-consuming, or are not quantitative, or require large amounts of DNA (>250 ng) as the starting material for measurement, or rely on the use of expensive equipment and radioisotope reagents.
These disadvantages lead to cost-ineffectiveness, low throughput and inconvenience for routine application in most health and medical institutions.

Method used

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  • Method of rapidly quantifying global DNA methylation
  • Method of rapidly quantifying global DNA methylation
  • Method of rapidly quantifying global DNA methylation

Examples

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example 1

[0030]The experiment was carried out to compare the effect of different nucleic acid binding substance coating on the detection of global DNA methylation. Polystyrene 8-well strips were coated with 0.01% poly-L-lysine, or 1% polycarbodiimide, respectively. After storage at 4° C. for 2 weeks, the coated and uncoated strips were used for DNA immobilization. 30 μl of naturally methylated NY-2A virus DNA at different concentrations were added into the strip wells. The strips were then incubated at 37° C. for 1.5 h followed by incubation at 60° C. for 30 min to dry the wells. The wells were blocked with 2% BSA solution at 37° C. for 30 min followed by washing 3 times. 50 μl of monoclonal anti-5-methylcytosine antibody was then added at 0.5 pg / ml and incubated at room temperature for 1 h. The wells were washed 3 times with PBS containing 0.1% tween-20 after the antibody solution was removed. 50 μl of anti-mouse antibody conjugated with HRP at 0.1 μg / ml was added into the wells and incubat...

example 2

[0031]The experiment was carried out to compare the effect of dry-capture method and conventional immobilization methods on the detection of global DNA methylation. Polystyrene 8-well strips were coated with 0.01% poly-L-lysine. After storage at 4° C. for 2 weeks, the coated strips were used for DNA immobilization. With the method of this invention, naturally methylated NY-2A virus DNA was diluted with 1× TE buffer. 30 μl of diluted virus DNA at different concentrations were added into the strip wells. The strips were then incubated at 37° C. for 1.5 h followed by incubation at 60° C. for 30 min to dry the wells. With the conventional method, 50 μl of diluted virus DNA at different concentrations were added into the wells and incubated at room temperature for 4 h or at 4° C. overnight. The remaining solution was removed after incubation. The DNA-immobilized wells with both methods were blocked with 2% BSA solution at 37° C. for 30 min followed by washing 3 times. 50 μl of monoclonal...

example 3

[0032]The experiment was carried out to examine the detection sensitivity of the method based on this invention on the quantification of global DNA methylation. Polystyrene 8-well strips were coated with 0.01% poly-L-lysine. After storage at 4° C. for 2 weeks, the coated strips were used for DNA immobilization. Naturally methylated NY-2A virus DNA was diluted with 1× TE buffer to different concentrations. 30 μl of diluted virus DNA were added into the wells at the amounts of 2 ng-100 ng / well. The strips were then incubated at 37° C. for 1.5 h followed by incubation at 60° C. for 30 min to dry the wells. The wells were then blocked with 2% BSA solution at 37° C. for 30 min followed by washing 3 times. 50 μl of monoclonal anti-5-methylcytosine antibody was then added at 0.5 μg / ml and incubated at room temperature for 1 h. The wells were washed 3 times with PBS containing 0.1% tween-20 after the antibody solution was removed. 50 μl of anti-mouse antibody conjugated with HRP at 0.1 μg / m...

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Abstract

This invention is related to a method and assay kit for rapidly quantifying global DNA methylation through immobilizing DNA by simple dry-capture on the plastic carrier followed by immunodetection of 5-methylcytosine structure that is the marker of DNA methylation.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]Not applicableSTATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT[0002]Not applicableREFERENCE TO A MICROFICHE APPENDIX[0003]Not applicableBACKGROUND OF THE INVENTION [0004]1. Field of the Invention[0005]This invention is related to a method and assay kit for rapidly quantifying global DNA methylation through immobilizing DNA by simple dry-capture on the plastic carrier followed by immunodetection of 5-methylcytosine structure that is the marker of DNA methylation.[0006]2. Description of the Related Art[0007]DNA methylation is an epigenetic modification which is catalyzed by DNA cytosine-5-methyltransferases (DNMTs) and occurs at the 5-position (C5) of the cytosine ring, within CpG dinucleotides. DNA methylation is essential in regulating gene expression in nearly all biological processes including development, growth, and differentiation (Laird P W et al: Annu Rew. Genet, 1996; Reik W et al: Science, 2001; Robertson K D et al...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6804Y10T436/143333C12Q1/6816
Inventor LI, WEIWEILI, JESSICA
Owner LI WEIWEI
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