Epigenase™ 5-mC Hydroxylase TET Activity/Inhibition
Contents
1. EpiQuik DNMT Activity Inhibition Assay Ultra Kit Fluorometric Base Catalog P 3010 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE Uses The EpiQuik DNMT Activity Inhibition Assay Ultra Kit Fluorometric is suitable for measuring total DNMT activity or inhibition using nuclear extracts or purified enzymes from a broad range of species such as mammalians plants fungi bacteria and viruses in a variety of forms including but not limited to cultured cells and fresh and frozen tissues Nuclear extracts can be prepared by using your own successful method For your convenience and the best results Epigentek also offers a nuclear extraction kit Cat No OP 0002 optimized for use with this kit Nuclear extracts can be used immediately or stored at 80 C for future use Purified enzymes can be active DNMTs from recombinant proteins or isolated from cell tissues Input Material Inout materials can be nuclear extracts or purified DNMT enzymes The amount of nuclear extracts for each assay can be between 0 2 ug to 10 ug with an optimal range of 5 yg to 10 ug The amount of purified enzymes can be 0 2 ng to 100 ng depending on the purity and catalytic activity of the enzymes Internal Control A positive enzyme control is provided in this kit Because DNMT activity can vary from tissue to tissue and from normal and diseased states it is advised to run replicate samples to ensure that the signal generated is validated Precautions T2. The controls and samples can be measured in duplicates Well Strip 1 Strip 2 Strip 3 Strip4 Strip 5 Strip 6 A Blank Blank Sample Sample Sample Sample B MK40 5ul MK40 5 ul Sample Sample Sample Sample Cc MK4 1 ul MK4 1 ul Sample Sample Sample Sample D Sample Sample Sample Sample Sample Sample E Sample Sample Sample Sample Sample Sample F Sample Sample Sample Sample Sample Sample G Sample Sample Sample Sample Sample Sample H Sample Sample Sample Sample Sample Sample TROUBLESHOOTING Problem Possible Cause Suggestion No signal or weak Reagents are added Check if reagents are added in the signal in both the incorrectly proper order with the right amount and positive control and if any steps in the protocol may have sample wells been omitted by mistake The well is incorrectly washed Ensure the well is not washed prior to before enzyme reaction adding the positive control and sample Incubation time and Ensure the incubation time and temperature are incorrect temperature described in the protocol are followed correctly Incorrect fluorescence reading Check if appropriate fluorescent 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 9 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2014 09 22 Epigentek Group Inc All rights reserved Products
3. Upon receipt 1 Store MK3 MK4 MK6 MK7 and MK8 at 20 C away from light 2 Store MK1 MK5 MK9 and the 8 Well Assay Strips at 4 C away from light 3 Store all remaining components MK2 MK10 and the Adhesive Covering Film at room temperature away from light All components of the kit are stable for 6 months from the date of shipment when stored properly Note 1 Check if MK1 10X Wash Buffer contains salt precipitates before use If so warm at room temperature or 37 C and shake the buffer until the salts are re dissolved MATERIALS REQUIRED BUT NOT SUPPLIED O Adjustable pipette or multiple cheannel pipette Multiple channel pipette reservoirs oO O Aerosol resistant pipette tips Oo Fluorescence microplate reader capable of reading fluorescence at 530 excitation and 590 emission nm O 1 5 ml microcentrifuge tubes 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Printed 20 Page 2 14 09 22 P 3010 Incubator for 37 C incubation Distilled water Nuclear extract or purified enzyme samples containing Dnmt activity o O o O GENERAL PRODUCT INFORMATION Parafilm M or aluminium foil Quality Control Each lot of this product is tested against predetermined specifications to ensure consistent product quality Epigentek guara
4. positive control to ensure that the signal generated is validated Carefully remove un needed strip wells from the plate frame and place them back in the bag seal the bag tightly and store at 4 C b Blank Wells Add 50 ul of Diluted MK3 per well c Positive Control Wells Add 50 ul of Diluted MK3 and 1 ul of MK4 per well 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 6 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Printed 2014 09 22 P 3010 d Sample Wells Without Inhibitor Add 45 ul to 49 ul of Diluted MK3 and 1 ul to 5 ul of nuclear extracts or 1 ul to 5 ul of purified DNMT enzymes per well Total volume should be 50 ul well e Sample Wells With Inhibitor Add 40 ul to 44 ul of Diluted MK3 1 ul to 5 ul of nuclear extracts or 1 ul to 5 ul of purified DNMT enzymes and 5 ul of inhibitor solution per well Total volume should be 50 pl well Note 1 Follow the suggested well setup diagrams 2 It is recommended to use 5 ug to 10 ug of nuclear extract per well or 10 ng to 100 ng of purified enzyme per well 3 The concentration of inhibitors to be added into the sample wells can be varied e g 1 uM to 1000 uM However the final concentration of the inhibitors before adding to the wells should be prepared with MK2 at a 1 10 ratio e g add 0 5 ul of inhibitor to 4 5 Ll of MK2 so that
5. the original solvent of the inhibitor can be reduced to 1 of the reaction solution or less f Tightly cover the strip well microplate with the Adhesive Covering Film to avoid evaporation and incubate at 37 C for 90 120 min Note 1 The incubation time may depend on intrinsic DNMT activity In general 60 90 min incubation is suitable for active purified DNMT enzymes and 90 120 min incubation is required for nuclear extracts 2 The Adhesive Covering Film can be cut to the required size to cover the strips based on the number of strips to be used g Remove the reaction solution from each well Wash each well with 150 ul of the Diluted MK1 1X Wash Buffer each time for three times This can be done by simply pipetting Diluted MK1 in and out of the wells 3 Antibody Binding amp Signal Enhancing a Add 50 ul of the Diluted MK5 to each well then carefully cover with Parafilm M or aluminium foil and incubate at room temperature for 60 min b Remove the Diluted MKS solution from each well c Wash each well with 150 ul of the Diluted MK1 each time for three times d Add 50 ul of the Diluted MK6 to each well then carefully cover with Parafilm M or aluminium foil and incubate at room temperature for 30 min e Remove the Diluted MK6 solution from each well f Wash each well with 150 ul of the Diluted MK1 each time for four times g Add 50 ul of the Diluted MK7 to each well then carefully cover with Parafilm M or aluminium foil
6. transcription is initiated In the bulk of genomic DNA most CpG sites are heavily methylated However CpG islands in germ line tissue and promoters of normal somatic cells remain unmethylated allowing gene expression to occur When a CpG island in the promoter region of a gene is methylated the expression of the gene is repressed The repression can be caused by directly inhibiting the binding of specific transcription factors and indirectly by recruiting methyl CpG binding proteins and their associated repressive chromatin remodeling activity In addition to the effect on gene transcription DNA methylation is also involved in genomic imprinting which refers to a parental origin specific expression of a gene and the formation of a chromatin domain DNA methylation is controlled at several different levels in normal and diseased cells The addition of methyl groups is carried out by a family of enzymes DNA methyltransferases DNMTs Chromatin structure in the vicinity of gene promoters also affects DNA methylation and transcriptional activity Three DNMTs DNMT1 DNMT3A and DNMT3B are required for the establishment and maintenance 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 3 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Printed 2014 09 22 P 3010 of DNA methylation patterns Two additional en
7. 10 Bi County Blvd Ste 122 Farmingdale NY 11735 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Page 12 Printed 2014 09 22 P 3010
8. For the best results it is advised to use Epigentek s Nuclear Extraction Kit Cat No OP 0002 Also use fresh cells or tissues for protein extraction as frozen cells or tissues could lose enzyme activity Sample amount added into the wells is insufficient Ensure a sufficient amount of purified enzymes or nuclear extracts is used as indicated in step 2 The sample can be titrated to determine the optimal amount to use in the assay Sample was not stored properly or has been stored for too long Ensure sample is stored in aliquots at 80 C with no more than 6 weeks for nuclear extracts and 6 months for purified enzymes Avoid repeated freezing thawing Little or no activity of DNMT contained in the sample This problem may be a result of many factors If the affecting factors cannot be determined use new or re prepared 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Page 10 Printed 2014 09 22 P 3010 nuclear extracts or purified enzymes Uneven fluorescent development Insufficient washing of the wells Ensure the wells are washed according to the protocol Ensure any residues from the wash buffer are removed as much as possible Delayed fluorescence development in the wells Ensur
9. K5 to 1000 ul of Diluted MK1 About 50 ul of Diluted MK5 will be required for each assay well Prepare Diluted MK6 Detection Antibody Solution Dilute MK6 Detection Antibody with Diluted MK1 at a ratio of 1 2000 i e add 1 ul of MK6 to 2000 ul of Diluted MK1 About 50 ul of this Diluted MK6 will be required for each assay well Prepare Diluted MK7 Enhancer Solution Dilute MK7 Enhancer Solution with Diluted MK1 at a ratio of 1 5000 i e add 1 ul of MK7 to 5000 ul of Diluted MK1 About 50 ul of this Diluted MK7 will be required for each assay well Prepare Fluorescence Development Solution Add 1 ul of MK8 Fluoro Developer and 1 ul of MK9 Fluoro Enhancer to every 500 ul of MK10 Fluoro Diluter About the MK4 DNMT Enzyme Control The MK4 DNMT Enzyme Control is an enzyme with activity of both maintenance and de novo DNMTs and is used as the positive control of the assay We do not recommend using this enzyme control to generate a standard curve for quantifying the activity of your samples as the amount of the enzyme is limited and catalytic activity unit is different Note Keep each of the diluted solutions except Diluted MK1 1X Wash Buffer on ice until use Any remaining diluted solutions other than Diluted MK1 should be discarded if not used within the same day 2 Enzymatic Reaction a Predetermine the number of strip wells required for your experiment It is advised to run replicate samples include blank and
10. ample is 5500 Average RFU of blank is 500 Protein amount is 5 pg Incubation time is 2 hours 120 min 5500 500 DNMT activity WWW x1000 500 000 RFU h mg 5 x 2 c Calculate DNMT inhibition using the following formula Inhibitor Sample RFU Blank RFU x 100 No Inhibitor Sample RFU Blank RFU 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 8 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Printed 2014 09 22 P 3010 SUGGESTED WORKING BUFFER AND SOLUTION SETUP Table 1 Approximate amount of required buffers and solutions for defined assay wells based on the protocol Reagents 1 well 8 wells 16 wells 48 wells 96 wells 1 strip 2 strips 6 strips 12 strips Diluted MK1 2 5 ml 20 ml 40 ml 120 ml 240 ml Diluted MK3 50 ul 400 ul 800 ul 2400 ul 4800 ul Diluted MK5 50 ul 400 ul 800 ul 2400 ul 4800 ul Diluted MK6 50 ul 400 ul 800 ul 2400 ul 4800 ul Diluted MK7 50 ul 400 ul 800 ul 2400 ul 4800 ul Fluorescence 0 05ml 0 4 ml 0 8 ml 2 4 ml 4 8 ml Development Solution DNMT Enzyme N A O 25ul tul jO5wl 2yuljipl 4uyl j2ul 8ul Conirol SUGGESTED STRIP WELL SETUP Table 2 The suggested strip well plate setup for the DNMT activity assay in a 48 assay format in a 96 assay format Strips 7 to 12 can be configured as Sample
11. and incubate at room temperature for 30 min h Remove the Diluted MK7 solution from each well i Wash each well with 150 ul of the Diluted MK1 each time for five times Note Ensure any residual wash buffer in the wells is thoroughly removed as much as possible at each wash step 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Page 7 Printed 2014 09 22 P 3010 4 Signal Detection a Add 50 ul of Fluorescence Development Solution to each well and incubate at room temperature for 1 to 3 min away from direct light The Fluorescence Development Solution will turn pink in the presence of sufficient methylated DNA Read the fluorescence on a fluorescence microplate reader within 2 to 10 min at 530 590 nm Note If the stripwell microplate frame does not fit in the microplate reader transfer the solution to a standard 96 well microplate 5 DNMT Activity Calculation a Calculate average duplicate readings for sample wells and blank wells b Calculate DNMT activity or inhibition using the following formula ies le RFU Blank RF DNMT Activity RFU h mg _GampleRFU Blank RFU iopo Protein Amount ug x hour Protein amount added into the reaction at step 2d in yg Incubation time at step 2f Example calculation Average RFU of s
12. are for research use only P 3010 wavelength 530 590 nm filter is used Kit was not stored or handled properly Ensure all components of the kit were stored at the appropriate temperature and caps are tightly capped after each opening or use No signal or weak signal in only the positive control wells The DNMT enzyme control is insufficiently added to the well in Step 2c Ensure a sufficient amount of DNMT enzyme control is added The quality of the DNMT enzyme control has been degraded due to improper storage conditions Follow the Shipping amp Storage guidance in this User Guide for storage instructions of MK4 DNMT Enzyme Control High background present in the blank wells Insufficient washing of wells Check if washing recommendations at each step is performed according to the protocol Contaminated by sample or positive control Ensure the well is not contaminated from adding sample or positive control accidentally or from using contaminated tips Incubation time with detection antibody is too long The incubation time at Step 3d should not exceed 45 min Over development of fluorescence Decrease the development time in Step 4a and measure fluorescence as quickly as possible No signal or weak signal only in sample wells Protein sample is not properly extracted or purified Ensure your protocol is suitable for DNMT protein extraction
13. distributed Do not stir Did not use the same pipette device throughout the experiment Use the same multi channel pipette device throughout the entire experiment as different pipette devices may have slight variations in performance Capture Antibody vial appears to be empty or insufficient in volume Buffer evaporated due to the very small volumes resulting in a higher concentrated antibody Add 1X PBS buffer into the Capture Antibody vial until you restore the correct intended volume according to the Kit Contents described in this User Guide Mix and centrifuge prior to use RELATED PRODUCTS 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Page 11 Printed 2014 09 22 P 3010 Nuclear Extract Preparation OP 0002 1 EpiQuik Nuclear Extraction Kit DNMT Activity Inhibition Assay and Content Quantification P 3009 EpiQuik DNMT Activity Inhibition Assay Ultra Kit Colorimetric P 3011 EpiQuik DNMT1 Assay Kit P 3012 EpiQuik DNMTS3A Assay Kit P 3013 EpiQuik DNMT3B Assay Kit DNMT Antibodies A 1001 DNMT1 Monoclonal Antibody A 1002 DNMT2 Polyclonal Antibody ABB 1002 DNMT2 Monoclonal Antibody A 1003 DNMTSA Polyclonal Antibody A 1004 DNMTS3B Polyclonal Antibody A 1005 DNMTS3L Polyclonal Antibody 1
14. e fluorescence development solution is added sequentially and consistent with the order you added the other reagents e g from well A to well G or from well 1 to well 12 Large variation between replicate wells Fluorescent reaction is not evenly occurring due to an inconsistency in pipetting time Ensure MK8 Fluoro Developer is added at the same time between replicates or otherwise maintain a consistent timing in between each addition of solutions Fluorescent reaction is not occurring evenly due to an inconsistent order of adding solutions Ensure all solutions particularly MK8 Fluoro Developer are added in the same order each time as all other solutions The solutions are not evenly added due to an inconsistency in pipetting volume Ensure the solution in each pipette tip is equal in the multi channel pipette Equilibrate the pipette tip in any solutions before adding them Ensure the solutions especially those with small volumes e g 1 ul are completely added into the wells Solutions or antibodies were not actually added into the wells Do not allow the pipette tip to touch the outer edges or inner sides of the wells in order to prevent solutions from sticking to the surface Did not sufficiently shake the solutions in the wells after adding sample or positive control at Step 2 Gently and evenly shake the plate frame across a flat surface so that the solutions in the wells are better
15. low throughput and or produce radioactive waste The original EpiQuik DNMT Activity Inhibition Assay Kit addressed this issue by introducing a simple method with an ELISA like 96 well plate format The EpiQuik DNMT Activity Inhibition Assay Ultra Kit Fluorometric is a further refinement of its predecessor kit by enhancing sample signals and significantly minimizing background signals in addition to being five times more sensitive e Fluorometric assay with easy to follow steps for convenience and speed The entire procedure can be completed within 3 hours and 45 minutes e Safe and innovative fluorometric assay without radioactivity extraction and chromatography e The ultra sensitive detection limit can be as low as 0 2 ug of nuclear extract or 0 2 ng of purified enzymes which is ten times better than the predecessor kit e Optimized antibody amp enhancer solutions allow high specificity to 5 mC without cross reactivity to unmethylated cytosine e 96 stripwell microplate format allows for either low or high throughput analysis PRINCIPLE amp PROCEDURE The EpiQuik DNMT Activity Inhibition Assay Ultra Kit Fluorometric contains all reagents necessary for the measurement of DNMT activity or inhibition In this assay a universal DNMT substrate is stably coated onto microplate wells DNMT enzymes transfer methyl group to cytosine from Adomet to methylate DNA substrate and the methylated DNA can be recognized with an anti 5 methylc
16. ntees the performance of all products in the manner described in our product instructions Product Warranty If this product does not meet your expectations simply contact our technical support unit or your regional distributor We also encourage you to contact us if you have any suggestions about product performance or new applications and techniques Safety Suitable lab coat disposable gloves and proper eye protection are required when working with this product Product Updates Epigentek reserves the right to change or modify any product to enhance its performance and design The information in this User Guide is subject to change at any time without notice Thus only use the User Guide that was supplied with the kit when using that kit Usage Limitation This product is for research use only and is not intended for diagnostic or therapeutic application Intellectual Property This product and methods of use contain proprietary technologies by Epigentek A BRIEF OVERVIEW DNA methylation occurs by a covalent addition of a methyl group at the 5 carbon of the cytosine ring resulting in 5 methylcytosine These methyl groups project into the major grooves of DNA and inhibit transcription In human DNA 5 methylcytosine is found in approximately 1 5 of genomic DNA primarily at CpG sites There are clusters of CpG sites at 0 3 to 2 kb stretches of DNA known as CpG islands that are typically found in or near promoter regions of genes where
17. o avoid cross contamination carefully pipette the sample or solution into the strip wells Use aerosol barrier pipette tips and always change pipette tips between liquid transfers Wear gloves throughout the entire procedure In case of contact between gloves and sample change gloves immediately 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 1 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com Web www epigentek com Printed 2014 09 22 Epigentek Group Inc All rights reserved Products are for research use only P 3010 KIT CONTENTS Component 48 Assays 96 Assays Storage Cat P 3010 48 Cat P 3010 96 Upon Receipt MK1 10X Wash Buffer 14 ml 28 ml 47 MK2 DNMT Assay Buffer 4ml 8 ml RT MK3 Adomet 50X 60 ul 120 ul 20 C MK4 DNMT Enzyme Control 50 ug ml 6 ul 12 ul 20 C MK5 Capture Antibody 1000 ug ml 5 ul 10 pl 4 MK6 Detection Antibody 400 ug ml 6 ul 12 ul 20 C MK7 Enhancer Solution 6 ul 12 ul 20 C MK8 Fluoro Developer 6 ul 12 ul 20 C MK9 Fluoro Enhancer 6 ul 12 ul 47 MK10 Fluoro Dilutor 4ml 8 ml RT 8 Well Assay Strips With Frame 6 12 47 Adhesive Covering Film 1 1 RT User Guide 1 1 RT Spin the solution down to the bottom prior to use SHIPPING amp STORAGE The kit is shipped in three parts the first part at ambient room temperature and the second and third part on frozen ice packs at 4 C
18. optimal range of 5 ug to 10 ug The amount of purified enzymes can be 0 2 ng to 200 ng depending on the purity and catalytic activity of the enzymes Nuclear Extraction You can use your own method of choice for preparing nuclear extracts Epigentek also offers a nuclear extraction kit Cat No OP 0002 optimized for use with this kit Nuclear Extract or Purified DNMT Storage Nuclear extract or purified DNMT enzymes should be stored at 80 C until use 1 Working Buffer and Solution Preparation a Prepare Diluted MK1 1X Wash Buffer 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 5 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com Web www epigentek com Printed 2014 09 22 Epigentek Group Inc All rights reserved Products are for research use only P 3010 48 Assay Kit Add 13 ml of MK1 10X Wash Buffer to 117 ml of distilled water and adjust pH to 7 2 7 5 96 Assay Kit Add 26 ml of MK1 10X Wash Buffer to 234 ml of distilled water and adjust pH to 7 2 7 5 This Diluted MK1 1X Wash Buffer can now be stored at 4 C for up to six months Prepare Diluted MK3 Working Buffer Freshly prepare the Diluted MK3 Working Buffer required for the assay by adding 2 ul of MK3 into 98 ul of MK2 DNMT Assay Buffer About 50 ul of this Diluted MK3 will be required for each assay well Prepare Diluted MK5 Capture Antibody Solution Dilute MK5 Capture Antibody with Diluted MK1 at a ratio of 1 1000 i e add 1 ul of M
19. ytosine antibody The ratio or amount of methylated DNA which is proportional to enzyme activity can then be measured by reading the fluorescence in a fluorescent microplate spectrophotometer at 530 excitation and 590 emission The activity of DNMT enzymes is proportional to the fluorescence intensity measured 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 4 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2014 09 22 Epigentek Group Inc All rights reserved Products are for research use only P 3010 Prepare nuclear extracts or purified enzymes Incubate with substrate amp assay buffer for 90 min Wash wells then add capture antibody i Wash wells then add RFU detection antibody and 0 20 40 60 80 100 enhancer solution Dnmt1 ng Demonstration of high sensitivity and specificity of DNMT activity inhibition assay achieved by using recombinant DNMT1 with the EpiQuik DNMT Activity Inhibition Assay Ultra Kit Add color developing Fluorometric solution for color develop ment then measure absorbance Schematic procedure of the EpiQuik DNMT Activity Inhibition Assay Ultra Kit Fluorometric PROTOCOL For the best results please read the protocol in its entirety prior to starting your experiment Starting Materials Input Amount The amount of nuclear extracts for each assay can be between 0 2 ug and 10 ug with an
20. zymes DNMT2 and DNMT3L may also have more specialized but related functions DNMT1 appears to be responsible for the maintenance of established patterns of DNA methylation while DNMT3A and DNMT3B seem to mediate the establishment of new or de novo DNA methylation patterns DNMT3L is found to be a catalytically inactive regulatory factor of DNA methyltransferases which is essential for the function of DNMT3A and DNMT3B Diseased cells such as cancer cells may be different in that DNMT1 alone is not responsible for maintaining abnormal gene hypermethylation and both DNMT1 and DNMT3B may be cooperative for this function The local chromatin structure also contributes to the control of DNA methylation yr HH DNA methyltransferase bas a o o n AdoMet AdoHcy c 5 C Fig 1 Methylation of cytosine in DNA via DNA methyltransferase and S adenosylmethionine The importance of DNA methylation is emphasized by the growing number of human diseases that are known to occur when DNA methylation information is not properly established and or maintained Abnormal DNA methylation associated with increased expression or the activity of DNMTs has been found in many different diseases especially in cancer Inhibition of DNMTs may lead to demethylation and expression of silenced genes DNMT inhibitors are currently being developed as potential anticancer agents Conventional DNMT activity inhibition assay methods are time consuming labor intensive have
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