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Methylamp DNA Modification Kit

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For robust, chemical-based bisulfite conversion of DNA for methylated cytosine analysis

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Suggested Workflow
DNA Isolation
 
 
DNA Bisulfite Conversion
 
 
DNA Size Selection
 
 
NGS Analysis
 
Two bisulfite-converted DNAs from six kits, including the Methylamp DNA Modification Kit (Cat #P-1001), were subjected to PCR with two amplicons (655 and 1109 bp) and three different extension temperatures (65°C, 68°C and 72°C) to assess capacity for long amplicon amplification. Bisulfite-converted DNA from Epigentek's Methylamp kit with a PCR extension temperature of 65°C had the most robust amplification of the longer 1109 bp amplicon. Yang Y, et al. BMC Genomics. 2015; 16(1): 350.
Schematic procedure of the Methylamp™ DNA Modification Kit.
Different amounts of DNA isolated from a serum sample were chemically modified using the Methylamp™ DNA Modification Kit. Real time PCR was then performed by using a pair of primers and a probe designed to amplify both methylated and unmethylated alleles of β-actin.
Input Type: DNA
Research Area: DNA Methylation
Target Application: Sample Modification
Vessel Format: Columns/Tubes
100% Guarantee: 6 months
Catalog No.SizePriceQty
P-1001-140 reactions $111.00 
P-1001-280 reactions $199.00 
Availability: Usually Ships in 1 Day or Same day delivery Same Day NY Delivery 
Product Overview

The Methylamp™ DNA Modification Kit is a complete set of optimized reagents designed to convert DNA so that 5-methylcytosines can be detected in downstream applications, for the purpose of gene-specific DNA methylation analysis. The kit has the following advantages and features:

  • Total protocol time is less than 2 hours.
  • 99.9% conversion rate of unmethylated cytosine into uracil/thymine
  • Extremely low degradation of DNA in the modification process, preventing over 90% of DNA loss.
  • Requires as little as only 50 pg of input DNA, equivalent to about 20 cells.
  • Extremely simple, robust, and reliable sodium bisulfite conversion protocol.
  • Suitable for bisulfite sequencing (direct and cloning), next-generation sequencing, pyrosequencing, PCR (real-time, end-point, methylation-specific), COBRA (combined bisulfite restriction analysis), and methylation-based microarrays.
  • Highly compatible with Illumina-based workflows.

Background Information
DNA methylation occurs by the covalent addition of a methyl group at the 5-carbon of the cytosine ring, resulting in 5-methylcytosine. DNA methylation is essential for the development of many species and is often associated with many key biological processes as well as gene regulation. There are various methods used to assess DNA methylation states. However, only bisulfite conversion of genomic DNA, followed by PCR amplification, cloning, and sequencing of individual PCR amplimers yields reliable information on the methylation states of individual cytosines on individual DNA molecules. By treating DNA with sodium bisulfite, cytosine residues are deaminated to uracil while leaving 5-methylcytosine intact, providing researchers with distinguishabe methylated positions in the DNA sequence during the downstream application. 

Principle & Procedure
DNA is chemically denatured to allow the sodium bisulfite reagent to react specifically with single-stranded DNA, thereby deaminating cytosine and creating a uracil residue. The unique DNA protection reagents contained in the modification buffer can prevent chemical and thermophilic degradation of DNA during the sodium bisulfite treatment process. The included non-toxic, modified DNA capture buffer enables the DNA to bind tightly to the column filter, permitting DNA cleaning to be carried out on the column to effectively remove residual sodium bisulfite and salts. Modified DNA can then be eluted for use in various methylation analysis applications or otherwise stored at -20°C for up to 2 months.

 
Fig. 3. Two bisulfite-converted DNAs from six kits, including the Methylamp DNA Modification Kit (Cat #P-1001), were subjected to PCR with two amplicons (655 and 1109 bp) and three different extension temperatures (65°C, 68°C and 72°C) to assess capacity for long amplicon amplification. Bisulfite-converted DNA from Epigentek's Methylamp kit with a PCR extension temperature of 65°C had the most robust amplification of the longer 1109 bp amplicon. Yang Y, et al. BMC Genomics. 2015; 16(1): 350.

 
Fig. 1. Schematic procedure of the Methylamp™ DNA Modification Kit. 


Fig. 2. Different amounts of DNA isolated from a serum sample were chemically modified using the Methylamp™ DNA Modification Kit. Real time PCR was then performed by using a pair of primers and a probe designed to amplify both methylated and unmethylated alleles of β-actin.

Product Components

R1 (DNA Denature Solution)
R2 (DNA Modification Powder)
R3 (DNA Modification Solution)
R4 (Modified DNA Capture Solution)
R5 (Modified DNA Cleaning Solution)
R6 (Modified DNA Elution Solution)
F-Spin Column
F-Collection Tube
User Guide 

Frequently Asked Q's

1. What is the difference between this DNA modification kit and your "one-step" DNA modification kit (Cat #P-1010)?
In the one-step DNA modification, DNA is denatured by heating, which allows DNA denaturation and bisulfite conversion to be carried out simultaneously. One-step DNA modification is suitable for the labs which are equipped with thermal cycler and requires simple and fast procedures for DNA modification. In the "two-step" DNA modification of this kit, DNA is denatured chemically followed by bisulfite treatment. It is suitable for general DNA modification using DNA isloated from various sources. Because one-step DNA modification may increase DNA degradation, a higher starting DNA amount may be required for one-step DNA modification than for two-step DNA modifcation.

2. How much starting DNA is required for DNA modification with this kit?
The starting DNA required for DNA modification can be as low as 50 pg. The signal of the modified DNA can be detected with real-time PCR.

3. Why are only 90 minutes required for the actual DNA modification?
With our unique modification composition, 90 minutes is sufficient for more than 99% C-T conversion while DNA degradation is greatly prevented. We have observed that an increase in modification time did not significantly improve C-T conversion, while yield of modified DNA was significantly reduced most likely due to increased DNA degradation.

4. Can the modified DNA with this kit be stored for a long time?
The modified DNA generated with this kit can be stored for 2 months at -20°C. The modified DNA can be stored for as long as 6 months at -80°C.

5. Can the kit be used for modifying DNA from formalin-fixed and paraffin-embedded (FFPE) samples?
DNA extracted from FFPE samples is often fragmented, but the kit can be used for FFPE samples as the modification solution included in the kit contains DNA stabilizing reagents that prevent a further fragmentation of DNA caused during the modification process.

6. Do I need to extract the DNA from blood first?
Yes. DNA isolation from blood is required and then DNA can be used for modification with the kit.

7. How much DNA can the columns actually handle?
The optimal range of DNA that can be used for each sample in each modification is 1 ng to 1 µg. The column can maximally bind about 5 µg DNA.

8. Has anyone tried adding carrier DNA to their reactions when they use your kits?
We tested the carrier DNA together with the use of our kits and it only slightly increased the yield of modified DNA.

9. How should I store the kit?
The kit can be stored at room temperature for at least 6 months or at 2-8°C for one year from the shipping date.

User Guide & MSDS

[User Guide]*
*Always use the actual User Guide that shipped with your product. Is the above file locked? You can also request user guides by emailing info@epigentek.com along with your contact information and institution name.

[Material Safety Data Sheet]
Product Citations

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Liu T et. al. (March 2009). Increased gamma-tubulin expression and P16INK4A promoter methylation occur together in preinvasive lesions and carcinomas of the breast. Ann Oncol. 20(3):441-8.

Aniagu SO et. al. (February 2009). Changes in gene expression and assessment of DNA methylation in primary human hepatocytes and HepG2 cells exposed to the environmental contaminants-Hexabromocyclododecane and 17-beta oestradiol. Toxicology. 256(3):143-51.

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