Your Position

NanoMeth - A Novel Quantitative Kit to Measure Genomic DNA G

Introduction

 

DNA methylation is essential for normal gene expression, X inactivation, genomic imprinting, chromatin modification, and silencing of endogenous retroviruses. Gene-specific DNA methylation analysis, a most common methodology to detect gene methylation, is difficult to find untargeted loci changes and unable to provide whole picture of DNA methylation changes in genome. The measurement of global methylation can lead to identify new disease biomarkers, which would not be accomplished by gene specific methylation techniques.
 
Several methods have been developed to measure global methylation, but numerous drawbacks exist. In general, those methods are time consuming, costly, imprecise, and less reliable. For example, recently developed bisulfate-conversion-based high throughput methods such as next generation sequencing and microarray share the similar shortcomings in addition to possible bisulfate conversion errors.  In broader areas of basic research and clinical applications, there is an urgent demand for the advancement in technologies for measurement of global methylation to overcome those disadvantages.
 
Here we report a novel approach, which directly measures the percentage of global methylation in DNA samples. NanoMeth, a unique kit with qPCR based technology, has been developed to measure global methylation of genomic DNA which conquers the technical limitations stated above.


 
Principle of the Harborgen Global Methylation
 
Genomic DNA is divided into 2 groups in equal amount then separately treated with Harborgen enzymes. Briefly, One group is digested with methylation sensitive restriction enzyme that digests the unmethylated CCGG and generates a unmethylated CCGG library (U-library).  Another group is digested with methylation non-sensitive restriction enzyme that digests all methylated and unmethylated CCGG and generates a CCGG library (M-library). The CCGG library is proportional to the amount of CpG (1/16) in the whole genome.
Harborgen invented a new technique that can cut all restriction site in genome and amplify all the enzyme fragments, so the methylation percentage of restriction site CCGG in genome can be calculated by realtime CT values from the two libraries (Comparative Quantitation), which represent the ratio of the fragment numbers in the two libraries.
 
 
 
Selected Performance Data
 


     
Left: qPCR standard curve. The starting DNA is 1.0 ng, serial dilutions of 10 times ranging from 1.0ng to 0.001pg.
Right: qPCR efficiencies are same in both libraries. PCR efficiency is 100.0% and R square=0.998.
 
     
Left: The gDNAs are isolated from C57BL/6 mice (2 day old). The data showed significant differences in levels of global methylation between different organs.
Right: RKO cell line was cultured using standard methods. Cells were treated with 5 µM 5-aza-2′deoxycytidine (DAC) for 3 days prior to being harvested. Our result showed that methylation percentage of RKO cells were decreased from 97.0% to 35.0% after DAC treatment.
 
Summary
Gene-specific DNA methylation analysis has many inherent problems and does not provide a complete picture of DNA methylation changes in genome. Measurement of global DNA methylation is cumbersome at present, often cost prohibitive, not accurate and not reliable, and needs large amount of samples.
 
NanoMeth is the only restriction enzyme and realtime PCR based global methylation quantitation method, which avoided the disadvantages caused by bisufite conversion. Compare with other method, NanoMeth, is robust and features as accurate, sensitive, simple, and fast. NanoMeth can be used to measure percentage of the global methylation with a single cell genome, which is the only one that enables researchers to study changes in stem cell global methylation between different developmental stages. NanoMeth is capable to identify 0.5% of global methylation difference between samples where any other existing methods are unable to do. In addition to the improved accuracy and sensitivity, the simplified process of NanoMeth will provide results in a few hours.
 
Related technology----Methylation loci Scan
 
NanoMeth is the most powerful tool to determine global DNA methylation at present. The amplicon generated with our method is a methylation library. To scan the methylation library, we have developed another new method, which is designed to isolate and identify the differences of specific methylation loci between two or more samples.