Whole genome methylation quantitative detection technology-LUMA
Cephalopods Series,Flower Octopus,Whole Clean Octopus,Frozen Baby Octopus Zhejiang Zhoushan Jiaze Aquatic Co., Ltd. , https://www.tianjia-aquatics.com
Introduction to LUMA <br> Changes in Genomic DNA Methylation (GDM) are thought to be important events in normal and pathological cellular processes, contributing to normal development and differentiation as well as cancer and other diseases. Here, we introduce a new method to evaluate whole-genome DNA methylation, called Luminometric Methylation Assay (LUMA). The method employs a methyl-sensitive restriction endonuclease DNA cleavage method in combination with pyrosequencing for polymerase extension analysis. The method is a quantitative detection technology with high reproducibility and easy to popularize.
The advantages are: no need for sulfite modification, no primers, no PCR process, no need for reagents such as magnetic beads for sequencing reaction, and sequencing reaction can be completed in about ten minutes, which is convenient and quick. Moreover, the experiment requires only 200-500 ng of genomic DNA and contains an internal control to eliminate the problem of different starting DNA numbers. The entire experimental process can be completed in 6 hours.
Technical Principles This method was established in 2006 by Karimiet et al., using DNA methylation-sensitive or insensitive restriction enzymes for DNA cleavage followed by bioluminescent polymerase extension assays to quantify the extent of restriction cleavage. . This experiment used a parallel reaction to the CpG methyl-sensitive restriction enzyme HpaII and its methyl-insensitive co-lyase MspI. EcoRI was used as an internal reference in all reactions.
The target sequence of MspI and HpaII is CCGG , and the number of CpG sites accounts for 8% of the CpG locus in the whole genome, which can be used as a marker to evaluate the level of methylation in the whole genome. MspI and HpaII cleave to form a 5' CG sticky end, while EcoRI produces a 5' AATT sticky end, and then gradually fills the glucosinolate by polymerase extension. With the successful extension of dNTPs, inorganic pyrophosphate (PPi) is released and converted to ATP by ATP sulfurylase and 5' phosphoryladenosine (APS). Fluorescein then produces a certain proportion of visible light by luciferase and ATP and is detected by CCD .
Experimental procedure
1. Digestion: Each sample was divided into 2 copies of DNA, which were digested with HpaII+EcoRI or MspI+EcoRI, respectively, and digested for about 4 hr.
2. Purification: The enzyme digestion product is purified and recovered.
3. Pyrosequencing, the reaction process is as follows:
dNTPs are added in four steps (step 1: dATPαS, second step: dGTP + dCTP, third step: dTTP and step 4: dGTP + dCTP). The addition of peaks corresponding to dATPαS (step 1) and dTTP (step 3), both represent EcoRI cleavage, so the two should be equivalent. Therefore, the dTTP peak was used as the quality control of the dATPαS peak. In the second step, dCTP is added together with dGTP, and the corresponding peak represents HpaII or MspI cleavage. In step 4, dCTP and dGTP are added again as internal control to complete the second step, so its corresponding peak should be zero or close to zero. 
Methylation rate ( %) calculation: as shown below 
Methylation rate={1-[HpaII(C+G)/EcoRI(A)]/[MspI(C+G)/EcoR(A)]}×100, the sample methylation GDM=57.12% in the above figure
Technology application and optimization
Since the establishment of the LUMA method, it has been widely used since Karimiet et al., especially the epigenetic research in recent years. This method often uses different techniques such as NGS and methylation chips to conduct epigenetic analysis of experimental samples at different levels. Detection. This method is also constantly being evaluated and improved during the application process.
references:
2. Carolina Soriano-Ta Ìrraga, et al, DNA Isolation Method Is a Source of Global DNA Methylation Variability Measured with LUMA.Experimental Analysis and a Systematic Review. PLOS ONE, April 2013, Volume 8, Issue 4, e60750
For technical advice and more references, please contact Shanghai Hippy Biotech Co., Ltd.!
phone,