MBD2b Protein Affinity Chromatography For Whole Genome Methyl-CpG Islands Enrichment

BackgroundAs we all know,cancer is caused by multiple abnormal genes,leading to uncontrolled cell growth of the pathological process.Its occurrence and development are due to the oncogene activation and inactivation of tumor suppressor genes.In the previous study,the mechanism of gene abnormal expression is confined to the analysis of its genetic mechanisms,including structural changes(such as mutation,deletion,insertion etc), chromosomal rearrangements and so on.Studies found that in tumor and normal cells with different methylation patterns can be divided into three types:hypermethylation,lower methylation and the intension of Mtase,The aberrant DNA methylation occur in the early cancer,cancer precancerous lesions and even some benign lesions,its over-methylation levels are usually lower than normal cells,and more with some degree of methylated CpG island increased,that is,oncogenes and tumor suppressor gene expression abnormalities,target cell proliferation access,and loss of function in inhibiting the growth of another important mechanism of cancer formation epigenetic mechanisms.Currently the most commonly used methylation detection technologies,such as sodium bisulfite/ sequencing,methylation-specific PCR,in situ-MSP hybridization,single nucleotide primer extension method, denaturing high-performance liquid chromatography etc can not complete the rapid and high-throughput detection of the whole genome methylated CpG islands,MBD2b protein column chromatography combined with the whole genome chip method can solve the problem.PurposeUse of protein MBD2b protein which has the characteristics of combination of a specific binding methylated DNA to establish an enrichment method which is based on DNA-immunoprecipitation technology that is used to genome-wide methylated CpG islands,respectively methylation and non-methylated fragment and patients with hepatocellular carcinoma and normal clinical samples for testing to prove that this method of enrichment efficiency and the feasibility of clinical application.At the same time,the difference of methylation research,cancer detection,diagnosis,staging and prognosis lay a good foundation for the specific molecular mechanism of tumor and clarify apparent in the control of human genetic diseases important in the role of significance.MethodExpression of the recombinant protein of GST-MBD2b in E.coli through Glutathione Sepharose 4B of the purified recombinant protein to prepare and get affinity chromatography column.According to the different ionic strength of salt has the different effect of MBD2b to the methylated DNA and non-methylated DNA,in order to get the enrichment of methylated DNA.By demethylase SssI dealing with the DNA fragments and non-methylation of DNA fragments enrichment condition exploration,through different concentrations of salt ions to determine the optimal elution extent of the enrichment conditions.The accumulated samples were tested by Real Time PCR and gene chip.Real Time PCR is to select tumor suppressor gene P16 to detect protein GST-MBD2b column chromatography enrichment efficiency as well as to join in the enrichment of different length of the methylation and non-methylation fragments in Arabidopsis. After the enrichment select arbitrary primers corresponding to a fragment of this method to detect the methylation of DNA and the accumulation of non-elution of DNA methylation results.Chip testing can be high-throughput by adding all these methylation and non-methylation of Arabidopsis fragments accumulation in the same test on a chip,will join the Arabidopsis fragment corresponding PCR products as probe,after cleaning and silane slides,arm molecular connectivity,and the probe sample preparation steps such as fixed on the chip.Methylation and will contain non-methylated fragments in Arabidopsis and the enrichment of chip hybridization analysis of fluorescence signal values to reflect accumulation.In addition,the protein can also column chromatography and genome-wide analysis of CpG island chip with normal liver,hepatocellular carcinoma and adjacent samples methylation of the situation.ResultThe experiment confirmed ion concentration of 0.5 M salt is the separated critical condition used MBD2b protein on methylated DNA and non-methylated DNA fragments.By Real Time PCR and gene chip analysis found that this method can be achieved in the whole genome DNA methylation of the effective concentration and the highest concentration multiplied 100 times can be achieved,the rate of methylation of enrichment/non-methylated washout rate could reach more than 4000 times,good stability and repeatability.ConcludeThis study established a new method about whole genome methylated CpG island,can achieve the detection of more points on the methylated DNA.In this study,GST-MBD2b protein column chromatography on methylated/non-methylated fragments and the enrichment of hepatocellular carcinoma specimens,it was confirmed that this method is effective,simple,fast and high-throughput after combined with gene chip and this research for the cancer genome-wide spectrum of alienation provides technical support.