The Improvement And Application Of MS-RDA For Study On High-altitude Adaption Associated Epigenetic DNA Methylation

Background: People reside in low altitude areas may probably suffer acute or chronic mountain sickness caused by hypoxia when they come into highland environment to which the high-land natives have already adapted for thousands of years. This adaptation is genetically hereditable because it has been formed by long term of natural selection. The study on the high-altitude adaptation mainly focused on the gene polymorphism based on DNA sequence variation. Yet neither the acclimatization nor the gene polymorphism caused by natural selection could explained the high-land adaptation appeared since embryonic and nurseling period. The advent of new theory and method in epigenetics these years provide the possibility to explain the developing adaptation. The epigenetic property is heritable and has proved to be important ways of gene regulation. It has not been reported whether hypoxia in high altitude could lead to epigenetic changes, which may offer the long-term adapation to high land environment. While some papers suggested that a kind of tubercle bacillus lacking DNA methylase died faster, that is to say that DNA methylase is important in hypoxia adaptation, the significance of DNA methylation in high-altitude adaption has not been reported. Thus, we focus on genomic methylation difference between the the highland and the plain population. Methods:1. The improvement of MS-RDA : MS-RDA is an important method to screen the differentially methylated DNA fragments from two kind of genomic DNA by sutraction hybridization. However, classical MS-RDA need to change DNA adaptor several times after digestion and DNA recovery, making it very fussy, low effiency and need a relatively larger amount of samples(at least 10). Thus, we improve the method based on the protocol to firstly amplify the driver DNA with d UTP containg d NTP mixture then use UDG and S1 nuclease to digest driver DNA or the driver and tester hybridizing complex, which greatly simplized the experiment process, reduced the necessary DNA amount of the sample and improved the chances to obtain the differentially methylated fragments. The specificity of the improved MS-RDA was firstly determined by using the same HUVEC genomic DNA as both tester and driver to exclude the false positive fragments. Then the sensitivity of the improved protocol was assessed by adding the linearized p UC19 plasmid treated with Hpa II methylase or not to the same HUVEC genomic DNA as driver and tester respectively. The enriched DNA fragments were then cloned into pc DNA3-T vector, transformed into DH5a competent cells and cultured on LB agarose plates. After the positive clones were picked out and identified by PCR, the target fragments were sequenced and analyzed. 2. The improvement of cloning the fragments: we firstly cloned the human histone H4 c DNA containing an Xcm I recognition site at both its 5’ and 3’ end into p CDNA3.0 vector unidirectionally and then generated T vector with p CDNA3.0 backbone by cutting the recombinant plasmid. To detect its usability, two c DNA fragments with the length of 312 bp and 1329 bp were ligated to it and the ligation mixture was transformed into E.coli DH5a competent cells and the positive rates of the transformants were evaluated by PCR and DNA agarose gel electrophoresis. 3. After the specificity and sensitivity of the improved MS-RDA has been confirmed, we screened the differentially methylated DNA fragments of Kirghiz people living in high land as well as plain. Eventually, the enriched fragments were sequenced with our own pc DNA3-T vector and blast with the human genomic DNA sequences. Results:The improvement of MS-RDA : We did not amplify any band by using the same genomic DNA as both tester and driver, suggesting that the improved MS-RDA has very good specificity. 2. Construction of T vector based on Xcm I recognition sites and optimization its ligation conditions to target PCR fragments. Our results showed that the T vector produced by Xcm I treatment can ligate to the target DNA fragments with high efficiency. Besides, the phosphorylation state of the primers used for PCR amplification is also an important factor determining the cloning efficiency. What’s more, as for longer DNA fragments, retreatment with Taq DNA polymerase and d ATP after PCR amplification and purification could improve the ligation efficiency significantly. 3. After adding the differentially methylated p UC19 plasmids to the tester and driver DNA and screening with the improved method, we finally amplified the right fragment with correct sequences, convincing the sensitivity and specificity of the improved method. After using the 5 Kirghiz people living in high land as driver and those people living in plainas tester to screen with our improve MS-RDA, 11 differentially methylated DNA sequences were obtained. After analysiswith online NCBI blast programme, 5 of them has been registed by Genbank and located in mitochondrion. In the 5 homologus sequences, 2 were highly similar to the genes encoding COX1, the other 3 were similar to the genes encoding NADH dehydrogenase2, 4 and 5. ConclusionThe improved MS-RDA has been successfully established with high specificity and sensitivity and isan ideal method to screen differentially methylated DNA.Our protocol may overcome the dependence on blue/white screening to get positive clones and provide a potent way to generate T vectors and ligate them to the gene of interesting.11 differentially methylated fragments have been isolated from Kirghiz people living in high land as driver and those people living in plainas tester. 5 of the obtained fragmentshas their homologus sequences, 2 were highly similar to the genes encoding COX1, the other 3 were similar to the genes encoding NADH dehydrogenase 2, 4 and 5.