| It is well known that DNA is the hereditary molecule and transmits information from generation to generation, which encodes the information to meet cells requirement for surviving, specializing and replication. The key to DNA function as an information molecule lies in the sequence of adenines, thymines, cytodines and guanines (A, T, C and G for short), the rungs of the DNA double helix consist of hydrogen-bongded complementary based pairs (A with T, and C with G). The rails are chains of alternating sugars and phosphates that run antiparallel to each other. All of these are the components of DNA backbones. In addition, DNA backbones can be modified in a number of ways, for instance, by replacing the phosphodiester groups with peptide groups, or with methyene methyl imino groups and so on. Therefore, DNA modification is the important component of DNA structure and encodes the numerous and mysterious genetic information, also.DNA modification is the covalent compound of DNA and other groups, which make the alternation of phenotypes occur without any change of the allelomorph gene sequence. This mechanism underlining is epigenetics that include DNA methylation, protein modification and non-encoding RNA. In another word, DNA modification is the main component of epigenetics. DNA modification is divided into two major type, DNA spontaneous chemical modification and modification from exogenous factors.Enzymatic methylation is most important DNA spontaneous chemical modification, and is also the most intensive epigenetics, which plays the important roles in both prokaryotes and eukaryotes. The simplified understanding of DNA methylation function is that the hypermethlation would down-regulate genes expression. Therefore. DNA methylation within the eukaryote species plays an important role in modulating genes expression, etiosis of tumor, genes imprinting, X chromosome inactivation and the maintenance of chromosome structure. Many studies have shown that the DNA alteration methylation in promoter occurs very early and frequently when the malignant tumor occurs. So the methylation of tumor related gene is expected to be an early, susceptive and promising index of tumor biomarker.In addition of methylation, another important type of DNA modification is DNA adducts, which can become permanent via the formation of mutations or other types of genomic instability. Environmental exogenous factors including alkylating carcinogens, ultraviolet (UV) -light and ionizing radiation (IR) as well as a large amount of anticancer drugs can induce DNA adducts. If not repaired prior to replication, DNA adducts is generally harmful resulting in effects detectable on the level of chromosomal aberrations, recombination as well as gene mutations and malignant transformation. Many different DNA base changes have been seen following some forms of oxidative stress, and these lesions are widely considered as instigators for the development of cancer and are also implicated in the process of aging. One of the most abundant forms of cellular stress is the constant exposure to reactive species of oxygen or radicals that cause protein and DNA damage.With the continuously development of molecular biology techniques in recent years, small molecular biomarker in vivo related with tumor occurrence, development and treatment have become the hotspot of tumor marker, especially the environmental problem. More perfect and intelligent techniques are required to examine the chemical carcinogen exigently so as to clarify the relationship between DNA modification and human diseases,heredity and aberrance.We used and developed bisulfite sequencing to detect the methylation of promoter of RASSF1A and E-cadherin in tumor cells, and the developed method likely can be applied to detect DNA methylation and to amplify other GC-rich genomic sequences. Otherwise, using parallel genotyping assay of SNPs and LMPCR for reference, we established a new method to detect DNA adducts in genomic DNAs. The results showed that the protocol of this new method is very reasonable and it is expected to be a high throughput method to detect the DNA adducts or other gene mutation in genomic DNAs.Part 1. Application and improvement of bisulfite sequencing in analysis of methylation of CpG island in gene promoter.Objective To Apply and improve bisulfite sequencing to analysis methylation of CpG island in gene promoter and to discuss the sensitivity and specificity of the improved polymerase chain reaction amplification in methylation analysis. Methods The genomic DNAs were extracted from MCF-7 and HepG2 cells and treated by bisulfite sodium, the methylation status at RASSF1A and E-cadherin amplified with regular, 'touchdown' and improved polymerase chain reaction respectively were detected by using busilfite sequencing, then the amplified DNA were subcloned into the TA cloning vector and sequencing. Results For HepG2, RASSF1A sequencing showed that there were 365 bases to be sequenced, in which 15 CG haven't changed, the percent of mCpG/CpG is 100% and the efficiency of bisulfite sodium treatment is 95.92 %. Therefore, The CpG islands of RASSF1A promoter in HepG2 are hypermethylated. For MCF-7, RASSF1A cloning sequencing show that there were 17 CG but only one has converted to TG, and the percent of mCpG/CpG is 94.2%. In addition, three CTG and one CAG haven't changed. Meanwhile, we got the different methylation feature of E-cadherin in MCF-7. TA cloning sequencing showed that there are 37 CG in this promoter but only 5 CG have maintained, and the percent of mCpG/CpG is only 13.5%. Besides that, three CTG and three CAG haven't changed too. Conclusion The improved PCR method can provide the powerful feature that the CpG islands of RASSF1A in MCF-7 and HepG2 are highly methylated but E-cadherin in MCF-7. Adding the primers at different time in PCR amplification and combining with 'touchdown' polymerase chain reaction can avoid nonspecific reactions and increase the specificity of PCR product, also the improved method can be applied to amplify other GC-rich genomic sequences.Part 2. A new method of detecting DNA adducts in genomic DNAs based on ligation-mediated PCRObjective To present a new method to detect DNA adducts in genomic DNAs based on Ligation-mediated PCR. Methods Firstly, HepG2 cells are treated with nitrogen mustard for 3h and the genomic DNAs was extracted and digested with Xbaâ… , and the adaptors(Linker3) were ligated to the ends of restriction fragments. The fragments were then amplified by using one of the strands of the adaptor as a primer. Secondly, the caused DNA strands were treated with alkaline phosphatase and piperidine to convert the sites of alkulation into single strand break (SSB). This step produces a 5'-phosphate group at the drug alkylation sites that is required for T4 DNA lagase reaction in a subsequent LMPCR. The single strand with 5'phosphoric group were extended for dual strands with 3'adenine (A), which is ligated with a linker(Linker1) with multiple enzyme-cleaving sites and 5'-phosphate group. The ligated products were amplified by PCR, and PCR products were subsequently captured by magnetic beads wrapped with streptavidin, then were cleaved with the enzyme Mmeâ… . The cleaved fragments were required to ligate with another linker (Linker2) that have multiple enzyme-cleaving sites and all sequence known. The ligated products amplified with PCR were electrophoresed with agarose gel. Results Restriction fragments of Xbaâ… in the size range of 250~1000 bp are preferentially amplified as shown in the gel image of PCR products, The ligated products of Mmeâ… Restriction fragments and Linker 2 amplified with PCR is 70 bp that is the fragments including DNA adducts. Conclusion The genomic DNAs have the DNA adducts. The new method can avoid the disadvantages of traditional LMPCR and is a new-style, simple and convenient detection method of DNA adducts in genomic DNAs.
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