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A Novel High Throughput Quantitative Method For DNA Methylation Based On Methylation-specific Primers And SAGE

Posted on:2007-06-18Degree:DoctorType:Dissertation
Country:ChinaCandidate:X L WangFull Text:PDF
GTID:1100360242963171Subject:Occupational and Environmental Health
Abstract/Summary:PDF Full Text Request
Heredity and variation are universal in the life world. After long march on scientific research, the knowledge about the essential of life has step to a new stage with finishing of the epoch-making event, human genome project. The classical genetics tell us the same genetic constitution would result in the unique phenotype, but now we find it is not so simple. A couple of mouse from the same zygote have two definitely different tails. The alternation of phenotypes can be transferred through the meiosis and mitosis manner without any change of the genomic DNA sequence, namely the so-called host of life. The mechanism underlining is the epigenetics, but the classical genetics. The most current interpretation of epigenetics is the study of changes in gene function that are mitotically and/or meiotically heritable and that do not entail a change in DNA sequence. The underlying mechanisms concern chemical modification of genetic make-ups, namely DNA methylation and histone methylation and acetylation. The Human Genome Project provided the blueprint for life, but the epigenome will tell us how this whole thing gets executed.The central task in the post-genome era is to elucidate the mechanism and network of genes modulation. DNA methylation occurs at much early stage than the modulation levels of transcription, translation and post translation for genes function and therefore it has become one of the key roles within the functional genomics. DNA methylation within the eukaryote species plays an important role in modulation genes expressing, etiosis of tumor, genes imprinting, X chromosome inactivation and the maintenance of chromosome structure. The simplified understanding of DNA methylation function is the hypermethylation would down-regulate genes expression. Now it is time for exploring this mysterious world within the eukaryote species, especially the human being.DNA methylation occurs mainly at the CpG dinucleotides. The tendency of 5-MeC to spontaneous deamination means that most CpGs are present only once in every 80 dinucleotides, representing a one-fifth reduction of that predicted from the average base composition (40% G+C), and they are usually methylated. In contrast, a small proportion of total CpGs are clustered into"CpG islands". The CpG islands often encompass the promoters and early exons of genes and occurs at almost all house-keeper genes and 40% tissue-specific genes. Each CpG site has its somewhat normal methylation level other than the complete yes or no. Mammalian genomic DNA methylation pattern varied in different tissues from different individuals in different time, termed tissue-specificity and inter-individual variation. Therefore, the epigenetic map, namely the scanning of DNA methylation within whole genome, will be invaluable for understanding gene regulation and disease etiosis. It would provide a revolutionary advancement for insight of epigenetics of eukaryote species.Human Epigenetic Project (HEP) with the objective of mapping of DNA methylation sites in all 30,000 human genes in around 200 samples, sponsored by the Human Epigenome Consortium (the Sanger Institute, Epigenomics AG, and the Centre National de Génotypage in Evry, France), is now underway. We are particularly interested in what we call methylation variable positions (MVPs), which will advance our ability to understand and diagnose human disease. Due to the unpreceded huge data to undermine, the bottle-neck for whether the accomplishment of HEP or not is the invention of high throughput method for genomic DNA methylation.Some difficulties for studies of DNA methylation exist due to poor performance of existing methods for DNA methylation quantitation. Till now, although many methods have been reported, they are only limited to qualitative measurement such as methylation-specific PCR or quantitation for methylation of one to several CpG sites such as MALDI method with mass spectrometry. The poor performance for studies on DNA methylation lies in the limitation of methods for DNA methylation measurement. The existing methods for DNA methylation can be classified into mainly two categories, based on restrictive enzyme to identify the specific methylated CpG sites or bisulfite conversion to identify the sequence differentiation between with and without bisulfite treatment. All these methods have many inborn flaws such as the limited CpG sites interrogating, involving of site-specific probes or site-specific primers and good laboratory equipment conditions. The project for DNA methylation mapping is much more complex than HGP, which cost human 13 years to be completed and therefore high throughput quantitation methods for mapping of genomic DNA methylation is needed urgently.Serial analysis of gene expression is a new high throughput method for total mRNA profiling of a certain organ with a brand-new schematic different from the existing mRNA microarray. This high-throughput method can detect not only whether a gene expresses or not but also its expressing density, which ignites us an idea to design a novel method based on methylation-specific primer and SAGE (MSP-SAGE) for high throughput quantitation of DNA methylation. We combine the advantages of several techniques such as bisulfite conversion, special characteristics of Mme I and methylation-specific primers.In MSP-SAGE assay, we used a kinased 6mer methylation-specific primer (PNNNNCG) to extend methylated CpG sequences rather than non-methylated CpG sequences. 17bp tags containing methylated CpG sequence are obtained by digestion of restriction endonuclease Mme I (the cognition site: TCCPuAC(N)20/18) from extended methylation sequence, and then the tags are concatenated and cloned for sequencing in accordance with SAGE method. Locations of methylation can be identified according to the sequences of tags and methylation status can be quantified from the frequency of the tags.The MSP-SAGE can be utilized for high throughput quantitation for genomic profiling of DNA methylation with a unique idea, and it is determined to shed light on epigenetic research, especially the upcoming life masterpiece HEP. The whole study is composed of the following three parts.PartⅠ: preparation of DNA methylation standards serial with definite methylation levelObjectives: To prepare DNA methylation standards serial with definite methylation level applicable in experiments on invention of new DNA methylation quantitation method and quantitation of specimens. Methods: to synthesize DNA sequences different from the existing any sequence within the human genome through BLAST retrieving. Methylated DNA was prepared from synthesized DNA with M.SssⅠmethyltransferase in presence of S-adenosylmethionine (SAM) according to the manufacture's instructions. The complete methylation modification by M.SssⅠwas checked with over digestion of restriction endonuclease (RE) HpaⅡfirst and then be sequenced after bisulfite treatment. For the preparation of mixtures with defined methylation status, a portion of the amplicons treated as above were mixed with the unmethylated amplicons to give methylation standards with 5, 20, 40, 60, 80, 100% methylation in both CpG alleles, respectively. Results: The complete methylation modification is checked by RE digestion and bisulfite sequencing and the results confirm the fully modification is success. Conclusions: The DNA methylation standards serial with definite methylation level is prepared successfully and it can be applied into the following experiments.PartⅡ: To invent a high-throughput method for genomic DNA methylation quantitation based on methylation-specific primer and SAGEObjectives: Mapping of genomic DNA methylation is a dispensable part of functional genomics in the post-genomic era. To develop a novel method based on methylation-specific primer and SAGE, called MSP-SAGE, for high throughput quantitation of genomic DNA methylation. Methods: We used a 6mer methylation-specific primer to extend the methylated CpG sequences other than non-methylated CpG sequences. The 17bp tags contained methylated CpG sequence, which were obtained from extensed methylation sequence by digestion of restriction endonuclease, and then the tags were concatenated and cloned for sequencing in accordance with SAGE method. We can identify the locations of methylation according to the sequences of tags and quantify the methylation status from the frequency of the tags. Results: MSP-SAGE has a calibration equation of Y=1.455x(R2=0.984,P<0.01). MSP-SAGE has a good linearity in a broad methylation range from 5 to 100% with good accuracy and high precision. At least 24 alleles can be accurately quantified simultaneously and the detection limit is approximately 3%. The methylation patterns of a sample assayed with MSP-SAGE are validated by bisulfite sequencing. Conclusions: The proof-of-principal study shows that MSP-SAGE is a reliable high throughput assay with many advantages for accurate analysis and quantitation of DNA methylation.PartⅢ: the application of MSP-SAGE to quantitate the artificial DNA methylation specimens from the CpG islands of human p16 geneObjectives: To test the applicability of MSP-SAGE for quantitation of genomic DNA methylation specimen. Methods:To amplify 5 fragments from the CpG islands of human p16 gene and then methylate them with M.SssⅠmethyltransferase. The artificial DNA methylation specimen with incremental methylation levels (5%, 20%, 40%, 60%, 80%, 100%,) can be prepared as above and be measured by MSP-SAGE and bisulfite sequencing simultaneously. Results: Each test has good linearity and the result differences of DNA methylation from these two methods are insignificant and the gross concordance rate is about 98.51%. Conclusions: The proof-of-principal study shows that MSP-SAGE is a reliable high throughput method for accurate analysis and quantitation of DNA methylation, and is a promising alternative method for genomic DNA methylation profiling.
Keywords/Search Tags:DNA methylation, Quantitation, High throughput, Epigenetics, Methylation-specific primer, SAGE, Bisulfite treatment, M.SssⅠ, Human Epigenetic Project
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