Multiplex and quantitative analysis system for regulatory genetic elements based on high-resolution CE-SSCP

Abstract

DNA methylation and microRNAs (miRNAs) are supposed as regulatory genetic elements which controls gene expression. As the diverse quantitative patterns of multiple DNA methylation and miRNA have been discovered in development and various physiological conditions, these regulatory genetic elements are believed to be critical in many aspects of bioprocess. Therefore it has been desired to develop the multiplex assay for quantifying DNA methylation and miRNA. Analyzing each DNA methylation and miRNA, however, is not simple due to the feature of itself. As DNA methylation is an epigenetic information in the form of CpG methylation patterns, traditional nucleic acid amplification assays could not analyze DNA methylation without specific pretreatments. (e.g., sodium bisulfite conversion and methylation-sensitive restriction enzyme digestion) Current DNA methylation analysis techniques usually based on sodium bisulfite treatment to discriminate between methylated and non-methylated CpG dinucleotides. Bisulfite conversion, however, is not always efficient because error can potentially introduced in quantitative analysis due to artifacts from bisulfite conversion such as incomplete bisulfite conversion and biased PCR efficiency. Analyzing miRNAs by traditional nucleic acid amplification assays has also limitations due to short fragment (~25bp) nature, which cause challenging in design of traditional PCR primers, the difficulty in selective pairing with miRNAs and increased possibility of cross-hybridization, etc. Isothermal exponential amplification (EXPAR) has attracted great interest as a miRNA assay with high amplification efficiency. This method, however, has significant limitation in broad application due to complexity of probe design. Furthermore, standard method for DNA methylation and miRNA is restricted to quantifying multiple targets due to limited multiplexing ability. Capillary electrophoresis (CE) has widely used as detection method for quantifying multiple genetic elements by length dependent separation. As conventional CE separate various targets in length dependent manner, design of probes to have length variations is necessary. However it makes also constrain a broad range of application of the method because long template frequently cause non-specific hybridization and often cause false-negative signals. In this thesis, we introduced capillary electrophoresis–single strand conformation polymorphism (CE-SSCP) method as a detection principle in analyzing multiple genetic elements. As CE-SSCP could separate similar length of targets with minor sequence difference, we could amplify multiple targets with similar efficiency without biased results. With developing each novel amplification method for DNA methylation and miRNAs, multiplex and quantitative analysis is enable based on CE-SSCP. We introduced various strategies to improve multiplexing power and increase the sensitivity of methods. Furthermore, CE-SSCP could be used as detection method to analyze multiple DNA methylations and microRNAs in quantitative manner. First, methylation-specific (MS)-multiplex ligation-dependent probe amplification (MLPA) -, which is free from artifacts of bisulfite conversion and can be used to analyze poor quality DNA, can analyze multiple CpG residues individually in a single reaction. MS-MLPA, however, is not without limitations, which make the custom design of assays impractical: the need for long probes containing stuffer sequences and reliance on only one methylation-sensitive restriction enzyme. To overcome the above-mentioned limitations, we developed stuffer-free MS-MLPA probes that are subsequently analyzed using high-resolution CE-SSCP instead of conventional length-dependent CE. Moreover, multiple methylation-sensitive restriction enzymes (HhaI, HpaII, and AciI) were used simultaneously to achieve desired probe design criteria for all targets. Using this assay concept, we analyzed 17 genes associated with hepatocellular carcinoma (HCC). Our results showed that custom-designed assay based on MS-MLPA-CE-SSCP provided robust multiplex quantification of DNA methylation levels. Second, isothermal exponential amplification reaction (EXPAR) was introduced to amplify multiple miRNAs with high amplification efficiency by combination of polymerase strand extension and single strand nicking. However, isothermal EXPAR also could not free from limitations: complexity of probe design for preventing background signal amplification and lack of multiplexing ability. Here, we developed a modified isothermal EXPAR method that are subsequently analyzed using high-resolution CE-SSCP. To enhance the specificity to the target miRNA, stem-loop structured probe was introduced instead of linear probe in isothermal EXPAR so that miRNAs could be specifically amplified with the minimized background signal. As a detection method, CE-SSCP, a conformation dependent separation method, was used since the size code could be removed from the probe and therefore, easier probe design and uniform amplification efficiency could be achieved. In addition, coupling isothermal EXPAR with CE-SSCP has another advantage that once constructed probe set could be used in different research models. As the prediction power of the single-strand amplicons’ mobility is limited in CE-SSCP, repetitive trial and error is inevitable for designing separated probes, which is distinguished by target sequence variation in each different researches. In isotherm EXPAR, on the other hand, once constructed probe-origin templates could be used in different research, which are separated by target-independent sequences.. Using this assay concept, easier probe design and uniform amplification efficiency could be achieved. 7 miRNAs related to development of Caenorhabditis elegans and two references were analyzed simultaneously by using the method developed in this study. Expression pattern was found to be almost identical to the previous studies and consequently the modified isothermal EXPAR combined with CE-SSCP allows a robust and multiplex quantification tool for miRNA expression levels.