| Background and ObjectivesLymphoma shares similarity with highly activated lymphoid tissues on histology and morphology and the differentiation diagnosis between them is very difficult, which results in frequent misdiagnosis. The pathologic diagnosis of lymphoma is one of the difficulty in clinical pathology. In the present China, the diagnosis depends on histomorphology as the main diagnostic means and immunohistology as an assistant one. With the development of molecular biology, the techniques for study and analysis of illness in genie level gradually appeared and matured with time, which provide effective tool for the diagnosis of lymphomas. The lymphoma cells are from the same monoclonal cell proliferation while the reactive lymphoid tissue develops from polyclonal lymphoid cells, which consists the primary difference between them two. After the rearrangement of the coding gene for immunoglobulin (Ig) and T cell receptor (TCR), it is different between one another for individual clones along with the rearranged genes. So, the gene rearrangement is specific valuable for the diagnosis of lymphoma. With its simplicity, high efficiency, low cost and wide fitness, techniques based on PCR are more and more regarded in the study of lymphoma gene rearrangements. Though lots of investigations have been carried out, the patterns applied in different literatures, including primers, PCR conditions and the ways in PCR products analysis are not all the same, resulting the diverse outcomes. So, it is difficult to introduce a suitable and standard process into the routine clinical pathological diagnosis. Jurkat cell line is frequently used as positive control in the research of gene rearrangement in T cell lymphomas, but the detailed gene rearrangement condition of the cell is not reported yet. pseudogenes commonly present in the germinal DNA genes of Ig and TCR, but the studies on the gene rearrangement of the pseudogenes and whether pseudogenes should be considered in the design of primers are not reported. In this study, we select TCR gene rearrangement as the research object. Through optimizing theprimers selection, PCR condition and the process of analyzing PCR products, we look for the most suitable process for the checkout of the gene rearrangements in T cell lymphoms. Meanwhile, by analyzing the characters of TCR gene rearrangements, we provide basic theories and technical supports for further study on TCR gene rearrangements. Methods1 We chose the TCRy gene rearrangement as the research subject, Collecting the consensus primers TVG, Vy I and family specific primers as the tools. Orthogonal experimental design was introduced into the optimization PCR conditions of Vy I primer. Another pair of consensus primers of TCRp was applied as the complement and control of TCRy primers.2 PCR and gene sequencing were conducted to analyze TCR gene rearrangement of Jurkat cell line while exploring the basic characters of TCR gene rearrangement.3 The methods to analize the PCR products ?TCRy consensus primers were applied to investigate the clonal gene rearrangement in T and B cell lymphomas, agarose gel electrophoresis and single-strand conformational polymorphism (SSCP) analysis were adopted to analyze the PCR products. Direct DNA sequencing and cloned DNA sequencing of the PCR products amplified by TCRy consensus primers were carried out to analyze the character of TCR gene rearrangement in T cell lymphomas. The methods and the importance of sequencing in the gene rearrangement were explored. The relationship between the numbers of DNA clones in PCR products and the numbers bands in SSCP analysis was analyzed.4 Various primer combinations were applied to analyze the condition of Multiplex PCRs and observe how much the positive ratio was improved along with the Multiplex PCRs. The condition for the consensus primers of TCRp and the ratio checked out by this pair of primers were studied.Results1 Orthogonal design experiment carried out to optimize the PCR conditions for Vy I...
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