Methods in molecular biology
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The aberrant DNA methylation has been noted to occur at promoter of tumor suppressor, cell adhesion, DNA repair, and other growth regulating genes during the progression of nonneoplastic esophageal mucosa to Barrett esophagus to esophageal adenocarcinoma. Methylation-mediated silencing of individual gene or concurrent loss of a number of genes plays crucial roles in dysplasia-metaplasia-neoplasia sequence of esophageal adenocarcinoma. ⋯ There are a number of methods including bead array, PCR and sequencing, pyrosequencing, methylation-specific PCR, and PCR with high-resolution melt curve available to determine the methylation status of particular gene of interest. Herein, we describe the polymerase chain reaction followed by sequencing-based protocol for identifying DNA methylation status in esophageal adenocarcinoma.
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Esophageal adenocarcinoma is heterogeneous and studies have reviewed many important mutations that contribute to the pathogenesis of the cancer. These discoveries have helped paved the way into identifying new gene markers or gene targets to develop novel molecular directed therapy for better patient outcomes in esophageal adenocarcinoma. Despite the recent bloom in next-generation sequencing, Sanger sequencing still represents the gold standard method for the study of the driver genes in esophageal adenocarcinoma. This chapter focuses on the sequencing techniques in identification of single gene mutations.
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Bisulfite sequencing (BS-seq) enables the detection of DNA methylation at cytosine residues (5mC) at single-nucleotide resolution. For many applications, a limiting factor of conventional BS-seq protocols is the high amount of DNA required, since the treatment with bisulfite causes severe DNA fragmentation. Here, we describe a post-bisulfite tagging method that accounts for this problem. ⋯ The method can also be used to analyze defined fractions of genomes from limited samples by Reduced Representation Bisulfite Sequencing (RRBS). This involves restriction digestion, gel separation and fragment elution prior to BS-seq library preparation to enrich certain areas of the genome. This reduction of represented genomic regions lowers the sequencing cost considerably while providing an accurate assessment of total genome-wide DNA methylation levels and assessment of DNA methylation in categorical genomic regions.
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DNA methylation is a covalent modification of DNA that plays important roles in processes such as the regulation of gene expression, transcription factor binding, and suppression of transposable elements. The use of whole genome bisulfite sequencing (WGBS) enables the genome-wide identification and quantification of DNA methylation patterns at single-base resolution and is the gold standard for analysis of DNA methylation. Computational analysis of WGBS data can be particularly challenging, as many computationally intensive steps are required. ⋯ Second, DNA methylation levels are estimated at each cytosine position using the aligned sequence reads of the bisulfite treated DNA. Third, regions of differential cytosine methylation between samples can be identified. Finally, these data need to be visualized and interpreted in the context of the biological question at hand.
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An esophagogastrectomy is a surgical procedure that is performed for treatment of confirmed localized esophageal and esophagogastric junction adenocarcinoma. Proper macroscopic assessment and cut-up technique is essential to ensure that the overall assessment is correct and reproducible. Here, we describe a standard for macroscopic assessment and dissection to be used for routine handling of esophagogastrectomy specimens in the clinical laboratory.