Methods in molecular biology
-
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.
-
Next-generation sequencing refers to the high-throughput DNA sequencing technologies, which are capable of sequencing large numbers of different DNA sequences in a single/parallel reaction. It is a powerful tool to identify inherited and acquired genetic alterations associated with the development of esophageal adenocarcinoma. ⋯ Thus, second-generation sequencing methods can provide a complete picture of the esophageal adenocarcinoma genome by detecting and discovering different type of alterations in the cancer. This would help in diagnostics and will further help in developing personalized medicine in esophageal adenocarcinoma.
-
The discovery of the CRISPR-Cas9 system raises hope for the treatment of many genetic disorders. We describe here an approach based on the use of a pair of single guide RNAs to form a hybrid exon that does not only restore the dystrophin gene reading frame but also results in the production of a dystrophin protein with an adequate structure of the central rod-domain, with a correct spectrin-like repeat. The therapeutic approach described here involved DMD patient cells having a deletion of exons 51-53 of the DMD gene.
-
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.
-
The discovery and adaptation of RNA-guided nucleases has resulted in the rapid development of efficient, scalable, and easily accessible synthetic biology tools for targeted genome editing and transcriptional control. In these systems, for example CRISPR-Cas9 from Streptococcus pyogenes, a protein with nuclease activity is targeted to a specific nucleotide sequence by a short RNA molecule, whereupon binding it cleaves the targeted nucleotide strand. To extend this genome-editing ability to the industrially important oleaginous yeast Yarrowia lipolytica, we developed a set of easily usable and effective CRISPR-Cas9 episomal vectors. ⋯ A second method demonstrates how the same CRISPR-Cas9 system can be used to induce markerless gene cassette integration into the genome by inducing homologous recombination after DNA cleavage by Cas9. Finally, we describe how a catalytically inactive form of Cas9 fused to a transcriptional repressor can be used to control transcription of native genes in Y. lipolytica. The CRISPR-Cas9 tools and strategies described here greatly increase the types of genome editing and transcriptional control that can be achieved in Y. lipolytica, and promise to facilitate more advanced engineering of this important oleaginous host.