Single-Strand Conformation Polymorphism Fingerprint Method for Dictyostelids

Abstract

Dictyostelid social amoebae are a highly diverse group of eukaryotic soil microbes that are valuable resources for biological research. Genetic diversity study of these organisms solely relies on molecular phylogenetics of the SSU rDNA gene, which is not ideal for large-scale genetic diversity study. Here, we designed a set of PCR–single-strand conformation polymorphism (SSCP) primers and optimized the SSCP fingerprint method for the screening of dictyostelids. The optimized SSCP condition required gel purification of the SSCP amplicons followed by electrophoresis using a 9% polyacrylamide gel under 4°C. We also tested the optimized SSCP procedure with 73 Thai isolates of dictyostelid that had the SSU rDNA gene sequences published. The SSCP fingerprint patterns were related to the genus-level taxonomy of dictyostelids, but the fingerprint dendrogram did not reflect the deep phylogeny. This method is rapid, cost-effective, and suitable for large-scale sample screening as compared with the phylogenetic analysis of the SSU rDNA gene sequences.

Circulating Nucleic Acids in Colorectal Cancer: Diagnostic and Prognostic Value

“…and a potential screening and predictive marker to immunotherapy [134] The wide spread of the mutations over variable codons [134] CBioportal, oncomine [132] RAS mutation analysis Excellent concordance with liver metastases in CRC patients who account for RAS mutations in exons 2 (codons 12 and 13), 3 (codons 59 and 61), and 4 (codons 117 and 146), predictive marker for monitoring the resistance to treatment with monoclonal antibodies (as monotherapy or combined with chemotherapy) [65] Not declared COSMIC, TCGA, GSEA, GO enrichment, KEGG, STRING, CPTAC, [125][126][127] OncoBEAMTM RAS One-step ultra-sensitive quantitative detection of plasmaderived KRAS and NRAS mutations for diagnosis, treatment, and immunotherapy monitoring of mCRC patients after surgery [84,95], significant low threshold detection [67], kinetical assaying of the mutated haploid GE quantity [67], prognostic value of MAF indicator in mCRC patients [67] Not declared MPprimer, Ultiplex [135,136] IdyllaTM RAS Accuracy sensitivity rate of ≤1% and ≤5 for KRAS mutations in exons 2, 3, and 4, respectively (potential assay for highly individualized anti-EGFR therapy and chemotherapy treatment decisions) [84] Two-step assay of RAS mutational status with lower clinical sensitivity than OncoBEAM assay [84] Biocartis Idylla™ System [84] SSCP method High sensitivity, specificity, for rapid diagnosis of hereditary nonpolyposis colorectal cancer (HNPCC) families based on hMLH1 and hMSH2 mutations with a good limit of detection (10%) [137][138][139][140] The longest turnaround time [137][138][139][140] GenBank, MUSCLE alignment program [141] Direct sequencing The gold standard, highly sensitive, and cost-effective, the quantitative measuring of an individual mutant allele in ctDNA for distinguishing CRC histological type [35,142,143] Limited potency for low amount mutant sequences detection in full wild-type DNA sequence context…”