What is the “modified” CTAB protocol? Characterizing modifications to the CTAB DNA extraction protocol

Abstract

Cetyltrimethylammonium bromide (CTAB)-based methods are widely used to isolate DNA from plant tissues, but the unique chemical composition of secondary metabolites among plant species has necessitated optimization. Research articles often cite a "modified" CTAB protocol without explicitly stating how the protocol had been altered, creating non-reproducible studies. Furthermore, the various modifications that have been applied to the CTAB protocol have not been rigorously reviewed and doing so could reveal optimization strategies across study systems. We surveyed the literature for modified CTAB protocols used for the isolation of plant DNA. We found that every stage of the CTAB protocol has been modified, and we summarized those modifications to provide recommendations for extraction optimization. Future genomic studies will rely on optimized CTAB protocols. Our review of the modifications that have been used, as well as the protocols we provide here, could better standardize DNA extractions, allowing for repeatable and transparent studies.

Enhancing the authenticity of animal by-products: harmonization of DNA extraction methods from novel ingredients

“…Despite the higher performance of conventional extraction methods regarding DNA yield, it is important to highlight that these methods are prone to contamination by chemical reagents during the extraction process. For instance, the presence of residual CTAB may increase the DNA solution’s absorbance at A260 nm and thus result in increased values of obtained concentration ( Drábková et al, 2002 ; Piskata et al, 2019 ; Schenk et al, 2023 ). This may occur due to less efficient purification steps when compared to commercial kits that applied silica-based columns, which enhance impurity removal, although lower yields are obtained ( Shetty, 2020 ).…”

Identification of Mycoviruses in the Pathogens of Fragrant Pear Valsa Canker from Xinjiang in China

“…strains was extracted by using a total RNA isolation kit (Sangon Biotech, Shanghai, China) according to the manufacturer’s instructions and subsequently treated with DNase I to remove DNA contamination ( Table S1 ). Fungal genomic DNA was extracted using the CTAB method [ 30 ]. The quality of the RNA and DNA was determined via agarose gel electrophoresis, and the total RNA and DNA samples were quantified via ultramicrospectrophotometry (NanoDrop 2000, Themo, Shanghai, China).…”

Emerging methods in botanical DNA/RNA extraction

“…However, researchers commonly refer to a "modified CTAB" approach, where various modifications address difficulties in extraction that are often taxon-specific and highly varied, but often without detailing what aspects of the protocol were adjusted. To better understand these alterations, the first paper in this issue (Schenk et al, 2023) reports the results of a literature survey and summary of these modified CTAB protocols. Schenk et al (2023) report and provide recommendations for modifications to eight steps in the CTAB protocol: tissue preparation, suspension, lysis, isolation, cleaning, elution, secondary cleanup, and quantification, with explanations as to why each step may require modification.…”

“…To better understand these alterations, the first paper in this issue (Schenk et al, 2023) reports the results of a literature survey and summary of these modified CTAB protocols. Schenk et al (2023) report and provide recommendations for modifications to eight steps in the CTAB protocol: tissue preparation, suspension, lysis, isolation, cleaning, elution, secondary cleanup, and quantification, with explanations as to why each step may require modification. Additionally, they provide four supplementary protocols as appendices, which detail the alterations to the lysis and/or extraction steps.…”