Comparing DNA Extraction Methods for Analysis of Botanical Materials Found in Anti-Diabetic Supplements

Abstract

A comparative performance evaluation of DNA extraction methods from anti-diabetic botanical supplements using various commercial kits was conducted, to determine which produces the best quality DNA suitable for PCR amplification, sequencing and species identification. All plant materials involved were of suboptimal quality showing various levels of degradation and therefore representing real conditions for testing herbal supplements. Eight different DNA extraction methods were used to isolate genomic DNA from 13 medicinal plant products. Two methods for evaluation, DNA concentration measurements that included absorbance ratios as well as PCR amplifiability, were used to determine quantity and quality of extracted DNA. We found that neither DNA concentrations nor commonly used UV absorbance ratio measurements at A(260)/A(280) between 1.7 and 1.9 are suitable for globally predicting PCR success in these plant samples, and that PCR amplifiablity itself was the best indicator of extracted product quality. However, our results suggest that A(260)/A(280) ratios below about 1.3 and above 2.3 indicated a DNA quality too poor to amplify. Therefore, A(260)/A(280) measurements are not useful to identify samples that likely will amplify but can be used to exclude samples that likely will not amplify reducing the cost for unnecessarily subjecting samples to PCR. The two Nucleospin(®) plant II kit extraction methods produced the most pure and amplifiable genomic DNA extracts. Our results suggest that there are clear, discernable differences between extraction methods for low quality plant samples in terms of producing contamination-free, high-quality genomic DNA to be used for further analysis.

Extraction of high purity genomic DNA from pine for use in a high-throughput Genotyping Platform

“…The N96 method has also performed well in other plant species and genotyping systems (Llongueras, et al, 2012). Comparable fail rates were also achieved with methods CQ and SQ, but the percentage of assays departing from HWE was nearly doubled.…”

“…However, purity alone or yield alone should not be the sole consideration when comparing methods (Llongueras, Nair, Salas-Leiva, & Schwarzbach, 2012), but should be considered in conjunction with assay performance.…”

Botanical origin authentication of dietary supplements by DNA‐based approaches

“…Although CTAB‐based methods can provide pure extracts from plant material and processed foods, they generally produce low DNA yields and the protocols are time consuming (Costa et al., 2015; Mafra, Silva, Moreira, da Silva, & Oliveira, 2008b). Accordingly, several commercial kits for DNA extraction have been preferred and extensively applied to botanicals and herbal dietary supplements, such as DNeasy plant mini kit (Qiagen), Nucleospin plant II kit (Macharey‐Nagel), Nucleospin food kit (Macharey‐Nagel), and Wizard ® Genomic DNA purification kit, among other (Cimino, 2010; Costa et al., 2015, 2016; Llongueras, Nair, Salas‐Leiva, & Schwarzbach, 2013; Lo & Shaw, 2018a; Newmaster et al., 2013; Sahu et al., 2012; Särkinen, Staats, Richardson, Cowan, & Bakker, 2012; Tan & Yiap, 2009; Wallace et al., 2012).…”

“…Cimino (2010) tested three DNA extraction kits (Qiagen, MoBio, and Phytopure) on 24 herbal products, including dietary supplements, containing Camellia , Citrus , Ephedra , Ginkgo , Hypericum , Serenoa , and Vaccinium , from which the Qiagen kit gave the highest amplification. The comparative study of 8 different DNA extraction kits applied to 13 medicinal plant products (DNeasy plant mini kit, Nucleospin plant II (PL1 and PL2/PL3), IBI genomic DNA mini kit (IBI, plant GP1, and GPX1), QuickExtract plant DNA extraction solution, QuickExtract seed DNA extraction solution (Epicentre Biotechnologies), and PowerPlant DNA isolation kit (MO BIO)) showed that both Nucleospin plant methods produced the most pure and amplifiable DNA extracts from degraded samples, being closely followed by the Dneasy kit, highlighting great differences among the methods and the need of further studies with other plant taxa (Llongueras et al., 2013).…”

Comparison of DNA extraction methods for entomological origin identification of honey using simple additive weighting method

“…Absorbance ratio (A 260 /A 280 ) was used to assess purity of extracted DNA, where values between 1.7 and 1.9 were considered as pure DNA (Llongueras et al, 2013). Values lower than 1.7 indicate contamination of DNA with proteins and/or other residual impurities carried over from DNA extraction such as phenol, ethanol, carbohydrates, or contamination from food matrix, whereas values above 1.9 indicate contamination with RNA (Llongueras et al, 2013). The A 260 /A 280 values of extracted DNA using CTAB method with the selective precipitation of DNA format ranged from 0.6 to 2.0 in Table 1.…”