Influence of DNA extraction methods, PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-derived traits

Abstract

Biotechnology-derived varieties of canola, cotton, corn and soybean are being grown in the USA, Canada and other predominantly grain exporting countries. Although the amount of farmland devoted to production of biotechnology-derived crops continues to increase, lingering concerns that unintended consequences may occur provide the EU and most grain-importing countries with justification to regulate these crops. Legislation in the EU requires traceability of grains/oilseeds, food and feed products, and labelling, when a threshold level of 0.9% w/w of genetically engineered trait is demonstrated to be present in an analytical sample. The GE content is routinely determined by quantitative PCR (qPCR) and plant genomic DNA provides the template for the initial steps in this process. A plethora of DNA extraction methods exist for qPCR applications. Implementing standardized methods for detection of genetically engineered traits is necessary to facilitate grain marketing. The International Organization for Standardization draft standard 21571 identifies detergent-based methods and commercially available kits that are widely used for DNA extraction, but also indicates that adaptations may be necessary depending upon the sample matrix. This review assesses advantages and disadvantages of various commercially available DNA extraction kits, as well as modifications to published cetyltrimethylammonium bromide methods. Inhibitors are a major obstacle for efficient amplification in qPCR. The types of PCR inhibitors and techniques to minimize inhibition are discussed. Finally, accurate quantification of DNA for applications in qPCR is not trivial. Many confounders contribute to differences in analytical measurements when a particular DNA quantification method is applied and different methods do not always provide concordant results on the same DNA sample. How these differences impact measurement uncertainty in qPCR is considered.

Impact of metagenomic DNA extraction procedures on the identifiable endophytic bacterial diversity in Sorghum bicolor (L. Moench)

“…DNA extraction constitutes one of the most critical components in any cultivation-independent study; particularly when determining microbial community diversity (Demeke and Jenkins, 2010). Accurate estimation of community profiles and diversity relies on the DNA's representativeness of the indigenous communities, and its usability in PCR (Demeke and Jenkins, 2010;Terrat et al, 2012).…”

“…T-RFLP, DGGE, pyrosequencing) (Demeke and Jenkins, 2010). The mDNA extraction must (i) ensure lysis of all microbial cells, (ii) provide sufficient genomic material (Terrat et al, 2012), and (iii) efficiently remove plant-derived phyto-chemicals, which may result in PCRinhibition (e.g.…”

“…polysaccharides, polyphenolic compounds, and secondary metabolites) and enzymes (e.g. DNases, proteinases) (Wilson, 1997;Demeke and Jenkins, 2010). Lysis of all microbial cells is particularly critical since the genomic mate-rial retrieved will dictate the bacterial diversity observed (Sène et al, 2001;Terrat et al, 2012).…”

Optimization of soybean dna extraction under different storage and development periods

“…Various extraction methods are used to obtain the DNA with high quality from leaf tissues (EUROPEAN COMMISSION, 2011) and from other parts of the plant (SHARMA; GILL; SINGHDNA, 2002;DEMEKE;JENKINS, 2010). The choice of the best development stage of the plant to collect the samples is a new variable, in which a bigger quantity and quality of the molecule is searched.…”

A simple method for extracting DNA from rhododendron plants infected with Phytophthora spp. for use in PCR

“…As has been proven here, the success of Phytophthora detection in rhododendron leaves depends on the DNA extraction procedure determining the quality, purity, and quantity of DNA. Thus, the extraction procedure affects the real-time PCR amplifications applied in diagnostics (Terry et al 2002;Demeke and Jenkins 2010). It is important to note, that in conventional PCR also the crucial factor is the quality of the DNA template and the possible absence of reaction inhibitors such as polysaccharides and other secondary metabolites.…”