Polymerase Chain Reaction Technology as Analytical Tool in Agricultural Biotechnology

Abstract

The agricultural biotechnology industry applies polymerase chain reaction (PCR) technology at numerous points in product development. Commodity and food companies as well as third-party diagnostic testing companies also rely on PCR technology for a number of purposes. The primary use of the technology is to verify the presence or absence of genetically modified (GM) material in a product or to quantify the amount of GM material present in a product. This article describes the fundamental elements of PCR analysis and its application to the testing of grains. The document highlights the many areas to which attention must be paid in order to produce reliable test results. These include sample preparation, method validation, choice of appropriate reference materials, and biological and instrumental sources of error. The article also discusses issues related to the analysis of different matrixes and the effect they may have on the accuracy of the PCR analytical results.

Quantification of Genetically Modified Soybeans Using a Combination of a Capillary-Type Real-Time PCR System and a Plasmid Reference Standard

“…[5][6][7][8][9][10][11][12][13][14][15][16][17][18] Furthermore, many real-time PCR systems based on fluorescent detection, such as TaqMan chemistry, have been developed to identify and quantify GM soybeans, GM maize, and GM varieties of other agricultural commodities. [19][20][21][22][23][24][25][26][27][28] Real-time PCR systems using TaqMan chemistry are based on the use of a fluorescent TaqMan probe that monitors the formation of the PCR product during each cycle of the reaction. In addition, most commonly, GMO quantification by quantitative real-time PCR methods is calculated from the ratio of the target transgenic specific DNA sequence copy number vs. the DNA sequence copy number of the respective target plant species (taxon gene sequence).…”

Detection limits of quantitative and digital PCR assays and their influence in presence–absence surveys of environmental DNA

“…We also observed the concentration plateau while performing standard serial dilution experiments with a synthetic gene to investigate qPCR and dPCR detection limits (see Materials and methods section for details). Additionally, at trace levels of DNA, the stochastic distribution of DNA molecules can induce large uncertainty associated with sampling variability of pipetting, heterogeneous clumping of DNA, inhibition of the PCR or degraded target DNA (Ellison et al 2006, Lipp et al 2005, Hromadnikova et al 2003. Sampling variability at low DNA concentrations can result from replicate PCRs containing zero, one or multiple DNA copies subsampled from the solution.…”

Methodological flaws introduce strong bias into molecular analysis of microbial populations

“…Thus, to produce reasonable PCR quantifications, both extracts had to be diluted (1 : 5; 1 : 100). The dilution of DNA extracts fosters minimization of inhibitor concentrations and enhances PCR efficiency, but, however, lower DNA concentrations can increase the PCR sensitivity (Lipp et al 2005;Corbisier et al 2007). Furthermore, different elution volumes specified by the manufacturer influenced the final genomic DNA concentrations as well (Table 1) what in turn can impact diversity results massively when subsequent PCR protocols are strictly applied.…”