Concordance Study: Methods of Quantifying Corn and Soybean Genomic DNA Intended for Real-Time Polymerase Chain Reaction Applications

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• 作者：G. Ronald Jenkins ; Jennifer T. Helber ; Larry D. Freese
• 刊名：Journal of Agricultural and Food Chemistry
• 出版年：2012
• 出版时间：August 29, 2012
• 年：2012
• 卷：60
• 期：34
• 页码：8323-8332
• 全文大小：261K
• 年卷期：v.60,no.34(August 29, 2012)
• ISSN：1520-5118

文摘

Quantitative real-time polymerase chain reaction (qPCR) is a technology commonly used for the detection and quantification of genetically engineered (GE) traits in grains and oilseeds. The method involves measuring copy numbers of taxon-specific, endogenous control genes exposed to the same manipulations as trait-specific target genes. Accurate DNA quantification is essential for successful and predictable results with qPCR. A systematic study of seven different DNA quantification methods, incorporating different chemistries and different instrumentation, were evaluated on corn and soy DNA that was extracted using two distinct extraction methods. A time course study showed that corn and soy DNA was stable under typical laboratory storage conditions. Corn_CTAB and corn_Qiagen DNA extracts produced statistically similar quantification values when measured by picogreen PG_TD700, PG_Lum20/20, Hoescht_TD700, and Hoescht_Lum20/20 methods, suggesting that these methods can be used interchangeably to quantify DNA in corn samples prior to initiation of qPCR. Soy_Qiagen provided greater stochastic measurement variability when quantification methods were compared, whereas soy_CTAB had statistically significant differences when a PG method was compared to a Hoescht method of DNA quantification. Finally, agarose gel electrophoresis data revealed more pronounced degradation for Qiagen-extracted DNA compared with CTAB extracts in both corn and soy. Consequently, C_t values generated by qPCR suggested that absolute copy numbers of PCR amplifiable targets were not concordant between Qiagen and CTAB DNA extracts. Understanding measurement uncertainty from component steps used in qPCR can contribute toward harmonizing methods for the detection of GE traits in grains and oilseeds.

Keywords:

p://pubs.acs.org/action/doSearch?action=search&searchText=DNA+quantification&qsSearchArea=searchText">DNA quantification; p://pubs.acs.org/action/doSearch?action=search&searchText=corn&qsSearchArea=searchText">corn; p://pubs.acs.org/action/doSearch?action=search&searchText=soy&qsSearchArea=searchText">soy; p://pubs.acs.org/action/doSearch?action=search&searchText=quantitative+PCR&qsSearchArea=searchText">quantitative PCR; p://pubs.acs.org/action/doSearch?action=search&searchText=Picogreen&qsSearchArea=searchText">Picogreen; p://pubs.acs.org/action/doSearch?action=search&searchText=Hoescht&qsSearchArea=searchText">Hoescht; p://pubs.acs.org/action/doSearch?action=search&searchText=A260&qsSearchArea=searchText">A₂₆₀; p://pubs.acs.org/action/doSearch?action=search&searchText=genetically+engineered+traits&qsSearchArea=searchText">genetically engineered traits