Effects of DNA extraction and purification methods on real-time quantitative PCR analysis of Roundup Ready soybean.
The quality of DNA affects the accuracy and repeatability of quantitative PCR results. Different DNA extraction and purification methods were compared for quantification of Roundup Ready (RR) soybean (event 40-3-2) by real-time PCR. DNA was extracted using cetylmethylammonium bromide (CTAB), DNeasy Plant Mini Kit, and Wizard Magnetic DNA purification system for food. CTAB-extracted DNA was also purified using the Zymo (DNA Clean & Concentrator 25 kit), Qtip 100 (Qiagen Genomic-Tip 100/G), and QIAEX II Gel Extraction Kit. The CTAB extraction method provided the largest amount of DNA, and the Zymo purification kit resulted in the highest percentage of DNA recovery. The Abs260/280 and Abs260/230 ratios were less than the expected values for some of the DNA extraction and purification methods used, indicating the presence of substances that could inhibit PCR reactions. Real-time quantitative PCR results were affected by the DNA extraction and purification methods used. Further purification or dilution of the CTAB DNA was required for successful quantification of RR soybean. Less variability of quantitative PCR results was observed among experiments and replications for DNA extracted and/or purified by CTAB, CTAB+Zymo, CTAB+Qtip 100, and DNeasy methods. Correct and repeatable results for real-time PCR quantification of RR soybean were achieved using CTAB DNA purified with Zymo and Qtip 100 methods.
Demeke
Tigst
T
Canadian Grain Commission, Grain Research Laboratory, 303 Main St, Winnipeg, MB, R3C 3G8, Canada. tigst.demeke@grainscanada.gc.ca
Ratnayaka
Indira
I
Phan
Anh
A
D000077286:Cetrimonium; D002593:Cetrimonium Compounds / Q000737:chemistry; D017931:DNA Primers; D018744:DNA, Plant / Q000235:genetics / Q000302:isolation & purification*; D003902:Detergents / Q000737:chemistry; D005998:Glycine / Q000031:analogs & derivatives*; D006540:Herbicides; D007202:Indicators and Reagents; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012639:Seeds / Q000737:chemistry; D013025:Glycine max / Q000737:chemistry* / Q000235:genetics; D000097797:Glyphosate
D000077286:Cetrimonium; D002593:Cetrimonium Compounds; D017931:DNA Primers; D018744:DNA, Plant; D003902:Detergents; D005998:Glycine; D006540:Herbicides; D007202:Indicators and Reagents; D000097797:Glyphosate