Evaluation of extraction methodologies for corn kernel (Zea mays) DNA for detection of trace amounts of biotechnology-derived DNA.
Sensitive and accurate testing for trace amounts of biotechnology-derived DNA from plant material requires pure, high-quality genomic DNA as template for subsequent amplification using the polymerase chain reaction (PCR). Six methodologies were evaluated for extracting DNA from ground corn kernels spiked with 0.1% (m/m) CBH351 (StarLink) corn. DNA preparations were evaluated for purity and fragment size. Extraction efficiency was determined. The alcohol dehydrogenase gene (adh1) and the CBH351 (cry9C, 35S promoter) genes in the genomic DNA were detected using PCR. DNA isolated by two of the methods proved unsuitable for performing PCR amplification. All other methods produced some DNA preparations that gave false negative PCR results. We observed that cornstarch, a primary component of corn kernels, was not an inhibitor of PCR, while acidic polysaccharides were. Our data suggest that amplification of an endogenous positive control gene, as an indicator for the absence of PCR inhibitors, is not always valid. This study points out aspects of DNA isolation that need to be considered when choosing a method for a particular plant/tissue type.
Holden
Marcia J
MJ
National Institute of Standards and Technology, Biotechnology Division, 100 Bureau Drive, MS-8312, Gaithersburg, MD 20899, USA. marcia.holden@nist.gov
Blasic
Joseph R
JR
Bussjaeger
Louis
L
Kao
Chuan
C
Shokere
Luke A
LA
Kendall
Donald C
DC
Freese
Larry
L
Jenkins
G Ronald
GR
D001709:Biotechnology; D018744:DNA, Plant / Q000302:isolation & purification*; D005188:False Negative Reactions; D005784:Gene Amplification; D010316:Particle Size; D030821:Plants, Genetically Modified / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D003313:Zea mays / Q000235:genetics*
D018744:DNA, Plant