Absolute quantification of plasmid DNA by real-time PCR with genomic DNA as external standard and its application to a biodistribution study of an HIV DNA vaccine.
Real-time quantitative PCR (QPCR) has been proven to be a powerful tool for quantifying specific target DNA sequences. Compared to relative quantification, absolute quantification has the advantage of determining the absolute copy number of a given target, such as pathogen or plasmid DNA in vivo. However, matrix or impurities remaining in a DNA sample after various sample treatment procedures may influence a subsequent DNA analysis. In this work, we have compared methods of sample processing and validated the use of tissue genomic DNA as a universal external standard to facilitate quantification of plasmid DNA in biological matrix, especially addressing the amplification inhibition due to matrix effect and sample complexity. Also, we applied our high-throughput sample preparation and absolute quantification method to determine the distribution of an HIV plasmid DNA vaccine in vivo. Successful application showed the validity and reliability of the method in absolute quantification of a particular gene in vivo.
Fu
Jie
J
Laboratory of Drug Metabolism and Pharmacokinetic, Beijing Institute of Radiation Medicine, PR China.
Li
Ding
D
Xia
Shaoyou
S
Song
Haifeng
H
Dong
Zengxiang
Z
Chen
Fang
F
Sun
Xiao
X
Tang
Zhongming
Z
D016915:AIDS Vaccines / Q000032:analysis / Q000493:pharmacokinetics*; D000818:Animals; D004247:DNA / Q000032:analysis* / Q000235:genetics; D004279:DNA, Viral / Q000032:analysis / Q000493:pharmacokinetics*; D005260:Female; D018628:Gene Dosage; D016678:Genome / Q000235:genetics*; D006678:HIV / Q000235:genetics / Q000276:immunology; D016014:Linear Models; D008297:Male; D051379:Mice; D010957:Plasmids / Q000032:analysis / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors; D014018:Tissue Distribution; D019444:Vaccines, DNA / Q000032:analysis / Q000493:pharmacokinetics*
D016915:AIDS Vaccines; D004279:DNA, Viral; D019444:Vaccines, DNA; D004247:DNA