title: Quantification of trace-level DNA by real-time whole genome amplification.
abstract: Quantification of trace amounts of DNA is a challenge in analytical applications where the concentration of a target DNA is very low or only limited amounts of samples are available for analysis. PCR-based methods including real-time PCR are highly sensitive and widely used for quantification of low-level DNA samples. However, ordinary PCR methods require at least one copy of a specific gene sequence for amplification and may not work for a sub-genomic amount of DNA. We suggest a real-time whole genome amplification method adopting the degenerate oligonucleotide primed PCR (DOP-PCR) for quantification of sub-genomic amounts of DNA. This approach enabled quantification of sub-picogram amounts of DNA independently of their sequences. When the method was applied to the human placental DNA of which amount was accurately determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES), an accurate and stable quantification capability for DNA samples ranging from 80 fg to 8 ng was obtained. In blind tests of laboratory-prepared DNA samples, measurement accuracies of 7.4%, -2.1%, and -13.9% with analytical precisions around 15% were achieved for 400-pg, 4-pg, and 400-fg DNA samples, respectively. A similar quantification capability was also observed for other DNA species from calf, E. coli, and lambda phage. Therefore, when provided with an appropriate standard DNA, the suggested real-time DOP-PCR method can be used as a universal method for quantification of trace amounts of DNA.
authors.affiliation: Center for Bio-Analysis, Korea Research Institute of Standards and Science, Daejon, Republic of Korea.
authors.forename: Min-Jung
authors.initials: MJ
authors.lastname: Kang
authors.forename: Hannah
authors.initials: H
authors.lastname: Yu
authors.forename: Sook-Kyung
authors.initials: SK
authors.lastname: Kim
authors.forename: Sang-Ryoul
authors.initials: SR
authors.lastname: Park
authors.forename: Inchul
authors.initials: I
authors.lastname: Yang
mesh_terms: D004247:DNA / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics*; D005260:Female; D015894:Genome, Human / Q000235:genetics*; D006801:Humans; D009841:Oligonucleotides / Q000235:genetics; D010920:Placenta / Q000378:metabolism; D011247:Pregnancy; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
chemical_list: D017931:DNA Primers; D009841:Oligonucleotides; D004247:DNA