Quantification of trace-level DNA by real-time whole genome amplification.

Min-Jung Kang,Sook-Kyung Kim,Sang-Ryoul Park,Hannah Yu,Inchul Yang,M Thomas P Gilbert

doi:10.1371/journal.pone.0028661

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.

Highlights

Quantification of trace amounts of DNA is of special importance in certain analytical applications where the concentration of a target DNA is very low or only limited amounts of samples are available for analysis
The Food and Drug Administration (FDA) guidelines suggest that the acceptable residual amount of host cell DNA in biopharmaceutical drugs should be below 100 pg/dose, while the acceptable limit of host cell DNA allowed by the European Union (EU) is up to 10 ng/ dose [5,6]
The degenerate oligonucleotide primed PCR (DOP-PCR) strategy has two potential advantages compared with ordinary PCR methods: its sequence independence, which enables universal applicability of the method for amplification of any arbitrary DNA species, and a potential sensitivity that is not limited by the requirement for one genome-equivalent amount of DNA as a template

Summary

Introduction

Quantification of trace amounts of DNA is of special importance in certain analytical applications where the concentration of a target DNA is very low or only limited amounts of samples are available for analysis. It is hard to achieve such a high sensitivity in quantification of practical samples containing only trace amounts of DNA since they would be generally not very concentrated to the level of 1 pg/ mL. Fluorescence-based techniques are widely used for quantification of DNA These methods show much higher sensitivity and accuracy compared with UV spectrophotometry for the quantification of DNA [10]. The fluorescence-based method was subject to interferences by contaminants, and was reported to be not effective for quantification of DNA samples lower than 50 pg/mL [9,11]

Objectives

Methods

Results