Performance of high-throughput DNA quantification methods

Kashif A Haque,Andrew W Bergen,Jeff P Struewing,Michael B Beerman,Ruth M Pfeiffer,Stephen J Chanock

doi:10.1186/1472-6750-3-20

Abstract

BackgroundThe accuracy and precision of estimates of DNA concentration are critical factors for efficient use of DNA samples in high-throughput genotype and sequence analyses. We evaluated the performance of spectrophotometric (OD) DNA quantification, and compared it to two fluorometric quantification methods, the PicoGreen® assay (PG), and a novel real-time quantitative genomic PCR assay (QG) specific to a region at the human BRCA1 locus. Twenty-Two lymphoblastoid cell line DNA samples with an initial concentration of ~350 ng/uL were diluted to 20 ng/uL. DNA concentration was estimated by OD and further diluted to 5 ng/uL. The concentrations of multiple aliquots of the final dilution were measured by the OD, QG and PG methods. The effects of manual and robotic laboratory sample handling procedures on the estimates of DNA concentration were assessed using variance components analyses.ResultsThe OD method was the DNA quantification method most concordant with the reference sample among the three methods evaluated. A large fraction of the total variance for all three methods (36.0–95.7%) was explained by sample-to-sample variation, whereas the amount of variance attributable to sample handling was small (0.8–17.5%). Residual error (3.2–59.4%), corresponding to un-modelled factors, contributed a greater extent to the total variation than the sample handling procedures.ConclusionThe application of a specific DNA quantification method to a particular molecular genetic laboratory protocol must take into account the accuracy and precision of the specific method, as well as the requirements of the experimental workflow with respect to sample volumes and throughput. While OD was the most concordant and precise DNA quantification method in this study, the information provided by the quantitative PCR assay regarding the suitability of DNA samples for PCR may be an essential factor for some protocols, despite the decreased concordance and precision of this method.

Highlights

The accuracy and precision of estimates of DNA concentration are critical factors for efficient use of DNA samples in high-throughput genotype and sequence analyses
The mean Optical Density Assay (OD)-D concentration estimate of 5.13 ng/uL was close to the expected value (p < 0.5464), while the distribution of the concentration estimates was positively skewed (Table 1 and Figure 1b)
The PicoGreen® assay (PG) method resulted in a mean concentration, 5.21 ng/uL, that was significantly greater than the expected value (p < 0.0148), and the concentration estimates were negatively skewed due to several low, but non-zero concentration estimates (Table 1 and Figure 1d)

Summary

Introduction

The accuracy and precision of estimates of DNA concentration are critical factors for efficient use of DNA samples in high-throughput genotype and sequence analyses. DNA concentration was estimated by OD and further diluted to 5 ng/uL. The effects of manual and robotic laboratory sample handling procedures on the estimates of DNA concentration were assessed using variance components analyses. One area of technological development essential for success in high-throughput genotyping is the quantification of DNA. Accurate and precise DNA quantification is necessary for efficient highthroughput genotyping and sample conservation. Conservation of the original DNA samples is important to validate previous findings and to allow for future studies, even if whole genome amplification technologies prove to be accurate and reliable [6,7]

Methods

Results

Discussion

Conclusion