Performance of high-throughput DNA quantification methods

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

Research output: Contribution to journalArticle

Abstract

Background: 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. 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. Results: The 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. Conclusion: The 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.

Original languageEnglish (US)
Article number20
JournalBMC Biotechnology
Volume3
DOIs
StatePublished - Oct 28 2003
Externally publishedYes

Fingerprint

DNA
Polymerase Chain Reaction
Workflow
Robotics
Sequence Analysis
Real-Time Polymerase Chain Reaction
Molecular Biology
Analysis of Variance
Genotype
Cell Line

ASJC Scopus subject areas

  • Medicine(all)
  • Biotechnology

Cite this

Haque, K. A., Pfeiffer, R. M., Beerman, M. B., Struewing, J. P., Chanock, S. J., & Bergen, A. W. (2003). Performance of high-throughput DNA quantification methods. BMC Biotechnology, 3, [20]. https://doi.org/10.1186/1472-6750-3-20

Performance of high-throughput DNA quantification methods. / Haque, Kashif A.; Pfeiffer, Ruth M.; Beerman, Michael B.; Struewing, Jeff P.; Chanock, Stephen J.; Bergen, Andrew W.

In: BMC Biotechnology, Vol. 3, 20, 28.10.2003.

Research output: Contribution to journalArticle

Haque, KA, Pfeiffer, RM, Beerman, MB, Struewing, JP, Chanock, SJ & Bergen, AW 2003, 'Performance of high-throughput DNA quantification methods', BMC Biotechnology, vol. 3, 20. https://doi.org/10.1186/1472-6750-3-20
Haque KA, Pfeiffer RM, Beerman MB, Struewing JP, Chanock SJ, Bergen AW. Performance of high-throughput DNA quantification methods. BMC Biotechnology. 2003 Oct 28;3. 20. https://doi.org/10.1186/1472-6750-3-20
Haque, Kashif A. ; Pfeiffer, Ruth M. ; Beerman, Michael B. ; Struewing, Jeff P. ; Chanock, Stephen J. ; Bergen, Andrew W. / Performance of high-throughput DNA quantification methods. In: BMC Biotechnology. 2003 ; Vol. 3.
@article{c15e1204d23d46a3a17302354dc9cf3d,
title = "Performance of high-throughput DNA quantification methods",
abstract = "Background: 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. We evaluated the performance of spectrophotometric (OD) DNA quantification, and compared it to two fluorometric quantification methods, the PicoGreen{\circledR} 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. Results: The 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. Conclusion: The 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.",
author = "Haque, {Kashif A.} and Pfeiffer, {Ruth M.} and Beerman, {Michael B.} and Struewing, {Jeff P.} and Chanock, {Stephen J.} and Bergen, {Andrew W.}",
year = "2003",
month = "10",
day = "28",
doi = "10.1186/1472-6750-3-20",
language = "English (US)",
volume = "3",
journal = "BMC Biotechnology",
issn = "1472-6750",
publisher = "BioMed Central",

}

TY - JOUR

T1 - Performance of high-throughput DNA quantification methods

AU - Haque, Kashif A.

AU - Pfeiffer, Ruth M.

AU - Beerman, Michael B.

AU - Struewing, Jeff P.

AU - Chanock, Stephen J.

AU - Bergen, Andrew W.

PY - 2003/10/28

Y1 - 2003/10/28

N2 - Background: 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. 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. Results: The 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. Conclusion: The 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.

AB - Background: 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. 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. Results: The 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. Conclusion: The 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.

UR - http://www.scopus.com/inward/record.url?scp=3042557900&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=3042557900&partnerID=8YFLogxK

U2 - 10.1186/1472-6750-3-20

DO - 10.1186/1472-6750-3-20

M3 - Article

C2 - 14583097

AN - SCOPUS:3042557900

VL - 3

JO - BMC Biotechnology

JF - BMC Biotechnology

SN - 1472-6750

M1 - 20

ER -