Efficacy of Bisulfite Modification and DNA Recovery Using Commercial Kits from Samples of Genomic and Circulating DNA

Olga Bryzgunova; Pavel Laktionov; Тatyana Skvortsova; Anna Bondar; Evgeniy Morozkin; Alena Lebedeva; Valentin Vlassov; Kurt Miller; Hans Krause

doi:doi:10.11648/j.cbb.20130106.11

Efficacy of Bisulfite Modification and DNA Recovery Using Commercial Kits from Samples of Genomic and Circulating DNA

Olga Bryzgunova, Pavel Laktionov, Тatyana Skvortsova, Anna Bondar, Evgeniy Morozkin, Alena Lebedeva, Valentin Vlassov, Kurt Miller, Hans Krause

Published in Computational Biology and Bioinformatics (Volume 1, Issue 6)

Received: 24 February 2013 Accepted: Published: 20 December 2013

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Abstract

This study had evaluated three commercially available bisulfite modification kits with regard to the procedural DNA loss and the efficacy of cytosine conversion to uracil. The efficacy of DNA conversion was estimated by pyrosequencing of eight CpG sites of unmethylated and fully enzymatically methylated templates of RARβ gene promoter region and cirDNAs of healthy donors and prostate cancer patients. The procedural DNA loss was calculated by methyl-independent qPCR for ubiquitously distributed L1RE1 retrotransposable elements. Our data demonstrates that all commercial kits displayed similar conversion efficacy of the unmethylated cytosines close to 99% independently of DNA concentration. However, when low DNA concentrations were used, the observed basic level of genomic DNA methylation increased to 11% depending on the position of CpG site. Qiagen and Chemicon kits recovered no more than 20% starting material at a high DNA input (500 ng/probe) and only 2.7–5.8% for low DNA input (10 ng/probe). EZ DNA Methylation-Gold Kit from Zymo Research provided the highest recovery regardless of the initial DNA input with average rates of no less than 86%. These results suggest that the EZ DNA Methylation-Gold Kit is the most appropriate tool for bisulfite modification of cirDNA when assaying DNA in low amounts.

Published in	Computational Biology and Bioinformatics (Volume 1, Issue 6)
DOI	10.11648/j.cbb.20130106.11
Page(s)	28-36
Creative Commons	This is an Open Access article, distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium or format, provided the original work is properly cited.
Copyright	Copyright © The Author(s), 2024. Published by Science Publishing Group

Keywords

DNA Methylation, Bisulfite Conversion, Pyrosequensing

References

[1]	K. Munson, J. Clark, K. Lamparska-Kupsik and S. Smith, "Recovery of bisulfite-converted genomic sequences in the methylation-sensitive QPCR," vol. 35, Nucleic Acids Research, 2007, pp. 2893–2903.
[2]	V. Vlassov, P. Laktionov and E. Rykova, "Extracellular nucleic acids," vol. 29, BioEssays, 2007, pp. 654–667.
[3]	M. Fleischhacker and B. Schmidt, "Circulating nucleic acids (CNAs) and cancer--a survey," vol. 1775, Biochim. Biophys. Acta, 2007, pp. 181-232.
[4]	M. Van der Vaart and P. Pretorius, "Is the role of circulating DNA as a biomarker of cancer being prematurely overrated," vol. 43, Clinical Biochemistry, 2010, pp. 26–36.
[5]	K. Jung, M. Fleischhacker and A. Rabien, "Cell-free DNA in the blood as a solid tumor biomarker--a critical appraisal of the literature", vol. 411, Clin Chim Acta, 2010, pp. 1611-1624.
[6]	P. Laktionov, S. Tamkovich, E. Rykova, O. Bryzgunova, A. Starikov, N. Kuznetsova and V. Vlassov, "Free and cell surface bound nucleic acids in blood of healthy donors and breast cancer patients," vol. 1022, Ann N Y Acad Sci, 2004, pp. 221-227.
[7]	P. Laktionov, S. Tamkovich, E. Rykovaand V. Vlassov, "Method of Nucleic acids isolation," Russian patent no. 2272072 from 20.03.2006.
[8]	T. Skvortsova, E. Rykova, S. Tamkovich, O. Bryzgunova, A. Starikov, N. Kuznetsova, V. Vlassov and P. Laktionov, "Cell-free and cell-bound circulating DNA in breast tumours: DNA quantification and analysis of tumour-related gene methylation," vol. 94, Br J Cancer, 2006, pp. 1492-1495.
[9]	V. Ngo, D. Gourdji, J, "LaverriereSite-specific methylation of rat prolactin and growth hormone promotors correlates with gene expression," vol. 16, Mol Cell Biol, 1996, pp. 3245-3254.
[10]	[10] S. Clark, J. Harrison and M. Frommer, "CpNpG methylation in mammalian cells" vol. 10, Nat Genet, 1995, pp. 20-27.
[11]	A. Padar, U. Sathyanarayana, M. Suzuki, R. Maruyama, J. Hsieh, E. Frenkel, J. Minna and A. Gazdar, "Inactivation of Cyclin D2 gene in Prostate Cancer by Aberrant Promoter Methylation," vol. 9, Clin Cancer Res, 2003, pp. 4730-4734.
[12]	C. Jerуnimo, R. Henrique, M. Hoque, F. Ribeiro, J. Oliveira, D. Fonseca, M. Teixeira, C. Lopes and D. Sidransky, "Quantitative RARbeta2 hypermethylation: a promising prostate cancer marker," vol. 10, Clin Cancer Res, 2004, pp. 4010-4014.
[13]	Y. Korshunova, R. Maloney, N. Lakey, R. Citek, B. Bacher, A. Budiman, J. Ordway, W. McCombie, J. Leon, J. Jeddeloh and J. McPherson, "Massively parallel bisulphite pyrosequencing reveals the molecular complexity of breast cancer-associated cytosine-methylation patterns obtained from tissue and serum DNA," vol. 18, Genome Res, 2008, pp. 19-29.
[14]	K. Reed, M. Poulin, L. Yan, A. Parissenti, "Comparison of bisulfite sequencing PCR with pyrosequencing for measuring differences in DNA methylation," vol. 397, Anal Biochem, 2010, pp. 96-106.
[15]	A. Yang, M. Estecio, K. Doshi, Y. Kondo, E. Tajara and J. Issa, "A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements," vol. 32, Nucl Acids Res, 2004, pp. e38.
[16]	K. Jeong and S. Lee, "Estimating the total mouse DNA methylation according to the B1 repetitive elements," vol. 335, Biochem Biophys Res Commun, 2005, pp. 1211-1216.
[17]	J. Issa, V. Gharibyan, J. Cortes, J. Jelinek, G. Morris, S. Verstovsek, M. Talpaz, G. Garcia-Manero and H. Kantarjian, "Phase II study of low-dose decitabine in patients with chronic myelogenous leukemia resistant to imatinib mesylate," vol. 23, Clin Oncol, 2005, pp. 3948-3956.
[18]	J. Dupont, J. Tost, H. Jammes and I. Gut, "De novo quantitative bisulfite sequencing using the pyrosequencing technology," vol. 333, Anal Biochem, 2004, pp. 119-127.
[19]	E. Dejeux, V. Audard, C. Cavard, I. Gut, B. Terris, J. Tost, "Rapid identification of promoter hypermethylation in hepatocellular carcinoma by pyrosequencing of etiologically homogeneous sample pools," vol. 9, J Mol Diagn, 2007, pp. 510-520.
[20]	E. Morozkin, T. Babochkina, V. Vlassov and P. Laktionov, "The Effect of Protein Transport Inhibitors on the Production of Extracellular DNA," vol. 1137, Ann N Y Acad Sci, 2008, pp. 31-36.
[21]	L. Li, J. Choi, K. Lee, H. Sung, S. Park, I. Oze, K. Pan, W. You, Y. Chen, J. Fang, K. Matsuo, W. Kim, Y. Yuasa, D. Kang, "DNA Methylation in Peripheral Blood: A Potential Biomarker for Cancer Molecular Epidemiology," vol. 22, J Epidemiol, 2012, pp. 384-394.

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APA Style

Olga Bryzgunova, Pavel Laktionov, Тatyana Skvortsova, Anna Bondar, Evgeniy Morozkin, et al. (2013). Efficacy of Bisulfite Modification and DNA Recovery Using Commercial Kits from Samples of Genomic and Circulating DNA. Computational Biology and Bioinformatics, 1(6), 28-36. https://doi.org/10.11648/j.cbb.20130106.11

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ACS Style

Olga Bryzgunova; Pavel Laktionov; Тatyana Skvortsova; Anna Bondar; Evgeniy Morozkin, et al. Efficacy of Bisulfite Modification and DNA Recovery Using Commercial Kits from Samples of Genomic and Circulating DNA. Comput. Biol. Bioinform. 2013, 1(6), 28-36. doi: 10.11648/j.cbb.20130106.11

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AMA Style

Olga Bryzgunova, Pavel Laktionov, Тatyana Skvortsova, Anna Bondar, Evgeniy Morozkin, et al. Efficacy of Bisulfite Modification and DNA Recovery Using Commercial Kits from Samples of Genomic and Circulating DNA. Comput Biol Bioinform. 2013;1(6):28-36. doi: 10.11648/j.cbb.20130106.11

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@article{10.11648/j.cbb.20130106.11,
  author = {Olga Bryzgunova and Pavel Laktionov and Тatyana Skvortsova and Anna Bondar and Evgeniy Morozkin and Alena Lebedeva and Valentin Vlassov and Kurt Miller and Hans Krause},
  title = {Efficacy of Bisulfite Modification and DNA Recovery Using Commercial Kits from Samples of Genomic and Circulating DNA},
  journal = {Computational Biology and Bioinformatics},
  volume = {1},
  number = {6},
  pages = {28-36},
  doi = {10.11648/j.cbb.20130106.11},
  url = {https://doi.org/10.11648/j.cbb.20130106.11},
  eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.cbb.20130106.11},
  abstract = {This study had evaluated three commercially available bisulfite modification kits with regard to the procedural DNA loss and the efficacy of cytosine conversion to uracil. The efficacy of DNA conversion was estimated by pyrosequencing of eight CpG sites of unmethylated and fully enzymatically methylated templates of RARβ gene promoter region and cirDNAs of healthy donors and prostate cancer patients. The procedural DNA loss was calculated by methyl-independent qPCR for ubiquitously distributed L1RE1 retrotransposable elements. Our data demonstrates that all commercial kits displayed similar conversion efficacy of the unmethylated cytosines close to 99% independently of DNA concentration. However, when low DNA concentrations were used, the observed basic level of genomic DNA methylation increased to 11% depending on the position of CpG site. Qiagen and Chemicon kits recovered no more than 20% starting material at a high DNA input (500 ng/probe) and only 2.7–5.8% for low DNA input (10 ng/probe). EZ DNA Methylation-Gold Kit from Zymo Research provided the highest recovery regardless of the initial DNA input with average rates of no less than 86%. These results suggest that the EZ DNA Methylation-Gold Kit is the most appropriate tool for bisulfite modification of cirDNA when assaying DNA in low amounts.},
 year = {2013}
}

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TY  - JOUR
T1  - Efficacy of Bisulfite Modification and DNA Recovery Using Commercial Kits from Samples of Genomic and Circulating DNA
AU  - Olga Bryzgunova
AU  - Pavel Laktionov
AU  - Тatyana Skvortsova
AU  - Anna Bondar
AU  - Evgeniy Morozkin
AU  - Alena Lebedeva
AU  - Valentin Vlassov
AU  - Kurt Miller
AU  - Hans Krause
Y1  - 2013/12/20
PY  - 2013
N1  - https://doi.org/10.11648/j.cbb.20130106.11
DO  - 10.11648/j.cbb.20130106.11
T2  - Computational Biology and Bioinformatics
JF  - Computational Biology and Bioinformatics
JO  - Computational Biology and Bioinformatics
SP  - 28
EP  - 36
PB  - Science Publishing Group
SN  - 2330-8281
UR  - https://doi.org/10.11648/j.cbb.20130106.11
AB  - This study had evaluated three commercially available bisulfite modification kits with regard to the procedural DNA loss and the efficacy of cytosine conversion to uracil. The efficacy of DNA conversion was estimated by pyrosequencing of eight CpG sites of unmethylated and fully enzymatically methylated templates of RARβ gene promoter region and cirDNAs of healthy donors and prostate cancer patients. The procedural DNA loss was calculated by methyl-independent qPCR for ubiquitously distributed L1RE1 retrotransposable elements. Our data demonstrates that all commercial kits displayed similar conversion efficacy of the unmethylated cytosines close to 99% independently of DNA concentration. However, when low DNA concentrations were used, the observed basic level of genomic DNA methylation increased to 11% depending on the position of CpG site. Qiagen and Chemicon kits recovered no more than 20% starting material at a high DNA input (500 ng/probe) and only 2.7–5.8% for low DNA input (10 ng/probe). EZ DNA Methylation-Gold Kit from Zymo Research provided the highest recovery regardless of the initial DNA input with average rates of no less than 86%. These results suggest that the EZ DNA Methylation-Gold Kit is the most appropriate tool for bisulfite modification of cirDNA when assaying DNA in low amounts.
VL  - 1
IS  - 6
ER  -

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Author Information

Olga Bryzgunova

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, pr. Lavrentieva 8, Novosibirsk, Russia
Pavel Laktionov

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, pr. Lavrentieva 8, Novosibirsk, Russia
Тatyana Skvortsova

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, pr. Lavrentieva 8, Novosibirsk, Russia
Anna Bondar

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, pr. Lavrentieva 8, Novosibirsk, Russia
Evgeniy Morozkin

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, pr. Lavrentieva 8, Novosibirsk, Russia
Alena Lebedeva

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, pr. Lavrentieva 8, Novosibirsk, Russia
Valentin Vlassov

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, pr. Lavrentieva 8, Novosibirsk, Russia
Kurt Miller

Charite-Universitatsmedizin Berlin, Campus Mitte (CCM), Department of Urology, Chariteplatz, Berlin, Germany
Hans Krause

Charite-Universitatsmedizin Berlin, Campus Mitte (CCM), Department of Urology, Chariteplatz, Berlin, Germany

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APA Style

Olga Bryzgunova, Pavel Laktionov, Тatyana Skvortsova, Anna Bondar, Evgeniy Morozkin, et al. (2013). Efficacy of Bisulfite Modification and DNA Recovery Using Commercial Kits from Samples of Genomic and Circulating DNA. Computational Biology and Bioinformatics, 1(6), 28-36. https://doi.org/10.11648/j.cbb.20130106.11

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ACS Style

Olga Bryzgunova; Pavel Laktionov; Тatyana Skvortsova; Anna Bondar; Evgeniy Morozkin, et al. Efficacy of Bisulfite Modification and DNA Recovery Using Commercial Kits from Samples of Genomic and Circulating DNA. Comput. Biol. Bioinform. 2013, 1(6), 28-36. doi: 10.11648/j.cbb.20130106.11

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AMA Style

Olga Bryzgunova, Pavel Laktionov, Тatyana Skvortsova, Anna Bondar, Evgeniy Morozkin, et al. Efficacy of Bisulfite Modification and DNA Recovery Using Commercial Kits from Samples of Genomic and Circulating DNA. Comput Biol Bioinform. 2013;1(6):28-36. doi: 10.11648/j.cbb.20130106.11

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@article{10.11648/j.cbb.20130106.11,
  author = {Olga Bryzgunova and Pavel Laktionov and Тatyana Skvortsova and Anna Bondar and Evgeniy Morozkin and Alena Lebedeva and Valentin Vlassov and Kurt Miller and Hans Krause},
  title = {Efficacy of Bisulfite Modification and DNA Recovery Using Commercial Kits from Samples of Genomic and Circulating DNA},
  journal = {Computational Biology and Bioinformatics},
  volume = {1},
  number = {6},
  pages = {28-36},
  doi = {10.11648/j.cbb.20130106.11},
  url = {https://doi.org/10.11648/j.cbb.20130106.11},
  eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.cbb.20130106.11},
  abstract = {This study had evaluated three commercially available bisulfite modification kits with regard to the procedural DNA loss and the efficacy of cytosine conversion to uracil. The efficacy of DNA conversion was estimated by pyrosequencing of eight CpG sites of unmethylated and fully enzymatically methylated templates of RARβ gene promoter region and cirDNAs of healthy donors and prostate cancer patients. The procedural DNA loss was calculated by methyl-independent qPCR for ubiquitously distributed L1RE1 retrotransposable elements. Our data demonstrates that all commercial kits displayed similar conversion efficacy of the unmethylated cytosines close to 99% independently of DNA concentration. However, when low DNA concentrations were used, the observed basic level of genomic DNA methylation increased to 11% depending on the position of CpG site. Qiagen and Chemicon kits recovered no more than 20% starting material at a high DNA input (500 ng/probe) and only 2.7–5.8% for low DNA input (10 ng/probe). EZ DNA Methylation-Gold Kit from Zymo Research provided the highest recovery regardless of the initial DNA input with average rates of no less than 86%. These results suggest that the EZ DNA Methylation-Gold Kit is the most appropriate tool for bisulfite modification of cirDNA when assaying DNA in low amounts.},
 year = {2013}
}

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TY  - JOUR
T1  - Efficacy of Bisulfite Modification and DNA Recovery Using Commercial Kits from Samples of Genomic and Circulating DNA
AU  - Olga Bryzgunova
AU  - Pavel Laktionov
AU  - Тatyana Skvortsova
AU  - Anna Bondar
AU  - Evgeniy Morozkin
AU  - Alena Lebedeva
AU  - Valentin Vlassov
AU  - Kurt Miller
AU  - Hans Krause
Y1  - 2013/12/20
PY  - 2013
N1  - https://doi.org/10.11648/j.cbb.20130106.11
DO  - 10.11648/j.cbb.20130106.11
T2  - Computational Biology and Bioinformatics
JF  - Computational Biology and Bioinformatics
JO  - Computational Biology and Bioinformatics
SP  - 28
EP  - 36
PB  - Science Publishing Group
SN  - 2330-8281
UR  - https://doi.org/10.11648/j.cbb.20130106.11
AB  - This study had evaluated three commercially available bisulfite modification kits with regard to the procedural DNA loss and the efficacy of cytosine conversion to uracil. The efficacy of DNA conversion was estimated by pyrosequencing of eight CpG sites of unmethylated and fully enzymatically methylated templates of RARβ gene promoter region and cirDNAs of healthy donors and prostate cancer patients. The procedural DNA loss was calculated by methyl-independent qPCR for ubiquitously distributed L1RE1 retrotransposable elements. Our data demonstrates that all commercial kits displayed similar conversion efficacy of the unmethylated cytosines close to 99% independently of DNA concentration. However, when low DNA concentrations were used, the observed basic level of genomic DNA methylation increased to 11% depending on the position of CpG site. Qiagen and Chemicon kits recovered no more than 20% starting material at a high DNA input (500 ng/probe) and only 2.7–5.8% for low DNA input (10 ng/probe). EZ DNA Methylation-Gold Kit from Zymo Research provided the highest recovery regardless of the initial DNA input with average rates of no less than 86%. These results suggest that the EZ DNA Methylation-Gold Kit is the most appropriate tool for bisulfite modification of cirDNA when assaying DNA in low amounts.
VL  - 1
IS  - 6
ER  -

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