#PAGE_PARAMS# #ADS_HEAD_SCRIPTS# #MICRODATA#

Circulating cell-free DNA from plasma undergoes less fragmentation during bisulfite treatment than genomic DNA due to low molecular weight


Autoři: Bonnita Werner aff001;  Nicole Laurencia Yuwono aff001;  Claire Henry aff001;  Kate Gunther aff001;  Robert William Rapkins aff001;  Caroline Elizabeth Ford aff001;  Kristina Warton aff001
Působiště autorů: School of Women’s and Children’s Health, University of New South Wales, Kensington, Sydney, Australia aff001;  Genomics and Epigenetics Division, Garvan Institute of Medical Research, Darlinghurst, Sydney, Australia aff002
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pone.0224338

Souhrn

Background

Methylation patterns in circulating cell-free DNA are potential biomarkers for cancer and other pathologies. Currently, bisulfite treatment underpins most DNA methylation analysis methods, however, it is known to fragment DNA. Circulating DNA is already short, and further fragmentation during bisulfite treatment is of concern, as it would potentially reduce the sensitivity of downstream assays.

Methods

We used high molecular weight genomic DNA to compare fragmentation and recovery following bisulfite treatment with 2 commercially available kits (Qiagen). The bisulfite treated DNA was visualised on an agarose gel and quantified by qPCR. We also bisulfite treated, visualised and quantitated circulating DNA from plasma.

Results

There was no difference in DNA fragmentation between the two kits tested, however, the Epitect Fast kit gave better recovery than the standard Epitect kit, with the same conversion efficiency. We also found that bisulfite treated circulating DNA migrates as distinct bands on agarose gels, suggesting that, in contrast to genomic DNA, it remains largely intact following treatment. Bisulfite treatment of 129 and 234 base PCR products confirmed that this was due to the short length of the circulating DNA fragments. Compared to double stranded DNA, bisulfite treated single stranded DNA gives a very weak signal on gel electrophoresis.

Conclusions

DNA fragmentation during bisulfite treatment does not contribute to loss of sensitivity in methylation analysis of circulating DNA. The absence of DNA fragments below approximately 170 bases from agarose gel images of purified circulating DNA raises the possibility that these fragments are single stranded following the DNA extraction step.

Klíčová slova:

Agarose gel electrophoresis – Blood plasma – DNA electrophoresis – DNA methylation – Elution – Gel electrophoresis – Polymerase chain reaction – DNA fragmentation


Zdroje

1. Warton K, Samimi G. Methylation of cell-free circulating DNA in the diagnosis of cancer. Front Mol Biosci. 2015;2:13. doi: 10.3389/fmolb.2015.00013 eCollection 2015. 25988180

2. Warton K, Mahon KL, Samimi G. Methylated circulating tumor DNA in blood: power in cancer prognosis and response. Endocr Relat Cancer. 2016;23(3):R157–71. doi: 10.1530/ERC-15-0369 Epub 2016 Jan 13. 26764421

3. Lehmann-Werman R, Neiman D, Zemmour H, Moss J, Magenheim J, Vaknin-Dembinsky A, et al. Identification of tissue-specific cell death using methylation patterns of circulating DNA. Proc Natl Acad Sci U S A. 2016;113(13):E1826–34. doi: 10.1073/pnas.1519286113 Epub 2016 Mar 14. 26976580

4. Moss J, Magenheim J, Neiman D, Zemmour H, Loyfer N, Korach A, et al. Comprehensive human cell-type methylation atlas reveals origins of circulating cell-free DNA in health and disease. Nat Commun. 2018;9(1):5068. doi: 10.1038/s41467-018-07466-6 30498206

5. Frommer M, McDonald LE, Millar DS, Collis CM, Watt F, Grigg GW, et al. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci U S A. 1992;89(5):1827–31. doi: 10.1073/pnas.89.5.1827 1542678

6. Clark SJ, Statham A, Stirzaker C, Molloy PL, Frommer M. DNA methylation: bisulphite modification and analysis. Nat Protoc. 2006;1(5):2353–64. doi: 10.1038/nprot.2006.324 17406479

7. Jahr S, Hentze H, Englisch S, Hardt D, Fackelmayer FO, Hesch RD, et al. DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells. Cancer Res. 2001;61(4):1659–65. 11245480

8. Devonshire AS, Whale AS, Gutteridge A, Jones G, Cowen S, Foy CA, et al. Towards standardisation of cell-free DNA measurement in plasma: controls for extraction efficiency, fragment size bias and quantification. Anal Bioanal Chem. 2014;406(26):6499–512. doi: 10.1007/s00216-014-7835-3 Epub 2014 May 24. 24853859

9. Warton K, Graham LJ, Yuwono N, Samimi G. Comparison of 4 commercial kits for the extraction of circulating DNA from plasma. Cancer Genet. 2018;228–229:143–150. doi: 10.1016/j.cancergen.2018.02.004 Epub Mar 6. 29567030

10. Andersen RF, Spindler KL, Brandslund I, Jakobsen A, Pallisgaard N. Improved sensitivity of circulating tumor DNA measurement using short PCR amplicons. Clin Chim Acta. 2015;439:97–101. doi: 10.1016/j.cca.2014.10.011 Epub Oct 20. 25446878

11. Warton K, Lin V, Navin T, Armstrong NJ, Kaplan W, Ying K, et al. Methylation-capture and Next-Generation Sequencing of free circulating DNA from human plasma. BMC Genomics. 2014;15:476. doi: 10.1186/1471-2164-15-476 24929644

12. Holmes EE, Jung M, Meller S, Leisse A, Sailer V, Zech J, et al. Performance evaluation of kits for bisulfite-conversion of DNA from tissues, cell lines, FFPE tissues, aspirates, lavages, effusions, plasma, serum, and urine. PLoS One. 2014;9(4):e93933. doi: 10.1371/journal.pone.0093933 eCollection 2014. 24699908

13. Olova N, Krueger F, Andrews S, Oxley D, Berrens RV, Branco MR, et al. Comparison of whole-genome bisulfite sequencing library preparation strategies identifies sources of biases affecting DNA methylation data. Genome Biol. 2018;19(1):33. doi: 10.1186/s13059-018-1408-2 29544553

14. Snyder MW, Kircher M, Hill AJ, Daza RM, Shendure J. Cell-free DNA Comprises an In Vivo Nucleosome Footprint that Informs Its Tissues-Of-Origin. Cell. 2016;164(1–2):57–68. doi: 10.1016/j.cell.2015.11.050 26771485


Článek vyšel v časopise

PLOS One


2019 Číslo 10
Nejčtenější tento týden
Nejčtenější v tomto čísle
Kurzy

Zvyšte si kvalifikaci online z pohodlí domova

KOST
Koncepce osteologické péče pro gynekology a praktické lékaře
nový kurz
Autoři: MUDr. František Šenk

Sekvenční léčba schizofrenie
Autoři: MUDr. Jana Hořínková

Hypertenze a hypercholesterolémie – synergický efekt léčby
Autoři: prof. MUDr. Hana Rosolová, DrSc.

Svět praktické medicíny 5/2023 (znalostní test z časopisu)

Imunopatologie? … a co my s tím???
Autoři: doc. MUDr. Helena Lahoda Brodská, Ph.D.

Všechny kurzy
Kurzy Podcasty Doporučená témata Časopisy
Přihlášení
Zapomenuté heslo

Zadejte e-mailovou adresu, se kterou jste vytvářel(a) účet, budou Vám na ni zaslány informace k nastavení nového hesla.

Přihlášení

Nemáte účet?  Registrujte se

#ADS_BOTTOM_SCRIPTS#