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: 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


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Článek vyšel v časopise

PLOS One


2019 Číslo 10