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

Autor:	Caroline E. Ford, Robert W. Rapkins, Kate Gunther, Claire Henry, Kristina Warton, Nicole Laurencia Yuwono, Bonnita Werner
Rok vydání:	2019
Předmět:	0301 basic medicine Physiology DNA electrophoresis Artificial Gene Amplification and Extension DNA fragmentation Polymerase Chain Reaction Biochemistry chemistry.chemical_compound 0302 clinical medicine Medicine and Health Sciences Gel Electrophoresis Multidisciplinary DNA methylation Middle Aged Chromatin Body Fluids Bisulfite Nucleic acids Separation Processes Blood 030220 oncology & carcinogenesis Agarose gel electrophoresis Medicine Female Epigenetics Anatomy DNA modification Cell-Free Nucleic Acids Chromatin modification Research Article Chromosome biology Adult Cell biology Science Agarose Gel Electrophoresis Research and Analysis Methods Blood Plasma 03 medical and health sciences Young Adult Electrophoretic Techniques Genetics Humans Sulfites Fragmentation (cell biology) Molecular Biology Techniques Molecular Biology Genome Human Biology and Life Sciences DNA Elution DNA extraction Molecular biology Circulating Cell-Free DNA Molecular Weight 030104 developmental biology chemistry Gene expression
Zdroj:	PLoS ONE PLoS ONE, Vol 14, Iss 10, p e0224338 (2019)
ISSN:	1932-6203
Popis:	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.
Databáze:	OpenAIRE
Externí odkaz:	https://explore.openaire.eu/search/publication?articleId=doi_dedup___::b9fb73400109b3befdc639e6d668ec14 https://pubmed.ncbi.nlm.nih.gov/31652288 Zobrazit plný text záznamu Plný text ve formátu PDF Plný text ve formátu HTML
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