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Optimized DNA extraction from neonatal dried blood spots: application in methylome profiling

Citation

Ghantous, A and Saffery, R and Cros, M-P and Ponsonby, A-L and Hirschfeld, S and Kasten, C and Dwyer, T and Herceg, Z and Hernandez-Vargas, H, Optimized DNA extraction from neonatal dried blood spots: application in methylome profiling, BMC Biotechnology, 14 Article 60. ISSN 1472-6750 (2014) [Refereed Article]


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Licensed under Creative Commons Attribution 4.0 International (CC BY 4.0) http://creativecommons.org/licenses/by/4.0/

DOI: doi:10.1186/1472-6750-14-60

Abstract

Background: Neonatal dried blood spots (DBS) represent an inexpensive method for long-term biobanking worldwide and are considered gold mines for research for several human diseases, including those of metabolic, infectious, genetic and epigenetic origin. However, the utility of DBS is restricted by the limited amount and quality of extractable biomolecules (including DNA), especially for genome wide profiling. Degradation of DNA in DBS often occurs during storage and extraction. Moreover, amplifying small quantities of DNA often leads to a bias in subsequent data, particularly in methylome profiles. Thus it is important to develop methodologies that maximize both the yield and quality of DNA from DBS for downstream analyses.

Results: Using combinations of in-house-derived and modified commercial extraction kits, we developed a robust and efficient protocol, compatible with methylome studies, many of which require stringent bisulfite conversion steps. Several parameters were tested in a step-wise manner, including blood extraction, cell lysis, protein digestion, and DNA precipitation, purification and elution. DNA quality was assessed based on spectrophotometric measurements, DNA detectability by PCR, and DNA integrity by gel electrophoresis and bioanalyzer analyses. Genome scale Infinium HumanMethylation450 and locus-specific pyrosequencing data generated using the refined DBS extraction protocol were of high quality, reproducible and consistent.

Conclusions: This study may prove useful to meet the increased demand for research on prenatal, particularly epigenetic, origins of human diseases and for newborn screening programs, all of which are often based on DNA extracted from DBS.

Item Details

Item Type:Refereed Article
Keywords:blood spot, DNA extraction, epigenetics, methylome, HM450, pyrosequencing, whole bisulfitome amplification, QIAamp, GenSolve, NucleoSpin
Research Division:Medical and Health Sciences
Research Group:Paediatrics and Reproductive Medicine
Research Field:Paediatrics
Objective Division:Health
Objective Group:Clinical Health (Organs, Diseases and Abnormal Conditions)
Objective Field:Clinical Health (Organs, Diseases and Abnormal Conditions) not elsewhere classified
UTAS Author:Ponsonby, A-L (Professor Anne Ponsonby)
UTAS Author:Dwyer, T (Professor Terry Dwyer)
ID Code:100391
Year Published:2014
Web of Science® Times Cited:18
Deposited By:Menzies Institute for Medical Research
Deposited On:2015-05-14
Last Modified:2017-11-06
Downloads:239 View Download Statistics

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