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Isolation of human genomic DNA for genetic analysis from premature neonates: A comparison between newborn dried blood spots, whole blood and umbilical cord tissue

Váradi, Anikó; Molnár, Elek; Rajatileka, Shavanthi; Luyt, Karen; El-Bokle, Manal; Williams, Maggie; Molnar, E.; Kemp, Helena; Varadi, Aniko

Authors

Anikó Váradi

Elek Molnár

Shavanthi Rajatileka shavanthi.rajatileka@uwe.ac.uk

Karen Luyt

Manal El-Bokle

Maggie Williams

E. Molnar

Helena Kemp

Aniko Varadi Aniko.Varadi@uwe.ac.uk
Professor in Biomedical Research



Abstract

Background: Genotyping requires biological sample collection that must be reliable, convenient and acceptable for patients and clinicians. Finding the most optimal procedure of sample collection for premature neonates who have a very limited blood volume is a particular challenge. The aim of the current study was to evaluate the use of umbilical cord (UC) tissue and newborn dried blood spot (DBS)-extracted genomic DNA (gDNA) as an alternative to venous blood-derived gDNA from premature neonates for molecular genetic analysis. All samples were obtained from premature newborn infants between 24-32weeks of gestation. Paired blood and UC samples were collected from 31 study participants. gDNA was extracted from ethylenediaminetetraacetic acid (EDTA) anticoagulant-treated blood samples (~500μl) and newborn DBSs (n = 723) using QIAamp DNA Micro kit (Qiagen Ltd., Crawley, UK); and from UC using Qiagen DNAeasy Blood and Tissue kit (Qiagen Ltd., Crawley, UK). gDNA was quantified and purity confirmed by measuring the A260:A280 ratio. PCR amplification and pyrosequencing was carried out to determine suitability of the gDNA for molecular genetic analysis. Minor allele frequency of two unrelated single nucleotide polymorphisms (SNPs) was calculated using the entire cohort.Results: Both whole blood samples and UC tissue provided good quality and yield of gDNA, which was considerably less from newborn DBS. The gDNA purity was also reduced after 3years of storage of the newborn DBS. PCR amplification of three unrelated genes resulted in clear products in all whole blood and UC samples and 86%-100% of newborn DBS. Genotyping using pyrosequencing showed 100% concordance in the paired UC and whole blood samples. Minor allele frequencies of the two SNPs indicated that no maternal gDNA contamination occurred in the genotyping of the UC samples.Conclusions: gDNAs from all three sources are suitable for standard PCR and pyrosequencing assays. Given that UC provide good quality and quantity gDNA with 100% concordance in the genetic analysis with whole blood, it can replace blood sampling from premature infants. This is likely to reduce the stress and potential side effects associated with invasive sample collection and thus, greatly facilitate participant recruitment for genetic studies. © 2013 Rajatileka et al.; licensee BioMed Central Ltd.

Citation

Váradi, A., Molnár, E., Rajatileka, S., Luyt, K., El-Bokle, M., Williams, M., …Varadi, A. (2013). Isolation of human genomic DNA for genetic analysis from premature neonates: A comparison between newborn dried blood spots, whole blood and umbilical cord tissue. BMC Genetics, 14(105), https://doi.org/10.1186/1471-2156-14-105

Journal Article Type Article
Publication Date Oct 29, 2013
Journal BMC Genetics
Electronic ISSN 1471-2156
Publisher BMC
Peer Reviewed Not Peer Reviewed
Volume 14
Issue 105
DOI https://doi.org/10.1186/1471-2156-14-105
Keywords isolation, DNA, premature neonates, newborn, blood, umbilical cord tissue
Public URL https://uwe-repository.worktribe.com/output/939931
Publisher URL http://dx.doi.org/10.1186/1471-2156-14-105