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

Shavanthi Rajatileka, Karen Luyt, Manal El-Bokle, Maggie Williams, Helena Kemp, Elek Molnár, Anikó Váradi

Research output: Contribution to journalArticle (Academic Journal)

13 Citations (Scopus)
306 Downloads (Pure)

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 24-32 weeks of gestation. Paired blood and UC samples were collected from 31 study participants. gDNA was extracted from ethylenediaminetetraacetic acid (EDTA) anticoagulant-treated blood samples (~500 μl) 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.ResultsBoth 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 3 years 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.ConclusionsgDNAs 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.

Original languageEnglish
Article number105
Number of pages9
JournalBMC Genetics
Volume14
DOIs
Publication statusPublished - 29 Oct 2013

Keywords

  • Alleles
  • Cohort Studies
  • DNA
  • Dried Blood Spot Testing
  • Gene Frequency
  • Genetic Techniques
  • Genetic Testing
  • Genome, Human
  • Genotype
  • Gestational Age
  • Humans
  • Infant, Newborn
  • Polymerase Chain Reaction
  • Single nucleotide polymorphism
  • Premature newborns
  • Sequence Analysis, DNA
  • Time Factors
  • Umbilical Cord
  • Newborn dried blood spots
  • Genomic DNA
  • Pyrosequencing
  • Sample storage

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