title: Isolation of human genomic DNA for genetic analysis from premature neonates: a comparison between newborn dried blood spots, whole blood and umbilical cord tissue.
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.
  
  
  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 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.
  
  
  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.
authors.forename: Shavanthi
authors.initials: S
authors.lastname: Rajatileka
authors.forename: Karen
authors.initials: K
authors.lastname: Luyt
authors.forename: Manal
authors.initials: M
authors.lastname: El-Bokle
authors.forename: Maggie
authors.initials: M
authors.lastname: Williams
authors.forename: Helena
authors.initials: H
authors.lastname: Kemp
authors.forename: Elek
authors.initials: E
authors.lastname: Molnár
authors.affiliation: Centre for Research in Biosciences, Department of Biological, Biomedical and Analytical Sciences, Faculty of Health and Applied Sciences, University of the West of England, Bristol BS16 1QY, UK. Aniko.Varadi@uwe.ac.uk.
authors.forename: Anikó
authors.initials: A
authors.lastname: Váradi
mesh_terms: D000483:Alleles; D015331:Cohort Studies; D004247:DNA / Q000032:analysis* / Q000097:blood / Q000302:isolation & purification; D059788:Dried Blood Spot Testing / Q000592:standards; D005787:Gene Frequency; D005821:Genetic Techniques / Q000592:standards*; D005820:Genetic Testing / Q000379:methods*; D015894:Genome, Human; D005838:Genotype; D005865:Gestational Age; D006801:Humans; D007231:Infant, Newborn; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D047928:Premature Birth; D017422:Sequence Analysis, DNA; D013997:Time Factors; D014470:Umbilical Cord / Q000378:metabolism*
chemical_list: D004247:DNA