Heat-transfer-based detection of SNPs in the PAH gene of PKU patients

Natalie Vanden Bon, Bart van Grinsven, Mohammed Sharif Murib, Weng Siang Yeap, Ken Haenen, Ward De Ceuninck, Patrick Wagner, Marcel Ameloot, Veronique Vermeeren, Luc Michiels*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

Conventional neonatal diagnosis of phenylketonuria is based on the presence of abnormal levels of phenylalanine in the blood. However, for carrier detection and prenatal diagnosis, direct detection of disease-correlated mutations is needed. To speed up and simplify mutation screening in genes, new technologies are developed. In this study, a heat-transfer method is evaluated as a mutation-detection technology in entire exons of the phenylalanine hydroxylase (PAH) gene. This method is based on the change in heat-transfer resistance (Rth) upon thermal denaturation of dsDNA (double-stranded DNA) on nanocrystalline diamond. First, ssDNA (single-stranded DNA) fragments that span the size range of the PAH exons were successfully immobilized on nanocrystalline diamond. Next, it was studied whether an Rth change could be observed during the thermal denaturation of these DNA fragments after hybridization to their complementary counterpart. A clear Rth shift during the denaturation of exon 5, exon 9, and exon 12 dsDNA was observed, corresponding to lengths of up to 123 bp. Finally, Rth was shown to detect prevalent single-nucleotide polymorphisms, c.473G > A (R158Q), c.932T > C (p.L311P), and c.1222C > T (R408W), correlated with phenylketonuria, displaying an effect related to the different melting temperatures of homoduplexes and heteroduplexes.

Original languageEnglish
Pages (from-to)1629-1640
Number of pages12
JournalInternational Journal of Nanomedicine
Volume9
DOIs
Publication statusPublished - 2014
Externally publishedYes

Keywords

  • mutation detection
  • heat-transfer resistance
  • melting temperature
  • nanocrystalline diamond
  • persistence length
  • PHENYLALANINE-HYDROXYLASE GENE
  • GRADIENT GEL-ELECTROPHORESIS
  • POLYMERASE-CHAIN-REACTION
  • LABEL-FREE
  • CLASSICAL PHENYLKETONURIA
  • PRENATAL-DIAGNOSIS
  • REAL-TIME
  • DNA
  • MUTATIONS
  • SURFACE

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