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

• Published in: International journal of nanomedicine 2014-01, Vol.9 (Issue 1), p.1629-1640
• Main Authors: Vanden Bon, Natalie, van Grinsven, Bart, Murib, Mohammed Sharif, Yeap, Weng Siang, Haenen, Ken, De Ceuninck, Ward, Wagner, Patrick, Ameloot, Marcel, Vermeeren, Veronique, Michiels, Luc
• Format: Article
• Language: English
• Subjects: Base Sequence; Diagnosis; DNA Mutational Analysis - methods; Genetic aspects; Genetic Markers - genetics; Genetic Predisposition to Disease - genetics; heat-transfer resistance; Humans; melting temperature; Molecular Sequence Data; mutation detection; nanocrystalline diamond; Nanoparticles; Original Research; persistence length; Phenylalanine; Phenylalanine Hydroxylase - genetics; Phenylketonuria; Phenylketonurias - diagnosis; Phenylketonurias - genetics; Physiological aspects; Polymorphism, Single Nucleotide - genetics; Reproducibility of Results; Sensitivity and Specificity; Single nucleotide polymorphisms; Thermography - methods; Transition Temperature
• ISSN: 1178-2013; 1176-9114; 1178-2013
• DOI: 10.2147/IJN.S58692

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 (R(th)) 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 R(th) change could be observed during the thermal denaturation of these DNA fragments after hybridization to their complementary counterpart. A clear R(th) shift during the denaturation of exon 5, exon 9, and exon 12 dsDNA was observed, corresponding to lengths of up to 123 bp. Finally, R(th) 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.