Multiplexed detection of SNPs using electrochemical melting curve analysis

Identificador:  TDX:3403

Handle: https://hdl.handle.net/20.500.11797/TDX3403

Autores:  Chahin, Nassif

Resumen:
The overall objective of this PhD thesis is to develop low cost geno-sensor platforms for multiplexed detection of single nucleotide polymorphism. The determination of single nucleotide polymorphisms (SNP) is of great importance in life sciences, having application in personalised medicine, patient stratification, forensics, as well as providing information regarding predisposition to disease. Nowadays electrochemistry-based methods are revealed as attractive alternatives to the most commonly used techniques for determining DNA melting point, because of their high sensitivity, simplicity, cost-effective and compatibility with microfabrication. The first platform was based on an electrochemical primer extension reaction, where the redox active Keggin and Dawson polyoxymetalates were used to modify dideoxynucleotides through amide bond formation. The second platform is based on the detection of single nucleotide polymorphisms using electrochemical melting curve analysis. The approach is based on solid phase melting curve analysis that exploits probes immobilised on gold electrodes. We developed a home-made electrochemical melting curve analysis device, able to detect a DNA target that carries a SNP related to cardiomyopathy from others with only one base in difference. Finally, this device was applied to SNPs detection in real blood samples. Osteoporosis related SNP sequence target was extracted from a finger prick and amplified using asymmetric Recombinase Polymerase Amplification. Also, the amplification conditions were optimised for best product yield, and the SNP from a blood sample was defined.