Multiplexed detection of snps using electrochemical melting curve analysis
- Autores: Nassif Chahin
- Directores de la Tesis: Ciara K. O'Sullivan (dir. tes.), Mayreli Ortiz Rodríguez (codir. tes.)
- Lectura: En la Universitat Rovira i Virgili ( España ) en 2021
- Idioma: español
- Tribunal Calificador de la Tesis: Jan Vanfleteren (presid.), Miriam Jauset Rubio (secret.), Luis Antonio Tortajada-Genaro (voc.)
- Programa de doctorado: Programa de Doctorado en Nanociencia, Materiales e Ingeniería Química por la Universidad Rovira i Virgili
- Materias:
- Enlaces
- Tesis en acceso abierto en: TDX
- Resumen
SNP detection technology is used to scan the new polymorphism in target sequences. SNP detection technology is developed to become efficient, automated process and less expensive. Usually, SNP scanning is based on either direct sequencing of the DNA or by (dHPLC) denaturing high performance liquid chromatograph. Although, many of the efficient genotyping methods are available nowadays, a new approach is needed to reduce the cost and increase the speed of SNP scanning.
The overall objective of this PhD thesis is to develop a low cost geno-sensor platform for multiplexed SNPs detection using electrochemical melting curve analysis, to simplify the amplification and the hybridisation process steps and applying electrochemical detection.
This thesis is divided into 5 chapters: Chapter 1 gives an introduction about single nucleotide polymorphisms (SNPs) and the methods used for SNP detection. It also gives some explanation about DNA sensors, focusing on the method of electrochemical melting curve analysis as a basis for the thesis work.
Chapter 2 gives an overview of the demonstration of an electrochemical primer extension based on polyoxometalate electroactive labels for multiplexed detection of single nucleotide polymorphisms, by the exploitation of ddNTPs labelled with Keggin and Dawson POMs for solid-phase primer extension and the electrochemical detection of SNPs.The work demonstrated successfully the incorporation of the POM-labelled ddNTPs enzymatically to the ssDNA tethered to the gold surface by using the quite inexpensive Therminator DNA polymerase. In this work, an array based primer extension (APEX) variant reaction called solid phase éPEX was used.The éPEX reaction uses an electroactive label instead of fluorescent detection offers low cost and easy detection platforms as an alternative to colorietry, fluorescence, or radioactive detection.
In this reaction, an isothermal reaction that takes advantage of immobilised single stranded DNA probes designed to hybridise to a single stranded PCR target one base downstream from the SNP site being interrogated. Following hybridisation, labelled ddNTPs, modified nucleotides that lack the 2′ and 3′-hydroxyl groups, are added. Using either ligation or elongation with a ligase or polymerase enzyme, the immobilised probe is extended by a single labelled ddNTP, complementary to the SNP being addressed. Following incorporation of the electroactive labelled ddNTP, no further phosphodiester bonds can be formed due to the lack of the 2′/3′-OH group, and therefore no elongation can take place after the SNP location. The éPEX reaction with ddNTPs labelled with different electroactive markers: ferrocene, anthraquinone phenothiazine and methylene blue was performed successfully, and the possibility of using POMs to label ddNTPs for use in éPEX were explored.
In Chapter 3, We have explored low cost and easy platform fabricated device with integrated homemade Peltier to control the temperature, microfluidic, and electrochemical measurements for multiplex SNPs detections using the melting curve analysis method. The homemade Peltier consists of two Aluminum blocks that can be heated in a controlled way using Arduino UNO board. It allows us to select from different resolutions: 0.2, 0.5 and 1 °C/step.The heating system characterized and the functionality of heating was validated. Primarily, four different amplicons of cardiomyopathy target model (each one has SNP at a different position) were hybridised to a capture probe tethered on a gold surface of an electrodes array, followed by generating the melting profiles and determining the melting point for each target successfully. We discovered that the more accurate approach for the electrochemical discrimination of the SNP from the wild type sequence is to design the probe to capture the SNP in the middle of the sequence. The Tm calculated under a range of experiments showed the capability of the setup to differentiate between different samples based on their melting point. The ongoing work is focusing on testing real genomic DNA samples like osteoporosis, TB MDR, as well as to replace the PCR amplification by the RPA (Recombinase Polymerase Amplification) reducing the amplification time and eliminating the need for using a thermocycler.
In Chapter 4, we investigated the ability of our device for multiplexed SNP detection as mentioned in previous work, but for detecting osteoporosis as a model system target, trying to discriminate between different SNPs from the wild type sequence. In this work, the ssDNA target of osteoporosis amplified and labelled with ferrocene using the Asy-RPA kit. Later, the target hybridised to four probes each one has a different SNP in the middle pr-immobilised on gold electrodes arrays and finally the melting profiles generated and the Tm was calculated successfully. Based on the amplification optimization results for generating a single strand labelled-Fc using Asy-RPA; the best amplification time found was 15 minutes and the best concentration ratio between forward and reverse primers was (5 to 1) as respectable.
The second part of this work was to test a real blood sample. As previously, osteoporosis target was hybridised to 4 different capture probes tethered on gold electrodes was done, followed by generating the melting profiles.The calculated melting points from the blood sample showed one high Tm besides three lower and matched Tm. The higher Tm is reflecting the full complementary between the target and the capture probe and thus referring to the real SNP on the target in blood sample.
Concluding remarks and an outlook for future work are described in Chapter 5.
Overall, my Ph.D. thesis focused on developing an electrochemical detections methods that use low-cost platforms as biosensors to be used in disease diagnosis, by exploration different possibilities of multiplexed SNP detection, using the primer extension method and electrochemical melting curve analysis and, for the first time, we could use isothermal amplification in real samples to detect SNPs that are related to osteoporosis by using a homemade Peltier device offering further simplification and inexpensive detection process.
