טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentPatkin Michelle
SubjectAssay Design of Single Molecule Barcoding Method for
Ultra-Fast Pathogen-Typing Using Nanopores
DepartmentDepartment of Biomedical Engineering
Supervisor Professor Amit Meller


Abstract

According to a World Health Organization (WHO) report, the number of patients severely affected by antimicrobial resistant (AR) infections has steadily grown in past years and reached staggering levels in industrialized countries. 1 Since this trend has been attributed to the common and improper use of antibiotics in clinical care, our approach is the development of an early and accurate method for the detection of pathogens. Genetic features such as single nucleotide variations (SNVs), insertions and deletions can be used for genotyping. We develop a direct, solid-state nanopore (ssNP) and ligation-based, genotyping technique that circumvents DNA amplification and sequencing, and uniquely transforms genetic variations to molecular-encoded barcodes and allows for unique classification of the bacterial genome. The signal acquired by the nanopore is interpreted as molecularly-encoded barcodes, which allow for unique classification of the source genome with high diagnostic accuracy and high level of multiplexing ability. Our DNA barcode formation method utilizes a sequence-specific ligation reaction using a set of probes designed to target specific sequences in the genome. Following a biotin bead separation step, the ssDNA ligation products are then hybridized to oligonucleotides to the characteristic barcode sequence and are analyzed using an electro-optical ssNP sensor. The pathogens are then determined based on the difference in dwell-time which results from the different number of beacons on each barcode molecule. This nanopore/ligation method provides an ultra-fast, high specificity and high-sensitivity detection and classification of a large variety of pathogens in the same mixture.  

The focus of this work is the design of the biochemical assay for the development of a method to differentiate between pathogen strains based on mutation patterns. Shown is the high specificity of the ligation reaction and the ability to target a wide variety of SNVs, the ability to achieve a high level of purification, the barcode design and verification, and nanopore optimization using test DNAs. The barcodes readout themselves are currently underway and is part of a PhD project.