Preliminary Studies Of Single-molecule DNA Sequencing Technologies Based On Nanopores And Nanoarray

Nanopore sequencing is among the most convincing technologies to achieve thegoals of the―$1,000genome.‖Two major types of nanopores, protein and silicon-basedsolid-state pores, have been extensively investgated. However, protein pores areshort-lived and the solid-state nanopores are deeper than the distance between adjacentbases, resulting in incapability to discriminate individual bases along the single-strandedDNA (ssDNA) molecules. In this dissertation, we report λ-DNA translocations throughgraphene nanopores. Silicon nitride substrates were first fabricated using focused ionbeam (FIB), individual graphene membranes were suspended onto the substrates, andnanopores with a diameter less than10nm are sculpted by focused electron beam (FEB)from a transmission electron microscope (TEM). The signals of DNA translocationthrough nanopores had been recorded, leading a step toward single-molecule DNAsequencing using graphene nanopores.We have previously designed an single-molecule DNA sequencing method usingnanopores made of single-walled carbon nanotube (SWNT) assisted with exonucleases.The four kinds of enzymatically released2-deoxyribonucleoside5‘-monophosphates, ordNMPs—dAMP, CMP, dGMP, and dTMP—which are negatively charged in a solutionunder neutral to higher pH, will sequentially pass through the nanopore while migratingfrom the cathode to the anode, when they are driven by an applied electric field. Differentkinds of dNMPs will leave a distinct―signature‖because of their distinctive spatialconfigurations. We used SWNT nanopores to control the two dimensions (diameter andlength) of the nanopores. The advantages of such pores includes as high as thousandtimes of aspect ratio, which, in combination with atomic smoothness of the inside wall ofthe pore, would pomise a high detection accuracy, and resovle the incapability ofbase-by-base discrimination in DNA sequencing by threading with solid-state nanopore,which is too deep for individual base identification. We have obtained some preliminaryresults in this experiment.Another part of this dissertation, fabrication of single-molecule chips was performed in order to upgrade chips used in second-generation sequencing into those in thethird-generation. Biotinylated DNA molecules were conjugated to streptavidin (STV).Conjugates containing single DNA molecule were separated from the mixture throughelectrophoresis in agarose gels, and were labeledd with DNA dyes. Nanowell arrays withdiameter of about30nm to hold single DNA-STV conjugates were fabricated on quartzsubstrates, which was covered with a layer of ion-sputtered gold film (around10nm ofthickness), by using FIB to drill nanoholes through the gold film till quartz substrate witha center-to-center periodic distance of500nm. Then, bottom quartz in nanowells werefunctionalized with biotin. Conjugates were loaded onto the nanowell array followed bycharacterization by Total Internal Reflection Fluorescence Microscopy (TIRFM).