Progress toward Ultrafast DNA Sequencing Using Solid-State Nanopores

Abstract

Background:Measurements of the ionic current flowing through nanometer-scale pores (nanopores) have been used to analyze single DNA and RNA molecules, with the ultimate goal of achieving ultrafast DNA sequencing. However, attempts at purely electronic measurements have not achieved the signal contrast required for single nucleotide differentiation. In this report we propose a novel method of optical detection of DNA sequence translocating through a nanopore. Methods: Each base of the target DNA sequence is 1st mapped onto a 2-unit code, 2 10-bp nucleotide sequence, by biochemical conversion into Designed DNA Polymers. These 2-unit codes are then hybridized to complementary, fluorescently labeled, and self-quenching molecular beacons. As the molecular beacons are sequentially unzipped during translocation through a <2-nm-wide nanopore, their fluorescent tags are unquenched and are detected by a custom-built dual-color total internal reflection fluorescence (TIRF) microscope. The 2-color optical signal is then correlated to the target DNA sequence. Results: A dual-color TIRFM microscope with singlemolecule resolution was constructed, and controlled fabrication of 1-dimensional and 2-dimensional arrays of solid-state nanopores was performed. A nanofluidic cell assembly was constructed for TIRF-based optical detection of voltage-driven DNA translocation through a nanopore. Conclusions: We present a novel nanopore-based DNA sequencing technique that uses an optical readout of DNA translocating unzipping through a nanopore. Our technique offers better single nucleotide differentiation in sequence readout, as well as the possibility of largescale parallelism using nanopore arrays.

Synchronous optical and electrical detection of biomolecules traversing through solid-state nanopores

“…Multicolor measurements also enrich attempts toward realizing nanopore based genome sequencing, espe- cially when combined with the wide-field imaging capabilities, which permit simultaneous signal probing from multiple nanopores. 17 In summary, we show for the first time single-molecule optical visualization of labeled DNA and DNA-protein complexes translocating through ϳ4 nm synthetic pores, with a temporal resolution of ϳ1 ms. We show that we can easily localize the pore on membrane with good signal to noise and extract intensity traces of single DNA translocation events. Intensity distributions of these events showed multiple levels of fluorescence intensities corresponding to the varying degree of labeling.…”

“…One particular application of note is the development of the "nextgeneration" single-molecule DNA sequencing methods. [15][16][17][18] Solely probing the ionic current flowing through nanopores, while proven to be highly useful, has a number of clear shortcomings. First and foremost, it provides temporal information rather than positional information on the process or the biomolecule under study.…”

“…Additionally, the optical detection open up the possibility of tagging biopolymers with multicolor fluorescent markers, which can be programmed to report on the DNA sequence. 17 Thus the method is applicable for emerging nanopore sequencing approaches. We demonstrate the feasibility of our system by optically probing labeled dsDNA and DNA-protein complexes inside a ϳ4 nm pore.…”

Enhanced translocation of single DNA molecules through α-hemolysin nanopores by manipulation of internal charge

“…The newly engineered pores or derivatives of them will be useful for enhancing the sensitivity of ␣HL as a biosensor of nucleic acids (2,(17)(18)(19)(20)(21)(22)(23)(24)(25). An additional potential application is in nanopore sequencing, where the bases in a single DNA strand are read off one by one during translocation (16,30,(32)(33)(34)(35)(36)(37). In this area, progress has been made on base identification (9,54) and in controlling the rate at which DNA moves through the pore (29,30).…”

“…Possible means of nanopore sequencing include the direct reading of bases in single strands as they pass through the pore (16), the reading of strands moved through the pore with the aid of an enzyme (30) and the detection or stripping of oligonucleotide probes on single strands as they are translocated (36,37). All of these approaches require the efficient capture of DNA strands from solution.…”