As DNA Analysis has become more prevalent and necessary in modern time, so have methods of sequencing.

Genome.gov holds programs and grants for those with the most rewarding possible future methods of DNA Sequencing. Below are a few that have some great potential.

Microfluidic DNA Sequencing

A single molecule detection method leveraging droplet-based microfluidics. This should limit the amount of reagent required to sequence DNA to less than several milliliters, while still retaining the ability to amplify the template that thereby enables us to use relatively inexpensive and robust detection. The method is simple and does not require enzymes.

Polony Sequencing

Ultra-high throughput polony genome sequencing, generating raw data to re-sequence the human genome in one week. The goal of the method is to increase the polony sequencing read length using a cyclic ligation strategy that involves enzymatic cleavage, and increase read density by using different clonal amplification strategies.

Millikan Sequencing by Nucleotide

This novel sequencing-by-synthesis approach measures the increased charge as nucleotides are added to DNA templates attached to a tethered bead. Opposing electrical, hydrodynamic and entropic forces will be used to measure the bead displacement, which is a function of the length of DNA attached to the bead. The much lower per-bead copy number required compared to the 454 system should enable amplification options other than emulsion PCR, such as bridge PCR, making initial sample preparation easier and cheaper.

Single-Molecule DNA Sequencing with Engineered Nanopores

In nanopore strand sequencing, a single strand of DNA moves through a narrow pore and the bases are identified as they pass a reading head. Here, we focus on the remaining tasks required to put into practice strand sequencing with the ±-hemolysin (±HL) protein nanopore. Nanopore sequencing is a rapid real-time technology; it does not require the time-consuming cyclic addition of reagents. After implementing a chip with 106 pores, we expect nanopore sequencing to achieve a 15-minute genome by 2014 with a very short sample preparation time. In addition, nanopore sequencing will be able to identify modified bases and to sequence RNA directly. The latest goal is to refine base recognition by using ±HL nanopores, engineered by conventional mutagenesis, unnatural amino acid mutagenesis and targeted chemical modification, to produce DNA reading heads fit for real-time sequencing.

Direct Real-time Single Molecule DNA Sequencing

Direct real-time sequencing of single DNA molecules from genomic DNA at the speed and accuracy of the natural DNA polymerases using native nucleotides. Unlike the difficult to engineer man-made nanostructures used in nanopore sequencing to distinguish the 4 base types in close proximity and constant fluctuation, DNA polymerases have precise atomic-resolution 3D structures and can synthesize very long DNA molecules with high fidelity and velocity. The strategy is to engineer sensors onto the surface of the polymerase by protein engineering to monitor the subtle yet distinct conformational changes accompanying the incorporation of each base type.

Tunnel Junction for Reading All Four Bases with High Discrimination

Distinct tunneling signals can be generated for all four nucleosides (and 5-methyldeoxycytidine) using one pair of tunneling electrodes functionalized with a simple reagent containing a hydrogen-bond donor and a hydrogen bond acceptor. The goals of this proposal are to extend the measurements to nucleotides in aqueous electrolyte, and then to small oligomers.

Single Molecule Sequencing by Nanopore-induced Photon Emission (SM-SNIPE)

Nanopore induced photon emission (SNIPE), utilizes optical detection rather than the more ubiquitous electrical detection. Dramatically increase the throughput, speed and accuracy of SNIPE. Develop and optimize our proprietary DNA conversion approach, Circular DNA conversion (CDC).

Modeling Macromolecular Transport for Sequencing Technologies

In Nanopore-based electrophoretic experiments, translocation of single molecules of DNA is monitored as they pass through protein channels and solid-state nanopores under an external electric field. This proposed method deals with a fundamental understanding of the behavior of DNA in nanopore environments under the influence of electrical and hydrodynamic forces.

Base-selective Heavy Atom Labels for Electron Microscopy-based DNA Sequencing

Since efficient electron scattering to a detector is highly dependent on atomic number (Z), it is possible to label single stranded DNA (ssDNA) with heavy atoms. To test the limits of this trend, this method proposes a multipronged approach to selectively prepared metal-DNA base pair complexes, focusing on the selective labeling of DNA bases and the development of an appropriate assay to evaluate our success.