Single-molecule electronic DNA sequencing

September 24, 2012

Schematic of single molecule DNA sequencing by a nanopore with phosphate-tagged nucleotides. Each of the four nucleotides will carry a different tag. During sequencing by synthesis (SBS), these tags, attached via the terminal-phosphate of the nucleotide, will be released into the nanopore one at a time, where they will produce unique current blockade signatures for sequence determination. A large array of such nanopores will lead to high throughput DNA sequencing. (Credit: Columbia University)

A team of researchers at Columbia University, have developed a novel approach to potentially sequence DNA in nanopores electronically at the single molecule level with single-base resolution.

This work, entitled “PEG-Labeled Nucleotides and Nanopore Detection for Single Molecule DNA Sequencing by Synthesis,” is now available in the open access online journal, Scientific Reports.


The major roadblock in DNA sequencing has been the cost and speed of obtaining highly accurate DNA sequences. Most current high-throughput sequencing instruments depend on optical techniques for the detection of the four building blocks of DNA: A, C, G and T.

To further advance the measurement capability, electronic DNA sequencing of an ensemble of DNA templates has also been developed.

Recently, it has been shown that DNA can be threaded through protein nanoscale pores under an applied electric current to produce electronic signals at single molecule level.

However, because the four nucleotides are very similar in their chemical structures, they cannot easily be distinguished using this technique.

So research and development of a single-molecule electronic DNA sequencing platform is an active area of investigation and has the potential to produce a hand-held DNA sequencer capable of deciphering the genome for personalized medicine and basic biomedical research.

How nanopore-based sequencing by synthesis works

The reported nanopore-based sequencing by synthesis (Nano-SBS) strategy can accurately distinguish four DNA bases by detecting 4 different sized tags released from 5’-phosphate-modified nucleotides at the single-molecule level for sequence determination.

As each nucleotide analog is incorporated into the growing DNA strand during the polymerase reaction, its tag is released by phosphodiester bond formation. The tags will enter a nanopore in the order of their release, producing unique ionic current blockade signatures due to their distinct chemical structures, thereby determining DNA sequence electronically at single molecule level with single base resolution.

The system uses four differently tagged nucleotides, which upon incorporation by DNA polymerase, release four different size tags that are distinguished from each other at the single molecule level when they pass through the nanopore. This approach overcomes any constraints imposed by the small differences among the four nucleotides, a challenge which most nanopore sequencing methods have faced for decades.

With further development of this Nano-SBS approach, such as the use of large arrays of protein or solid nanopores, this system has the potential to accurately sequence an entire human genome rapidly and at low cost, thereby enabling it to be used in routine medical diagnoses.