DNA sequencing is the driving force behind key discoveries in medicine
and biology. For instance, the complete sequence of an individual's
genome provides important markers and guidelines for medical diagnostics
and healthcare. Up to now, the major roadblock has been the cost and
speed of obtaining highly accurate DNA sequences. While numerous
advances have been made in the last 10 years, 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. Thus, the research and development
of a single-molecule electronic DNA sequencing platform is the most
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.
A team of researchers at Columbia University, headed by Dr. Jingyue
Ju (the Samuel Ruben-Peter G. Viele Professor of Engineering, Professor
of Chemical Engineering and Pharmacology, Director of the Center for
Genome Technology and Biomolecular Engineering), with colleagues at the
National Institute of Standards and Technology (NIST) led by Dr. John
Kasianowicz (Fellow of the American Physical Society), have developed a
novel approach to potentially sequence DNA in nanopores electronically
at 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, from Nature Publishing Group.
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. The basic
principle of the Nano-SBS strategy is described as follows. 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.
As proof-of-principle, the research team attached four different
length polymer tags to the terminal phosphate of
2'-deoxyguanosine-5'-tetraphosphate (a modified DNA building block) and
demonstrated efficient incorporation of the nucleotide analogs during
the polymerase reaction, as well as better than baseline discrimination
among the four tags at single molecule level based on their nanopore
ionic current blockade signatures. This approach coupled with polymerase
attached to the nanopores in an array format should yield a
single-molecule electronic Nano-SBS platform.
In previous work, the Center of Genome Technology & Biomolecular
Engineering at Columbia University, led by Professor Ju and Dr. Nicholas
J. Turro (William P. Schweitzer Professor of Chemistry), developed a
four-color DNA sequencing by synthesis (SBS) platform using cleavable
fluorescent nucleotide reversible terminators (NRT), which is licensed
to Intelligent Bio-Systems, Inc., a QIAGEN company. SBS with cleavable
fluorescent NRTs is the dominant approach used in the next generation
DNA sequencing systems. Dr. Kasianowicz and his group at NIST pioneered
the investigation of nanopores for single molecule analysis. They
previously reported that different length polymers, polyethylene glycols
(PEGs), could be distinguished by their unique effects on current
readings in a α-hemolysin protein nanopores at single molecule level and
subsequently developed a theory for the method. Their results provide
the proof-of-concept for single molecule mass spectrometry. The
combination of the SBS concept with the distinct nanopore-detectable
electronic tags to label DNA building blocks led to the development of
the single-molecule electronic Nano-SBS approach described the current Scientific Reports article (09/21/2012).
As lead author Dr. Shiv Kumar points out, "The novelty of our
approach lies in the design and use of 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." Moreover, the technique is quite flexible; with
PEG tags as prototypes, other chemical tags can be chosen to provide
optimal separation in different nanopore systems.
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.
The authors of the Scientific Reports article were Shiv
Kumar, Chuanjuan Tao, Minchen Chien, Brittney Hellner, Arvind
Balijepalli, Joseph W.F. Robertson, Zengmin Li, James J. Russo, Joseph
E. Reiner, John J. Kasianowicz, and Jingyue Ju. The study was supported
by a grant from the National Institutes of Health, a National Research
Council/NIST/NIH Research Fellowship, and a grant from the NIST Office
of Law Enforcement Standards.
Journal Reference:
- Shiv Kumar, Chuanjuan Tao, Minchen Chien, Brittney Hellner, Arvind Balijepalli, Joseph W. F. Robertson, Zengmin Li, James J. Russo, Joseph E. Reiner, John J. Kasianowicz, Jingyue Ju. PEG-Labeled Nucleotides and Nanopore Detection for Single Molecule DNA Sequencing by Synthesis. Scientific Reports, 2012; 2 DOI: 10.1038/srep00684
Courtesy: ScienceDaily
No comments:
Post a Comment