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Purely chemical process to sequence DNA

03/10/2010
Erin Podolak

A new dynamic chemistry method is the first non-enzymatic sequencing method based on analyzing a reversible reaction between a PNA-DNA duplex.

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Researchers in the laboratory of Mark Bradley at the University of Edinburgh (United Kingdom) have developed a dynamic chemistry approach to sequencing DNA. Dynamic chemistry refers to reversible covalent chemical reactions carried out under conditions of equilibrium control. Bradley’s team applied dynamic chemistry to DNA analysis of a peptide nucleic acid (PNA) strand that was engineered to mimic a strand of DNA with an unknown sequence.

A dynamic chemistry approach using a DNA template has previously been used for preparing stimuli-responsive polymers and to gain insight into the chemistry of primordial self-replicating systems (1). Recent research, however, has demonstrated PNA’s ability to be used as a template for identifying missing bases on a complementary DNA strand (2). Therefore, Bradley’s team hypothesized that a PNA template could be used with dynamic chemistry to establish a non-enzymatic platform for sequencing DNA.

According to Juan Diaz-Mochon—a researcher in the department of chemistry at the University of Edinburgh and an author on the recent paper describing the technique in Angewandte Chemie—the researchers hope to improve the speed and costs associated with other genome sequencing methods, including sequencing by ligation and nanopores. “What our technology provides is a completely new way of doing genotyping and sequencing, by using a purely chemical process,” Diaz-Mochon told BioTechniques.

PNA’s got potential

Watson-Crick base pairing was used to create a template for PNA. Source: Wikipedia Commons.


To apply dynamic chemistry to DNA analysis, the researchers created a PNA-DNA duplex that contained a blank position on the PNA strand opposite the nucleobase of interest in the DNA strand. A dynamic chemistry reaction selects for the four nucleobases using Watson-Crick base pairing (Adenine with Thymine, Guanine with Cytosine) and uses base stacking to create a nucleobase template. Base stacking is the interaction between the pi bonds—covalent chemical bonds where two lobes of one involved electron orbital overlap two lobes of another involved electron orbital—and the nucleobases’ aromatic rings (stabilized bonds). The template drives the selection of a complementary iminium (salt- or cation-based) nueclobase. Subsequent reduction and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry allows the rapid determination of the unknown base on the DNA strand.

Although PNA was first described in 1991 (3), Diaz-Mochon said that its full potential has yet to be realized. “PNA is a wonderful DNA mimic as it hybridizes complementary DNA strands with higher efficiency than DNA itself,” said Diaz-Mochon. “We have become one of the world leaders in this field with the development and successful application of our PNA-encoding libraries. Therefore, reading DNA with PNA was an intellectual challenge we set ourselves.”

So many choices, for such a low cost

Dynamic chemistry for DNA analysis is not a replacement for other sequencing methods. Diaz-Mochon said the new method was designed for targeted genetic analysis for personalized medicine instead of rapid whole-genome sequencing. “Rather than eliminating current sequencing platforms, our technology will be a great addition to them,” said Diaz-Mochon.

Sequencing systems that are based on chemical ligation typically have high reagent costs. The large quantity of reagent consumption has been reduced in recent years, and according to Diaz-Mochon, the dynamic chemistry system is comparable to the “current best in class level for reagent costs.” He noted, however, that reagent costs are not the only thing to consider when calculating the cost of sequencing. Bio-informatics systems, time to run the sequencing instruments, and time spent conducting analyses all impact the total cost.

“Cost seemed to be the holy grail of sequencing, with the $1000 genome project target set some time ago as an inspirational target for all technology advancement,” said Diaz-Mochon. “I believe the $1000 goal will be achieved within five years. Third-generation technologies are going to make this possible and they should be applauded when delivering this remarkable achievement.”

The future is in bits and pieces

The power of third-generation sequencing technology should not be undervalued. “I see these ‘third-generation’ technologies fundamentally changing the way we will treat diseases in the future,” said Diaz-Mochon. Methods such as the dynamic chemistry approach that are quick, cost effective, and easy to use, however, will still have a role in the spectrum of sequencing technologies by providing target-specific genetic analysis for medical applications.

According to Diaz-Mochon, once comprehensive connections have been established between genetic profiles and diseases the need for sequencing the entire human genome will be reduced.“This is when technologies like ours will be pivotal," he said.

“No one travels from London’s East End to the West End by Concorde [Airplane],” said Diaz-Mochon. “Similarly no one will need to use a Pacific Bioscience–like platform to sequence just a short region of DNA for genotyping.”

A patent for the technology was filed by the researchers in the United Kingdom in 2007. The paper, “DNA analysis by dynamic chemistry,” was published Feb. 12, in Angewandte Chemie.

References

1. Goodwin, J. T. and D. G. Lynn (1992). "Template-directed synthesis: use of a reversible reaction." Journal of the American Chemical Society 114(23): 9197-8.

2. L. Marcus Wilhelmsson, Bengt Norden, Kaushik Mukherjee, Maria T. Dulay, Richard N. Zare “Genetic screening using the colour change of a PNA-DNA hybrid-binding cyanine dye” Nucleic Acids Research 30:2 2002

3. Peter E. Nielsen, Michael Egholm, Rolf H. Berg, and Ole Buchardt “Sequence specific inhibition of DNA restriction enzyme cleavage by PNA” Nucleic Acids Res., 25 January 1993; 21: 197 - 200.