Organic Chemists Contributing to Whole Genome Sequencing
Background - Why is this important?
How such knowledge will be managed and used is an ongoing and important ethical debate; however, for the individual patient, the opportunity to better understand disease, apply the most appropriate existing therapies or to discover new ones provides real hope.
What did the organic chemists do?
Progress in genome sequencing is underpinned by extensive and concerted application of powerful technologies from many scientific disciplines including genetics, molecular biology, engineering, and chemistry.
Using synthetic fluorophores
Synthetic organic chemists working at Pacific Biosciences developed an innovative DNA sequencing technology which relies upon the availability of DNA bases uniquely labelled with synthetic fluorophores. The sequencing technology used DNA polymerase; the same enzyme that builds the DNA double helix.
The enzyme is tethered to the bottom of micro-plate wells. DNA fragments are loaded into the wells, which are then washed with the uniquely labelled DNA bases. A digital camera records the sequence that these labelled bases are incorporated into the growing DNA strand by the DNA polymerase. The phosphodiester bond formation catalyzed by DNA polymerase results in release of the fluorophore from the incorporated nucleotide generating natural, unmodified DNA and a unique fluorescence identifier. The sequence of light emissions can be decoded to identify the structure of the growing strand.1
Synthesis of a fluorophore-labelled DNA base
Each nucleotide base component has a unique fluorophore linked through an aminoalkyl chain to the terminal phosphate of a pentaphosphate linker moiety. This long linker has been shown to increase the efficiency of incorporation into the growing DNA strand. Successful implementation of the technology required the development of an efficient synthetic chemistry method for fluorophore incorporation which would be applicable to all 4 nucleotide bases.2 The synthetic route applies the well known carbonyldiimidazole (CDI) coupling reagent, which was discovered in the 1960's, to form the phosphoester linkers;3 the second coupling applies a novel variation using MgCl24 to increase the coupling efficiency.
UK scientists working at Oxford Nanopore Technologies synthesised stable nanopores based upon the heptameric protein alpha-hemolysin (AHL) by protein engineering techniques.5,6 In this example, each uniquely engineered nanopore incorporates a different nucleotide recognition sequence.
Nucleotide interaction with these sequences triggers a detectable current modulation across the nanopore. As the stand passes over the nanopore, individual bases on a single DNA strand can, in principle, be identified. This is a powerful exemplification of molecular recognition linked to analytical detection. This impressive recognition technology is equally applicable to the detection of small molecules or proteins.7,8
What is the impact?
The scientific community has thrown its weight behind the human genome sequencing challenge and progress has been dramatic. The first “human genome” sequence cost over $1 billion and was an assembly of DNA sequences taken from several volunteers. In recent years there has been significant progress in reducing the cost, according to the National Genome Research Institute the cost per genome is currently $7,666.
The impact of many scientific disciplines including genetics, molecular biology, engineering, and chemistry has already impacted upon our ability to sequence individual genomes and the implementation of the methods described here, as well as others, will herald a new era of truly personalised medicine where individual disease susceptibility and response to treatment can be monitored in real time.
1 J Eid et al., Science, 2009, 323, 133
2 J Korlach et al., Nucleosides, Nucleotides and Nucleic Acids, 2008, 27, 1072
3 A Sood et al., J.Am.Chem. Soc., 2005, 127, 2394
4 M Kadokura et al., Tetrahedron Lett., 1997, 38, 8359
5 K R Liberman et al., J. Am. Chem. Soc., 2010, 132, 17961
6 D Branton et al., Nat. Biotechnol., 2008, 26, 1146
7 X-F Kang, S Cheley, X Guan and H Bayley, J. Am. Chem. Soc., 2006, 128, 10684
8 S Cheley, H Xie and H Bayley, Chem. BioChem., 2006, 7, 1923
Also of interest
With the relentless rise of DNA nanotechnology’s popularity, Emma Davies explores the role chemistry has played in its success
Molecular information is vital for personalised medicine. Chemistry plays an important role in developing ways to obtain this information faster and more efficiently.
The first draft sequence of the human genome, announced 10 years ago, was time-consuming and expensive
Sequencing in the fast lane
Advances in DNA sequencing technology are changing the way scientists look at genomes. Phillip Broadwith gets up to speed with the latest developments
Chemical Society Review: DNA-based nanotechnology
A themed issue of Chemical Society Reviews on advances in DNA-based nanotechnology
Jeff Hrkach talks about how he got into polymer chemistry
Contact and Further Information
Dr Anne Horan
Programme Manager, Life Sciences
Royal Society of Chemistry, Thomas Graham House, Science Park, Milton Road, Cambridge, CB4 0WF
Tel: 01223 432699