Synthetic Biology and Molecular Diagnostics


The introduction of thermostable polymerases revolutionized PCR and biotechnology. However, many GC-rich genes cannot be PCR-amplified with high efficiency in water, irrespective of temperature. Although polar organic cosolvents can enhance nucleic acid polymerization and amplification by destabilizing duplex DNA and secondary structures, nature has not selected for the evolution of solvent-tolerant polymerase enzymes. Here, we used ultrahigh-throughput droplet-based selection and sequencing along with computational free energy and binding affinity calculations to evolve Taq polymerase into RNA- and DNA-dependent DNA polymerases that are both stable and highly active in the presence of organic cosolvents, resulting in up to 10% solvent resistance and over 100-fold increase in stability at 97.5℃ in the presence of 1, 4-butanediol (BD), as well as up to 10 times enhanced tolerance to the potent cosolvents sulfolane and 2-pyrrolidone. Using these polymerases, we successfully amplified a broad spectrum of GC-rich templates containing regions with over 90% GC content, including templates recalcitrant to amplification with existing polymerases, even in the presence of cosolvents. We also demonstrated dramatically reduced GC bias in the amplification of genes with widely varying GC content in target enrichment for next-generation sequencing, as well as reverse transcription in aqueous-organic media and high temperatures. By expanding the scope of solvent systems compatible with nucleic acid polymerization, these organic solvent-resistant polymerases enable a dramatic reduction of sequence bias not achievable through thermal resistance alone, with significant implications for a wide range of downstream applications including NGS sequencing and differential gene expression analysis.

Working Papers:

  • Evolution of solvostable DNA polymerases. Alok Upadhyay, Xiangying Guan, Devin Hudson, Rahul Bose, M Elias, J Kwak, Ioanna Petrounia, M Lorilliere, Gauthier Errasti, Thomas Delacroix, Anisha Ghosh and Raj Chakrabarti.  (2022)
  • Applying ultrahigh-throughput screening and massively parallel data analysis to identify improved polymerase variants in the presence of organic solvents. Xiangying Guan, Devin Hudson, Rahul Bose, M Elias, J Kwak, Ioanna Petrounia, K Touah, S Mansour, Gauthier Errasti, Thomas Delacroix, Anisha Ghosh and Raj Chakrabarti. (2022)


DNA amplification - the replication of genetic material in a test tube - is arguably the central technology of the modern era of molecular bioscience. The most common DNA amplification reaction is the Nobel prize-winning polymerase chain reaction (PCR), which can in principle produce millions of copies of DNA molecules, starting from a single molecule, through repeated cycles of heating and cooling.

For every DNA sequence, there is an ideal schedule for heating and cooling that maximizes the amount of DNA produced. However, no prescription for computing this ideal temperature cycling protocol has been available since the invention of PCR. The operating conditions for PCR reactions are instead selected based on qualitative analysis of their kinetics and thermodynamics. Over the past two decades, many variants of DNA amplification have been invented based on the notions of DNA denaturation, annealing and polymerization, each tailored to a particular amplification objective, but a unified framework for the design of new reactions has been lacking.

Based on fundamental biophysical modeling, chemical process control engineering, and advanced computational optimization algorithms, we have developed a generalized framework for next-generation PCR called Optimally Controlled DNA Amplification. This framework enables the automated computation of the ideal temperature cycling protocol for any DNA sequence and desired amplification objective. Unlike other next-generation PCR technologies, this patented methodology can be used in any PCR machine and for any PCR reaction, and hence possesses the generality required to serve as a core technology in the rapidly growing diagnostics and sequencing markets.

Selected Publications:



The Polymerase Chain Reaction (PCR) is an in vitro method for rapid replication of DNA that is the workhorse of genome sequencing, genetic profiling, and forensic science. The applications of PCR are central to life science experiments and many potential breakthrough technologies. In addition, the COVID pandemic has highlighted the importance of PCR-based diagnostic tests as the gold standard for pathogen and mutation detection. However, PCR reactions often generate inadequate yield of the target DNA sequence and amplification of undesired nonspecific products. These problems can be especially severe in case of targets with high-GC content (more GC than AT base pairs). The difficulties encountered in amplifying high-GC targets are hindering genetic diagnostics, since many DNAs of interest, particularly those that are relevant to disease risk, reside in high-GC containing segments of the genome. We have internally developed intellectual property for sequencing GC-rich segments of the genome essential for diagnosing a variety of genetically inherited diseases. In a series of papers, we developed chemical techniques whereby small molecules could be employed to control the progress of the PCR reaction so that the amplification could be achieved for virtually any genomic template.

Our patents have been licensed by companies such as Celera/Abbott Diagnostics, Quest Diagnostics (the leading clinical IVD test for autism), and New England Biolabs (the market leading Q5 polymerase used worldwide in DNA sequencing applications), and have also been the basis of a partnership with Toyobo/Sekisui Diagnostics. The annual sales of products based on these patents is close to $40M.

We are also applying microfluidics and microelectronics to DNA-based molecular diagnostics.

Selected Publications:

  • "Novel PCR-enhancing compounds and their modes of action," R. Chakrabarti In: PCR Technology: Current Innovations, 2nd Edition. Ed. T. Weissensteiner, H.G. Griffin and A. Griffin, CRC Press: Boca Raton, FL (2003) [invited article]

  • Label-free microelectronic PCR quantification. Chih-Sheng Johnson Hou, Nebojsa Milovic, Michel Godin, Peter R. Russo, Raj Chakrabarti and Scott R. Manalis. Anal. Chem. 78 2526-2531 (2006)

  • Celera licenses DNA biotechnology platform