Synthetic Biology and Molecular Diagnostics
POLYMERASE
ENZYME ENGINEERING
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 Organic Solvent-Resistant DNA Polymerases. Mohammed Elias, Xiangying Guan, Devin Hudson, Rahul Bose, Joon Kwak, Ioanna Petrounia, Kenza Touah, Sourour Mansour, Peng Yue, Gauthier Errasti, Thomas Delacroix, Anisha Ghosh, and Raj Chakrabarti ACS Synthetic Biology
10.1021/acssynbio.2c00515 (2023)
- Synthetic 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)
NEXT-GENERATION
DNA AMPLIFICATION AND SEQUENCING
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:
ENABLING
THE MOLECULAR DIAGNOSTICS OF GENETIC MARKERS FOR
DISEASES
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
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