A superior DNA polymerase for use in PCR
Submitting InstitutionNewcastle University
Unit of AssessmentBiological Sciences
Summary Impact TypeTechnological
Research Subject Area(s)
Biological Sciences: Biochemistry and Cell Biology, Genetics
Medical and Health Sciences: Medical Microbiology
Summary of the impact
Through their study of DNA polymerases from organisms of the domain
archaea, researchers at Newcastle University and University College London
identified the mechanism by which these organisms avoid potentially
damaging mutations in their DNA. As a consequence of this work they
invented a novel genetically-engineered DNA polymerase. This enzyme has
been patented and is the world's only high-fidelity, proofreading DNA
polymerase that efficiently reads through uracil in the polymerase chain
reaction (PCR). PCR is a very widely used technique in biomedical
research. An international bioscience company [Text removed for
publication, EV d] signed a licensing agreement with Newcastle University
in 2008 to market the enzyme, and total sales since 2008 exceed [Text
removed for publication, EV d]. Further commercial exploitation has begun
through licensing agreements with other major companies.
Key Newcastle University researcher
Professor Bernard Connolly conceived and led the research in Newcastle and
contributed significantly to collaborative work, detailed where
Four chemical bases make up DNA: adenine (A), thymine (T), cytosine (C)
and guanine (G). Cytosine is prone to a deamination reaction (the loss of
an amine moiety), the product of which is uracil. Unless it is repaired to
cytosine uracil remains in the DNA strand and, when this is replicated
using a normal DNA polymerase, the uracil forms a base pair with adenine.
When the DNA strand including this adenine is replicated in turn, it will
form a pair with thymine. Thus, the presence of uracil eventually results
in the replacement of the original C-G base pair with T-A. This mutation
(error) in the DNA is irreversible and scrambles the genetic code
(Hofreiter et al. 2001 PMID: 11726688).
The deamination of cytosine is the most common reason for mutation in
DNA. The reaction proceeds at a low rate at room temperature, but rises
significantly at higher temperatures, such as those that are used during
the polymerase chain reaction (PCR).
Archaea are single-celled organisms that are classified in their own
domain, having been shown to have life-processes that are neither
exclusively like bacteria nor eukaryotes (the other domains). Many archaea
are found in extreme environments such as the high temperature water
around deep-sea thermal vents. When they were discovered, it was suggested
that such hyperthermophilic archaea would be particularly vulnerable to
mutations produced through cytosine deamination caused by the high
temperatures to which they are exposed in their natural habitat. Like all
other organisms whose genetics is based on DNA, archaea have an absolute
requirement for some type of uracil detection and repair system. However,
no homologues of the two known families of uracil base excision-repair
enzymes ubiquitous in the other domains have so far been identified in
archaeal genomes, suggesting that a novel mechanism of error checking
might be discovered in the archaea.
Connolly's research at Newcastle University concerns the nature of
protein-DNA interactions. In the late 1990s his group began to study
organisms in the domain archaea, aiming to identify the molecular
mechanism by which they recognise uracil in their DNA. This involved the
study of enzymes, termed DNA polymerases, which are involved in the
replication of DNA.
In 1999, Connolly and co-investigator Professor Laurence Pearl
(University College London) published results which showed that DNA
polymerases from several archaea stop DNA replication (stall) at uracil in
the strand being copied (the template strand; R1). This stalling pointed
to a possible mechanism that had evolved to protect archaea from harmful
rates of mutation caused by cytosine deamination in the high temperature
environments where they live. Stalling was achieved by the DNA polymerase
recognising uracil in the strand ahead of the point at which it was
currently copying. Importantly, this was the first publication to show the
existence of a read-ahead template-checking function in any DNA
polymerase, from any type of organism.
Further studies of the structure of these DNA polymerases by Connolly and
colleagues revealed the molecular basis of the template-checking function.
The researchers demonstrated the presence of a `pocket' in the region of
the DNA polymerase away from the active site that interacts with the
template DNA strand. By introducing mutations into the gene sequence
coding for the DNA polymerase they demonstrated that chemical groups in
the `pocket' discriminate uracil from the other bases in DNA and,
moreover, that it is this specificity that causes the DNA polymerase to
stall at uracil but still progress unimpeded past the four normal bases in
DNA. Significantly, these studies led Connolly and colleagues to
genetically engineer the uracil binding pocket in the DNA polymerase to
create a new type of DNA polymerase that could read through template
strand uracil (R2).
An advantage of archaeal DNA polymerases is that, unlike bacterial DNA
polymerases, they have a 3'-5' exonuclease III (proofreading) activity
(Cline et al., 1996 PMID: 883618). This activity together with the
Newcastle research into uracil recognition resulted in the production of a
high-fidelity proofreading DNA polymerase, stable at high temperatures,
which could read through uracil. These properties of the unique DNA
polymerase make it a valuable laboratory tool.
References to the research
(Newcastle researchers in bold. Citations from Scopus as at July 2013.)
R1. Greagg MA, Fogg MJ, Panayotou G, Evans SJ, Connolly BA
and Pearl LH (1999) A read-ahead function in archaeal DNA polymerases
detects promutagenic template-strand uracil. Proceedings of the
National Academy of Sciences USA 96(16):9045-50. DOI:
10.1073/pnas.96.16.9045. 75 citations.
R2. Fogg MJ, Pearl LH and Connolly BA (2002) Structural
basis for uracil recognition by archaeal family B DNA polymerases. Nature
Structural & Molecular Biology 9(12):922-7. DOI: 10.1038/nsb867.
Key research grants
MRC. 1993-6. £101,000. Joint award with Laurence Pearl, UCL. Mapping
of the DNA binding site.
BBSRC. 2000-3. £193,000. Recognition of DNA containing DU bases by
archaeal DNA polymerases.
BBSRC. 2004-6. £222,000. Improving archaeal DNA polymerases for
molecular biology applications.
Details of the impact
The DNA polymerases of hyperthermophilic archaea were of interest to
molecular biologists because of their inherent ability to withstand high
temperatures. Such a property was valuable for the amplification of DNA in
the laboratory, using the polymerase chain reaction (PCR). PCR makes use
of DNA polymerases to replicate DNA and involves repeated heating and
cooling cycles, amplifying a small quantity of DNA into one large enough
for meaningful analysis and for many experimental approaches. However, the
high temperature phases of the PCR increase the rate of the deamination of
cytosine and lead to a build-up of DNA strands incorporating uracil during
the amplification process. The commonly used heat-stable DNA polymerase,
Taq (from Thermus aquaticus)
cannot proofread, so uracil generated during PCR using Taq polymerase
leads to the accumulation of T-A for G-C substitutions and loss of
fidelity. Archaeal polymerases have a proof-reading activity, but the fact
that archaeal polymerases stall at uracil make them less suited to PCR in
their native form.
In the course of their work, not only had Newcastle researchers and their
colleagues identified a unique property of certain hyperthermophilic
archaeal DNA polymerases but, through their discovery that the uracil
recognition function was not present at the active site, they were able to
engineer a change in the structure of the uracil binding pocket to create
a novel DNA polymerase that was capable of reading through DNA strands
that included uracil. This combination of properties — both the high
fidelity and stability at high temperatures — was recognised as having
potential for commercial exploitation.
Patenting a new enzyme
The new DNA polymerase, hereafter referred to as V93Q Pol, was co-invented
by Connolly and Mark Fogg (Connolly's PhD student) at Newcastle
University, and Professor Laurence Pearl at UCL, in 2003. The invention
was underpinned by insights gained through the researchers' studies into
how archaeal DNA polymerases recognise the base uracil. A patent to
protect the invention, Mutation of DNA polymerases from archaebacteria,
was filed in April 2003, and it has now been granted in many countries
worldwide (Australia, Canada, Japan, USA, UK and other European countries;
Ev a). The enzyme is unique.
Some DNA polymerases used in PCR have been derived from hyperthermophilic
archaea but they become less efficient in the presence of even small
amounts of uracil in the reaction mixture, because their structure causes
them to stall at uracil. V93Q Pol was engineered to be uracil-insensitive
and is therefore not affected in the same way. Because of its broad
tolerance to the presence of uracil, V93Q Pol is often used for reactions
in which high-fidelity copying is required but reaction conditions have
not been optimised, or where the template is relatively long and contains
many cytosine residues (which can be deaminated to uracil, as described
above). Under equivalent reaction conditions, V93Q Pol typically yields
more DNA product than standard enzymes used in PCR.
The V93Q Pol enzyme is particularly useful in DNA methylation studies,
which are important in understanding why genes are switched off in cells
(Ev b). Methods that target DNA methylation sites (bisulphite sequencing)
lead to degradation of much of the DNA, leaving very few intact copies of
the DNA sequence. Amplifying these few copies using PCR requires many
cycles of heating and cooling before sufficient DNA is available for
analysis and generates large amounts of uracil that other polymerases
The V93Q Pol enzyme has been on the market since 2003, but the substantial
commercial impact has only occurred since an exclusive licensing agreement
was signed between Newcastle University and [Text removed for publication,
EV d] on 1 November 2008. [Company name removed for publication] sell V93Q
Pol using the brand name [Text removed for publication] (Ev b and c).
Income to Newcastle University from [Company name removed for
publication, EV d] in 2008 and 2009 was in the form of an agreed fee. From
2010 onwards income has been in the form of royalties on sales. Royalties
are shared 75% to Newcastle University and 25% to UCL, reflecting the fact
that Professor Pearl at UCL was an important collaborator in the early
stages. Most of the research that underpinned the development of the
enzyme was carried out at Newcastle University.
[Sales figures removed for publication, EV d]
Expanding commercial impact
At the end of 2011, the licensing agreement with [Company name removed for
publication] became non-exclusive. Two further licensing arrangements have
now been signed, one in November 2012 and one in February 2013, expanding
the commercial impact of the invention [Text removed for publication, EV
Sources to corroborate the impact
Ev a. Patent: Mutation of DNA polymerases from archaeobacteria.
Ev b. [Text removed for publication]
Ev c. [Text removed for publication]
Ev d. The contact details for the Newcastle University lawyer responsible
for managing the agreements can be made available on request, as can
confidential information confirming the commercial agreements.