Note: Descriptions are shown in the official language in which they were submitted.
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2050276
IMPROVED PRIMER EXTENSION REACTIONS
Background of the Invention
This invention was made with government support
including a grant from Department of Energy Grant No. DE-SG02-
88ER60688 and U.S. Public Health Service Grant No. A1-06045.
The U.S. government has certain rights to the invention.
This invention relates to methods for performing a
primer extension reaction, such as a DNA sequencing reaction, or
a polymerase chain reaction.
In a primer extension reaction an oligonucleotide
primer having homology to a single-stranded template DNA, e.g.,
genomic DNA, is caused to anneal to the template DNA. The
annealed mixture is then provided with a DNA polymerase in the
presence of nucleoside triphosphates under conditions in which
the DNA polymerase extends the primer to form a complementary
DNA strand to the template DNA. In a DNA sequencing reaction,
the primer is extended in the presence of a chain-terminating
agent, e.g., a dideoxynucleoside triphosphate, to cause base-
specific termination of the primer extension. Sanger et al., 74
Proc. Nat'1. Acad. Sci. 5463, 1977. In a polymerase chain
reaction two primers are provided, each having homology to
opposite strands of a double-stranded DNA molecule. After the
primers are extended, they are separated from their templates,
and additional primers caused to anneal to the templates and the
extended primers. The additional primers are then extended.
The steps of separating, annealing, and extending are repeated
in order to amplify the number of copies of template DNA. Saiki
et al., 239 Science 487, 1988.
Summary of the Invention
In a first aspect, the invention features a solution or
kit for use in extension of an oligonucleotide primer having a
first single-stranded region on a template molecule having a
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second single-stranded region, the first and second regions
being homologous. The solution or kit includes a first agent
able to cause extension of the first single stranded region of
the primer on the second single-stranded region of the template
in a reaction mixture, and a second agent able to reduce the
level of pyrophosphate in the reaction mixture below the level
produced during extension in the absence of the second agent.
As a result, in accordance with one aspect of the
invention, there is provided a solution or kit for use in a DNA
sequencing reaction, comprising a DNA polymerase, a chain-
terminating agent, and an agent able to reduce the amount of
pyrophosphate in a DNA polymerization reaction mixture.
By solution is meant any aqueous and/or buffered
liquid containing the components described above. These
components are present in the solution at concentrations
sufficient to perform their desired function. For example, the
second agent is present at a concentration sufficient to reduce
the level of pyrophosphate in the solution. By kit is meant a
container which holds one or more of the components of the
solution separately. For example, the first and second agents
are held in separate containers in solutions adapted to be
mixed together.
By causing extension of the oligonucleotide primer
is meant performing a reaction in which an oligonucleotide
primer having a single-stranded region is annealed, or
naturally occurs in the annealed state, with another
nucleic acid molecule which acts as a template upon which
the oligonucleotide primer can be
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extended by addition of nucleoside triphosphate~ to form
nucleic acid homologous to the template nucleic acid.
Generally, externsion entails providing a DNA polymerase
or RNA polymerise to covalently add nucl:eot:des,to the
primer.
A reaction mixture is any solution or solid
phase suitable for performing an extension reaction.
Generally, it is a licuid buffer containing nucleoside
or deoxynucleoside triphosphates and metal ions required
for an extension reaction. The mixture may also contain
any standard bufferirg~ agents and, for a DNA sequencing
reaction, one or more dideoxy:~ucleos:de triphosphates,
or an equivalent chain-terminating agent.
By reducing the level of pyrophosphate is meant
that the amount of pyrophosphate .n the reaction mixture
is reduced to an amount which has latle or no
significant effect on the extension of the primer on she
template. That is, the level of pyrophosphate is low
enough to reduce pyrophosphorolysis to an insignificant
level (less than 10~ the level of pyrophosphorolysis in
the presence of 300 uM pyrophosphate). Preferably.
the level of pyrophosphate is reduced to below 25uM,
even more preferably to below 5PM. This phase is
meant to include use of an agent, such as a
pyrophosphatase, which acts to prevent the build-up of
pyrophosphate, as well as remove it from a solution.
Hy homologous is meant that the two
single-stranded regions are able to form sufficient
non-covalent bonds between their respective nucleotides
to form a~stable double-stranded structure under
conditions normally used for annealing nucleic acids,
and for performing a primer extension reaction.
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In preferred embodiments, the first agent is a DNA
polymerase, most preferably chosen from Klenow, Taq polymerase,
a T7-type DNA polymerase (i.e., a polymerase similar to that in
a phage in which the DNA polymerase requires host thioredoxin
as a subunit, e.g., T7 DNA polymerase of the DNA polymerase of
T3, ~I, III, H, W31, gh-1, Y, AA1122, or Sp6), T4 DNA
polymerase, T5 DNA polymerase, X29 DNA polymerase and reverse
transcriptase; the second agent is an enzyme, most preferably a
pyrophosphatase, for example, a pyrophosphatase resistant to
heating at between 60°C and 95°C.
In a further aspect, the invention features an
improved method for extending an oligonucleotide primer having
a first single-stranded region on a template molecule having a
second single-stranded region, including providing a first
agent able to cause extension of the primer on the template.
The improvement is provision of a second agent able to reduce
the amount of pyrophosphate below the amount produced during
extension in the absence of the second agent.
As a result, according to a further aspect of the
present invention, there is provided an improved method for a
DNA sequencing reaction, including providing a DNA polymerase,
and a chain-terminating agent the improvement comprising:
providing an agent able to reduce the level of pyrophosphate
below the amount produced during said extension in the absence
of said agent.
In preferred embodiments, the method includes the
steps of providing at least one or two oligonucleotide primers
having single-stranded regions and at least one or two template
molecules having single-stranded regions, and annealing the
single-stranded regions of the primers and the templates to
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form an annealed mixture. The resulting annealed mixture is
provided with the first and second agents to cause extension of
the primers. The annealed mixture may also be provided with a
dideoxynucleoside triphosphate. The method may further include
5 the step of separating the primers from the templates after
their extension, and repeating the steps of providing primers,
extending the primers, and separating the primers.
In a related aspect, the invention features a method
for amplifying DNA, including performing a polymerase chain
l0 reaction in the presence of an agent able to reduce the amount
of pyrophosphate in the reaction below the amount produced
during a polymerase chain reaction in the absence of the agent.
Preferably, the agent is a pyrophosphatase.
In another related aspect, the invention features a method
for amplifying DNA comprising the step of performing a
polymerase chain reaction with a DNA polymerase, in the
presence of a agent able to reduce the amount of pyrophosphate
in said reaction below the amount produced during said reaction
in the absence of said agent.
In another related aspect, the invention features a
method for amplifying DNA including providing a solution of X29
DNA polymerase, a DNA to be amplified, and an agent able to
reduce the amount of pyrophosphate in the solution below that
amount produced in the absence of the agent.
In another related aspect, the invention features a
method for amplifying DNA comprising providing in a solution of
DNA polymerase and a DNA to be amplified an agent able to
reduce the amount of pyrophosphate in said solution below the
amount produced in said solution in the absence of said agent.
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5a
Applicants have determined that pyrophosphorolysis,
where an oligonucleotide chain is reduced in length, is
detrimental to a primer extension reaction. The
pyrophosphorolysis is caused by the availability of
pyrophosphate. For example, a polymerase chain reaction, as
described by Cetus (European Patent Application 0,258,017) and
by Saiki et al., 239 Science 487, 1988, is inhibited by
addition of pyrophosphate even at very low concentrations.
This pyrophosphorolysis can be prevented by providing an agent,
for example, a pyrophosphatase, capable of removing
pyrophosphate. Addition of pyrophosphatase to a polymerase
chain reaction greatly enhances the progress of that reaction,
and provides superior results compared to use of the method
without a pyrophosphatase. Similarly addition of a
pyrophosphatase to a DNA sequencing reaction provides more
uniformity in
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intensities of bands formed in a polyacrylamide gel used
to identify products of the sequencing reaction. This
unirormity is due to prevention of degradation of
specific DNA products by pyrophosphorolysis.
Other features and advantages of the invention
will be apparent from the following description of the
preferred embodiment thereof, and from the claims.
Description of the Preferred Embodiments
Any agent which is capable of inhibiting a
pyrophosphorolysis reaction is useful in this
invention. One way to inhibit pyrophosphorolysis .s to
break down any pyrophosphate that is generated during a
polymerase reaction, by adding the enzyme
pyrophosphatase. Even trace addition of a
pyrophosphatase (one thousanth the molar ratio of ANA
polymerase molecules in a solution) to a primer
extension reaction completely stabilizes oligonucieot:de
fragments produced in a polymerase reaction, by
preventing pyrophosphorolysis. The agent should be
added at a concentration sufficient to either catalyze
the hydrolysis of pyrophosphate in the reaction mixture
at a rate that will prevent accumulation of
pyrophosphate to a level that will lead to
~5 pyrophosphorolysis, or prevent accumulation of
pyrophosphate in any other manner. The amount of agent
needed is readily determined by standard techniques.
There follows an example of the use of
pyrophosphatase in a polymerase chain reaction. Tris
example is not limiting to this invention; those skilled
in the art will reccgnize that any primer extension
reaction will be benefited by ~he addition of an agent
as described above. Similarly, the use of
pyrophosphatase in the examples below is not limiting to
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this invention, other agents suitable for reducing the
effect of excess pyrophosphate in a primer extension
reaction are readily identified by those skilled in the
art. The relative ccncentrations of primer, DNA
polymerase, and pyrophosphatase suitable in the
. invention are readily determined by routine
experimentation, and are well known to those in the art.
It is preferable that a pyrophosphatase used is
this invention be resistant to heating at high
temperatures, since high temperatures are used in a
polymerase chain reac~:on, for example, _emgeratures
between 95°C to 100°C, althoucr te~r~neratures between
65°C and 95°C are also cemmoniy used. T:~us, it is
advantageous to provide a pyrophosphate resistant to
heating at 65°C to 95°C. Such a pyrophosphatase can ~e
readily obtained :rom any bacteriu.~~ that is naturally
able to grow and flourish at high temperatures. e.g.,
Thermus acuaticus. Most bacteria have
naturally-occurring pyrophosphatases, and those existing
in natural environments at high te:npera~ures will
therefore be suitable sources of this enzyme.
Use of a pyrophosphatase in a polymerase chain
reaction as described below with Taq polymerase allows
the reaction to run to completion--that is, to cause
depletion of all the provided deoxynucleoside
triphosphates. This allows diagnostic techniques which
make use of a polymerase chain reaction to be
automated. Assay for progress of -he reaction can
entail measurement of the generation of phosphate or the
generation of DNA from the deoxynucleoside triphosphates
(for example, by acid precipitation?, both of which are
simple and quick assays, instead of the necessity to run
a gel to detect the product of the polymerase chain
reaction.
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Example 1: PCR Reaction with Pyrophophatase
In this example DNA termed M13 Trx-F (the actual DNA
used is not critical in this invention) was amplified by
provision of a forward and reverse primer using a polymerase
chain reaction as follows: This method is generally described
in Saiki et al., supra. Trx-F DNA at a concentration of 0.4
picomoles was mixed with 1 ~1 Tris (1M, pH 8.5), 10 ~1 magnesium
chloride (15 mM), 6.7 ~1 of four deoxynucleoside triphosphates
(3 mM), 10 ~1 of forward primer (10 picomole; from ALN), 20 ~.,
reverse primer (10 picomole, New England BioLabs), 2 ~1 gelatin
(0.5%), and 55 ~1 distilled water. 0.5 ~,l of Taq polymerase (12
units, U.S. Biochemicals, Cleveland, Ohio) was then added and
the solution heated to 94°C for one minute, 50°C for one minute,
and 72°C, for two minutes and this cycle of heating repeated 40
times. Identical reactions were run in the absence or presence
of pyrophosphate at various concentrations (12 ~M, 37 ~M,
333 ~M, and 1 mM) and in the presence of pyrophosphatase (yeast
inorganic pyrophosphatase from Sigma, Catalog No. I-4503, used
without purification, or used after purification on an FPLC Mono
Q* column). Another source of pyrophosphatase is Worthington
yeast inorganic pyrophosphatase without further purification.
Generally, 0.001 units of yeast inorganic pyrophosphate (4ng)
are suitable in a reaction as described above. This amount may
of course be considerably greater, and may be less. The range
of concentrations is readily determined by routine
experimentation. The concentration need only be enough to lower
the level of pyrophosphate below about 5-50 ~M.
In the above reaction, pyrophosphate inhibited the
polymerase chain reaction at levels of 25 ~M or
*Trade-mark
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_ g _
greater. Pyrophosphatase reversed this inhibition and
stimulated production of the polymerase chain reaction
products by approximately two fold.
Example 2: Preparation of Heat Resistant Pvroohos~hatase
This is an example of purification of an
inorganic pyrophos~hatase from cells 4f Ther~us
aawaticus. Cells of T. aQUaticus were obtainl f:om ;.'.:e
American Type Culture Collection. 10 lute-rs of cells
were grown at 70°C using the growth medium of-Chien et
al. 127 J. Bacteriol. 1550 (1976). The cells were
harvested (-20 gm), resuspended in 40 m1 of~ i0%
sucrose, 50 mM ':ris riCl , pH 7.5, 5 :nM EDTA: '_y'sed by
three passages through a r=erch press: and cell debris
removed by centrifugation at 30,000 rpm, for 60-min in a
Hecicman 50Ti rotor. The supernatant was. treated with
streptomycin sulfate ~o remove DNA. 4 m1 of a 40%
streptomycin solution was added to 40 rnl supernatant.
mixed for 30 min., and centrifuged for 30 min at 8.000
rpm. The resulting supernatant was then treated wits
ammonium sulfate. No pyrophosphatase activity was
precipitated at 60% ammonium sulfate, but all was
precipitated by'70% ammonium sulfate: To 19 mI of
supernatant 7.2 gm ammonium sulfate (60%) was added.
mixed for 30 min., and spun for 30 min. at 8.000 rpm.
To the supernatant 3-gm ammonium sulfate (70%) was
added, mixed for 30 min., and spun far 3~ min. at 8,000
rpm. The pellet was resusperded in 20 ml 20 m~ ~Tr is-HC1
pH _7.5. 1 mM EDTA. 10% glycerol, 10 mM 2-mer~aotoethanol
(Buffer A) and then dialyzed overnight against 2 liters
of Suffer A. The dialysate was passed over a DEAE DE52
column (100 m1) ecuilibrated in Buf~er A, washed witz
300 ml of Buffer A + 50 mM NaCl, and-then run in a liter
gradient of buffer A containing from 50 mM to 54'0 mM
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NaCl. The pyrophophatase eluted at buffer A containing
125 mM NaCl. The eluate (60 mL) was dialyzed against 2
liters of 20 mM KP04 pH 7.4, 1 mM EDTA, 10 mM
2-mercaptoethanol, l0% glycerol (Buffer H) and loaded
onto a phosphocellulose column (100 ml) equilibrated is
buffer B. All of the pyrophosphatase activity flowed
through the column. This flow-through was then dialyzed
against 20 mM Tris HCl pH 7Ø 1 mM EDTA, 10% glycerol
(Buffer C), and applied to an FPLC monoQ column in
buffer C. A gradient, in Buffer C, containing 100 mM
NaCl to 250 mM NaCI was :un and the pyrophosphatase
activity eluted at :80 ~.M NaCl. Fractions with
pyrophosphazase activity were dialyzed against 20 :nM
KP04 pH 7.4, 0.1 mM EDTr, 50% glycerol, and stored at
-20°C.
This pyrophosphatase activity was not affected
by 40 cycles of a polymerase chain reaction, with eacz
cycle containing a 95°C. 1 min. heating step. Further,
the pyrophosphatase did not hydrolyze dNTPs, nor was it
inhibited by dNTPs in the reaction mixture. The
pyrophosphatase activity was assayed generally as
described by Chen et al. 28 Anal. Chem. 1756 (1956), and
Josse. 241 J. Biol. Chem. 1938 (1966).
Other Embodiments
Other embodiments are within the following
claims. For example, enzymes which use a protei~ primer
rather than a DNA primer, e.g., X29 DNA polymerase
which polymerizes double stranded DNA, can be used to
amplify DNA without need for denaturing heating steps or
reannealing steps. Blanco et al., DNA replication and
mutagenesis, A.S.:~. Chapter 12, 1988. Inclusion of a
pyrophosphatase, or its equivalent, in such an
amplification reaction will enhance the yield of DNA
amplified in this system.
