U.S. patent application number 13/729566 was filed with the patent office on 2013-05-23 for contamination-free reagents for nucleic acid amplification.
This patent application is currently assigned to GENERAL ELECTRIC COMPANY. The applicant listed for this patent is GENERAL ELECTRIC COMPANY. Invention is credited to Robert Scott Duthie, Gregory Andrew Grossman, John Richard Nelson.
Application Number | 20130130352 13/729566 |
Document ID | / |
Family ID | 40404832 |
Filed Date | 2013-05-23 |
United States Patent
Application |
20130130352 |
Kind Code |
A1 |
Nelson; John Richard ; et
al. |
May 23, 2013 |
CONTAMINATION-FREE REAGENTS FOR NUCLEIC ACID AMPLIFICATION
Abstract
Methods and kits for generating contamination-free reagents and
reagent solutions for use in nucleic acid amplification are
provided. Methods include processing of polymerase solutions,
nucleotide solutions and primer solutions to render contaminating
nucleic acid inert. The methods employ the proofreading activity of
the polymerase and/or exonucleases to de-contaminate the reagents
and reagent solutions. Methods and kits for contamination-free
nucleic acid amplification are provided.
Inventors: |
Nelson; John Richard;
(Clifton Park, NY) ; Duthie; Robert Scott;
(Schenectady, NY) ; Grossman; Gregory Andrew;
(Halfmoon, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GENERAL ELECTRIC COMPANY; |
Schenectady |
NY |
US |
|
|
Assignee: |
GENERAL ELECTRIC COMPANY
Schenectady
NY
|
Family ID: |
40404832 |
Appl. No.: |
13/729566 |
Filed: |
December 28, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11957534 |
Dec 17, 2007 |
8361712 |
|
|
13729566 |
|
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Current U.S.
Class: |
435/194 |
Current CPC
Class: |
C12Q 1/6848 20130101;
C12N 9/1241 20130101; C12Q 1/6848 20130101; C12Q 2521/319 20130101;
C12Q 2525/125 20130101 |
Class at
Publication: |
435/194 |
International
Class: |
C12N 9/12 20060101
C12N009/12 |
Claims
1. A kit for DNA amplification comprising: (a) a proofreading DNA
polymerase; (b) a nuclease resistant primer; and (c) an
exonuclease.
2. The kit of claim 1, further comprising a single stranded
DNA-binding protein.
3. The kit of claim 1, wherein the proofreading DNA polymerase
comprises a Phi29 DNA polymerase.
4. The kit of claim 1, wherein the exonuclease is selected from the
group consisting of exonuclease I, exonuclease III and combinations
thereof.
5. The kit of claim 1, wherein the proofreading DNA polymerase is a
decontaminated proofreading DNA polymerase.
6. The kit of claim 5, wherein the decontaminated proofreading DNA
polymerase is generated by a method comprising the steps of: (a)
providing a polymerase solution consisting essentially of a
proofreading DNA polymerase that is contaminated with a
contaminating nucleic acid; (b) contacting the polymerase solution
with a divalent cation to form a polymerase-cation mixture; and (c)
incubating the polymerase-cation mixture whereby the contaminating
nucleic acid is rendered inert by the proofreading DNA polymerase
to generate the decontaminated proofreading DNA polymerase, wherein
the contacting and the incubating steps are performed in the
absence of any substantial amount of free nucleotides (dNTPs), and
wherein the decontamination is performed in absence of an
exonuclease or DNAse.
7. The kit of claim 1, wherein the nuclease-resistant primer is a
decontaminated nuclease-resistant primer, and wherein the
decontaminated nuclease-resistant primer is generated by a method
comprising the steps of: (a) providing a contaminated
nuclease-resistant primer solution that consists of a
nuclease-resistant primer and a contaminating nucleic acid; (b)
contacting the primer solution with a nuclease and a divalent
cation; and (c) incubating the primer solution whereby the
contaminating nucleic acid is rendered inert by the nuclease to
generate the decontaminated nuclease-resistant primer.
8. The kit of claim 1, wherein the nuclease-resistant primer is an
exonuclease-resistant primer.
9. The kit of claim 1, further comprising decontaminated free
nucleotides (dNTPs), wherein the decontaminated free nucleotides
(dNTPs) is generated by: (a) contacting a nucleotide solution with
a nuclease and a divalent cation, wherein the nucleotide solution
comprises free nucleotides and a contaminating nucleic acid; and
(b) incubating the nucleotide solution to allow the nuclease to
render the contaminating nucleic acid inert.
10. The kit of claim 1, further comprising a non-proofreading DNA
polymerase.
11. A kit for DNA amplification comprising: a decontaminated
proofreading DNA polymerase, which is generated by a method
comprising the steps of: (a) providing a polymerase solution
consisting essentially of a proofreading DNA polymerase that is
contaminated with a contaminating nucleic acid; (b) contacting the
polymerase solution with a divalent cation to form a
polymerase-cation mixture; and (c) incubating the polymerase-cation
mixture whereby the contaminating nucleic acid is rendered inert by
the proofreading DNA polymerase to generate the decontaminated
proofreading DNA polymerase, wherein the contacting and the
incubating steps are performed in the absence of any substantial
amount of free nucleotides (dNTPs), and wherein the decontamination
is performed in absence of an exonuclease or DNAse.
12. The kit of claim 11, wherein the decontaminated proofreading
DNA polymerase is a decontaminated Phi29 DNA polymerase.
13. The kit of claim 11, further comprising a decontaminated
non-proofreading DNA polymerase.
14. A decontaminated proofreading DNA polymerase made by incubating
a proofreading DNA polymerase with a divalent cation in the absence
of any substantial amount of free nucleotides, wherein the
incubation is performed in the absence of any exonuclease or
DNAse.
15. The decontaminated proofreading DNA polymerase of claim 14,
wherein the polymerase is a Phi29 DNA polymerase.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 11/957,534, which was filed on Dec. 17, 2007, and entitled
CONTAMINATION-FREE REAGENTS FOR NUCLEIC ACID AMPLIFICATION.
BACKGROUND
[0002] A variety of techniques are currently available for
efficient amplification of nucleic acids even from a few starting
nucleic acid templates resulting in a large number of amplified
products. These include polymerase chain reaction (PCR), rolling
circle amplification (RCA) and strand displacement amplification
(SDA). Due to higher amplification efficiencies of these
techniques, even the slightest contamination of the
reagents/reagent solutions employed in such amplification reactions
with an undesired nucleic acid molecule may result in a huge amount
of false amplification products. If such an amplification were used
for diagnostic applications, this would likely result in a
false-positive diagnosis.
[0003] Reagents or reagent solutions that are used in nucleic acid
amplification reactions may get contaminated in various ways. For
example, contamination may arise from carry-over amplification
product (amplicons) of previous amplification reactions, from the
site from which the sample for amplification is collected, by
exogenous DNA in the laboratory environment or from reagents or
reagent solutions used for amplification reaction.
[0004] Various pre-amplification sterilization procedures have been
developed to minimize amplicon carry-over. For example,
deoxythymidine triphosphate (dTTP) is substituted for deoxyuridine
triphosphate (dUTP) in PCR amplifications to make PCR products
distinguishable from template DNA. Use of enzyme
uracil-N-glycosylase (UNG) in a pre-amplification step cleaves the
carry-over amplicons at the incorporated uracil residues. In
amplification reactions using the same primers and the same target
sequences, enzymatic removal of amplicons from previous similar
amplification reactions has also been reported. These methods take
advantage of the fact that the contaminant amplicon carries its
primer sites at or near the ends of the molecule whereas virtually
all other template DNA molecules not arising themselves from a
previous PCR reaction, do not have their primer sites so
located.
[0005] Single strand-specific exonuclease has been used for
amplicon de-contamination during strand displacement amplification
(SDA) reaction wherein either (or both) the target nucleic acid or
the amplicons are in single stranded form. In such methods, even
though both the target and amplicons are attacked, due to the short
length of amplicons (25-2,000 nucleotides) and their lack of
secondary structures, the amplicons are preferentially cleaved.
[0006] Use of selectively activable enzymes such as micrococcal
nuclease and of DNA-binding agents have been employed to
de-contaminate the reagent solution. Enzymatic, physical or
chemical pre-treatment of the sample has also been employed to
remove or inactivate a contaminating DNA that is originating from
the site from where the sample is collected.
[0007] Apart from amplicon carry-over, reagents and reagent
solutions commonly used to amplify nucleic acids may contain
unwanted nucleic acid contaminants that could potentially interfere
with standard nucleic acid amplification protocols and procedures.
Contaminating DNA may be much longer than that of a primer or an
amplicon and specific information about the contaminating DNA may
often be minimal. During amplification reactions, false
amplification products may also be formed by the inherent
contamination of the reagents used for such reactions. For example,
polymerization enzymes such as DNA polymerases that are used in
amplification reactions may inherently carry contaminating nucleic
acids. Standard protein purification techniques might not be
sufficient enough to de-contaminate such nucleic acid-binding
proteins effectively. So, there exists a need for specific
treatments to de-contaminate the reagents and the reagent solutions
used for amplification reactions.
FIELD OF INVENTION
[0008] The present invention relates generally to methods and kits
for removing contaminating nucleic acids from reagents or reagent
solutions used in nucleic acid amplification reactions. By
de-contaminating the reagents or reagent solutions, the present
methods help reduce the artifacts in DNA amplification reactions
and improve the amplification efficiency.
BRIEF DESCRIPTION
[0009] One or more of the embodiments of the present invention are
directed to methods for removing, digesting, degrading or otherwise
inactivating nucleic acids (e.g., contaminating nucleic acids),
which may be present in a reagent (e.g., a nucleic acid polymerase)
or a reagent solution (e.g., a buffer solution used in a reaction
mixture) used for nucleic acid amplification reactions. Generally,
these nucleic acids are referred as contaminating nucleic acids,
however, the methods of the present invention are generally
applicable to all nucleic acids that are desired to be removed,
digested, degraded or otherwise inactivated. According to the
methods of the present invention, the nucleic acids are altered by
digestion or degradation in a manner to prevent their further
activity or reactivity in nucleic acid amplification reactions. The
contaminating nucleic acids are either removed or inactivated to
render them inert. One or more of the embodiments of the present
invention help to increase the amplification efficiency of target
nucleic acids (e.g., a target DNA). For example, during
amplification reactions involving small amounts of target nucleic
acids (e.g., single molecule DNA amplification), it is advisable to
begin with reagents that are nucleic acid contamination-free to
reduce false-positives.
[0010] In some embodiments, the present invention provides methods
for processing a polymerase solution. The processing of the
polymerase solution comprises steps for de-contaminating the
polymerase solution. It may further comprise steps of using the
processed polymerase solution for amplification reactions. In one
embodiment, the method comprises the steps of contacting the
polymerase solution comprising a proofreading DNA polymerase and a
contaminating nucleic acid with a divalent cation and incubating
the polymerase solution to allow the proofreading DNA polymerase to
render the contaminating nucleic acid inert. In some embodiments,
the methods for self-cleaning the polymerase solution utilize the
intrinsic proofreading activity of the polymerase to degrade the
contaminating nucleic acid. In some other embodiments, additional
exonucleases are added to the polymerase solution to render the
contaminating nucleic acid inert.
[0011] In some embodiments, the present invention provides methods
to degrade a contaminating nucleic acid in a proofreading DNA
polymerase. The method comprises the steps of contacting the
proofreading DNA polymerase, comprising the contaminating nucleic
acid, with a divalent cation to form a polymerase-cation mixture
having a proofreading activity, and incubating the
polymerase-cation mixture to allow the proofreading DNA polymerase
to degrade the contaminating nucleic acid. In some embodiments, the
contacting step and the incubating step are performed in the
absence of any substantial amount of free nucleotides (dNTPs). In
some specific embodiments, an exonuclease such as exonuclease I or
exonuclease III or a combination of exonuclease I and exonuclease
III may be added to the solution comprising proofreading DNA
polymerase. In some embodiments, the method comprises
de-contaminating a Phi29 DNA polymerase solution.
[0012] In some embodiments, the present invention provides methods
for processing a primer solution comprising a nuclease-resistant
primer. The method comprises the steps of contacting a primer
solution with a nuclease and a divalent cation, wherein the primer
solution comprises a contaminating nucleic acid; and incubating the
primer solution to allow the nuclease to render the contaminating
nucleic acid inert. The primer solution may further comprise free
nucleotides and/or a single stranded DNA-binding protein. In some
embodiments, one or more of exonuclease is used in the reaction,
wherein one or more of the exonucleases is selected from the group
consisting of exonuclease I, exonuclease III and combinations
thereof.
[0013] In some embodiments, the present invention also provides
methods to amplify a target nucleic acid. The method comprises the
steps of (a) incubating a first solution with a first divalent
cation to render a first contaminating nucleic acid inert, wherein
the first solution comprises a proofreading DNA polymerase and the
first contaminating nucleic acid (b) incubating a second solution
with an exonuclease and a second divalent cation to render a second
contaminating nucleic acid inert, wherein the second solution
comprises a nuclease-resistant primer and the second contaminating
nucleic acid (c) inactivating the exonuclease in the second
solution (d) mixing the first solution and the second solution with
a third solution comprising the target DNA and (d) amplifying the
target DNA. The incubation of the first solution may be performed
in the absence of free nucleotides (dNTPs). The first solution may
further comprise an exonuclease and/or a single stranded
DNA-binding protein (SSB protein). The second solution may further
comprise free nucleotides (dNTPs). The target DNA may be amplified
using other known methods such as, but not limited to, isothermal
DNA amplification techniques such as rolling circle amplification
(RCA) or multiple displacement amplification (MDA).
[0014] In some embodiments, the present invention provides a kit
for DNA amplification. The kit may provide contamination-free
reagents for DNA amplification or may provide components that could
be used for making the reagents used in amplification reactions
contamination-free. In one example embodiment, the kit comprises a
proofreading DNA polymerase, a nuclease-resistant primer and an
exonuclease. The kit may further comprise a single stranded
DNA-binding protein and/or a buffer solution suitable for
performing a reaction for degrading a contaminating DNA. In one
example embodiment, the kit comprises a Phi29 DNA polymerase and a
nuclease-resistant primer comprising at least one phosphorothioate
nucleotide. In one specific example embodiment, the kit comprises
an exonuclease chosen from exonuclease I, exonuclease III and
combinations thereof.
DRAWINGS
[0015] These and other features, aspects and advantages of the
present invention will become better understood when the following
detailed description is read with reference to the accompanying
figures.
[0016] FIG. 1 is a schematic representation of the general
embodiment of an amplification reaction of the invention
[0017] FIG. 2 depicts the exonuclease treatment of reagent/buffer
solutions comprising free nucleotides to remove contaminating
nucleic acid. Lambda DNA is used as a non-limiting example of a
contaminating nucleic acid.
[0018] FIG. 3 illustrates an embodiment of the method for
processing a polymerase solution over time in which Phi29
polymerase is incubated with a divalent cation to degrade the
contaminating nucleic acid.
[0019] FIG. 4 illustrates an embodiment of the method for
processing a primer solution over time using exonuclease I or
exonuclease III or a combination of exonuclease I and exonuclease
III to degrade the contaminating nucleic acid and render it
inert.
[0020] FIG. 5 illustrates the effect of an embodiment of the
exonuclease treatment of a primer solution and a proofreading DNA
polymerase solution on DNA amplification
[0021] FIG. 6 illustrates the effect of an embodiment of the step
of incubating the proofreading DNA polymerase with an exonuclease
and magnesium ion on template DNA amplification. In this example
embodiment, the exonucleases were not deactivated prior to template
DNA amplification reaction.
[0022] FIG. 7 illustrates the effect of an embodiment of the step
of incubating Phi29 DNA polymerase and a primer solution with an
exonuclease on a template DNA titration. In this example
embodiment, pUC DNA is used as a template DNA and a
nuclease-resistant hexamer sequence, NNNN*N*N is used as a primer.
The template DNA is amplified using rolling circle
amplification.
[0023] FIG. 8 illustrates the effect of an embodiment of the step
of processing the reagents or reagent solutions with an exonuclease
on a template DNA amplification
[0024] FIG. 9 illustrates the effect of an embodiment of the step
of processing the reagents or reagent solutions with an exonuclease
and a single stranded DNA-binding protein (SSB protein) on a
template DNA amplification
[0025] FIG. 10 illustrates the effect of an embodiment of the step
of processing the proofreading DNA polymerase solution with an
exonuclease at 30.degree. C. for 60 min. on a template DNA
amplification
[0026] FIG. 11 illustrates the effect of an embodiment of the step
of processing the proofreading DNA polymerase solution with an
exonuclease at 4.degree. C. for 24 h. on a template DNA
amplification
DETAILED DESCRIPTION
[0027] One or more embodiments of the present invention are
directed at methods, reagents and kits useful for inactivating
nucleic acids or otherwise rendering them inert in, for example,
amplification reactions (e.g., DNA amplification reactions).
Inactivation of nucleic acids (e.g., contaminating nucleic acids),
may be desired in applications such as analytical, diagnostic,
prognostic or forensic applications and the like. The precise use,
including the choice of variables such as concentrations, volumes,
incubation times, incubation temperatures, and the like will depend
in large part on the particular application for which it is
intended. It is to be understood that one of skill in the art will
be able to identify suitable variables based on the present
disclosure. For convenience, in this detailed description of
various embodiments, the disclosed methods generally relate, but
are not limited to, the removal of contaminating DNA in reagents
and reagent solutions, such as those reagents and solutions for use
in amplification reactions (e.g., PCR or isothermal DNA
amplification methods). It will be within the ability of those
skilled in the art, however, given the benefit of this disclosure,
to select and optimize suitable conditions for using the methods in
accordance with the principles of the present invention, suitable
for these and other types of applications.
[0028] To more clearly and concisely describe and point out the
subject matter of the claimed invention, the following definitions
are provided for specific terms, which are used in the following
description and the appended claims. Throughout the specification,
exemplification of specific terms should be considered as
non-limiting examples.
[0029] The singular forms "a", "an" and "the" include plural
referents unless the context clearly dictates otherwise.
Approximating language, as used herein throughout the specification
and claims, may be applied to modify any quantitative
representation that could permissibly vary without resulting in a
change in the basic function to which it is related. Accordingly, a
value modified by a term such as "about" is not to be limited to
the precise value specified.
[0030] As used herein, the term "contaminating nucleic acid" refers
to nucleic acid, which is present in a reagent, a reagent solution,
or an apparatus, but is otherwise undesirable. That is,
contaminating nucleic acid is any nucleic acid, which is not
intended to be amplified, further characterized or present in an
assay to be performed. In some embodiments, the contaminating
nucleic acid is a deoxyribonucleic acid (DNA). For example, a DNA
that is present in a reagent or reagent solution suitable for
performing a DNA synthesis reaction, prior to adding a DNA template
to be amplified, is considered to be a contaminating nucleic acid.
The contaminating nucleic acid in a DNA synthesis reaction may act
as a potential DNA template or a primer and thus participate in the
DNA synthesis reaction, resulting in unwanted amplification
products. So, it is desirable to remove such contaminating nucleic
acid prior to addition of the DNA template to be amplified such
that when the DNA template to be amplified is added to the
solution, the contaminating nucleic acid will not interfere with
the DNA synthesis reaction. Prior removal of contaminating DNA from
the reagents and reagent solutions is generally desired to reduce
artifacts during DNA synthesis reaction if the DNA template to be
amplified is available only in limited amounts. In some
embodiments, the contaminating nucleic acid is a ribonucleic acid
(RNA).
[0031] As used herein, the term "render inert" refers to the
altering or modifying of nucleic acid(s) such that the nucleic
acid(s) cannot interfere with any subsequent chemical and/or
biological reactions. The nucleic acid(s) can be rendered inert by
chemical modification of the nucleic acid, for example, by removal
of one or more functional groups such that the nucleic acid is
unable to react with a DNA template or polymerase, for example.
Nucleic acid may also be rendered inert by degrading or digesting
the nucleic acid, for example, using an enzyme. Depending on the
nature of the reagents (e.g., an enzyme), the mechanism by which
the nucleic acid is rendered inert may vary. For example, in
embodiments where the proofreading DNA polymerase is rendering the
nucleic acid inert, the DNA may be rendered inert by the
exonuclease activity of the proofreading DNA polymerase. In
embodiments involving Phi29 DNA polymerase, the contaminating DNA
may be rendered inert by the 3'.fwdarw.5' exonuclease activity of
the Phi29 DNA polymerase. Here, the nucleic acid is rendered inert
by digesting the nucleic acid to produce free nucleotides having a
3'-hydroxyl group and a 5'-phosphate group. Such nucleotides do not
participate in subsequent DNA synthesis reaction.
[0032] As used herein, the term "render contaminating nucleic acid
inert" refers to the process of modifying contaminated nucleic
acids so that they cannot substantially interfere with subsequent
chemical analysis and/or procedures, such as, but not limited to,
isothermal DNA amplification or PCR. In some embodiments, this is
achieved by degrading or digesting the contaminating nucleic acid.
In some embodiments, rendering contaminating nucleic acid inert
refers to a complete removal or a reduction in the amount of
contaminating nucleic acid so that the contaminating nucleic acid
does not interfere with the further biological/chemical
reactions.
[0033] As used herein the terms "digesting" or "degrading" refer to
breaking of bonds, for example, phosphodiester bonds, between two
or more chemical groups. Preferably, digesting refers to breaking
bonds between two or more nucleotides, such that the free
nucleotides or nucleotide fragments are produced. In some
embodiments, the digesting refers to breaking of bonds between two
or more nucleotides such that the products are rendered inert. The
digestion may lead to products, which can no longer react with the
other components in the solution or participate in subsequent
reactions, for example, in a DNA polymerization reaction. For
example, digestion of contaminating nucleic acids may lead to
nucleotides or nucleotide fragments, which cannot act as a primer
or a template for a subsequent DNA synthesis reaction.
[0034] As used herein, the terms "reagent solution" or "solution
suitable for performing a DNA synthesis reaction" refer to any or
all solutions, which are typically used to perform an amplification
reaction/DNA synthesis. It includes, but is not limited to,
solutions used in isothermal DNA amplification methods and/or PCR.
The solution suitable for DNA synthesis reaction may comprise
buffer, salts, and nucleotides. It may also further comprise
primers and a DNA template to be amplified.
[0035] As used herein, the term "incubating" refers to the process
of keeping a solution or reaction mixture at a pre-determined
temperature and pressure for a pre-determined period of time to
achieve a specific reaction. The temperature and the period of
incubation are suitably selected such that the purpose of the
incubation (e.g., rendering contaminating nucleic acid inert) is
achieved at the end of incubation. The incubation time and
temperature may vary depending on the kinetic properties of the
reagents/enzyme that are involved in the reaction. Depending on the
nature and properties of the reagents/solutions involved, one
skilled in the art, given the benefit of this disclosure, will be
able to select suitable temperature and time period for
incubation.
[0036] As used herein the term "reaction mixture" refers to the
combination of reagents or reagent solutions, which are used to
carry out one or more chemical analysis or biological assays. In
some embodiments, the reaction mixture includes all necessary
components to carry out a DNA synthesis/amplification reaction.
[0037] As used herein, the term "amplification" or the term
amplifying refers to the production of multiple copies of a target
nucleic acid sequence or the production of multiple nucleic acid
sequence copies that are complementary to the target nucleic acid
sequence.
[0038] As used herein, the term "nucleotide" refers to both natural
and modified nucleoside phosphates. The term "nucleoside" refers to
a compound having a purine, deazapurine, pyrimidine or a modified
base linked at the 1' position or at an equivalent position to a
sugar or a sugar substitute (e.g., a carbocyclic or an acyclic
moiety). The nucleoside may contain a 2'-deoxy, 2'-hydroxyl or
2',3'-dideoxy forms of sugar or sugar substitute as well as other
substituted forms. The sugar moiety in the nucleoside phosphate may
be a pentose sugar, such as ribose, and the phosphate
esterification site may correspond to the hydroxyl group attached
to the C-5 position of the pentose sugar of the nucleoside. A
nucleotide may be, but not limited to, a deoxyribonucleoside
triphosphate (dNTP).
[0039] The term "free nucleotides", as used herein, refers to the
nucleotides that are free in the solution or reaction mixture. The
free nucleotides may or may not participate in a polymerization
reaction (e.g., DNA synthesis reaction). The chances of
participation or reactivity of the free nucleotides in a
polymerization reaction depends on the chemical nature of the
nucleotides that are freely available. For example, if the free
nucleotide is dNTP, it is capable of participating in a
polymerization reaction. The term "substantial amount of free
nucleotides" refers to the minimum amount of nucleotides that is
required to switch the primary activity of a proofreading DNA
polymerase from its exonuclease activity/proofreading activity to
its DNA synthesis activity.
[0040] The term "oligonucleotide", as used herein, refers to
oligomers of nucleotides or derivatives thereof. Throughout the
specification, whenever an oligonucleotide is represented by a
sequence of letters, the nucleotides are in 5'3' order from left to
right. In the letter sequence, letter A denotes adenosine, C
denotes cytosine, G denotes guanosine, T denotes thymidine, W
denotes A or T and S denotes G or C. N represents a random nucleic
acid base (for example, N may be any of A, C, G, U or T). +N
represents a synthetic, locked random nucleotide and *N represents
a phosphorothioate modified random nucleotide.
[0041] As used herein, the term "primer" refers to a short linear
oligonucleotide that hybridizes to a target nucleic acid sequence
(e.g., a DNA template to be amplified). Primers may be specific
primers or random primers. The specific primers are designed to
have a sequence, which is the reverse complement of a
pre-determined region of the target nucleic acid to which it
anneals. Both the upper and lower limits of the length of the
primer are empirically determined. The lower limit on primer length
is the minimum length that is required to form a stable duplex upon
hybridization with the target nucleic acid. Very short primers
(usually less than 3 nucleotides long) do not form
thermodynamically stable duplexes with target nucleic acid under
hybridization conditions. Upper limit is determined by the
possibility of having a duplex formation in a region other than the
pre-determined nucleic acid sequence in the target nucleic acid.
Suitable primer lengths are in the range of about 3 to about 100
nucleotides long. Suitable primer lengths may be about 3 to about
40 nucleotides long or may be about 3 to about 25 nucleotides long.
In some embodiments, suitable primers are 6 nucleotides-long
hexamers.
[0042] As used herein the term "DNA polymerase" refers to any
enzyme that catalyzes the production/synthesis of a new DNA. DNA
polymerase uses an existing DNA or RNA as a template for DNA
synthesis and catalyzes the polymerization of deoxyribonucleotides
alongside the template strand, which they `read`. The newly
synthesized DNA strand is complementary to the template strand. DNA
polymerase can add free nucleotides only to the 3-hydroxyl end of
the newly forming strand. It synthesizes oligonucleotides via
transfer of a nucleoside monophosphate from a nucleoside
triphosphate (NTP) or deoxyribonucleoside triphosphate (dNTP) to
the 3'-hydroxyl group of a growing oligonucleotide chain. This
results in elongation of the new strand in a 5'.fwdarw.3'
direction. DNA polymerase can only add a nucleotide onto a
pre-existing 3'-OH group. So, to begin a DNA synthesis reaction, a
DNA polymerase needs a primer at which it can add the first
nucleotide. Suitable primers include RNA and DNA.
[0043] As used herein the term "proofreading DNA polymerase" refers
to any DNA polymerase that is capable of correcting its errors
while performing DNA synthesis. Proofreading DNA polymerase
possesses a 3'.fwdarw.5' exonuclease activity apart from its
polymerase activity and this exonuclease activity is referred here
as proofreading activity. Proofreading activity of such polymerases
correct mistakes in the newly synthesized DNA. During DNA
synthesis, when an incorrect base pair is recognized, the
proofreading DNA polymerase reverses its direction by one base pair
of DNA. The 3'.fwdarw.5' exonuclease activity of the enzyme allows
the incorrect base pair to be excised (proofreading activity).
Following base excision, the polymerase re-inserts the correct base
and DNA synthesis continues. When free dNTPs are present in the
solution/reaction mixture suitable for DNA synthesis, the primary
activity of the proofreading DNA polymerase is DNA synthesis.
However, when dNTPs are not available for DNA synthesis reaction,
the primary activity of the proofreading DNA polymerase is its
3'.fwdarw.5' exonuclease activity. So, when dNTPs are not available
for DNA synthesis, the proofreading DNA polymerases, due to their
exonuclease activity, are capable of digesting/degrading DNA.
Proofreading DNA polymerases may be or may not be specific toward
digestion of DNA single-strands. In some embodiments, proofreading
DNA polymerase include any DNA polymerase that is capable of
digesting/degrading nucleic acids in the absence of substantial
amounts of dNTPs in the reaction mixture.
[0044] Some of the proofreading DNA polymerases require the
presence of a divalent cation for their proofreading activity as
well as for their polymerase activity. Suitable divalent cations
that can switch on the proofreading activity of the proofreading
polymerases include, but are not limited to, magnesium and
manganese.
[0045] In accordance with one or more embodiments, a method for
processing a polymerase solution is disclosed. Processing may
involve steps for de-contamination of the polymerase as well as its
subsequent optional use in further reactions, for example, nucleic
acid amplification. In some embodiments, the processing of the
polymerase solution is performed to render a contaminating nucleic
acid inert.
[0046] Nucleic acid contamination of a polymerase may either be an
inherent contamination or an extraneous contamination. Inherent
contamination refers to the contamination of the polymerase by
itself; i.e., the polymerase is intrinsically contaminated with a
contaminating nucleic acid. Being a nucleic acid-binding enzyme,
conventional methods of protein purification is often insufficient
to devoid the polymerase with contaminating nucleic acids. Thus,
the polymerase used for nucleic acid amplification may inherently
be contaminated with a contaminating nucleic acid, for example a
contaminant DNA. The nucleic acid thus inherently present in the
polymerase could interfere with a subsequent biological/chemical
reaction, such as a template DNA amplification. For example, such
contaminating nucleic acids may act as a primer or as a template
DNA during subsequent DNA amplification reaction. In embodiments
where the contaminant DNA is present in the polymerase itself, the
methods may comprise self-cleaning the polymerase by rendering the
contaminating DNA inert. A solution comprising the polymerase may
also be contaminated by extraneous means even though the polymerase
by itself was devoid of any contaminating nucleic acids. For
example, the polymerase solution may become contaminated by
contaminants arising from lab environments, from the
buffers/solutions used for making polymerase solution and the like.
In such embodiments, the processing of the polymerase solution may
comprise rendering the contaminating nucleic acid inert.
[0047] In some embodiments, the polymerase solution comprises a
proofreading DNA polymerase. In such embodiments, the method for
processing the polymerase solution comprises the steps of
contacting the polymerase solution comprising a proofreading DNA
polymerase and a contaminating nucleic acid with a divalent cation
and incubating the polymerase solution to allow the proof-reading
DNA polymerase to render the contaminating nucleic acid inert. In
some embodiments, the contaminating nucleic acid may comprise a
DNA.
[0048] In some embodiments, a method to degrade a contaminating
nucleic acid in a proofreading DNA polymerase comprises the steps
of contacting the proofreading DNA polymerase containing the
contaminating nucleic acid with a divalent cation to form a
polymerase-cation mixture having a proofreading activity and
incubating the polymerase-cation mixture to allow the proofreading
DNA polymerase to degrade the contaminating nucleic acid. In one
example embodiment, the proofreading DNA polymerase comprises a
contaminating DNA and the processing of the proofreading DNA
polymerase solution is performed to render the contaminating DNA
inert. Incubating with divalent cation activates the proofreading
activity of the proofreading DNA polymerase and the nucleic acid is
rendered inert by the intrinsic exonuclease activity of the
proofreading DNA polymerase. In some embodiments, the exonuclease
activity of the proofreading DNA polymerase may be specific to
single stranded DNA. The contacting and the incubating steps are
performed in the absence of any substantial amount of free
nucleotides (dNTPs). In one embodiment, the contacting and the
incubating steps are performed in the absence of dNTPs. When dNTPs
are absent in the solution, the primary activity of the
proofreading DNA polymerase in presence of the divalent cation is
not the DNA synthesis but is its exonuclease activity/proof reading
activity. The term "substantial amount of free nucleotides" refers
to the minimum amount of nucleotides that is required to switch the
primary activity of the proofreading DNA polymerase from the
exonuclease activity/proofreading activity to the DNA synthesis
activity. In some embodiments, the contaminating nucleic acid is
digested or degraded to make them inert. The digestion or
degradation may result in a complete removal of the contaminating
nucleic acid or may result in a reduction of its amount to an
extent that it does not interfere with subsequent steps, for
example, a DNA amplification reaction.
[0049] In some embodiments, the proofreading DNA polymerase
comprises a thermally stable DNA polymerase. Proofreading DNA
polymerase may be a thermophilic DNA polymerase or a mesophilic DNA
polymerase. Any proofreading DNA polymerase known in the art could
be used in the present invention. Examples of proofreading
polymerases that are suitable for use in the present invention
include, but not limited to, Phi29 DNA polymerase, hi-fidelity
fusion DNA polymerase (for e.g., Pyrococcus-like enzyme with a
processivity-enhancing domain from New England Biolabs, MA), Pfu
DNA polymerase from Pyrococcus furiosus (Strategene, Lajolla,
Calif.), Klenow fragment from DNA polymerase I of E. coli, T7 DNA
polymerase, T4 DNA polymerase, DNA polymerase from Pyrococcus
species GB-D (New England Biolabs, MA) and DNA polymerase from
Thermococcus litoralis (New England Biolabs, MA).
[0050] Any divalent cation that can activate the
exonuclease/proofreading activity of a proofreading DNA polymerase
may be used in the methods. Suitable divalent cations include, but
are not limited to manganese and magnesium ions. Depending upon the
proofreading polymerase and the divalent cation used, the
concentration of the divalent cation that is required for the
proofreading DNA polymerase to render the contaminating DNA may
vary. Typically a molar excess of divalent cations are used with
respect to the proofreading DNA polymerase. Under such conditions,
majority of the proofreading DNA polymerases are proofreadingly
active. One skilled in the art given the benefit of this disclosure
will be able to select and optimize the concentration of the
divalent cations.
[0051] In some embodiments, magnesium ions are used as a suitable
divalent cation. Usually, the concentration of the magnesium ions
may range from about 5 mM to about 50 mM. In some embodiments, the
concentration of the magnesium ions ranges from about 10 mM to
about 30 mM. In some embodiments, 20 mM magnesium ions are
used.
[0052] Proofreading DNA polymerase solution is incubated with the
divalent cation for a specified amount of time that is sufficient
to render the contaminating nucleic acid inert. The incubation time
typically varies with the kinetic properties of the proofreading
DNA polymerase and the divalent cation that are being used. The
incubation time may also depend on the temperature at which the
incubation is performed. The incubation time that is required to
render the contaminating nucleic acid inert may be optimized by
analyzing the extent of de-contamination. The extent of
de-contamination can be tested by various techniques known in the
art for characterizing the presence of nucleic acids. For example,
after the incubating step, a DNA polymerase synthesis reaction may
be carried out without adding any target template DNA to determine
if contaminant DNA is amplified (false-positive signal). Extent of
the reduction of DNA contamination could be assessed either by the
absence of false-positive signal (no contaminant DNA getting
amplified) or by the extended kinetics of contaminant DNA
amplification (slower rate of contaminant DNA amplification).
Suitable incubation time may range from about 5 min to about 24 h.
In some embodiments, the incubation time may range from about 1 min
to 100 min.
[0053] The temperature at which the incubation is performed depends
primarily on the nature of the proofreading DNA polymerase used.
The maximum temperature that could be used in particular reaction
is limited by the stability of the proofreading DNA polymerase and
the minimum temperature that could be employed for the incubation
is limited by the proofreading activity of the proofreading DNA
polymerase at that temperature. For example, when a thermally
stable DNA polymerase is used, the incubation may be performed at a
temperature as high as 105.degree. C. In some embodiments, the
incubation temperature may range from about -20.degree. C. to about
95.degree. C. In some embodiments, the suitable incubation
temperature ranges from about 4.degree. C. to about 45.degree. C.
In some embodiments, the incubation is performed at a temperature
between about 10.degree. C. to about 35.degree. C. In one
embodiment, the incubation is performed at about 30.degree. C. for
about 1 h.
[0054] In some embodiments, a single stranded DNA-binding protein
(SSB protein), which binds preferentially to single stranded DNA,
is added to the solution comprising the proofreading DNA
polymerase. The SSB protein may either be added to the polymerase
solution prior to the addition of the divalent cation or it may be
added to the polymerase-cation mixture. In some embodiments, the
additions of SSB proteins assist the intrinsic exonuclease activity
of the proofreading DNA polymerase. Suitable SSB proteins that
could be used in the present invention include, but not limited to,
extremely thermostable single stranded DNA-binding protein (ET SSB
from New England Biolabs, MA), E. coli RecA, Tth RecA (RecA homolog
isolated from Thermus thermophilus from New England Biolabs, MA),
phage T4 gene-32 protein and E. coli SSB protein.
[0055] In some embodiments, an exonuclease is added to the solution
comprising the proofreading DNA polymerase. The exonuclease may
either be added to the polymerase solution prior to the addition of
the divalent cation or it may be added to the polymerase-cation
mixture. In some embodiments, the added exonuclease is a double
strand-specific exonuclease. In some embodiments, the added
exonuclease is a DNA double strand-specific exonuclease and it
degrades a double-stranded DNA preferentially. The added
exonuclease may either be a 5'.fwdarw.3' (i.e., degrades a DNA from
the 5' end) or a 3'.fwdarw.5' exonuclease. In some embodiments, a
combination of exonucleases is used. Non-limiting examples of
suitable exonucleases that could be used in the present invention
include exonuclease I, exonuclease III, exonuclease VII,
exonuclease T, Mung Bean nuclease, Nuclease BAL-31, T7 gene 6
exonuclease, spleen exonuclease, T5 D15 exonuclease and lambda
exonuclease.
[0056] In yet another embodiment, an endonuclease is added to the
solution comprising the proofreading DNA polymerase. This is
particularly useful in embodiments wherein the contaminating
nucleic acid may include a circular DNA. Endonucleases act on
circular DNA and nick them. Once the nick is made, the proofreading
DNA polymerase or the exonuclease can act on the contaminant,
nicked DNA and degrade them to make them inert. Nonlimiting
examples of suitable endonucleases include DNAses such as DNAse
I.
[0057] In some embodiments, the solution comprising the
proofreading DNA polymerase may further comprise a non-proofreading
DNA polymerase. Suitable examples of non-proofreading DNA
polymerase that could be used include, but not limited to Taq DNA
polymerase, large fragment of Bst DNA polymerase, exo (-) DNA
Polymerase gene from Pyrococcus species GB-D (New England Biolabs,
MA), exo (-) DNA Polymerase from Thermococcus litoralis (New
England Biolabs, MA).
[0058] In some embodiments, the processing of the proofreading DNA
polymerase solution include the steps of (a) contacting the
proofreading DNA polymerase containing the contaminating nucleic
acid with a divalent cation to form a polymerase-cation mixture
having a proofreading activity (b) optionally adding at least one
or more of exonuclease (c) optionally adding one or more of SSB
protein (d) optionally adding one or more of endonuclease and (e)
incubating the polymerase-cation mixture to degrade the
contaminating nucleic acid. The addition of the divalent cation,
the exonuclease, the SSB protein and the exonuclease to the
solution comprising proofreading DNA polymerase and the
contaminated nucleic acid may either be performed sequentially or
simultaneously. In embodiments where the sequential addition is
performed, the addition may be carried out in any particular order.
For example, in some embodiments, the exonuclease and the divalent
cation may be mixed first and then added to the proofreading DNA
polymerase solution followed by the SSB protein. In some other
embodiments, the proofreading DNA polymerase solution may be
contacted with the SSB protein first and then the exonuclease and
the divalent cation could be added. One skilled in the art, given
the benefit of this disclosure, will be able to optimize these
conditions.
[0059] In some embodiments, the proofreading DNA polymerase
solution comprises a Phi29 DNA polymerase. In some embodiments, the
Phi29 DNA polymerase solution containing a contaminated nucleic
acid is incubated with a divalent cation at a specified temperature
for a period of time that is sufficient to render the contaminating
DNA inert. In some embodiments, the contaminating nucleic acid is a
DNA. In some embodiments, magnesium ions are used as a suitable
divalent cation. In some embodiments, the Phi29 DNA polymerase
comprises a contaminating DNA; i.e., the contaminating DNA is
inherently present with the Phi29 DNA polymerase. In some
embodiments, the contaminating DNA is rendered inert by the
intrinsic 3'.fwdarw.5' exonuclease activity of the Phi29 DNA
polymerase upon incubation with magnesium ions. The contaminating
DNA is digested to produce free nucleotides having a 3'-hydroxyl
group and a 5'-phosphate group (e.g.,
deoxyribonucleoside-5'-monophosphate). In some embodiments, the
Phi29 DNA polymerase is incubated with the divalent cation at a
temperature that ranges between about -20.degree. C. to about
42.degree. C. In some embodiments, the Phi29 DNA polymerase is
incubated with magnesium ions at a temperature ranging from about
4.degree. C. to about 40.degree. C. In some embodiments, the
incubation is performed at a temperature between about 25.degree.
C. to about 35.degree. C. In some embodiments, the incubation is
performed at 30.degree. C. The concentration of the magnesium ions
required for the reaction depends on the concentration of the Phi29
DNA polymerase used. Typically, a molar excess of magnesium ions is
used for the de-contamination reaction so that substantially all
the Phi29 DNA polymerase is activated toward proofreading. In some
embodiments, the range of magnesium concentration varies from about
5 mM to about 50 mM. Incubation period may ranges from about 1 min.
to about 24 h. In some embodiments, the incubation period ranges
from about 10 min. to 100 min. In some embodiments, the Phi29 DNA
polymerase is incubated with magnesium ions at 4.degree. C. for
about 24 h. In some embodiments, the Phi29 DNA polymerase is
incubated with magnesium ions at 37.degree. C. for about 80 min. In
some other embodiments, the Phi29 DNA polymerase is incubated with
20 mM magnesium ions at 30.degree. C. for about 60 min.
[0060] The effectiveness of the removal of a contaminating DNA from
a polymerase solution may depends on various factors such as (i)
the specific proofreading DNA polymerase used (ii) the amount
and/or length of the contaminating DNA (iii) the nature and/or
amount of SSB protein used, if any (iv) the nature and/or amount of
exonuclease used, if any (v) the reaction conditions (e.g., pH,
salt and temperature) (vi) the amount and/or type of the divalent
cation used and the like. One skilled in the art, given the benefit
of this disclosure, will be able to empirically optimize the system
by varying one or more of these variables and arrive at conditions
that render the contaminating nucleic acid inert most
efficiently.
[0061] The processing of the polymerase solution may further
comprise the steps of using the processed polymerase solution for
specific applications, for example, a nucleic acid polymerization
reaction. In some embodiments, after the incubation with a divalent
cation, once the contaminating nucleic acid is rendered inert, the
incubated mixture can be directly used for a target DNA
amplification reaction. In specific examples, a primer is added to
the processed polymerase solution along with free nucleotides
(dNTPs) and a DNA template. No removal of the divalent cation from
the processed polymerase solution is necessary since the primary
activity of the proofreading DNA polymerase automatically switches
from proofreading to polymerization (DNA synthesis) once free dNTPs
are present in the reaction mixture. In contrast, the presence of
divalent cations may also be a necessary requirement for a
subsequent reaction in some specific embodiments. Removal of
degraded contaminating nucleic acids from the processed polymerase
solution is also not needed since they have been rendered inert and
cannot interfere with the subsequent DNA synthesis reaction. Even
in the embodiments where an exonuclease is employed for processing
the proofreading DNA polymerase solution, removal or inactivation
of the exonuclease may not be required prior to a subsequent DNA
amplification reaction if the concentration of the exonucleases is
selected in such a way that they does not significantly interfere
in the subsequent amplification reaction. So, no purification steps
are necessary prior to the use of the processed polymerase solution
in the subsequent DNA synthesis reaction. The methods provided
herein to process a polymerase solution and/or its subsequent use
in a DNA amplification reaction can either be manually performed or
be automated. The embodiments of the present invention for removing
contaminated nucleic acids, without the need to separate the
digestion products prior to performing a DNA synthesis reaction,
may be particularly useful for automating the entire process.
[0062] The DNA template to be amplified (target DNA template) may
either be single-stranded or double-stranded. The DNA template can
be a circular DNA, a linear DNA or a nicked DNA. The DNA template
may be a genomic DNA or a cDNA. The free nucleotides used for the
DNA template amplification may (dNTPs) include, but are not limited
to, dATP, dGTP, dCTP and dTTP. Other components such as suitable
buffers, salts and the like may also be added to the processed
polymerase solution to allow the DNA amplification to occur
efficiently. In embodiments, where an exonuclease is used in the
processing of the polymerase solution, the deactivation of the
exonuclease may or may not be required prior to the subsequent DNA
template amplification.
[0063] The DNA template may be amplified using any of a variety of
DNA amplification methods known in the art. For example, the
amplification of the DNA template may be performed using thermal
cycling methods or using isothermal DNA amplification methods.
Non-limited examples for DNA amplification methods that could be
used in the present invention include polymerase chain reaction
(PCR), ligase chain reaction (LCR), self-sustained sequence
replication (SSR), nucleic acid sequence-based amplification
(NASBA) and amplification with Q.beta.-replicase. In some specific
embodiments, a DNA template is amplified using rolling circle
amplification (RCA) method. The RCA may either be a linear RCA
(LRCA) or an exponential RCA (ERCA). In some embodiments,
multiply-primed rolling circle amplification (MPRCA) is employed.
In some other embodiments, a DNA template is amplified using strand
displacement amplification reaction (SDA). In another embodiment,
the DNA template is amplified using multiple displacement
amplification (MDA).
[0064] The primers used in the amplification reaction typically
depend on the sequence of the DNA template to be amplified and the
selected amplification method. One skilled in the art, given the
benefit of this disclosure, will be able to design and select
suitable primers depending on the sequence and the nature of the
DNA template to be amplified. Either a single primer or multiple
primers could be used for amplification. The primer employed in the
present disclosure may either be a specific primer or a random
primer. Specific primers have or are engineered to have, a
nucleotide sequence that is complementary, in the Watson-Crick
sense, to a sequence present in the target DNA template. Random
primers have nucleotide sequences unrelated to the nucleotide
sequences of the DNA template resulting in hybridization of the
primers with the DNA template at random locations. In some
embodiments, the primer comprises a nuclease-resistant primer, for
example, a primer resistant to an exonuclease.
Exonuclease-resistant primers useful in the methods disclosed
herein may include modified nucleotides to make them resistant to
the exonuclease digestion. For example, a primer may possess one,
two, three or four phosphorothioate linkages between nucleotides at
the 3' end of the primer sequence. In some embodiments, the present
invention relate to processes wherein the primers contain at least
one nucleotide that makes the primer resistant to degradation,
particularly by an exonuclease and more particularly by a
3'.fwdarw.5' exonuclease. The modified nucleotide may be a
phosphorothioate nucleotide. The modified nucleotide is commonly a
3'-terminal nucleotide but the method of the present invention also
relates to embodiments wherein such a nucleotide is located at a
position other than the 3'-terminal position. When the modified
nucleotide is located at positions other than the 3'-terminal end
of a primer sequence, the 3'-terminal nucleotide of said primer may
be removed by the 3'.fwdarw.5' exonuclease activity. In some
embodiments, a random hexamer primer is used that is resistant to
3'.fwdarw.5' exonuclease activity. In some embodiments, primers
comprising the sequences such as WWNN*N*S or NNNN*N*N is used as a
suitable primer. In these cases, the primer sequences may have two
phosphorothioate nucleotides at the 3'-terminal end (* represents a
phosphorothioate bond between the nucleotides). In some specific
embodiments, multiple primers are used for the DNA template
amplification. In some embodiments, the multiple primers are
selected from the group consisting of primers sensitive to
exonuclease activity, primers resistant to exonuclease activity and
a mixture of primers sensitive to exonuclease activity and
resistant to exonuclease activity.
[0065] The present invention further provides embodiments of
methods to process reagent solutions other than a polymerase
solution (for e.g., nucleotide solution and primer solution) that
are commonly used in DNA amplification reactions.
[0066] In some embodiments, the methods comprise processing a
nucleotide solution comprising free nucleotides (dNTPs) and a
contaminating nucleic acid. In some embodiments, the processing of
the nucleotide solution is performed to render the contaminating
nucleic acid inert (de-contamination of the nucleotide solution) to
yield a processed nucleotide solution. The method comprises the
steps of contacting the nucleotide solution with a nuclease and a
divalent cation and incubating the nucleotide solution to allow the
nuclease to render the contaminating nucleic acid inert. In some
embodiments, the nucleotide solution may further comprise a
nuclease-resistant primer. In some embodiments, the processing of
the nucleotide solution further comprises steps describing the use
of the processed nucleotide solutions in specific applications, for
example, a DNA amplification reaction.
[0067] Any divalent cation that can activate the nuclease may be
used in the processing of the nucleotide solution. Some
non-limiting examples include magnesium and manganese. The
concentration of the divalent cation primarily depends on the
concentration of the nuclease. Some of the parameters that
determine the concentration of the nuclease include the amount of
contaminating nucleic acid, the turn-over of the particular
nuclease used and other kinetic parameters for the nuclease
activity. In some embodiments, a molar excess of the divalent
cation with respect to the nuclease is used in the processing of
the nucleotide solution.
[0068] The nucleotide solution is incubated with the nuclease and
the divalent cation for a period of time that is sufficient to
render the contaminating nucleic acid inert. The incubation time
may vary with the kinetic properties of the nuclease and the
divalent cation that is being used. The incubation time may also
depend on the temperature at which the incubation is performed.
Incubation time may be optimized by analyzing the efficiency of the
de-contamination process. The efficiency can be tested by various
techniques known in the art for characterizing the presence of
nucleic acids. Suitable incubation time may range from about 5 min.
to about 3 h. In some embodiments, the incubation time may ranges
from about 1 min to about 100 min. In some embodiments, the
nucleotide solution is incubated with the exonuclease and the
divalent cation at 37.degree. C. for about 60 min.
[0069] The temperature, at which the incubation of the nucleotide
solution is performed, may vary by the nature of the particular
nuclease used. The maximum temperature that may be used for the
incubation is limited by the stability of the exonuclease and the
minimum temperature that may be employed for the incubation is
decided by the nuclease activity at that temperature. In some
embodiments, the incubation is performed at a temperature at or
below 50.degree. C. In some embodiments, the suitable incubation
temperature ranges from about 0.degree. C. to about 45.degree. C.
In some specific embodiments, the incubation is performed at a
temperature between about 10.degree. C. to about 40.degree. C.
[0070] In some embodiments, methods for processing the nucleotide
solution may further comprise adding a single-stranded DNA
binding-protein (SSB protein). Suitable SSB proteins that may be
used in the present invention include, but not limited to, extreme
thermostable single stranded DNA-binding protein (ET SSB from New
England Biolabs, MA), E. coli RecA, Tth RecA (RecA homolog isolated
from Thermus thermophilus from New England Biolabs, MA), phage T4
gene-32 protein and E. coli SSB protein. The addition of
exonuclease, divalent cation and/or the SSB to the solution
comprising free nucleotides and contaminated nucleic acid may
either be performed sequentially or simultaneously. In embodiments
where the sequential addition is performed, the addition may be
carried out in any particular order. For example, in some
embodiments, the exonuclease and the divalent cation may be mixed
first and then added to the nucleotide solution followed by the SSB
protein. In some other embodiments, the nucleotide solution may be
contacted with the SSB protein first and then the exonuclease and
the divalent cation could be added. In some embodiments, the
nucleotide solution may further comprise a circular DNA template
that is to be amplified.
[0071] A single exonuclease or a combination of exonucleases may be
used to de-contaminate the nucleotide solution. Suitable
exonucleases that may be used in the present invention include, but
not limited, to exonuclease I, exonuclease III, exonuclease VII, T7
gene-6 exonuclease, spleen exonuclease, T5 D15 exonuclease, and
lambda exonuclease. In one embodiment, a combination of exonuclease
I and exonuclease III is used in the processing of the nucleotide
solution.
[0072] The processing of a nucleotide solution may further comprise
the steps of using such processed nucleotide solution for specific
application, for example, a target DNA amplification. In some
embodiments, after incubation of the nucleotide solution with a
divalent cation and a nuclease, once the contaminating nucleic acid
is rendered inert, the nuclease in the nucleotide solution may be
inactivated prior to its use in a subsequent polymerization (e.g.,
DNA amplification) reaction. The nuclease may be inactivated by a
variety of methods that is available in the art. In one example
embodiment, the nuclease may be inactivated by thermal denaturation
of the nuclease. The thermal denaturation of the nuclease may be
achieved by incubating the processed nucleotide solution at a
temperature at which the nuclease is not stable. The incubation is
performed for a specified period of time that is sufficient to
inactivate the nuclease. In some embodiments, this may be achieved
by incubating the processed nucleotide solution at temperature at
or above 65.degree. C. In some embodiments, the processed
nucleotide solution may be incubated at a temperature between
65.degree. C. and about 95.degree. C. The time that is sufficient
to thermally inactivate the nuclease may vary depending on the
temperature used and the type of nuclease involved. Typically, the
thermal inactivation is performed for a time span of about 30 sec.
to about 2 h. In some embodiments, the processed nucleotide
solution may be incubated at about 85.degree. C. for 15 min and
then at about 95.degree. C. for 5 min. One skilled in the art,
given the benefit of this disclosure, will be able to optimize the
time span and the temperature required for thermally inactivating
the nuclease.
[0073] In some embodiments, methods for processing a primer
solution comprising a nuclease-resistant primer contaminated and a
contaminating nucleic acid are described. In some embodiments, the
processing of the primer solution is performed to render the
contaminating nucleic acid inert (de-contamination of the primer
solution) to yield a processed primer solution. The method
comprises the steps of contacting the primer solution with a
nuclease and a divalent cation and incubating the primer solution
to allow the nuclease to render the contaminating nucleic acid
inert. The primer solution may further comprise free nucleotides
(dNTPs). In some embodiments, the processing of the primer solution
further comprises steps describing the use of the processed primer
solutions in specific applications, for example, a DNA
amplification reaction.
[0074] Any divalent cation that can activate the nuclease could be
used in the processing of the primer solution. Some non-limiting
examples include magnesium and manganese. The concentration of the
divalent cation primarily depends on the concentration of the
nuclease. Some of the parameters that determine the concentration
of the nuclease include the amount of contaminating nucleic acid,
the turn-over of the particular nuclease and other kinetic
parameters for the nuclease activity. In some embodiments, a molar
excess of the divalent cation with respect to the nuclease is used
in the processing of the primer solution.
[0075] The primer solution is incubated with the nuclease and the
divalent cation for a period of time that is sufficient to render
the contaminating nucleic acid inert. The incubation time may vary
with the kinetic properties of the nuclease and the divalent cation
that is being used. The incubation time may also depend on the
temperature at which the incubation is performed. Incubation time
may be optimized by analyzing the efficiency of the
de-contamination process. The efficiency may be tested by various
techniques known in the art for characterizing the presence of
nucleic acids. Suitable incubation time may range from about 5 min.
to about 3 h. In some embodiments, the incubation time may ranges
from about 1 min to about 100 min. In some specific embodiments,
the primer solution may be incubated with the exonuclease and the
divalent cation at 37.degree. C. for about 60 min.
[0076] The temperature at which the incubation of the primer
solution is performed may vary by the nature of the particular
nuclease used. The maximum temperature that may be used for the
incubation is limited by the stability of the exonuclease and the
minimum temperature that may be employed for the incubation is
decided by the nuclease activity at that temperature. In some
embodiments, the incubation is performed at a temperature at or
below 50.degree. C. In some embodiments, the suitable incubation
temperature ranges from about 0.degree. C. to about 45.degree. C.
In some specific embodiments, the incubation may be performed at a
temperature between about 10.degree. C. to about 40.degree. C.
[0077] In some embodiments, methods for processing the primer
solution may further comprise adding a single-stranded DNA
binding-protein (SSB protein). Suitable SSB proteins that may be
used in the present invention include, but not limited to, extreme
thermostable single stranded DNA-binding protein (ET SSB from New
England Biolabs, MA), E. coli RecA, Tth RecA (RecA homolog isolated
from Thermus thermophilus from New England Biolabs, MA) phage T4
gene-32 protein and E. coli SSB protein. The addition of
exonuclease, divalent cation and/or the SSB to the solution
comprising nuclease-resistant primer and contaminated nucleic acid
may either be performed sequentially or simultaneously. In
embodiments where the sequential addition is performed, the
addition may be carried out in any particular order. For example,
in some embodiments, the exonuclease and the divalent cation may be
mixed first and then added to the primer solution followed by the
SSB protein. In some other embodiments, the primer solution may be
contacted with the SSB protein first and then the exonuclease and
the divalent cation could be added. In some embodiments, the primer
solution may further comprise a circular DNA template that is to be
amplified.
[0078] In some embodiments, the primer is an exonuclease-resistant
primer and the nuclease used in processing the primer solution is
an exonuclease. In some preferred embodiments, the primer is
resistant to 3'.fwdarw.5' exonuclease activity.
Exonuclease-resistant primers useful in the methods disclosed
herein may include modified nucleotides to make them resistant to
exonuclease digestion. For example, a primer may possess one, two,
three or four phosphorothioate linkages between nucleotides at the
3' end of the primer. In some embodiments, the present invention
relate to processes wherein the primers contain at least one
nucleotide that makes the primer resistant to degradation,
particularly by an exonuclease and more particularly by a
3'.fwdarw.5' exonuclease. The at least one modified nucleotide may
be a phosphorothioate nucleotide. Other nucleotide modifications
known in the art that make a nucleotide sequence resistant to an
exonuclease may be used as well. Modified nucleotide may be
commonly located at 3'-terminal end of the primer sequence but the
method of the present invention also relates to embodiments wherein
such a nucleotide is located at positions other than the
3'-terminal position. When the modified nucleotides are present in
positions other than the 3'-terminal position of a primer sequence,
the 3'-terminal nucleotide of said primer may be removed by
3'.fwdarw.5' exonuclease. The primers employed in the present
invention may either be a specific primer or a random primer. In
some example embodiments, the primer comprises a random hexamer
primer.
[0079] A single exonuclease or a combination of exonucleases may be
used to de-contaminate the primer solution. Suitable exonucleases
that may be used in the present invention include, but not limited,
to exonuclease I, exonuclease III, exonuclease VII, T7 gene-6
exonuclease, spleen exonuclease, T5 D15 exonuclease and lambda
exonuclease. In one embodiment, a combination of exonuclease I and
exonuclease III is used in the processing of the primer
solution.
[0080] The processing of a primer solution may further comprise the
steps of using such processed primer solution for specific
application, for example, a target DNA amplification. In some
embodiments, after incubation of the primer solution with a
divalent cation and a nuclease, once the contaminating nucleic acid
is rendered inert, the nuclease in the primer solution may be
inactivated prior to its use in a subsequent polymerization (e.g.,
DNA amplification) reaction. The nuclease may be inactivated by a
variety of methods that is available in the art. In one specific
embodiment, the nuclease may be inactivated by thermal denaturation
of the nuclease. The thermal denaturation of the nuclease may be
achieved by incubating the processed primer solution at a
temperature at which the nuclease is not stable. The incubation is
performed for a specified period of time that is sufficient to
inactivate the nuclease. In some embodiments, this may be achieved
by incubating the processed primer solution at temperature at or
above 65.degree. C. In some embodiments, the processed primer
solution may be incubated at a temperature between 65.degree. C.
and about 95.degree. C. The time that is sufficient to thermally
inactivate the nuclease may vary depending on the temperature used
and the type of nuclease involved. Typically, the thermal
inactivation may be performed for a time span of about 30 sec. to
about 2 h. In some embodiments, the processed primer solution may
be incubated at about 85.degree. C. for 15 min and then at about
95.degree. C. for 5 min. One skilled in the art, given the benefit
of this disclosure, will be able to optimize the time span and the
temperature required for thermally inactivating the nuclease.
[0081] In accordance with certain embodiments, once substantially
all the contaminating nucleic acid has been rendered inert, a DNA
template to be amplified, a DNA polymerase and free nucleotides if
not already present may be added to the processed primer solution
to amplify the DNA template. Removal of degraded contaminating
nucleic acids from the processed primer solution may not be
required since they have been rendered inert and cannot interfere
with the DNA synthesis reaction. The DNA polymerase that could be
employed for amplifying the DNA template may be a proofreading DNA
polymerase or a non-proofreading DNA polymerase. In some specific
embodiments, a combination of a proofreading DNA polymerase and a
non-proofreading DNA polymerase may be used for efficient
amplification of the DNA template. The DNA template to be amplified
may either be a single-stranded DNA template or a double-stranded
DNA template. The DNA template may either be a circular DNA
template, a linear DNA template or a nicked DNA template. The DNA
template may be a genomic DNA template or a cDNA template. The DNA
template may be amplified using any of a variety of DNA
amplification methods known in the art. The amplification of the
DNA template may be performed by using thermal cycling methods or
by using isothermal DNA amplification methods. Non-limiting
examples of DNA amplification methods that may be used in the
present invention include polymerase chain reaction (PCR), ligase
chain reaction (LCR), self-sustained sequence replication (SSR),
nucleic acid sequence-based amplification (NASBA) and amplification
using Q.beta.-replicase. In some specific embodiments, the DNA
template is amplified using rolling circle amplification (RCA). The
RCA may either be a linear RCA (LRCA) or an exponential RCA (ERCA).
In some embodiments, multiply-primed rolling circle amplification
(MPRCA) may be employed for amplifying the DNA template. In some
other embodiments, the DNA template may be amplified using a strand
displacement amplification reaction (SDA). In yet another
embodiment, the DNA template may be amplified using a multiple
displacement amplification (MDA).
[0082] In one embodiment, the DNA polymerase that is added to the
processed primer solution comprises a proofreading DNA polymerase.
In some specific embodiments, the solution of the proofreading DNA
polymerase may also be processed to render any contaminating DNA
inert prior to its addition to the processed primer solution. The
polymerase solution may be processed by any of the methods that are
described in the present disclosure for processing a polymerase
solution. In one embodiment, a Phi29 DNA polymerase is used as a
proofreading DNA polymerase. In one embodiment, the Phi29 DNA
polymerase solution is processed by incubating the Phi29 DNA
polymerase with magnesium ions at a specified temperature for a
sufficient period of time to render the contaminating nucleic acid
inert prior to its addition to the processed primer solution to
carry out the template DNA amplification.
[0083] In some embodiments, the free nucleotides that are added to
the processed primer solution may also be processed to render any
contaminating DNA inert. The nucleotide solution may be processed
by any of the methods that are described in the present disclosure
for processing a nucleotide solution prior to its use in DNA
amplification reaction. In one embodiment, the nucleotide solution
may be processed by incubating the nucleotide solution with an
exonuclease and magnesium ions at a specified temperature for a
sufficient period of time to render the contaminating nucleic acid
inert.
[0084] In some embodiments, the present invention provides methods
for amplifying a target template DNA. The method uses reagents and
reagent solutions that are substantially free of any contaminating
nucleic acids. In some embodiments, the reagents and reagent
solutions are processed by using the methods described in the
present invention prior to their use in amplification reactions to
amplify the template DNA. In some embodiments, a proofreading DNA
polymerase solution may be processed to render the contaminating
nucleic acid inert. In some other embodiments, a primer solution
may be processed to render the contaminating nucleic acid inert. In
some other embodiments, a nucleotide solution may be processed to
render the contaminating nucleic acid inert. In some specific
embodiments, the proofreading DNA polymerase solution, the
nucleotide solution and the primer solution that are to be used in
DNA amplification reaction are all processed to render the
contaminating nucleic acid inert.
[0085] In a specific embodiment, a method to amplify a target DNA
comprises the steps of (a) incubating a first solution with a first
divalent cation to render a first contaminating nucleic acid inert,
wherein the first solution comprises a proofreading DNA polymerase
and the first contaminating nucleic acid, (b) incubating a second
solution with an exonuclease and a second divalent cation to render
a second contaminating nucleic acid inert, wherein the second
solution comprises a nuclease resistant primer and the second
contaminating nucleic acid, (c) inactivating the exonuclease in the
second solution, (d) mixing the first solution and the second
solution with a third solution comprising the target DNA and (e)
amplifying the target DNA. The first divalent cation and the second
divalent cation may or may not be the same. Similarly, the first
contaminating nucleic acid and the second contaminating nucleic
acid may or may not be the same. In one embodiment, the
contaminating nucleic acid comprises a DNA.
[0086] In some embodiments, the incubation of first solution may be
performed in the absence of any free nucleotides. The first
solution may further comprise an exonuclease and/or a single
strand-binding protein. In one embodiment the first solution
comprises a Phi29 DNA polymerase. In yet another embodiment, the
first solution comprises a mixture of proofreading DNA polymerase
and a non-proofreading DNA polymerase. In a specific embodiment,
the first solution comprises a combination of a Phi29 DNA
polymerase and a Taq DNA polymerase. In some embodiments wherein
the first solution comprises a Phi29 DNA polymerase, the first
solution is incubated with magnesium ions at a specified
temperature for a period of time that is sufficient to render the
contaminating DNA inert. Here, the contaminating DNA may be
rendered inert by the intrinsic 3'.fwdarw.5' exonuclease activity
of the Phi29 DNA polymerase upon incubation with magnesium ions.
The contaminating nucleic acid may be digested to produce free
nucleotides having a 3'-hydroxyl group and a 5'-phosphate group
(e.g., deoxyribonucleoside-5'-monophosphate). In some embodiments,
the first solution comprising a Phi29 DNA polymerase may be
incubated with the divalent cation at a temperature range between
about -20.degree. C. to about 42.degree. C. In some embodiments,
the first solution comprising the Phi29 DNA polymerase may be
incubated with magnesium ions at a temperature ranging from about
10.degree. C. to about 30.degree. C. In some embodiments the
incubation is performed at 25.degree. C. The concentration of the
magnesium required for the reaction may depend on the concentration
of the Phi29 DNA polymerase used. Typically, a molar excess of
magnesium ions are used for the de-contamination reaction so that
substantially all the Phi29 DNA polymerase is activated toward
proofreading by the magnesium ions. In some embodiments, the range
of magnesium concentration varies from about 5 mM to about 50 mM
and the incubation period may range from about 1 min. to about 24
h. In some embodiments, the incubation period ranges from about 10
min. to 100 min. In some embodiments, the first solution comprising
the Phi29 DNA polymerase is incubated with magnesium ions at
30.degree. C. for about 60 min.
[0087] In some embodiments, the second solution may further
comprise free nucleotides. In some embodiments, the second solution
may further comprise a circular DNA template. In one embodiment,
the second solution comprises an exonuclease-resistant primer. In
some embodiments, the second solution comprising the
exonuclease-resistant primer may be incubated with the divalent
cation in presence of one or more of exonucleases. In some
embodiments, the second solution may be incubated with a
combination of a single strand-specific exonuclease and a double
strand-specific exonuclease. In some preferred embodiments, the
second solution may be incubated with a mixture of exonuclease I
and exonuclease III. Suitable exonucleases that may be used
include, but not limited, to exonuclease I, exonuclease III,
exonuclease VII, T7 gene-6 exonuclease, spleen exonuclease, T5 D15
exonuclease and lambda exonuclease. In some embodiments, magnesium
ions are used as a suitable divalent cation.
[0088] In some embodiments, the second solution further comprises a
single stranded DNA-binding protein (SSB protein). Suitable SSB
proteins that may be used in the present invention include, but not
limited to, extreme thermostable single stranded DNA-binding
protein (ET SSB from New England Biolabs, MA), E. coli RecA, Tth
RecA (RecA homolog isolated from Thermus thermophilus from New
England Biolabs, MA) phage T4 gene-32 protein and E. coli SSB
protein.
[0089] The exonuclease in the second solution may be inactivated by
a variety of methods that is available in the art. In one specific
embodiment, the exonuclease may be inactivated by thermal
denaturation of the exonuclease. The thermal denaturation of the
exonuclease may be achieved by incubating the second solution at a
temperature at which the exonuclease is not stable. The incubation
may be performed for a specified period of time that is sufficient
to inactivate the exonuclease. In some embodiments, this may be
achieved by incubating the second solution at temperature at or
above 65.degree. C. In some embodiments, the second solution may be
incubated at a temperature between 65.degree. C. and about
95.degree. C. The time that is sufficient to thermally inactivate
the exonuclease may vary depending on the temperature used and the
type of exonuclease involved. Typically, the thermal inactivation
is performed for a time span of about 30 sec. to about 2 h. In some
embodiments, the second solution may be incubated at about
80.degree. C. for 15 min and then at about 95.degree. C. for 5 min.
One skilled in the art, given the benefit of this disclosure, will
be able to optimize the time span and the temperature required for
thermally inactivating the exonuclease.
[0090] The target DNA in the third solution may either be a
single-stranded or a double-stranded DNA. The target DNA may be in
a circular form, a linear form or a nicked form. The target DNA may
be a genomic DNA or a cDNA. In some embodiments, the target DNA may
be obtained or derived from trace sources, such as single human
cells, forensic samples, single bacterial cells, especially
difficult to culture cells and single DNA molecule sources.
[0091] The target DNA in the third solution may be amplified using
any of a variety of DNA amplification methods known in the art. The
amplification of the target DNA may be performed by using thermal
cycling methods or by using isothermal DNA amplification methods.
Non-limiting examples of DNA amplification methods that may be used
to amplify the target DNA include polymerase chain reaction (PCR),
ligase chain reaction (LCR), self-sustained sequence replication
(SSR), nucleic acid sequence-based amplification (NASBA) and
amplification using Q.beta.-replicase. In some specific
embodiments, the target DNA may be amplified using rolling circle
amplification (RCA) method. The RCA may either be a linear RCA
(LRCA) or an exponential RCA (ERCA). In some embodiments,
multiply-primed rolling circle amplification (MPRCA) may be
employed for amplifying the target DNA. In some other embodiments,
the target DNA may be amplified using a strand displacement
amplification reaction (SDA). In yet another embodiment, the target
DNA may be amplified using a multiple displacement amplification
(MDA).
[0092] Also provided herein are kits containing reagents required
to practice the presently described inventive methods that permit
de-contamination of a polymerase solution and a primer solution and
their subsequent use in DNA amplification reactions. In some
embodiments, the kit comprises a proofreading DNA polymerase, a
nuclease resistant primer and an exonuclease. The kit may be used
to process a polymerase solution or a nuclease resistant primer
solution to render the contaminating nucleic acids inert. In some
embodiments, the kit comprises a Phi29 DNA polymerase, a primer
that is resistant to an exonuclease and the exonuclease. In some
embodiments, the kit may further comprise reagents or reagent
solution required for performing a DNA synthesis reaction.
[0093] Exonuclease-resistant primers in the kit may include
modified nucleotides to make them resistant to exonuclease
digestion. For example, the exonuclease-resistant primer may
possess one or more of phosphorothioate linkages between the
nucleotides. In some embodiments, the kit includes primers that
contain at least one modified nucleotide that makes the primer
resistant to exonuclease digestion. Modified nucleotide may be
commonly located at 3'-terminal end of the primer sequence but it
may also be located at positions other than the 3'-terminal
position. The exonuclease-resistant primer included in the kit may
either be a specific primer or a random primer. In some specific
embodiments, the primer comprises a random hexamer primer. In some
embodiments, the kit includes multiple exonuclease-resistant
primers.
[0094] The kit may further comprise a single stranded DNA-binding
protein (SSB protein). Suitable SSB proteins that may be included
in the kit include, but not limited to, extreme thermostable single
stranded DNA-binding protein (ET SSB from New England Biolabs, MA),
E. coli RecA, Tth RecA (RecA homolog isolated from Thermus
thermophilus from New England Biolabs, MA) phage T4 gene-32 protein
and E. coli SSB protein.
[0095] In some embodiments, the kit further comprises at least one
buffer that is suitable for performing a reaction for degrading
contaminating DNA. The buffer comprises divalent cations. In some
specific embodiments, the kit comprises at least one buffer
comprising magnesium ions. Kit may comprise the pre-made buffer
comprising the magnesium ions or it may comprise the necessary
reagents required to produce the said buffer. In some specific
embodiment, kit may comprise a salt of magnesium, for example, but
not limited to, magnesium chloride (MgCl.sub.2).
[0096] Suitable exonucleases that the kit may include are, for
example, but not limited to, exonuclease I, exonuclease III,
exonuclease VII, T7 gene-6 exonuclease, spleen exonuclease, T5 D15
exonuclease and lambda exonuclease. In some embodiments, the kit
comprises exonuclease III. In some other embodiments, the kit may
comprise a mixture of exonuclease I and exonuclease III. The
combination of exonucleases may be provided in a single vessel or
in multiple vessels. The kit provided herein may further include an
instruction manual detailing the specific components included in
the kit and the protocols for using them in a de-contamination
reaction or in a DNA amplification reaction or both.
[0097] While only certain features of the invention have been
illustrated and described herein, one skilled in the art, given the
benefit of this disclosure, will be able to make
modifications/changes to optimize the parameters. It is therefore,
to be understood that the appended claims are intended to cover all
such modifications and changes as fall within the true spirit of
the invention. The examples are provided for illustrative purposes
only, and should not be construed as limiting the scope of the
present invention as defined by the appended claims. Some
abbreviations used in the examples section are expanded as follows:
"mg": milligrams; "ng": nanograms; "pg": picograms; "fg":
femtograms; "mL": milliliters; "mg/mL": milligrams per milliliter;
"mM": millimolar; "mmol": millimoles; "pM": picomolar; "pmol":
picomoles; ".mu.L": microliters; "min.": minutes and "h.":
hours.
[0098] FIG. 1 is a schematic representation of one of the general
embodiments of an amplification reaction of present invention.
Contaminating nucleic acids in individual reagents or reagent
solutions are rendered inert using the methods in accordance with
certain embodiments of the present invention. Figure schematically
represents the method for processing of a polymerase solution and a
nuclease-resistant primer solution to render the contaminating
nucleic acid inert. A proofreading DNA polymerase solution is
incubated with a divalent cation in the absence or presence of
additional exonucleases to render the contaminating nucleic acid
inert. The primer solution is incubated with a nuclease in presence
of divalent cations to degrade the contaminating nucleic acid. The
primer solution may also contain free nucleotides (dNTPs). The
nuclease in the primer solution is then deactivated prior to its
use in a subsequent amplification reaction. FIG. 1 also illustrates
the use of these de-contaminated reagents or reagent solutions for
subsequent reactions such as amplification.
Example 1
[0099] Optimization of the exonuclease treatment of a reagent or
reagent solution is achieved by analyzing the exonuclease activity
in the reagent or reagent solution (buffer solution) used for DNA
amplification reaction in comparison with its activity in buffers
regularly used in exonuclease treatments. Varying concentrations of
a mixture of exonuclease I and exonuclease III (0.25 unit and 1
unit) were incubated with 50 ng of lambda DNA at 37.degree. C. for
1.8 h in presence of 20 mM MgCl.sub.2 and 15 mM KCl in different
buffer solutions. Some of the buffer solutions used include
TempliPhi buffer having 75 mM NaCl (GE Healthcare), 25 mM HEPES
(pH=8.0, no salt) and 25 mM HEPES (pH=8.6, no salt). TempliPhi
buffer is commonly used in rolling circle amplification reactions.
The TempliPhi buffer used included 400 .mu.M of free nucleotides
(dNTPs) and 40 .mu.M exonuclease-resistant hexamer primer, NNNN*N*N
(*N represents a phosphorothioate modified random nucleotide). The
total reaction volume was kept constant at 10 .mu.L. At the end of
incubation, the remaining lambda DNA present in the reaction
mixture was quantified to understand the exonuclease efficiency in
different buffers.
[0100] FIG. 2 illustrates the exonuclease treatment of
reagent/buffer solutions comprising free nucleotides to remove
contaminating nucleic acid. Lambda DNA is used as a non-limiting
example of a contaminating nucleic acid. As shown in FIG. 2, the
exonuclease was inefficient in digesting the lambda DNA in
TempliPhi buffer, whereas complete digestion of the lambda DNA by
the exonuclease was achieved in exonuclease buffers (25 mM HEPES,
no NaCl) at pH=8.0 and pH=8.6. High salt concentration (>75 mM
of NaCl) in TempliPhi buffer inhibited the exonuclease activity.
So, in embodiments where high salt (NaCl) concentrations are to be
used, the reagents/reagent solutions should be treated with
exonuclease prior to the addition of salt solution. The salt (NaCl)
solution, in such circumstances, may be separately treated with
ultraviolet rays to render any contaminating nucleic acid in the
salt solution inert.
Example 2
[0101] Use of a proofreading DNA polymerase, either alone or in
combination with an exonuclease, to render the contaminating
nucleic acid inert is illustrated by the following example. A
non-denatured, linear DNA (pUC DNA) was used as a contaminating DNA
to illustrate the efficiency of the proofreading DNA polymerase
(Phi29 DNA polymerase) to render the contaminating nucleic acid
inert. Typically 400 ng of Phi29 DNA polymerase (20 ng/.mu.L) was
incubated with 200 ng of pUC DNA in Tris-HCl buffer (50 mM
Tris-HCl, pH=8.0, 0.01% Tween-20, 1 mM TCEP) containing 20 mM
MgCl.sub.2. The incubation was performed at 30.degree. C. for a
period of 0 min. to 60 min. The same step was performed with
exonuclease III and also with a combination of Phi29 DNA polymerase
and exonuclease III. One unit/reaction of exonuclease was used.
After the incubation, the concentration of the remaining DNA was
estimated by staining the double stranded DNA (ds DNA) with
picogreen and quantifying the stain using a Tecan fluorescent plate
reader.
[0102] FIG. 3 illustrates an embodiment of the method for
processing a polymerase solution over time in which Phi29
polymerase is incubated with a divalent cation to degrade the
contaminating nucleic acid. As shown in FIG. 3, the Phi29 DNA
polymerase degraded the pUC DNA upon incubation with magnesium
ions. The efficiency of rendering the contaminating DNA inert was
greater when the incubation of the Phi29 DNA polymerase was
performed along with exonuclease III. FIG. 3 also shows a graph
illustrating the effect of exonuclease III treatment on DNA
degradation (positive control).
Example 3
[0103] Use of an exonuclease to render a contaminating nucleic acid
inert in a primer solution is illustrated. A non-denatured, linear
DNA (pUC DNA) was used as a contaminating DNA. The primer solution
contained an exonuclease-resistant, thioated hexamer primer,
NNNN*N*N, having one phosphorothioate linkage between the
nucleotides at the terminal 3' position of the primer sequence (*N
represents a phosphorothioated random nucleotide). Free nucleotides
(dNTPs) were also present in the primer solution during the
processing. Typically, a solution of 400 pmoles of the thioated
hexamer primer was mixed with 16,000 pmoles of free nucleotides
(dNTPs) in 2.5 .mu.L of 2.times. buffer (50 mM Tris-HCl, pH=8.0, 20
mM MgCl.sub.2, 0.01% Tween-20 and 1 mM TCEP) and a mixture of 1
unit of exonuclease I and 1 unit of exonuclease III. The solution
was then incubated at 37.degree. C. for about 80 min. After the
incubation, the concentration of the remaining DNA was estimated by
staining the double stranded DNA (ds DNA) with picogreen and
quantifying the stain using a Tecan fluorescent plate reader.
[0104] FIG. 4 illustrates an embodiment of the method for
processing a primer solution over time using exonuclease I or
exonuclease III or a combination of exonuclease I and exonuclease
III to degrade the contaminating nucleic acid and render it inert.
As shown in FIG. 4, the processing of the primer solution by the
incubation with the exonucleases and magnesium ions degraded the
pUC DNA. Higher efficiencies of de-contamination were observed when
a combination of exonuclease III and exonuclease I was used. Use of
exonuclease I alone is generally not sufficient to remove the
contaminating double stranded DNA.
Example 4
[0105] Effect of exonuclease treatment of reagents or reagent
solution (primer and proofreading DNA polymerase solutions) on
rendering a contaminating nucleic acid inert is illustrated by a
DNA amplification reaction in which varying amounts of a target
template DNA (DNA to be amplified) is amplified. The DNA
amplification was performed using the reagents or the reagent
solutions (a primer solution and a proofreading DNA polymerase
solution) that are pre-treated or processed with the exonuclease as
per one embodiment of the invention. The results were then compared
with a control DNA amplification reaction in which the primer
solution and the proofreading DNA polymerase solution had not
undergone any pre-treatment/process. For processing the polymerase
solution lambda exonuclease was used. For processing the primer
solution a mixture of exonuclease I and exonuclease III was
used.
[0106] To render the contaminating nucleic acid inert, the
polymerase solution containing 200 ng of Phi29 DNA polymerase was
incubated with 1 unit of lambda exonuclease in 5 .mu.L of 1.times.
reaction buffer (50 mM Tris-HCl, pH=8.0) containing 75 mM KCl, 20
mM MgCl.sub.2, 0.01% Tween-20 and 1 mM TCEP (Pierce Biotechnology)
at 23.degree. for about 80 min. The processed Phi29 DNA polymerase
solution was then used for target DNA amplification without prior
inactivation of the lambda exonuclease.
[0107] An exonuclease-resistant, thioated hexamer primer, NNNN*N*N,
having phosphorothioate linkages between the nucleotides at the
terminal 3' position of the primer sequence, was selected as a
suitable primer for the target template DNA amplification.
Approximately 25% of the primer in the primer solution may be
sensitive to the exonuclease. Free nucleotides (dNTPs) were also
present in the primer solution during the processing. Typically, a
solution of 400 pmoles of the thioated hexamer primer was mixed
with 16,000 pmoles of free nucleotides (dNTPs) in 2.5 .mu.L of
2.times. buffer (50 mM Tris-HCl, pH=8.0 containing 75 mM KCl, 20 mM
MgCl.sub.2, 0.01% Tween-20 and 1 mM TCEP). To this was added a
mixture of 1 unit of exonuclease I and 1 unit of exonuclease III.
The solution was then incubated at 37.degree. C. for about 80
min.
[0108] For the positive control DNA amplification reaction, the
polymerase solution and the primer solution is treated the same way
as described above without adding any exonucleases.
[0109] For the amplification of target template DNA, varying
concentrations of target template pUC DNA (0 ng, 1 ng, 10.sup.-2
ng, 10.sup.-4 ng, 10.sup.-6 ng, 10.sup.-8 ng) was used. The DNA
amplification reaction at 0 ng of target DNA (zero concentration of
target DNA) illustrated the false-positives, i.e., amplification of
contaminated DNA. In a typical target DNA amplification reaction,
2.5 .mu.L of target DNA in HET (H=10, E=0.1 and T=0.01%) was used
with 400 .mu.M of dNTP, 40 .mu.M of thioated primer and 400 ng of
Phi29 DNA polymerase (20 ng/.mu.L) in a reaction volume of 20
.mu.L. The same Tris-HCl buffer as mentioned above (50 mM Tris-HCl,
pH=8.0 containing 75 mM KCl, 20 mM MgCl.sub.2, 0.01% Tween-20 and 1
mM TCEP) was used for the target DNA amplification reaction.
[0110] FIG. 5 illustrates the effect of an embodiment of the
exonuclease treatment of a primer solution and a polymerase
solution on DNA amplification. FIG. 5 and FIG. 6 together
illustrate the effect of exonuclease treatment of the primer
solution and the proofreading DNA polymerase solution on rolling
circle DNA amplification. Primer solution comprising free
nucleotides are incubated with a mixture of exonuclease I and
exonuclease III along with magnesium ions. Phi29 DNA polymerase is
treated with lambda exonuclease. The graphs labeled as "without
exonuclease" refers to the control DNA amplification reactions in
which no exonuclease pre-treatment/processing was performed. The
graphs labeled as "with exonuclease I, exonuclease III, Lambda
exonuclease" or "with exonuclease" represent the DNA amplification
reactions in which the polymerase or primer solutions have been
processed with the exonuclease.
[0111] The graphs in FIG. 5 and FIG. 6 show the kinetics of the %
yield of amplified DNA with time, with or without the exonuclease
treatments. The amount of the target template DNA present in each
reaction is indicated in the label of the graph. When the
polymerase and the primer solutions are pre-treated/processed with
exonuclease, an increase in kinetics of the target DNA
amplification was observed. The increased kinetic rates were more
prominent when the target template DNA concentrations were above
10.sup.-6 ng. It is possible that at least some of the exonucleases
were active for the duration of the target DNA amplification
reaction. When the target template DNA concentration was only
10.sup.-2 ng, the exonuclease treatment of either the Phi29 DNA
polymerase, the primer solution or the free nucleotide solution did
not increase the rate of kinetics of target DNA amplification (FIG.
6)
Example 5
[0112] Effect of processing (exonuclease treatment) of reagents or
reagent solution on rendering a contaminating nucleic acid inert is
illustrated by a real time DNA amplification reaction in which
varying amounts of a target template DNA (DNA to be amplified) are
amplified. The DNA amplification was performed using the reagents
or the reagent solutions (a primer solution and a proofreading DNA
polymerase solution) that are pre-treated or processed with the
exonuclease as per some embodiments of the invention. The results
were then compared with a control DNA amplification reaction in
which the primer solution and the proofreading DNA polymerase
solution had not undergone any pre-treatment/process. pUC DNA was
used as target template DNA. The concentration of the target DNA
used ranged from 1 ng to 1 fg pUC DNA was prepared in HET buffer
that was filter-sterilized using 0.2.mu. sterile filter. An
exonuclease-resistant, locked nucleic acid primer, NNNN*N*N having
phosphorothioate linkages between the nucleotides at the terminal
3' position of the primer sequence was selected as a suitable
primer for the reaction. Real-time data was collected in Tecan
fluorescent plate reader with 1:10,000 SYBR green in the assay.
[0113] To render the contaminating nucleic acid inert, the
polymerase solution containing 200 ng of Phi29 DNA polymerase was
incubated with 1 unit of exonuclease III in 5 .mu.L of Tris-HCl
buffer (pH=8.0) containing 20 mM MgCl.sub.2, 0.01% Tween-20 and 1
mM TCEP at 30.degree. C. for about 60 min. The processed Phi29 DNA
polymerase solution was then used as such in the target DNA
amplification assay without prior inactivation of the exonuclease
III.
[0114] A solution containing 40 .mu.M of the primer, 400 .mu.M
dNTPs and 1:10,000 diluted SYBR green I dye in 50 mM Tris-HCl (50
mM Tris-HCl, pH=8.0, 20 mM MgCl.sub.2, 0.01% Tween-20 and 1 mM
TCEP) was incubated with 1 unit of exonuclease I and exonuclease
III. The solution was then incubated at 30.degree. C. for about 30
min. The exonuclease was then thermally inactivated (by incubating
the solution at 85.degree. C. for 15 min followed by an incubation
at 95.degree. C. for 5 min.)
[0115] For the control DNA amplification reaction, the polymerase
solution and the primer solution is treated the same way as
described above without adding any exonucleases.
[0116] Real-time amplification reaction was performed with varying
concentrations of target pUC DNA (10.sup.8, 10.sup.6, 10.sup.4,
10.sup.2, or 0 copies of pUC DNA circles). The DNA amplification
reaction at 0 ng of target DNA depicted the false-positives, i.e.,
amplification of contaminated DNA or non-templated DNA
amplification.
[0117] FIG. 7 illustrates the effect of an embodiment of the step
of incubating Phi29 DNA polymerase and a primer solution with an
exonuclease on a template DNA titration. It illustrates the effect
of exonuclease treatment of the primer solution and the
proofreading DNA polymerase solution on real time rolling circle
DNA amplification. In this example, pUC DNA is used as a template
DNA. The graphs labeled as "without exonuclease cleaned reagents`
refers to the control DNA amplification reactions in which no
exonuclease pre-treatment/processing was performed. The graphs
labeled as "with exonuclease cleaned reagents" represent the DNA
amplification reactions in which the polymerase or primer solutions
have been processed with the exonuclease. From the graph, it can be
observed that, DNA amplification at zero target DNA concentration
(the false positives) was considerably reduced when the polymerase
solution and the primer solution were processed as per some of the
embodiments of the present invention to render the contaminating
nucleic acid inert. In this particular reaction, a 50-times
reduction in the background false-positive signal (amplification of
contaminated nucleic acid) was observed when the reagents or
reagent solutions were processed with exonucleases prior to their
use in the DNA amplification reaction.
Example 6
[0118] Effect of addition of a single stranded DNA binding protein
(SSB protein) in the processing of reagents or reagent solution for
rendering a contaminating nucleic acid inert as per some
embodiments of the present invention is illustrated by a real time
DNA amplification reaction. The primer solution used in the DNA
amplification reaction was de-contaminated as per some embodiments
of the invention by using a combination of exonuclease I,
exonuclease III and a single stranded DNA binding protein (SSB
protein). The DNA polymerase solution used was also pre-treated or
processed with the exonuclease as per some embodiments of the
invention. Varying amounts of a target template DNA was then
amplified using the processed reagents or reagent solutions. The
results were then compared with a control DNA amplification
reaction in which the primer solution and the proofreading DNA
polymerase solution had not undergone any pre-treatment or process
to make the contaminating nucleic acid inert.
[0119] Fish DNA was used as target template DNA. The concentration
of the target DNA used ranged from 1 ng to 10 fg Fish DNA was
prepared in HET buffer that was filter-sterilized using 0.2.mu.
sterile filter. An exonuclease-resistant, locked nucleic acid
primer, W+W+N*N*S (+N represents a synthetic, locked random
nucleotide, W=A or T, S=G or C) having one phosphorothioate linkage
between the nucleotides at the terminal 3' position of the primer
sequence was selected as a suitable primer for the reaction.
Real-time data was collected in Tecan fluorescent plate reader with
1:10,000 SYBR green in the assay.
[0120] As shown in Table 1, to render the contaminating nucleic
acid inert, the polymerase solution containing 200 ng of Phi29 DNA
polymerase was incubated with 0.1 unit of exonuclease III in 5
.mu.L of 50 mM HEPES buffer (pH=8.0) containing 15 mM KCl, 20 mM
MgCl.sub.2, 0.01% Tween-20 and 1 mM TCEP. The incubation was
performed either at 30.degree. C. for about 60 min. or at 4.degree.
C. for 12 h. The processed Phi29 DNA polymerase solution was
transferred to an ice-bath and then was used in the target DNA
amplification assay without prior inactivation of the exonuclease
III.
TABLE-US-00001 TABLE 1 Conditions for the de-contamination
processing of the DNA polymerase and primer solutions. DNA
Primer/Nucleotide polymerase Mix (Each (Enzyme) Mix reaction) (Each
reaction) 2X Reaction buffer (Reaction buffer 2.5 .mu.L 2.5 .mu.L
is 50 mM HEPES buffer (pH = 8.0), 15 mM KCl, 20 mM MgCl.sub.2,
0.01% Tween-20 and 1 mM TCEP) Water -- 2.2 .mu.L 10 mM dNTP mix 0.4
.mu.L -- 1 mM primer 0.4 .mu.L -- Exonuclease I (20 unit/.mu.L) 0.5
.mu.L -- Exonuclease III (10 unit/.mu.L) 0.1 .mu.L -- Exonuclease
III (1 unit/.mu.L) -- 0.1 .mu.L SSB protein (100 ng/.mu.L) 1 .mu.L
-- 1:100 SYBR Green I 0.1 .mu.L -- Phi29 DNA polymerase (5 mg/ml)
-- 0.2 .mu.L Total Volume 5 .mu.L 5 .mu.L
[0121] To decontaminate the primer solution, it was incubated with
a combination of exonuclease I, exonuclease III and SSB protein as
shown in Table 1. E. coli SSB protein was used in this example for
the processing of the primer solution. The primer/nucleotide mix
was incubated at 37.degree. C. for about 60 min. The exonuclease
was then thermally inactivated by incubating the primer/nucleotide
solution at 85.degree. C. for 15 min followed by incubation at
95.degree. C. for 5 min.
[0122] For the control DNA amplification reaction, the polymerase
solution and the primer solution was treated the same way as
described above without adding any exonucleases or SSB protein.
[0123] Real-time amplification reaction was then performed with
varying concentrations (1 ng to 10 fg of DNA) of target Fish DNA (0
ng, 1 ng, 10.sup.-10 g, 10.sup.-11 g, 10.sup.-12 g, 10.sup.-13 g
and 10.sup.-14 g). For DNA amplification, the target DNA was added
to the primer/nucleotide mix. Enzyme mix (DNA polymerase mix) was
then added to the DNA-primer/nucleotide mix and the DNA
amplification reaction was performed for about 260 min. The DNA
amplification reaction at 0 ng of target DNA depicted the
false-positives, i.e., amplification of contaminated DNA.
Composition for the DNA amplification reaction (Total volume 20
.mu.L) included 50 mM HEPES (pH=8.0), 15 mM KCl, 20 mM MgCl2, 0.01%
Tween-20, 1 mM TCEP, 400 ng Phi29 DNA polymerase, 2.5 mM TRIS-HCl
(pH=7.2), 10 mM NaCl, 0.5 mM DTT, 0.3 mM EDTA, 2.5% (v/v) Glycerol,
0.2 unit exonuclease III, 400 mM dNTP, 800 pm exonuclease-resistant
primer (W+W+N*N*S), 2 unit exonuclease III (heat-inactivated), 20
unit exonuclease I (heat-inactivated) and 200 ng E. coli SSB
protein (heat-inactivated).
[0124] FIG. 8, FIG. 9, FIG. 10 and FIG. 11 illustrate the effect of
processing of Phi29 DNA polymerase and a primer solution with an
exonuclease on the template DNA titration. Figures also illustrate
the effect of SSB protein on the exonuclease treatment of the
primer solution. As shown in FIG. 8, processing of the
nucleotide/hexamer solution eliminated 0.1 pg of DNA to completion
using 10 units exonuclease I and one unit exonuclease III. The
addition of 100 ng SSB increased the de-contamination efficiency to
10 pg of DNA de-contamination (FIG. 9). As shown in FIG. 10 and
FIG. 11, the de-contamination of the Phi29 DNA polymerase
eliminated 1 pg to 10 pg of contaminating DNA to completion using
200 ng of Phi29 DNA polymerase and 0.1 unit exonuclease III.
[0125] The invention may be embodied in other specific forms
without departing from the spirit or essential characteristics
thereof. The foregoing embodiments are therefore to be considered
in all respects as illustrative rather than limiting on the
invention described herein. The scope of the invention is thus
indicated by the appended claims rather than by the foregoing
description, and all changes that come within the meaning and range
of equivalency of the claims are therefore intended to be embraced
therein.
* * * * *