U.S. patent application number 15/297348 was filed with the patent office on 2017-02-09 for multi-component inhibitors of nucleic acid polymerases.
The applicant listed for this patent is LIFE TECHNOLOGIES CORPORATION. Invention is credited to John BISHOP, Jun LEE.
Application Number | 20170037458 15/297348 |
Document ID | / |
Family ID | 36741093 |
Filed Date | 2017-02-09 |
United States Patent
Application |
20170037458 |
Kind Code |
A1 |
BISHOP; John ; et
al. |
February 9, 2017 |
MULTI-COMPONENT INHIBITORS OF NUCLEIC ACID POLYMERASES
Abstract
The present invention provides multi-component inhibitors of
nucleic acid polymerases, methods of making, and methods of using
same. One component of the multi-component inhibitor is a molecule
that binds to a polymerase (i.e., a polymerase-binding molecule
(PBM)), but does not thereby substantially inhibit its polymerase
activity. Another component is a molecule or complex of molecules
that binds to a PBM (i.e., a PBM-binding molecule). The combination
of the PBM and PBM-binding molecule/complex substantially inhibits
polymerase activity. The disclosed multi-component inhibitors are
useful for DNA sequencing, nucleic acid amplification, cloning and
synthesis, and the like.
Inventors: |
BISHOP; John; (Carlsbad,
CA) ; LEE; Jun; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LIFE TECHNOLOGIES CORPORATION |
Carlsbad |
CA |
US |
|
|
Family ID: |
36741093 |
Appl. No.: |
15/297348 |
Filed: |
October 19, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11044620 |
Jan 28, 2005 |
9505846 |
|
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15297348 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/40 20130101;
C12P 19/34 20130101; C12N 9/93 20130101; C07K 2317/24 20130101;
C12Q 2527/127 20130101; C12Q 1/6848 20130101; C12Q 1/6848 20130101;
C12Q 2521/119 20130101; C07K 2317/76 20130101; C12Q 2521/101
20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12N 9/00 20060101 C12N009/00; C07K 16/40 20060101
C07K016/40; C12P 19/34 20060101 C12P019/34 |
Claims
1. A composition comprising: (a) a nucleic acid polymerase; (b) a
polymerase-binding molecule (PBM) that binds to said polymerase and
does not substantially inhibit the polymerase activity of said
polymerase; and (c) a PBM-binding molecule or complex or molecules
that binds to said PBM, wherein binding of the PBM and PBM-binding
molecule or complex together substantially inhibits the polymerase
activity of said polymerase.
2. The composition of claim 1, wherein said PBM is an antibody
(PBA).
3. The composition of claim 2, wherein said antibody is a
monoclonal antibody.
4. The composition of claim 1, wherein said PBM-binding
molecule/complex is selected from the group consisting of: an
antibody; protein G; protein A; a derivatized antibody; a
derivatized protein G; a derivatized protein A; IgG and a
derivatized protein G; sir22; sibA; and a complex comprising any
one of the foregoing.
5. The composition of claim 2, wherein said derivatized PBM-binding
molecule or complex of molecules comprises a moiety selected from
the group consisting of: a detectable label; a protein; and a
polymer.
6. The composition of claim 5, wherein said detectable label is
selected from the group consisting of: rhodamine; biotin;
fluorescein; horseradish peroxidase; alkaline phosphatase; and
AlexaFluor488.
7. The composition of claim 5, wherein said protein is selected
from the group consisting of: horseradish peroxidase; alkaline
phosphatase; and albumin.
8. The composition of claim 5, wherein said polymer is selected
from the group consisting of: a polyethylene glycol; a
polyoxyethylene; a polyoxypropylene; and a
polyoxyethylene/polyoxyethylene copolymer.
9. The composition of claim 2, wherein said antibody is selected
from the group consisting of (a) IgA; (b) IgG; (c) IgM; (d) IgD;
(e) IgE; (f) IgY; (g) a fragment of any of (a)-(f); (h) a
derivative of any of (a)-(f); (i) a derivatized fragment of any of
(a)-(h); and (j) a complex of any of (a)-(i).
10. The composition of claim 2, wherein said PBM-binding
molecule/complex is a derivatized or underivatized: monoclonal
antibody, polyclonal antibody, Fc antibody fragment, chimeric
antibody or recombinant antibody.
11. The composition of claim 10, wherein said derivatized antibody
is derivatized by attachment of a moiety selected from the group
consisting of: a detectable label; a protein; and a polymer.
12. The composition of claim 11, wherein said detectable label is
selected from the group consisting of: rhodamine; biotin;
fluorescein; horseradish peroxidase; alkaline phosphatase; and
AlexaFluor488.
13. The composition of claim 11, wherein said protein is selected
from the group consisting of: horseradish peroxidase; alkaline
phosphatase; and albumin.
14. The composition of claim 11, wherein said polymer is selected
from the group consisting of: a polyethylene glycol; a
polyoxyethylene; a polyoxypropylene; and a
polyoxyethylene/polyoxypropylene copolymer.
15. The composition of claim 2, wherein said PBM-binding molecule
or complex of molecules is selected from the group consisting of: a
goat anti-mouse IgG antibody coupled to horseradish peroxidase; a
complex of a goat anti-mouse IgG antibody and protein G-horseradish
peroxidase; protein G-AlexaFluor488; a goat anti-mouse IgG
antibody; a complex of streptavidin and an antibody to mouse IgG
coupled to biotin; and Protein G.
16. The composition of claim 1, wherein said nucleic acid
polymerase is selected from the group consisting of: a
DNA-dependent DNA polymerase; and an RNA-dependent DNA
polymerase.
17. The composition of claim 16, wherein said polymerase is
thermolabile.
18. The composition of claim 16, wherein said polymerase is
thermostable.
19. The composition of claim 18, wherein said polymerase is
selected from the group consisting of: Thermus aquaticus (Taq);
Thermus thermophilus (Tth); Thermus filiformis (Tfi); Thermus
flavus (Tfl); Pyrococcus furiosus (Pfu); Thermococcus litoralis
(Tli); Thermococcus zilligi (Tzi); Thermatoga neopolitana (Tne);
Thermatoga maritime (Tma); VENT.RTM.; DEEPVENT.RTM.;
THERMOSCRIPT.RTM.; SUPERSCRIPT I.RTM.; SUPERSCRIPT II.RTM.;
SUPERSCRIPT III.RTM.; and a mutant of any of the above.
20. The composition of claim 18, wherein said nucleic acid
polymerase is recombinant.
21. A composition comprising: (a) a thermostable nucleic acid
polymerase; (b) a polymerase-binding antibody (PBA) that binds to
said polymerase and does not substantially inhibit the polymerase
activity of said polymerase; and (c) a PBA-binding molecule or
complex or molecules that binds to said PBA, wherein binding of the
PBA and PBA-binding molecule or complex together substantially
inhibits the polymerase activity of said polymerase at a
temperature less than about 40.degree. C., and wherein binding of
the PBA and PBA-binding molecule or complex together does not
substantially inhibit the polymerase activity of said polymerase at
a temperature greater than about 40.degree. C.
22. The composition of claim 21, wherein said nucleic acid
polymerase is selected from the group consisting of: a
DNA-dependent DNA polymerase; and an RNA-dependent DNA
polymerase.
23. The composition of claim 22, wherein said nucleic acid
polymerase is selected from the group consisting of: Thermus
aquaticus (Taq); Thermus thermophilus (Tth); Thermus filiformis
(Tfi); Thermus flavus (Tfl); Pyrococcus furiosus (Pfu);
Thermococcus litoralis (Tli); Thermococcus zilligi (Tzi);
Thermatoga neopolitana (Tne); Thermatoga maritime (Tma); VENT.RTM.;
DEEPVENT.RTM.; THERMOSCRIPT.RTM.; SUPERSCRIPT I.RTM.; SUPERSCRIPT
II.RTM.; SUPERSCRIPT III.RTM.; and a mutant of any of the
above.
24. The composition of claim 21, wherein said thermostable
polymerase is recombinant.
25. A method of inhibiting the polymerase activity of a nucleic
acid polymerase comprising contacting said polymerase with a PBM
and a PBM-binding molecule or complex of molecules, wherein the
binding of said PBM does not substantially inhibit polymerase
activity of said polymerase, and wherein the binding of said PBM
and said PBM-binding molecule or complex of molecules together
substantially inhibits the polymerase activity of said
polymerase.
26. The method of claim 25, wherein said inhibition is
irreversible.
27. The method of claim 25, wherein said inhibition is reversible
by heating to a temperature of at least about 40.degree. C.
28. The method of claim 25, wherein said PBM is a PBA.
29. The method of claim 25, wherein said PBM-binding molecule is an
antibody.
30. The method of claim 25, wherein said nucleic acid polymerase is
selected from the group consisting of: a DNA-dependent DNA
polymerase; and an RNA-dependent DNA polymerase.
31. The method of claim 30, wherein said nucleic acid polymerase is
selected from the group consisting of: Thermus aquaticus (Taq);
Thermus thermophilus (7th); Thermus filiformis (Tfi); Thermus
flavus (Tfl); Pyrococcus furiosus (Pfu); Thermococcus litoralis
(Tli); Thermococcus zilligi (Tzi); Thermatoga neopolitana (Tne);
Thermatoga maritime (Tma); VENT.RTM., DEEPVENT.RTM.;
THERMOSCRIPT.RTM.; SUPERSCRIPT I.RTM.; SUPERSCRIPT II.RTM.;
SUPERSCRIPT III.RTM.; and a mutant of any of the above.
32. The method of claim 30, wherein said polymerase is
recombinant.
33. A method for synthesizing a nucleic acid molecule, comprising:
(a) contacting a template nucleic acid with a composition
comprising a thermostable nucleic acid polymerase, one or more
nucleoside and/or deoxynucleoside triphosphates and an inhibitor of
said polymerase, wherein said inhibitor comprises: (i) a Polymerase
binding molecule (PBM) that does not substantially inhibit the
polymerase activity of said polymerase; and (ii) a PBM-binding
molecule or complex of molecules, wherein the combination of said
PBM and said PBM-binding molecule/complex substantially inhibits
the polymerase activity of said polymerase; and (b) bringing the
resulting mixture to a temperature sufficient to inactivate said
inhibitor, but that does not inactivate said polymerase.
34. The method of claim 33, wherein said PBM is a Polymerase
Binding Antibody (PBA).
35. The method of claim 33, wherein said PBM-binding molecule is an
antibody.
36. The method of claim 33, wherein said inhibition is
irreversible.
37. The method of claim 33, wherein said inhibition is reversible
by heating to a temperature of at least about 40.degree. C.
38. The method of claim 33, wherein said nucleic acid polymerase is
selected from the group consisting of: a DNA-dependent DNA
polymerase; and an RNA-dependent DNA polymerase.
39. The method of claim 38, wherein said nucleic acid polymerase is
selected from the group consisting of: Thermus aquaticus (Taq);
Thermus thermophilus (Tth); Thermus filiformis (Tfi); Thermus
flavus (Tfl); Pyrococcus furiosus (Pfu); Thermococcus litoralis
(Tli); Thermococcus zilligi (Tzi); Thermatoga neopolitana (Tne);
Thermatoga maritime (Tma); VENT.RTM., DEEPVENT.RTM.;
THERMOSCRIPT.RTM.; SUPERSCRIPT I.RTM.; SUPERSCRIPT II.RTM.;
SUPERSCRIPT III.RTM.; and a mutant of any of the above.
40. The method of claim 38, wherein said nucleic acid polymerase is
recombinant.
Description
BACKGROUND OF THE INVENTION
[0001] Field of the Invention
[0002] This present invention relates to compositions and methods
for inhibiting nucleic acid polymerases. These compositions and
methods may be used for nucleic acid synthesis, amplification,
sequencing and cloning.
[0003] Related Art
[0004] Nucleic acid polymerases ("polymerases") are enzymes that
catalyze the synthesis of nucleic acid molecules that are
complementary to a nucleic acid template. Template-directed nucleic
acid synthesis is an important aspect of many molecular biology
research and diagnostic techniques and assays. Such techniques and
assays typically involve extension of a nucleic acid primer
designed to hybridize to a specific region of the template.
[0005] The yield and homogeneity of primer extension products made
by polymerases can be adversely affected by "mispriming," i.e.,
hybridization of primers to inappropriate regions of the template,
or to non-template nucleic acids. Extension of misprimed nucleic
acids can produce high background and obscure detection of properly
primed primer extension products. In addition, diversion of nucleic
acid synthesis reaction constituents to extend misprimed nucleic
acids can reduce the yield of properly primed primer extension
products, reducing the sensitivity of detection. The yield of
primer extension products also can be adversely affected by
template or primer degradation (e.g., by a nuclease activity of a
polymerase). Mispriming and template or primer degradation can
occur, e.g., when nucleic acid synthesis mixtures containing
template, primers and polymerase are maintained at temperatures
associated with manufacture, shipping, storage or bench top
assembly of such mixtures.
BRIEF SUMMARY OF THE INVENTION
[0006] The invention provides methods and materials for
synthesizing nucleic acids. The methods and materials of the
invention can enhance the yield and/or homogeneity of primer
extension products made by polymerases.
[0007] One embodiment of the present invention is a composition
comprising a nucleic acid polymerase; a polymerase-binding molecule
(PBM) that binds to the polymerase and does not substantially
inhibit the polymerase activity of the polymerase; and a
PBM-binding molecule or complex of molecules that binds to the PBM
such that binding of the PBM and PBM-binding molecule or complex
together substantially inhibits the polymerase activity of the
polymerase. In one aspect of this embodiment, the PBM is an
antibody (PBA). The antibody may be a monoclonal antibody. In
another aspect, the PBM-binding molecule/complex is an antibody,
protein G, protein A, a derivatized antibody, a derivatized protein
G, a derivatized protein A, IgG and a derivatized protein G, sir22,
sib A or a complex comprising any one of the foregoing. In one
embodiment, the derivatized PBM-binding molecule or complex of
molecules comprises a detectable label, protein or polymer. The
detectable label may be rhodamine, biotin, fluorescein, horseradish
peroxidase, alkaline phosphatase or AlexaFluor488. The protein may
be horseradish peroxidase, alkaline phosphatase or albumin. The
polymer may be a polyethylene glycol, a polyoxyethylene, a
polyoxypropylene or a polyoxyethylene/polyoxyethylene copolymer. In
one embodiment, the antibody is (a) IgA; (b) IgG; (c) IgM; (d) IgD;
(e) IgE; (f) IgY; (g) a fragment of any of (a)-(f); (h) a
derivative of any of (a)-(f); (i) a derivatized fragment of any of
(a)-(h); and (j) a complex of any of (a)-(i). In another
embodiment, the PBM-binding molecule/complex is a derivatized or
underivatized monoclonal antibody, polyclonal antibody, Fe antibody
fragment, chimeric antibody or recombinant antibody. In yet another
embodiment, the derivatized antibody is derivatized by attachment
of a detectable label, protein or polymer. In one aspect of this
embodiment, the detectable label is rhodamine, biotin, fluorescein,
horseradish peroxidase, alkaline phosphatase or AlexaFluor488. In
another aspect of this embodiment, the protein is horseradish
peroxidase, alkaline phosphatase or albumin. In yet another aspect
of this embodiment, the polymer is a polyethylene glycol, a
polyoxyethylene, a polyoxypropylene or a
polyoxyethylene/polyoxyethylene copolymer. In another embodiment,
the PBM-binding molecule or complex of molecules is a goat
anti-mouse IgG antibody coupled to horseradish peroxidase; a
complex of a goat anti-mouse IgG antibody and protein G-horseradish
peroxidase; protein G-AlexaFluor488; a goat anti-mouse IgG
antibody; a complex of streptavidin and an antibody to mouse IgG
coupled to biotin; and Protein G. The nucleic acid polymerase may
be a DNA-dependent DNA polymerase of an RNA-dependent DNA
polymerase. In one embodiment, the polymerase is thermolabile. In
another embodiment, the polymerase is thermostable. The polymerase
may be Thermus aquaticus (Taq), Thermus thermophilus (TTh), Thermus
flliformis (Tfi), Thermus flavus (Tfl), Pyrocoecus furiosus (Pfu),
Thermococcus litoralis (Tli), Thermococcus zilligi (Tzi),
Thermatoga neopolitana (Tne), Thermatoga maritime (Tma), VENT.RTM.,
DEEPVENT.RTM., THERMOSCRIPT.RTM., SUPERSCRIPT I.RTM., SUPERSCRIPT
II.RTM., SUPERSCRIPT III.RTM. polymerase and a mutant of any of the
above. In one embodiment, the polymerase is recombinant.
[0008] The present invention also provides a composition comprising
a thermostable nucleic acid polymerase; a polymerase-binding
antibody (PBA) that binds to the polymerase and does not
substantially inhibit the polymerase activity of the polymerase;
and a PBA-binding molecule or complex of molecules that binds to
the PBA, such that binding of the PBA and PBA-binding molecule or
complex together substantially inhibits the polymerase activity of
the polymerase at a temperature less than about 40.degree. C., and
such that the binding of the PBA and PBA-binding molecule or
complex together does not substantially inhibit the polymerase
activity of the polymerase at a temperature greater than about
40.degree. C. The nucleic acid polymerase may be a DNA-dependent
DNA polymerase of an RNA-dependent DNA polymerase. In one
embodiment, the polymerase is thermolabile. In another embodiment,
the polymerase is thermostable. The polymerase may be Thermus
aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis
(Tfi), Thermus flavus (Tfl), Pyrococcus furiosus (Pfu),
Thermococcus litoralis (Tli), Thermococcus zilligi (Tzi),
Thermatoga neopolitana (Tne), Thermatoga maritime (Tma), Vent.RTM.,
DeepVent.RTM., Thermoscript.RTM., Superscript I.RTM., Superscript
II.RTM., Superscript III.RTM. polymerase and a mutant of any of the
above. In one embodiment, the polymerase is recombinant.
[0009] There is also provided a method of inhibiting the polymeras
activity of a nucleic acid polymerase comprising contacting the
polymerase with a PBM and PBM-binding molecule or complex of
molecules, wherein the binding of the PBM does not substantially
inhibit the polymerase activity of the polymerase, and in which the
binding of the PBM and the PBM-binding molecule or complex of
molecules together substantially inhibits the polymerase activity
of the polymerase. The inhibition may be irreversible, The
inhibition may also be reversible by heating to a temperature of at
least about 40.degree. C. In one embodiment, the PBM is a PBA. In
another embodiment, the PBM-binding molecule is an antibody. The
nucleic acid polymerase may be a DNA-dependent DNA polymerase or an
RNA-dependent DNA polymerase. The polymerase may be Thermus
aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis
(Tfi), Thermus flavus (Tfl), Pyrococcus furiosus (Pfu),
Thermococcus litoralis (Tli), Thermococcus zilligi (Tzi),
Thermatoga neopolitana (Tne), Thermatoga maritime (Tma), Vent.RTM.,
DeepVent.RTM., Thermoscript.RTM., Superscript I.RTM., Superscript
II.RTM., Superscript III.RTM. polymerase and a mutant of any of the
above. In one embodiment, the polymerase is recombinant.
[0010] The present invention also provides a method for
synthesizing a nucleic acid molecule, comprising contacting a
template nucleic acid with a composition comprising a thermostable
nucleic acid polymerase, one or more nucleoside and/or
deoxynucleoside triphosphates and an inhibitor of said polymerase,
in which the inhibitor comprises a PBM that does not substantially
inhibit the polymerase activity of the polymerase; and a
PBM-binding molecule or complex of molecules, such that the
combination of the PBM and the PBM-binding molecule/complex
substantially inhibits the polymerase activity of the polymerase;
and bringing the resulting mixture to a temperature sufficient to
inactivate the inhibitor, but that does not inactivate the
polymerase. In one embodiment, the PBM is a PBA. The PBM-binding
molecule may be an antibody. In one embodiment, the inhibition is
irreversible. In another embodiment, the inhibition is reversible
by heating to a temperature of at least about 40.degree. C. The
nucleic acid polymerase may be a DNA-dependent DNA polymerase or an
RNA-dependent DNA polymerase. The polymerase may be Thermus
aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis
(Tfi), Thermus flavus (Tfl). Pyrococcus furiosus (Pfu),
Thermococcus litoralis (Tli), Thermococcus zilligi (Tzi),
Thermatoga neopolitana (Tne), Thermatoga maritime (Tma), Vent.RTM.,
DeepVent.RTM., Thermoscript.RTM., Superscript I.RTM., Superscript
II.RTM., Superscript III.RTM. polymerase and a mutant of any of the
above. In one embodiment, the polymerase is recombinant.
[0011] Other preferred embodiments will be apparent to one of
ordinary skill in light of the drawings, description and
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1. Bar graph showing the effect of multicomponent
inhibitors on SSIII activity. SSIII activity is depicted in the
presence of each specified anti-SSIII primary antibody alone (light
bars) or with anti-SSIII primary antibody plus goat
anti-mouse-IgG-horse radish peroxidase (dark bars). All other
activities are normalized to reaction 1. Column 1: No primary mAb
or inhibitor components. SSIII activity set to 100% by definition.
Columns 2-9: Anti-SSIII mAb clone #4 in molar ratios of SSIII: mAb
clone #4 of 1:10 to 2:1. Columns 10-15: Anti-SSIII mAb clone #2 in
molar ratios of SSIII: mAb clone #2 of 1:10 to 2:1. Columns 16-17:
Anti-SSIII mAb clones #2 and #4 in molar ratios of SSII: mAb clones
#2 and #4 of 1:2. Column 18: Anti-SSIII mAb clones #2 and #4 in
molar ratios of SSIII: mAb clones #2 and #4 of 1:2, where the
anti-SSIII mAbs were heated to 96.degree. for 7 minutes prior to
adding to the SSIII.
[0013] FIG. 2. Bar graph showing SSIII activity in the presence of
different anti-SSIII primary mAb, each used as part of a
multicomponent inhibitor. SSIII activity in reactions is normalized
to that in Column 1. Column 1: No primary mAb or inhibitor
components. Activity set to 100% by definition. Column 2:
Anti-ThermoScript mAb DE11 with goat-anti-mouse-IgG-horse radish
peroxidase. Columns 3-6: Decreasing amounts of anti-SSIII mAb with
constant amount of goat-anti-mouse-IgG-horse radish peroxidase.
Columns 7-10: Four separate anti-SSIII clones with
goat-anti-mouse-IgG-horse radish peroxidase. Columns 11-13:
Constant amount of anti-SSIII mAb clone #4 with decreasing amounts
of goat-anti-mouse-IgG-horse radish peroxidase.
[0014] FIG. 3. Bar graph showing the effect of anti-SSIII primary
mAb and several different inhibitor components on SSIII activity.
SSIII activity is depicted in the presence of each specified
inhibitor component either without anti-SSIII primary mAb (light
bars), or with anti-SSIII primary mAb (dark bars). SSIII activity
in reactions is normalized to that in Column 1. Column 1: No
primary mAb or inhibitor components. Column 2:
Goat-anti-mouse-IgG-Horse radish peroxidase. Columns 3-4:
Concavalin A (ConA). Columns 5-6: Protein G-AlexaFluor 488. Columns
7-8: Goat-anti-mouse-IgG-biotin+Streptavidin. Column 9: Goat
anti-mouse-IgG. Column 10: Goat anti-mouse-IgG+ConA. Column 11:
Goat anti-mouse-IgG+Protein G-AlexaFluor 488.
[0015] FIG. 4. Bar graph showing the effect of multicomponent
inhibitors at different temperatures on SSIII activity. Light bars
depict polymerase activity at 27.degree. and dark bars depict
activity at 55.degree.. SSIII polymerase activity in each reaction
is normalized to that in Column 1. Columns 1-2: No primary mAb or
inhibitor components. SSIII activity at 27.degree. and 55.degree.
are each independently set to 100% by definition. Columns 3-4:
Anti-ThermoScript primary mAb with Gmix
(Goat-anti-mouse-IgG+Protein G-Alexafluor 488). Columns 5-6:
Anti-SSIII primary mAb with goat-anti-mouse-IgG-horse radish
peroxidase. Columns 7-8: Anti-SSIII primary mAb with Gmix.
[0016] FIG. 5. Bar graph showing the effect of multicomponent
inhibitors on Thermococcus zilligi (Tzi) thermostable DNA
polymerase activity. Tzi activity is depicted in the presence of
each specified component with the addition of buffer only (light
bars) or Gmix (goat anti-mouse-IgG+Protein G-AlexaFluor 488, dark
bars). Tzi polymerase activity in each reaction is normalized to
that in Column 1. Column 1: No primary anti-Tzi antibody or
inhibitor components. Set to 100% by definition. Column 2: Gmix
only. Columns 3-4: Primary anti-Tzi antibody clone 8C9.2. Columns
5-6: Primary anti-Tzi antibody clone 3811.2. Columns 7-8: Primary
anti-Tzi antibody clone 8F3.2. Columns 9-10: Primary anti-Tzi
antibody clone 9G2.2. Columns 11-12: Primary anti-Tzi antibody
clone 802.2. Columns 13-14: Primary anti-Tzi antibody clone
10F7.3.
[0017] FIG. 6. Bar graph showing the effect of multicomponent
inhibitors at different temperatures on Tzi activity. Light bars
depict Tzi activity at 37.degree. C. and dark bars depict activity
at 74.degree. C. Tzi polymerase activity in reactions is normalized
to that in Column 1. Columns 1-2: No primary anti-Tzi mAb or
inhibitor components. Tzi activity at 37.degree. and 74.degree. are
each independently set to 100% by definition. Columns 3-4: Gmix
(goat-anti-mouse-IgG+Protein G-AlexaFluor 488) without primary
anti-Tzi mAb. Columns 5-6: Primary anti-Tzi mAb clone 1011.3 with
Gmix.
[0018] FIG. 7. Line graph showing Tzi polymerase activity in the
presence of increasing concentrations of anti-Tzi polymerase
monoclonal antibodies in the presence of absence of secondary
inhibitors.
[0019] FIG. 8: Line graph showing RT-41 polymerase activity in the
presence of constant concentration of anti-RT-41 mAb #10 and
increasing concentrations of anti-RT-41 mAb #5 with or without
rabbit anti-mouse IgG secondary antibody.
[0020] FIG. 9: Line graph showing Taq polymerase activity in the
presence of constant concentration of anti-Taq mAb #10 and
increasing concentrations of anti-Taq mAb #5 with or without rabbit
anti-mouse IgG secondary antibody.
[0021] FIG. 10: Line graph showing Tzi polymerase activity in the
presence of increasing concentrations of rabbit-anti-mouse IgG in
the presence or absence of anti-Tri polymerase monoclonal
antibodies.
[0022] FIG. 11: Line graph showing Tzi polymerase activity after 2
minutes of preincubation at various temperatures in the presence of
absence of anti-Tzi polymerase monoclonal antibody and secondary
inhibitors.
DETAILED DESCRIPTION OF THE INVENTION
General Definitions
[0023] "A," "An" and "One" include both the singular and plural,
unless otherwise indicated or unless it is clear from the context
in which the term is used that one or the other is intended.
[0024] "About" refers to a value that is within plus or minus 10%
of a reference value. For example, a value of about 50.degree. C.
would encompass a range of values between 45.degree. C. and
55.degree. C.
[0025] "Amplification" refers to any in vitro method for increasing
the number of copies of a nucleotide sequence with the use of a
polymerase. Amplification results in the incorporation of
nucleotides into a nucleic acid (e.g., DNA) molecule or primer
thereby forming a new nucleic acid molecule complementary to the
nucleic acid template. Typically, the template and newly formed
nucleic acid molecule can be used as templates to synthesize
additional nucleic acid molecules. As used herein, one
amplification reaction may consist of many rounds of nucleic acid
synthesis. Amplification reactions include, for example, polymerase
chain reactions (PCR). One PCR-type amplification may consist of 5
to 100 or more rounds of denaturation and synthesis of a nucleic
acid molecule.
[0026] "Antibody" refers to molecule(s) that are capable of binding
an epitope or antigenic determinant. The term is meant to include
whole antibodies and antigen-binding fragments thereof, including
Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv), single-chain
antibodies, disulfide-linked Fvs (sdFv) and fragments comprising
either a V.sub.L or V.sub.H domain. The antibodies can be from any
animal origin including birds such as chicken, and mammals such as
human, murine, rat, rabbit, goat, guinea pig, sheep, cow, camel and
horse. The term "antibodies" also includes genetically prepared
equivalents thereof, and chemically or genetically prepared
fragments of antibodies (such as Fab fragments), recombinant
antibodies, chimeric antibodies, monoclonal, polyclonal, affinity
purified polyclonal, and the like. Antibodies and fragments thereof
can be used singly or in mixtures in the practice of this
invention.
[0027] "Bound" means to be coupled via covalent or non-covalent
interactions. Covalent binding can occur via chemically coupling
and the formation of, e.g., ester, ether, phosphoester, thioester,
thioether, methane, amide, amine, peptide, imide, hydrazone,
hydrazide, carbon-sulfur, carbon-phosphorus, and like bonds.
Non-covalent binding can occur via, e.g., ionic interactions,
hydrophobic interactions, hydrogen bonds, etc.
[0028] "Exonuclease activity" relates to enzymatic activity
resulting in the removal of nucleotides from a polynucleotide, in
either the 3'-to-5' direction ("3'-to-5' exonuclease activity") or
the 5'-to-3' direction ("5'-to-3' exonuclease activity"). A
polymerase may exhibit either or both 3'-to-5' and 5'-to-3'
exonuclease activity. Modified or recombinant polymerases are
available in which either or both have been substantially reduced
or eliminated.
[0029] "Hybridization" and "hybridizing" refer to the pairing of
two complementary single-stranded nucleic acid molecules (RNA
and/or DNA) to give a double-stranded molecule. Two nucleic acid
molecules may be hybridized, although the base pairing is not
completely complementary. Accordingly, mismatched bases do not
prevent hybridization of two nucleic acid molecules provided that
appropriate conditions, well known in the art, are used.
Hybridization refers in some contexts to pairing of an
oligonucleotide with a DNA template molecule.
[0030] "Inactivated" refers to a reduction of a specified property
or activity to less than 10%, 7.5%, 5%, 2.5%, 1%, 0.5% or 0.1% of
its original property or activity including the polymerase activity
of a polymerase or the inhibitory activity of an inhibitor.
[0031] "Nucleotide" refers to a base-sugar-phosphate combination.
Nucleotides are monomeric units of a nucleic acid (DNA and RNA).
The term nucleotide includes deoxyribonucleoside triphosphates
("dNTPs"), such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or
derivatives thereof. Such derivatives include, for example,
[.alpha.S]dATP, 7-deaza-dGTP and 7-deaza-dATP. The term nucleotide
also includes dideoxyribonucleoside triphosphates ("ddNTPs") and
their derivatives, such as ddATP, ddCTP, ddGTP, ddITP, and ddTTP.
The term nucleotide also includes ribonucleoside triphosphates
(rNTPs) such as rATP, rCTP, rITP, rUTP, rGTP, rTTP and their
derivatives, which are analogous to the above-described dNTPs and
ddNTPs except that the rNTPs comprise ribose instead of deoxyribose
or dideoxyribose in their sugar-phosphate backbone. The term "NTP"
is more general and may encompass rNTP, dNTP, ddNTP or nucleotide
analogs. A "nucleotide" may be unlabeled or detectably labeled by
well known techniques. Detectable labels include, for example,
radioactive isotopes, fluorescent labels, chemiluminescent labels,
bioluminescent labels and enzyme labels.
[0032] "Nucleic acid" and "Nucleic acid molecule" refer to a series
of contiguous nucleotides which may encode a full-length
polypeptide or a fragment of any length thereof, or which may be
non-coding.
[0033] "Nucleic acid polymerase" and "Polymerase" refer to any
polypeptide, protein or enzyme with nucleic acid polymerase
activity.
[0034] "Oligonucleotide" refers to a synthetic or natural molecule
comprising a covalently linked sequence of nucleotides which are
joined by a phosphodiester bond between the 3' position of the
pentose of one nucleotide and the 5' position of the pentose of the
adjacent nucleotide.
[0035] "Polymerase activity" is an enzymatic activity, whereby a
polymerase synthesizes polynucleotides in the 5' to 3' direction by
addition of a new nucleotide to the 3' end of a the previous
nucleotide, according to an RNA or DNA template that directs the
synthesis of the polynucleotide. For example, a DNA polymerase can
synthesize the formation of a DNA molecule complementary to a
single-stranded DNA or RNA template by extending a primer in the
5'-to-3' direction. Polymerases include DNA-dependant DNA
polymerases; DNA-dependant RNA polymerases, also known as
transcriptases; RNA-dependant DNA polymerases, also known as
reverse transcriptases; and, more often seen in certain viruses,
RNA-dependant RNA polymerases. A given polymerase enzyme may have
more than one polymerase activity. For example, some DNA-dependent
DNA polymerases, such as Taq, also exhibit reverse transcriptase
polymerase activity.
[0036] "Polypeptide," "Peptide" and "Protein" refer to series of
contiguous amino acids, of any length.
[0037] "Primer" refers to a single-stranded oligonucleotide that is
extended by covalent bonding of nucleotide monomers during
amplification or polymerization of a DNA molecule.
[0038] "Stable" and "Stability" refer to the retention by an enzyme
of at least about 70%, at least about 80%, or at least about 90%,
of the original enzymatic activity (in units) after the enzyme or
composition containing the enzyme has been subjected to a condition
which might otherwise have resulted in loss of activity for an
enzyme that was not stable. Labile is the opposite of stable.
[0039] "Substantially pure" means that the desired purified
molecule such as a protein or nucleic acid is essentially free from
contaminants typically associated with the desired molecule.
Contaminating components include compounds or molecules that may
interfere with the inhibitory or synthesis reactions of the
invention, and/or that degrade or digest the molecules of the
invention and/or that degrade or digest the synthesized or
amplified nucleic acid molecules produced by the methods of the
invention.
[0040] "Template" refers to a double-stranded or single-stranded
nucleic acid molecule that is to be amplified, synthesized or
sequenced. In the case of a double-stranded RNA or DNA molecule,
denaturation of its strands to form a first and a second strand is
performed before these molecules may be amplified, synthesized or
sequenced. A primer complementary to a portion of a template is
hybridized to the template under appropriate conditions and a
polymerase of the invention may then synthesize a nucleic acid
molecule complementary to the template or a portion thereof. The
newly synthesized DNA molecule, according to the invention, may be
equal or shorter in length than the original DNA template. Mismatch
incorporation or strand slippage during the synthesis or extension
of the newly synthesized DNA molecule may result in one or a number
of mismatched base pairs. Thus, the synthesized nucleic acid
molecule need not be exactly complementary to the template.
[0041] "Thermostable" refers to an enzyme (such as a polypeptide
having polymerase activity) that is resistant to inactivation by
beat. A "thermostable" enzyme is in contrast to a "thermolabile"
polymerase, which can be inactivated by heat treatment.
Thermolabile proteins can be inactivated at physiological
temperatures, and can be categorized as mesothermostable
(inactivation at about 45.degree. C. to 65.degree. C.), and a
thermostable (inactivation greater than about 65.degree. C.). For
example, the activities of the thermolabile T5 and T7 DNA
polymerases can be totally inactivated by exposing the enzymes to a
temperature of about 90.degree. C. for about 30 seconds. A
thermostable polymerase activity is more resistant to heat
inactivation than a thermolabile polymerase. However, a
thermostable polymerase does not mean to refer to an enzyme that is
totally resistant to heat inactivation; thus heat treatment may
reduce the polymerase activity to some extent. A thermostable
polymerase typically will also have a higher optimum temperature
than thermolabile DNA polymerases.
[0042] "Unit" refers to the activity of an enzyme. When referring
to a thermostable polymerase (e.g., Taq and Pfx), one unit of
activity is the amount of enzyme that will incorporate 10 nanomoles
of NTPs into acid-insoluble material (i.e., DNA or RNA) in 30
minutes under standard primed DNA synthesis conditions at
74.degree. C. When referring to reverse transcriptases (e.g.
SuperScript III), one unit is defined as the amount of enzyme which
incorporates 1 nmole of dTTP into acid insoluble material in 10 min
at 37.degree. C.
[0043] "Working concentration" refers to the concentration of a
reagent that is at or near the optimal concentration used in a
solution to perform a particular function (such as amplification or
digestion of a nucleic acid molecule). The working concentration of
a reagent is also described equivalently as a "IX concentration" or
a "1.times. solution" (if the reagent is in solution) of the
reagent. Accordingly, higher concentrations of the reagent may also
be described based on the working concentration; for example, a
"2.times. concentration" or a "2.times. solution" of a reagent is
defined as a concentration or solution that is twice as high as the
working concentration of the reagent; a "5.times. concentration" or
a "5.times. solution" is five times as high as the working
concentration of the reagent; and so on.
Compositions
[0044] The invention provides compositions that include a
polymerase-binding molecule (PBM) and a PBM-binding molecule or
complex, where binding of the PBM to a polymerase does not
substantially inhibit the polymerase activity of the polymerase,
but where binding of the PBM and PBM-binding molecule/complex
together substantially inhibit the polymerase activity of the
polymerase. The PBM and PBM-binding molecule/complex may also
inhibit the 3'-5' exonuclease activity, 5'-3' exonuclease activity
and/or RNase H activity of a polymerase.
[0045] The PBM does not substantially inhibit the polymerase
activity of a polymerase in the absence of the PBM-binding
molecule/complex. Accordingly, a polymerase bound by a PBM in the
absence of a PBM-binding molecule/complex exhibits at least about
70%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, 99.75%, 100% or >100%
of the polymerase activity observed in the absence of the PBM.
[0046] Binding of a PBM together with a PBM-binding
molecule/complex substantially inhibits the polymerase activity of
a polymerase. Accordingly, a polymerase bound by a PBM in the
presence of a PBM-binding molecule/complex exhibits less than about
30%, 25%, 20%, 15%, 10%, 5%, 2.5% 1% or 0.25% of the polymerase
activity observed either in the absence of both the PBM and
PBM-binding molecule/complex, or in the presence of the PBM but in
the absence of the PBM-binding molecule/complex.
[0047] Polymerase inhibition by a PBM and PBM-binding
molecule/complex may be irreversible or reversible. Inhibition of
polymerase activity may be reversed by any means known in the art
including, e.g., dilution, competition, physical or ionic
disruption, or temperature change. For example, heating a
composition containing a polymerase, PBM and PBM-binding
molecule/complex may reverse polymerase inhibition. Such heating
typically involves a shift to a higher temperature that does not
substantially reduce the polymerase's polymerase activity (e.g.,
temperatures up to 40.degree. C., 50.degree. C., 55.degree. C.,
60.degree. C., 65.degree. C., 70.degree. C., 75.degree. C.,
80.degree. C., 90.degree. C., 95.degree. C. or 99.degree. C.). Such
heating may be sufficient to cause denaturation of the PBM (e.g.,
antibody); denaturation of the PBM-binding molecule/complex;
dissociation of the PBM from the nucleic acid polymerase;
dissociation of the PBM-binding molecule/complex from the PBM;
dissociation of a PBM-binding complex, or a combination of these
effects.
Polymerase Binding Molecules (PBMs)
[0048] A PBM can be an antibody, antibody fragment, chemical
compound, acid, antibiotic, heavy metal, metal chelator, nucleotide
analog, sulfhydryl reagent, anionic detergent, polyanion, captan
((N-[trichloromethyl]-thio)-4-cyclohexene-1,2-dicarboximide),
acidic polysaccharide or lectin.
[0049] A PBM can be specific for a DNA-dependent a DNA polymerase,
a DNA-dependent RNA polymerase, a RNA-dependent DNA polymerase
(reverse transcriptase) and/or a RNA-dependent RNA polymerase.
Thus, compositions in accord with the invention can include a PBM
that binds a DNA polymerase such as Taq DNA polymerase, Tzi DNA
polymerase, Tne DNA polymerase, Tma DNA polymerase, Pfu DNA
polymerase, Tfl DNA polymerase, Tth DNA polymerase, Pwo DNA
polymerase, Bst DNA polymerase, Bca DNA polymerase, VENT.TM. DNA
polymerase, DEEPVENT.TM. DNA polymerase, T7 DNA polymerase, T5 DNA
polymerase, DNA polymerase III, Klenow fragment DNA polymerase,
Stoffel fragment DNA polymerase, and/or mutants, fragments or
derivatives thereof. Compositions in accord with the invention also
can include a PBM that binds a polymerase having reverse
transcriptase activity, such as an M-MLV reverse transcriptase, an
RSV reverse transcriptase, an AMV reverse transcriptase, an RAV
reverse transcriptase, an MAV reverse transcriptase, an HIV reverse
transcriptase (any of which may be reduced in, substantially
reduced in, or have no detectable RNase H activity) and/or mutants,
fragments or derivatives thereof.
[0050] Compositions in accord with the invention can include a PBM
that binds to a thermolabile polymerase, and/or to a thermostable
nucleic acid polymerase, such as a Thermus aquaticus polymerase, a
Thermus thermophilus polymerase, a Thermus filiformis polymerase, a
Thermus flavus polymerase, a Pyrooccus furiosus polymerase, a
Thermococcus litoralis, Thermococcus zilligi or a Thermotoga
species polymerase, or a recombinant variant thereof.
[0051] Certain exemplary compositions of the invention include
antibody PBMs ("PBAs"). Accordingly, the invention provides
compositions that include a polymerase-binding antibody (PBA) or
derivative and a PBA-binding molecule/complex, where binding of the
PBA or derivative to a polymerase does not substantially inhibit
the polymerase activity of the polymerase, but where binding of the
PBA and PBA-binding molecule/complex together substantially inhibit
the polymerase activity of the polymerase.
[0052] A PBA can be a monoclonal antibody, a polyclonal antibody,
an Fc antibody fragment, a chimeric antibody or a recombinant or
other derivative thereof. A PBA can be an IgA antibody, an IgG
antibody, an IgM antibody, an IgD antibody, an IgE antibody, an IgY
antibody, an IgE antibody or fragment thereof. Specific monoclonal
PBAs include: Anti-SSIII mAb clone #4, Anti-SSM mAb clone #2,
Primary anti-Tzi antibody clone 8C9, Primary anti-Tzi antibody
clone 3B11.2, Primary anti-Tzi antibody clone 8F3.2, Primary
anti-Tzi antibody clone 9G2.2, Primary anti-Tzi antibody clone
802.2, Primary anti-Tzi antibody clone 10F7.3, Primary anti-Tzi mAb
clone 1 G11.3, anti-ThermoScript mAb DE11, anti-Rt41A mAb #5,
anti-Rt41A mAb #10, anti-Taq mAb #5 and anti-Taq mAb #10.
PBM-Binding Molecules/Complexes
[0053] A PBM-binding molecule/complex can be any molecule/complex
known in the art to be capable of binding a PBM.
[0054] When the PBM is an antibody (i.e., a PBA), the PBM-binding
molecule/complex is capable of binding an antibody, and can be
referred to as a PBA-binding molecule/complex. Many
molecules/complexes capable of binding antibodies are known to
those skilled in the art. Thus, a PBA-binding molecule/complex can
include an antibody, an antibody fragment (e.g., Fab fragment),
protein G, protein A, a derivatized antibody, a derivatized protein
G, a derivatized protein A, protein H, ARP or a complex including
any of the foregoing with another moiety.
[0055] In some embodiments, a PBA-binding molecule/complex
comprises an antibody. The antibody may be monoclonal, polyclonal,
chimeric, or an Fc fragment. The antibody may also be recombinant.
In another embodiment, the antibody may be IgA, IgG, IgM, IgE, IgY,
IgD or derivative thereof. A PBA-binding antibody may be from any
species (including humans, monkeys, mice, rats, rabbits, horses,
goats, sheep, cows, pigs, chickens, fish, etc.) and may bind to
antibodies from any species (including humans, monkeys, mice, rats,
rabbits, horses, goats, sheep, cows, pigs, chickens, fish, etc.).
PBA-binding antibodies may be derivatized or underivatized
monoclonal antibodies, derivatized or underivatized polyclonal
antibodies, derivatized or underivatized Fe or Fab antibody
fragments, derivatized or underivatized chimeric antibodies, or
derivatized or underivatized recombinant antibodies. PBA-binding
antibodies can be IgA, IgG, IgM, IgD, IgE, IgY antibodies or
fragments or derivatives thereof.
[0056] In some embodiments, a PBA-binding molecule/complex
comprises a bacterial immunoglobulin binding protein (IgBP) such
as, e.g., Staphylococcus aureus protein A, which binds to all
immunoglobulin molecules, and streptococcal protein G, which binds
specifically to IgG. Other bacterial IgBPs are also available and
may be used in the compositions and methods described herein
including, for example, Haemophilus somnus high molecular weight
IgBPs, and Peptostreptococcus magnus protein L, which binds
immunoglobulin (Ig) light chains. A variety of eukaryotic
antibody-binding proteins may also be used, such as Fc receptors on
cells of the immune system. In addition, other immunoglobulin
binding proteins may be used, including sir22 (Stenberg et al., J.
Biol. Chem. 269:13458-13464, 1994), sibA (Fagan et al., Infect.
Immun. 69:4851-4857, 2001) and ARP (U.S. Pat. No. 5,180,810;
European Patent Application No. 0367890 A1).
[0057] PBM-binding molecules (including PBA-binding molecules such
as antibodies) and molecules comprising PBM-binding complexes, may
be derivatized with one or more molecules or moieties using
well-known procedures, such as chemical coupling or recombinant DNA
technology. Derivatization moieties include, e.g., detectable
labels, signaling groups, proteins, chemical groups, ligands,
haptens or polymers. A detectable label can be enzymatic,
chemiluminescent, bioluminescent, radioactive or fluorescent.
Exemplary detectable labels include rhodamine, biotin, fluorescein,
horseradish peroxidase, alkaline phosphatase, and AlexaFluor488.
Exemplary derivatization proteins include horseradish peroxidase,
alkaline phosphatase, protein G, and albumin. Exemplary
derivatization polymers include polyethylene glycol,
polyoxyethylene, polyoxypropylene, and
polyoxyethylene/polyoxypropylene copolymer.
[0058] Derivatization typically alters the properties of a
PBM-binding molecule. For example, large moieties including
polymers, proteins and large chemical groups will increase the
bulkiness of a PBM-binding molecule/complex. Attached moieties may
also alter the net charge, solubility, ionic strength or other
physical or chemical property of a PBM. Attached moieties may also
serve as targets for other binding molecules.
[0059] PBM-binding molecules and PBM-binding complexes of molecules
may be used in the compositions and methods described herein. By
way of example, a PBA-binding complex may include a PBA-binding
antibody, to which is bound Protein G. Other exemplary PBA-binding
complexes include an antibody coupled to horseradish peroxidase; an
antibody-protein G-horseradish peroxidase complex; a protein
G-AlexaFluor488 complex; and an antibody-biotin-streptavidin
complex. Yet another exemplary PBA-binding complexes include a
PBA-binding antibody derivatized with a hapten, which can bind an
antibody against the hapten. In this manner, the number of
antibodies, moieties and the like associated with a given target
polymerase to be inhibited can be varied as desired.
Polymerases
[0060] Compositions of the invention may include one or more DNA
and/or RNA polymerases. The polymerases may be thermolabile or
thermostable. DNA polymerases include, but are not limited to,
Thermus thermophilus (Tth) DNA polymerase, Thermus aquaticus (Taq)
DNA polymerase, Thermococcus zilligi (Tzi), Thermotoga neopolitana
(Tne) DNA polymerase, Thermotoga maritima (Tma) DNA polymerase,
Thermococcus litoralis (Tli or VENT.TM.) DNA polymerase, Pyrococcus
furiosus (Pfu) DNA polymerase, DEEPVENT.TM. DNA polymerase,
Pyrococcus woosii (Pwo) DNA polymerase, Pyrococcus sp KOD2 (KOD)
DNA polymerase, Bacillus stearothermophilus (Bst) DNA polymerase,
Bacillus caldophilus (Bca) DNA polymerase, Sulfolobus
acldocaldarius (Sac) DNA polymerase, Thermoplasma acidophilum (Tac)
DNA polymerase, Thermus flavus (Tfl/Tub) DNA polymerase, Thermus
ruber (Tru) DNA polymerase, Thermus brockianus (DYNAZYME.TM.) DNA
polymerase, Methanobacterium thermoautotrophicum (Mth) DNA
polymerase, a mycobacterium DNA polymerase (e.g. Mtb, Mlep); and
generally Pol I and Pol III type polymerases.
[0061] Thermolabile Pol I and Pol III type nucleic acid polymerases
and their respective Klenow fragments include, but are not limited
to, those which may be isolated from organisms such as E. coli, H.
influenzae, D. radiodurans, H. pylori, C. aurantiacus, R.
prowazekii, T. pallidum, Synechocysts sp., B. subtlis, L. lactis,
S. pneumoniae, M. tuberculosis, M. leprae and M. smegmatis
bacteria; L5, phi-C31, T7, T3, T5, SP01, and SP02 bacteriophage; S.
cerevisiae MIP-1 mitochondria; cukaryotes C. elegans, and D.
melanogaster (Astatke, M. et al., 1998, J. Mol. Biol. 278,
147-165); and viral nucleic acid polymerases; and any mutants,
variants and derivatives thereof.
[0062] Thermostable DNA polymerases that may be used in the methods
and compositions of the invention include Tfi; Tfl; Tzi; Tne; Tma;
Pfu; Pwo; Vent.RTM.; KOD, Stoffel fragment, DeepVent.RTM.;
Thermoscript.RTM.; Superscript I.RTM.; Superscript II.RTM.;
Superscript III.RTM.; and mutants or variant thereof. Such
polymerases are described, for example, in U.S. Pat. No. 5,436,149;
U.S. Pat. No. 4,889,818; U.S. Pat. No. 4,965,188; U.S. Pat. No.
5,079,352; U.S. Pat. No. 5,614,365; U.S. Pat. No. 5,374,553; U.S.
Pat. No. 5,270,179; U.S. Pat. No. 5,047,342; U.S. Pat. No.
5,512,462; WO 92/06188; WO 92/06200; WO 96/10640; WO 97/09451;
Barnes, W. M. Gene 112:29-35 (1992); Lawyer, F. C., et al, PCR
Meth. Appl. 2:275-287 (1993); Flaman, J.-M, et al., Nucl. Acids
Res. 22(15):3259-3260 (1994)). In one embodiment, DNA polymerase
from Thermus Rt41A (herein called "Rt41A"), a species of Thermus
filiformis, is used in the compositions and methods described
herein. The Rt41A nucleotide and protein sequences are disclosed in
SEQ ID NOS: 9 and 21, respectively, in WO03/025132 and copending
U.S. patent application Ser. No. 10/244,081, the entire contents of
which are incorporated herein by reference. Isolation and
characterization of Tzi polymerase is described in copending U.S.
Provisional Patent Application Ser. No. ______, entitled "DNA
Polymerase from Thermococcus zilligi and Mutants Thereof" listing
inventors Jun. E. Lee, Kyusung Park, Katherine R. Griffiths,
Moreland D. Gibbs, and Peter L. Bergquist, filed on the same date
as the present application, the entire contents of which are
incorporated herein by reference.
[0063] RNA polymerases suitable for use in the compositions and
methods described herein include any enzyme having RNA polymerase
activity, including both DNA-dependent and RNA-dependant RNA
polymerases. More typically, RNA polymerases used in the present
invention will be DNA-dependent RNA polymerases, also known as
reverse transcriptases. Transcriptases may be isolated from any
source, including E. coli, T3, T5, SP-6, T7 and Xenopus, and
mutants, variants and derivatives thereof.
[0064] Reverse transcriptases include any enzyme having reverse
transcriptase activity. Such enzymes include, but are not limited
to, retroviral reverse transcriptase, retrotransposon reverse
transcriptase, hepatitis B reverse transcriptase, cauliflower
mosaic virus reverse transcriptase, bacterial reverse
transcriptase, Tth DNA polymerase, Taq DNA polymerase (Saiki, R.
K., et al, Science 239:487-491 (1988); U.S. Pat. Nos. 4,889,818 and
4,965,188; Shandilya et al., Extremophiles 8(3):243-251, 2004), The
DNA polymerase (WO 96/10640 and WO 97/09451), Tma DNA polymerase
(U.S. Pat. No. 5,374,553) and mutants, variants or derivatives
thereof (see, e.g., WO 97/09451 and WO 98/47912).
[0065] In one embodiment, reverse transcriptases include those that
have reduced, substantially reduced or eliminated RNase H activity.
By an enzyme "substantially reduced in RNase H activity" is meant
that the enzyme has less than about 20%, 15%, 10%, 5%, or 2%, of
the RNase H activity of the corresponding wild type or RNase
H+enzyme such as wild type Moloney Murine Leukemia Virus (M-MLV),
Avian Myeloblastosis Virus (AMV) or Rous Sarcoma Virus (RSV)
reverse transcriptases. The RNase H activity of any enzyme may be
determined by a variety of assays, such as those described, for
example, in U.S. Pat. No. 5,244,797, in Kotewicz, M. L., et al.
Nucl. Acids Res. 16:265 (1988) and in Gerard, G. F., et al., FOCUS
14(5):91 (1992), the disclosures of all of which are fully
incorporated herein by reference. Polypeptides suitable for use in
the compositions and methods described herein include, but are not
limited to, M-MLV H.sup.- reverse transcriptase, RSV H.sup.-
reverse transcriptase, AMV H.sup.- reverse transcriptase, RAV
(Rous-associated virus) H.sup.- reverse transcriptase, MAV
(myeloblastosis-associated virus) H.sup.- reverse transcriptase and
HIV H.sup.- reverse transcriptase (See U.S. Pat. No. 5,244,797 and
WO 98/47912), and superscript III. It will be understood by one of
ordinary skill, however, that any enzyme capable of producing a DNA
molecule from a ribonucleic acid molecule (i.e., having reverse
transcriptase activity) may be equivalently used in the
compositions, methods and kits described herein, including those
described in PCT WO03/025132, the entire disclosure of which is
incorporated herein by reference.
[0066] The enzymes having polymerase may be obtained commercially,
for example from Invitrogen (Carlsbad, Calif.), Perkin-Elmer
(Branchburg, N.J.), New England BioLabs (Beverly, Mass.) or
Boehringer Mannheim Biochemicals (Indianapolis, Ind.).
Alternatively, polymerases or reverse transcriptases having
polymerase activity may be isolated from their natural viral or
bacterial sources according to standard procedures for isolating
and purifying natural proteins that are well-known to one of
ordinary skill in the art (see, e.g., Houts, G. E., et al., J.
Virol. 29:517 (1979)). In addition, such polymerases/reverse
transcriptases may be prepared by routine recombinant DNA
techniques well know to those skilled in the art (see, e.g.,
Kotewicz, M. L., et al., Nucl. Acids Res. 16:265 (1988); U.S. Pat.
No. 5,244,797; WO 98/47912; Soltis, D. A., and Skalka, A. M., Proc.
Natl. Acad. Sci. USA 85:3372-3376 (1988)).
[0067] Recombinant, mutants and other variants of the polymerases
described herein may also be used. Recombinant variants may be
particularly useful in some embodiments described herein. For
example, using recombinant DNA technology, skilled artisans can
generate a polymerase that contains an amino acid epitope that is
the target of a polymerase-binding antibody. Alternatively, or in
addition, the epitope could bind other molecules such as ligands,
receptors, haptens and the like. As a result, by adding a
particular epitope to several polymerases, it is possible to
inhibit a range of different polymerases with a single PBM in the
presence of a PBM-binding molecule/complex. In another embodiment,
the conditions under which a given polymerase is inhibited by a
given inhibitor can be optimized by modifying the inhibitor and/or,
through recombinant technologies, the polymerase.
Antibodies
[0068] PBAs and PBA-binding antibodies may be polyclonal or
monoclonal, and may be prepared by any of a variety of methods
(see, e.g., U.S. Pat. No. 5,587,287). For example, polyclonal
antibodies may be made by immunizing an animal with one or more
polypeptides having polymerase activity or portions thereof
according to standard techniques (see, e.g., Harlow, E., and Lane,
D., Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.: Cold
Spring Harbor Laboratory Press (1988); Kaufman, P. B., et al., In:
Handbook of Molecular and Cellular Methods in Biology and Medicine,
Boca Raton, Fla.: CRC Press, pp. 468-469 (1995)). Alternatively,
anti-polymerase monoclonal antibodies (or fragments thereof) may be
prepared using hybridoma technology that is well-known in the art
(Kohler et al., Nature 256:495 (1975); Kohler et al., Eur. J.
Immunol. 6:511 (1976); Kohler et al., Eur. J. Immunol. 6:292
(1976); Hammerling et al., In: Monoclonal Antibodies and T-Cell
Hybridomas, New York: Elsovier, pp. 563-681 (1981); Kaufman, P. B.,
et al., In: Handbook of Molecular and Cellular Methods in Biology
and Medicine, Boca Raton, Fla.: CRC Press, pp. 444-467 (1995)).
Monoclonal PBAs typically have a polymerase association constant of
at least about 10.sup.7 molar.sup.-1, although antibodies having
lower affinities may also be used.
[0069] Exemplary antibodies include: Anti-SSIII mAb clone #4;
Anti-SSIII mAb clone #2; Primary anti-Tzi antibody clone 8C9;
Primary anti-Tzi antibody clone 3B11.2; Primary anti-Tzi antibody
clone 8F3.2; Primary anti-Tzi antibody clone 9G2.2; Primary
anti-Tzi antibody clone 802.2; Primary anti-Tzi antibody clone
10F7.3; Primary anti-Tzi mAb clone 1G11.3; anti-ThermoScript mAb
DE11; Primary anti-Tzi antibody clone 9G3.3, Primary anti-Tzi
antibody clone 6F3.3, anti-RT-41 mAb #5, anti-RT-41 mAb #10,
anti-Taq mAb #5 and anti-Taq mAb #10.
[0070] It will be appreciated that Fab, F(ab').sub.2 and other
fragments of the above-described antibodies may be used in the
methods described herein. Such fragments are typically produced by
proteolytic cleavage, using enzymes such as papain (to produce Fab
fragments) or pepsin (to produce F(ab').sub.2 fragments). Antibody
fragments may also be produced through the application of
recombinant DNA technology or through synthetic chemistry.
Formulation of Compositions
[0071] Compositions of the invention can include, in addition to a
PBM and a PBM-binding molecule/complex, one or more polymerases.
Some such compositions include one or more thermostable polymerases
in addition to a PBM and a PBM-binding molecule. Any thermostable
polymerase, PBM (e.g., PBA) that binds to the polymerase, and
PBM-binding molecule/complex are suitable for use in the invention,
including those described herein. In methods or compositions
involving the use or presence of polymerase, the polypeptides
having polymerase activity are used in the present methods at a
final concentration in solution of about 0.1-4000 units/ml, about
0.1-1000 units/ml, about 0.1-500 units/ml, about 0.1-250 units/ml,
about 0.1-100 units/ml, about 0.1-50 units/ml, about 0.1-40
units/ml, about 0.1-36 units/ml, about 0.1-34 units/ml, about
0.1-32 units/ml, about 0.1-30 units/ml, or about 0.1-20 units/ml.
In one embodiment, the polypeptides having nucleic acid polymerase
and/or reverse transcriptase activity are used at a final
concentration in solution of about 20 units/ml. Of course, other
suitable concentrations of reverse transcriptase enzymes and
nucleic acid polymerases will be apparent to one of ordinary skill
in the art.
[0072] Compositions in accord with the invention also can include
one or more DNA modifying enzymes (e.g., ligase, kinase,
phosphatase, nuclease, endonuclease, exonuclease, topoisomerase,
gyrase, terminal deoxynucleotidyl transferase), nucleic acid
templates, nucleic acid primers, nucleic acid substrates (e.g.,
rATP, rCTP, rGTP, rTTP, rUTP, rITP, dATP, dCTP, dGTP, dTTP, dUTP,
dITP, ddATP, ddCTP, ddGTP, ddTTP, ddUTP, ddITP, and derivatives
thereof, including labeled nucleosides and nucleotides), detectable
nucleic acid primers, and combinations thereof.
[0073] Compositions of the invention also can include one or more
detergents (e.g., TRITON X-100.RTM., Nonidet P-40, Tween 20, Brij
35, sodium deoxycholate and sodium dodecylsulfate), enzyme
cofactors, buffers (e.g., tris(hydroxymethyl)aminomethane,
N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid,
4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid,
N-(2-hydroxyethyl)piperazine-N'-(2-hydroxypropanesulfonic acid),
3-(N-morpholino)propanesulfonic acid and
N[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid, phosphate
salts (such as sodium phosphate (mono- or dibasic) and potassium
phosphate), sodium bicarbonate, and sodium acetate. Ammonium
sulfate, magnesium salt (e.g., magnesium chloride and magnesium
sulfate), manganese salt (e.g., manganese sulfate) and potassium
salts (e.g., potassium chloride) also may be included in
compositions of the invention. One or more chelating agents such as
ehylenediaminetetraacetate (EDTA) may also be included (e.g., at a
concentration of about 0.1 millimolar).
[0074] Compositions of the invention generally are at a pH in the
range of from about 7.5 to about 9.5 (e.g., at a pH of from about 8
to about 9).
[0075] The reagents described herein are provided and used in any
concentration suitable for a given use. Required amounts of
primers, cofactors and nucleotide-5'-triphosphates needed for
amplification or other reactions, and suitable ranges of each are
well known in the art. The amount of complex of polymerase and the
inhibitor is generally enough to supply at least about 1 unit of
enzyme per 100 .mu.l of reaction mixture once the inhibitor becomes
ineffective. In one embodiment, from about 1 to about 16 units of
polymerase per 100 .mu.l of reaction mixture are needed for PCR,
and depending upon the particular activity of a given enzyme, the
amount of complex is readily determined by one skilled in the art.
The amount of inhibitor present in the composition is generally
from about 0.5 to about 5 moles of inhibitor per mole of DNA
polymerase. In one embodiment, from about 1 to about 3 moles of
inhibitor per mole of DNA polymerase is used.
[0076] Compositions may be formulated at working concentrations, or
in solutions of higher reagent concentrations (e.g., 2.times.,
2.5.times., 5.times., 10.times., 20.times., 25.times., 50.times.,
100.times., 250.times., 500.times. and 1000.times.) that may then
be diluted before use.
Methods
[0077] The invention provides methods involving the use of
compositions that contain a PBM and a PBM-binding molecule/complex,
e.g., to inhibit a polymerase. The compositions described herein
are particularly useful for nucleic acid synthesis, sequencing,
amplification, and cloning. Such methods typically involve bringing
a polymerase into contact with a PBM (e.g., PBA) and a PBM-binding
molecule/complex, where binding of the PBM to the polymerase does
not substantially inhibit the polymerase activity of the
polymerase, but where binding of the PBM and PBM-binding
molecule/complex together substantially inhibit the polymerase
activity of the polymerase. Methods of the invention may also
involve reversing inhibition caused by a PBM and PBM-binding
molecule/complex.
Synthesis, Amplification and Sequencing
[0078] The compositions described herein are particularly useful in
methods for synthesizing, amplifying and sequencing nucleic acid
molecules. Nucleic acid synthesis methods of the invention can be
used to make any nucleic acid molecule from DNA or RNA templates,
including DNA molecules, RNA molecules, or hybrid DNA/RNA
molecules, and of which may be double-stranded or single-stranded.
As such, the methods and/or compositions of the invention may be
used in any technique including, but not limited to, primer
extension, transcription, amplification, PCR, reverse
transcription, sequencing and the like.
[0079] Nucleic acid synthesis and amplification methods in which
the present compositions may be used include PCR.RTM. (U.S. Pat.
Nos. 4,683,195 and 4,683,202), Strand Displacement Amplification
(SDA; U.S. Pat. No. 5,455,166; EP 0 684 315), Nucleic Acid
Sequence-Based Amplification (NASBA; U.S. Pat. No. 5,409,818; EP 0
329 822), and Abortive Transcription (Published U.S. Patent
Application No. 2003/0099950-A1). Nucleic acid sequencing
techniques include dideoxy sequencing methods such as those
disclosed in U.S. Pat. Nos. 4,962,022 and 5,498,523, as well as
more complex PCR-based nucleic acid fingerprinting techniques such
as Random Amplified Polymorphic DNA (RAPD) analysis (Williams, J.
G. K., et al., Nucl. Acids Res. 18(22):6531-6535, 1990),
Arbitrarily Primed PCR (AP-PCR; Welsh, J., and McClelland, M.,
Nucl. Acids Res. 18(24):7213-7218, 1990), DNA Amplification
Fingerprinting (DAF; Caetano-Anolles et al., Bio/Technology
9:553-557, 1991), microsatellite PCR or Directed Amplification of
Minisatellite-region DNA (DAMD; Heath, D. D., et al., Nucl. Acids
Res. 21(24): 5782-5785, 1993), and Amplification Fragment Length
Polymorphism (AFLP) analysis (EP 0 534 858; Vos, P., et al., Nucl.
Acids Res. 23(21):4407-4414, 1995; Lin, J J., and Kuo, J., FOCUS
17(2):66-70, 1995).
[0080] Nucleic acid amplification methods comprise contacting a
nucleic acid molecule to be amplified with one or more of the
compositions described herein, thus providing a population of
amplified copies of the nucleic acid molecule. Nucleic acid
sequencing methods comprise contacting the nucleic acid molecule to
be sequenced with one or more of the compositions described herein.
According to these methods, amplification and sequencing of the
nucleic acid molecule may be accomplished by any of the
above-described amplification and sequencing techniques. In one
embodiment, amplification and sequencing is performed by PCR. The
present amplification and sequencing methods may be used for
amplification and sequencing of nucleic acid molecules between
about 0.5 and 7 kb, 0.5-5 kb, 1-5 kb, 1-3 kb or 1-2 kb.
[0081] Nucleic acid synthesis, amplification and sequencing methods
typically involve contacting a template nucleic acid with a
composition comprising a polymerase (e.g., thermostable
polymerase), a PBM, a PBM-binding molecule/complex, and nucleotide
substrates under conditions where the polymerase activity of the
polymerase is inhibited. Such reaction mixtures typically include a
nucleic acid primer as well. Such nucleic acid synthesis,
amplification and sequencing methods typically involve bringing
such reaction mixtures to a condition (e.g., higher temperature)
that is sufficient to reverse inhibition of the polymerase but that
does not substantially reduce the polymerase's polymerase activity.
For example, such inhibition reversal may be accomplished by
shifting a reaction mixture to a temperature up to 40.degree. C.,
50.degree. C., 55.degree. C., 60.degree. C., 65.degree. C.,
70.degree. C., 75.degree. C., 80.degree. C., 90.degree. C.,
95.degree. C. or 99.degree. C. This heating may be sufficient to
cause denaturation of the PBM (e.g., antibody); denaturation of the
PBM-binding molecule/complex; dissociation of the PBM from the
nucleic acid polymerase; dissociation of the PBM-binding
molecule/complex from the PBM; dissociation of the PBM-binding
molecule/complex, or a combination of these effects.
Cloning
[0082] Methods of cloning nucleic acids also are provided. Such
cloning methods typically involve synthesizing or amplifying one or
more nucleic acid molecules using a polymerase; incubating the
synthesized nucleic acids with a PBM and a PBM-binding
molecule/complex, such that binding of the PBM to the polymerase
does not substantially inhibit the polymerase activity of the
polymerase, but such that binding of the PBM and PBM-binding
molecule/complex together substantially inhibit the polymerase
activity of the polymerase; and inserting the amplified or
synthesized nucleic acid molecules into one or more host cells.
[0083] The invention provides cloning methods that involve the use
of a PBM and PBM-binding molecule, whereby residual polymerase
activity remaining in the reaction mixture after nucleic acid
amplification or synthesis is inactivated or inhibited. By the
methods described herein, amplified, synthesized or digested
nucleic acid molecules may be quickly and efficiently ligated
(using ligases, topoisomerases, etc.) into cloning vectors, and
these vectors then inserted into host cells.
[0084] One exemplary cloning method involves: (a) amplifying or
synthesizing one more nucleic acid molecules in the presence of one
or more polymerases to produce amplified nucleic acid molecules;
and (b) incubating the nucleic acid molecules with a PBM and
PBM-binding molecule under conditions sufficient to inhibit or
inactivate the polymerase activity of the polymerase.
[0085] In one embodiment, the amplified nucleic acid fragments may
be cloned (ligated) directly into one or more vectors to produce
one or more genetic constructs. The genetic constructs then may be
transformed into one or more host cells.
[0086] In other exemplary cloning methods, amplified molecules
cleaved or digested with one or more restriction enzymes or one or
more recombination proteins as described in more detail below are
cloned into appropriate insertion sites of cloning vectors (see,
e.g., Ausubel, F. M., et al., eds., "Current Protocols in Molecular
Biology," New York: John Wiley & Sons, Inc., pp. 3.16.1-3.16.11
(1995)). Restriction enzymes used for cleavage of the amplified
molecules may include blunt-end cutters (e.g., SmaI, SspI, ScaI,
etc.) and sticky-end cutters (e.g., HindIII, BamHI, KpnI, etc.).
Such cloning methods also may involve the use of uracil DNA
glycosylase ((UDG); see U.S. Pat. No. 5,137,814, which is
incorporated herein by reference in its entirety). Such methods
typically involve: (a) forming a mixture comprising one or more
nucleic acid molecules, one or more PBMs and one or more
PBM-binding molecules; and (b) ligating the nucleic acid molecules
into one or more of the above-described vectors to form one or more
genetic constructs. Analogously, methods suitable for cloning a
nucleic acid molecule into one or more vectors typically involve:
(a) forming a mixture comprising nucleic acid molecules to be
cloned, cloning vectors and one or more polymerase inhibitors; and
(b) ligating the nucleic acid molecules into one or more vectors to
form one or more genetic constructs. Another exemplary cloning
method involves: (a) forming a mixture comprising the nucleic acid
molecules to be cloned, one or more PBMs, one or more PBM-binding
molecules and one or more restriction endonucleases; and (b)
ligating the nucleic acid molecules into one or more of the
above-described vectors to form one or more genetic constructs.
Another exemplary cloning method involves: (a) forming a mixture
comprising the nucleic acid molecules to be cloned, one or more
PBMs, one or more PBM-binding molecules, and one or more
recombination proteins; and (b) ligating the nucleic acid molecules
into one or more of the above-described vectors to form one or more
genetic constructs.
[0087] The mixture formed in the steps (a) of the above-described
methods may further comprise one or more additional components,
including, a polymerase, dNTPs or ddNTPs, one or more buffer salts,
and the like. A polymerase and restriction endonuclease or
recombination protein may be added to the mixture simultaneously,
or may be added sequentially, in any order.
[0088] The exemplary cloning methods may also comprise one or more
additional steps, such as the transformation of one or more of the
genetic constructs formed by these methods into host cells.
Target Nucleic Acids
[0089] Nucleic acid may be DNA (including cDNA), RNA (including
polyadenylated RNA (polyA+RNA), messenger RNA (mRNA), transfer RNA
(tRNA) and ribosomal RNA (rRNA)) or DNA-RNA hybrid molecules, and
may be single-stranded or double-stranded.
[0090] Nucleic acids to be cloned, or to serve as templates for
sequencing, synthesis, amplification may be derived from a variety
of sources. For example, target nucleic acids may be prepared
synthetically according to standard organic chemical synthesis
methods that will be familiar to one of ordinary skill or may be
obtained from natural sources, such as a variety of cells, tissues,
organs or organisms. Nucleic acids and cDNA libraries may be
obtained commercially, for example from Invitrogen (Carlsbad,
Calif.) and other commercial suppliers that will be familiar to the
skilled artisan.
[0091] A target nucleic acid may also be extracted in some manner
to make it available for contact with the primers and other
reagents. This may involve the removal of unwanted proteins and
cellular matter from the specimen. Various procedures for doing
this are known in the art, including those described by Laure et al
in The Lancet, pp. 538-540 (Sep. 3, 1988), Maniatis et al,
Molecular Cloning: A Laboratory Manual, pp. 280-281 (1982),
Gross-Belland et al in Eur. J. Biochem., 36, 32 (1973) and U.S.
Pat. No. 4,965,188. Extraction of DNA from whole blood or
components thereof are described, for example, in EP-A-0 393 744
(published Oct. 24, 1990), Bell et al, Proc. Natl. Acad. Sci. USA,
78(9), pp. 5759-5763 (1981) and Saiki et al, Bio/Technology, pp.
1008-1012 (1985).
Variations
[0092] Variations of the compositions and methods described herein
may be performed by one of ordinary skill in the art. For example,
in any of the described methods, a PBM and a PBM-binding
molecule/complex may be added to a nucleic acid synthesis reaction
mixture together or separately. As another example, molecules that
comprise a PBA-binding complex may be added to a nucleic acid
synthesis reaction mixture together or separately. As yet another
example, polymerase inhibition compositions or methods may include
or involve two or more PBMs (one or more of which can be an
antibody (i.e., PBA)), two or more PBM-binding molecules/complexes
(one or more of which can be or include an antibody), and/or two or
more polymerases.
[0093] It will be readily apparent to one of ordinary skill in the
art that other suitable modifications and adaptations to the
methods and applications described herein are obvious and may be
made without departing from the scope of the embodiments described
herein. Having now described the present embodiments in detail, the
same will be more clearly understood by reference to the following
examples, which are included herewith for purposes of illustration
only and are not intended to be limiting.
EXAMPLES
Example 1
Multicomponent Inhibition of Superscript III Reverse Transcriptase
and Tzi DNA-Dependent DNA Polymerase
Introduction
[0094] This example demonstrates that multicomponent inhibitors are
effective at inhibiting nucleic acid polymerase activity. Such
inhibition may be practiced on any polymerase. In the following
example, inhibitors were identified for inhibiting an RNA-dependent
DNA polymerase (reverse transcriptase) and a DNA-dependent DNA
polymerase. The degree of inhibition could be modulated by
selection of different polymerase binding antibodies as the first
molecule, and different antibody binding molecules or complexes of
molecules as the second component of the inhibitor. Inhibition of a
thermostable polymerase was reversed by heating or dilution.
Materials and Methods
[0095] Antibody production: Recombinant SSIII, Tzi, Rt41A and Taq
DNA polymerases were produced and purified (Invitrogen) and used to
raise monoclonal mouse anti-SSIII, anti-Tzi, anti-Rt41A, and
anti-Taq antibodies by commercial antibody producers (Chemicon
International, Temecula, Calif. and ProSci Incorporated, Poway
Calif.). All antibodies were verified by the manufacturer to be
antigen specific by ELISA. Monoclonal antibodies were purified from
hybridoma supernatants or ascites fluid using standard protein G
chromatography methods.
[0096] SuperScript III unit assay: The activity of SSIII with and
without inhibitors was measured using the MMLV reverse
transcriptase unit assay (Invitrogen SOP 30573.SOP) with slight
modification. Activity was measured in 25 .mu.l total volume of
assay buffer (50 mM Tris-HCl pH8.3, 75 mM KCl, 6 mM MgCl.sub.2, 1
mM DTT, 0.5 mM dTTP, 1 mM poly(A)-0.6 mM d(T).sub.25, .about.20
.mu.Cu/ml [.alpha.-.sup.32P]dTTP) containing 1 U of SSIII and
varying amounts of inhibitory components. Incubations were
performed at indicated temperatures for 15 minutes, and terminated
by spotting 20 .mu.l of each reaction onto Whatman GF/C fiber
filters. Unincorporated label was removed by TCA washes, and
incorporation assessed by liquid scintillation.
[0097] Tzi, Rt41A, and Taq unit assay: The activity of Tzi, RT41A,
or Taq with and without inhibitors was measured using the Taq/Tsp
unit assay (Invitrogen SOP 30382.SOP), with slight modification.
Activity was measured in 25 ul total volume in assay buffer (25 mM
TAPS pH 9.3, 2 mM MgCl.sub.2, 50 mM KCl, 1 mM DTT, 0.2 mM dNTP mix,
2.5 .mu.g nicked salmon testes DNA, and 21 .mu.Ci/ml
[.alpha.-.sup.32P]dCTP), containing 0.25 U Tzi, RT-41, or Taq, and
varying amounts of inhibitory components. Incubations were
performed at indicated temperatures for 15 minutes, and terminated
by spotting 20 .mu.l of each reaction onto Whatman GF/C fiber
filters. Unincorporated label was removed by TCA washes, and
incorporation assessed by liquid scintillation.
Inhibition of Reverse Transcriptase Activity by Multicomponent
Inhibitors
[0098] To determine if SuperScript III (SSIII) can be inhibited by
antibodies, SSIII activity was measured by unit assay in the
presence and absence of mouse anti-SSIII monoclonal antibodies
(mAb). Anti-SSIII clones #2 and #4 did not directly inhibit SSIII
activity, either alone or in combination, even to 10 fold molar
excess of mAb to SSIII (FIG. 1). However, the addition of
goat-anti-mouse-IgG-horse radish peroxidase (anti-IgG-HRP) in
combination with anti-SSII clones #4 and #2 strongly inhibited
SSIII activity (FIG. 1). This inhibition was specific to anti-SSIII
mAb components, as SSIII activity was inhibited by anti-IgG-HRP in
combination with any of an additional four independent anti-SSIII
mAbs, but not with an anti-ThermoScript mAb (DE11) (FIG. 2).
Denaturing the anti-SSIII mAbs before adding anti-IgG-HRP prevented
the majority of the SSIII inhibition (FIG. 1), as did titrating the
amount of anti-IgG-HRP (FIG. 2). Furthermore, anti-IgG-HRP by
itself produced little inhibition of SSIII activity (data not
shown). Taken together, these results indicate that both components
of the inhibitory complex (the SSIII-specific mAb and the
inhibitory anti-IgG mAb) must be present for strong inhibition of
SSIII activity.
[0099] To determine if components other than anti-IgG-HRP could
effectively inhibit SSIII activity in conjunction with anti-SSIII,
a variety of molecules that interact with antibodies were screened
for the ability to inhibit SSII in the presence and absence of
anti-SSIII. The lectin Concavalin A, AlexaFluor 488 conjugated
streptococcal antibody-binding protein G, anti-IgG, and the
anti-IgG-biotin+streptavidin complex all produced little or no
inhibition of SSIII activity by themselves (FIG. 3, and data not
shown for anti-IgG). However, in conjunction with anti-SSIII,
protein G-AlexaFluor 488, anti-IgG, and
anti-IgG-biotin+streptavidin each partially inhibited SSIII, while
anti-IgG+Protein G-AlexaFluor 488 very strongly inhibited SSIII
(FIG. 3). These results indicate that a variety of
antibody-interacting molecules can couple with the anti-SSIII to
effect SSIII inhibition.
[0100] To determine if inhibition of SSIII by the multi-component
complex is reversible, SSIII unit-activity was measured at both
room temperature and at a temperature where the antibody should be
destabilized/denatured (55.degree.). In agreement with previous
experiments, SSIII was strongly inhibited by both anti-IgG-HRP and
anti-IgG+Protein G-AlexaFluor 488 (Gmix) at room temperature in the
presence of anti-SSIII but not anti-ThermoScript (DE11) (FIG. 4).
As expected, the SSIII inhibition seen at room temperature was
strongly reversed at 55.degree.. These data demonstrate that these
SSIII-inhibitor formulations have the potential to be useful,
reversible, inhibitors of RT activity at room temperature.
Inhibition of Tzi, Rt41A and Taq Polymerase Activity by
Multicomponent Inhibitors
[0101] To determine if the principle of multi-component inhibitors
can be applied to enzymes other than SSIII, inhibition of the
thermostable DNA polymerases Tzi, RT-41 and Taq was assayed in the
presence and absence of multi-component inhibitors. Each of six
primary mouse anti-Tzi mAbs failed to directly inhibit Tzi by
themselves, as did Gmix by itself (FIG. 5). However, in combination
with Gmix, each of the six anti-Tzi mAbs produced moderate to
strong inhibition of Tzi (FIG. 5). Furthermore, as seen with SSIII
multicomponent inhibitors, the inhibition of Tzi at lower
temperatures was significantly reversed at elevated temperatures
(FIG. 6). Multi-component inhibitors have thus been demonstrated to
be effective and reversible inhibitors of reverse transcriptases
and DNA polymerase, and are likely to be broadly applicable
inhibitors of many classes of enzymes.
[0102] To determine the effect of the molar ratio of anti-Tzi
monoclonal antibody on Tzi polymerase activity, the Tzi unit assay
was performed with no anti-Tzi monoclonal antibody, or in the
presence of increasing molar ratios of anti-Tzi to Tzi (between
1:256 and 8:1). Tzi unit activity was measured at 37 C in the
presence of increasing concentrations of anti-Tzi mAbs 9G3.3+6F3.2,
either with or without a secondary inhibitor mixture comprising a
4:1 molar ratio rabbit-anti-mouse IgG:Tzi and 4:1 molar ratio
protein G:Tzi. At a 1:1 molar ratio of anti-Tzi mAbs:Tzi, in the
absence of secondary inhibitors, Tzi activity was potentiated,
whereas in the presence of secondary inhibitors, Tzi activity was
maximally inhibited (FIG. 7). Tzi inhibition began to be lost above
molar ratios of anti-Tzi mAB:Tzi of 2:1, most likely due to
titration of the secondary inhibitors by the excess of Tzi. Similar
results to the above were also obtained with RT-41 (FIG. 8) and Taq
(FIG. 9) DNA polymerases using anti-Rt41A and anti-Taq monoclonal
antibodies with rabbit-anti-mouse-IgG secondary antibody.
[0103] To determine the effect of the molar ratio of
rabbit-anti-mouse-IgG on Tzi polymerase activity, the Tzi unit
assay was performed in the presence of increasing concentrations of
rabbit-anti-mouse-IgG (between 1:16 and 8:1) either with or without
a 1:1 molar ratio of anti-Tzi mAbs (9G3.3+6F3.2):Tzi. Molar ratios
of rabbit-anti-mouse-IgG:Tzi as low as 4:1 maximally inhibited Tzi
activity in the presence but not the absence of anti-Tzi mAbs (FIG.
10).
[0104] To determine the effect of preincubation temperature on Tzi
activity, Tzi alone or (Tzi+anti-Tzi mAbs+rabbit-anti-mouse IgG)
was preincubated at various temperatures for 2 minutes prior to
performing the Tzi unit assay. Tzi activity was measured at
37.degree. C. after the preincubation, and nearly full activity was
regained after as little as 2 minutes preincubation at 94.degree.
C. (FIG. 11).
[0105] The multicomponent inhibitor methods described above also
improved the specificity and yield of specific product using
PCR.
[0106] Antibodies suitable for use in the present invention which
bind to nucleic acid polymerases and which do not substantially
inhibit the polymerase activity may be identified using the methods
described above. Similarly, antibody-binding molecules or complexes
of molecules which bind to the antibody and substantially inhibit
the polymerase activity may also be identified using the methods
described above. Thus, any such antibody which does not
substantially inhibit, and any antibody-binding molecule or complex
of molecules which substantially inhibits polymerase activity are
within the scope of the present invention.
[0107] Having now fully described the present invention in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious to one of ordinary skill in
the art that the same can be performed by modifying or changing the
invention within a wide and equivalent range of conditions,
formulations and other parameters without affecting the scope of
the invention or any specific embodiment thereof and that such
modifications or changes are intended to be encompassed within the
scope of the appended claims.
[0108] All publications, patents and patent applications mentioned
in this specification are indicative of the level of skill of those
skilled in the art to which this invention pertains, and are herein
incorporated by reference to the same extent as if each individual
publication, patent or patent application was specifically and
individually indicated to be incorporated by reference.
* * * * *