U.S. patent application number 09/757132 was filed with the patent office on 2001-08-16 for nucleic acid detection.
This patent application is currently assigned to Promega Corporation. Invention is credited to Leippe, Donna M., Lewis, Martin K., Nelson, Lisa S., Nelson, Michelle N., Shultz, John W..
Application Number | 20010014451 09/757132 |
Document ID | / |
Family ID | 21921054 |
Filed Date | 2001-08-16 |
United States Patent
Application |
20010014451 |
Kind Code |
A1 |
Shultz, John W. ; et
al. |
August 16, 2001 |
Nucleic acid detection
Abstract
This invention discloses methods, compositions and kits for the
detection of extremely low levels of nucleic acid, cells and
cellular material in biological samples. The nucleic acid detection
systems utilize either the pyrophosphorolysis reaction catalyzed by
various polymerases or nuclease digestion coupled with
pyrophosphorylation catalyzed by phosphoribosylpyrophosphate
synthetase to produce either deoxyribonucleoside triphosphates or
ribonucleoside triphosphates. dNTPs are transformed to ATP by the
action of nucleoside diphosphate kinase. The ATP produced by these
reactions may be detected by luciferase or NADH based detection
systems. If more sensitive detection is required, schemes for the
amplification of NTPs and dNTPS are provided. A detection system
for cells or cellular material in a sample is provided wherein AMP
and a high energy phosphate donor added to a sample are converted
to ATP by the action of endogenous enzymes, followed by detection
of the ATP.
Inventors: |
Shultz, John W.; (Verona,
WI) ; Nelson, Michelle N.; (Fitchburg, WI) ;
Leippe, Donna M.; (Madison, WI) ; Lewis, Martin
K.; (Madison, WI) ; Nelson, Lisa S.; (De
Forest, WI) |
Correspondence
Address: |
WELSH & KATZ, LTD
120 S RIVERSIDE PLAZA
22ND FLOOR
CHICAGO
IL
60606
US
|
Assignee: |
Promega Corporation
2800 Woods Hollow Road
Madison
WI
53711
|
Family ID: |
21921054 |
Appl. No.: |
09/757132 |
Filed: |
January 9, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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09757132 |
Jan 9, 2001 |
|
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09042287 |
Mar 13, 1998 |
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Current U.S.
Class: |
435/6.13 ;
536/23.1 |
Current CPC
Class: |
C12Q 1/66 20130101; C12Q
1/6827 20130101; C12Q 1/04 20130101; C12Q 2521/319 20130101; C12Q
2535/131 20130101; C12Q 2521/319 20130101; C12Q 2535/131 20130101;
C12Q 2535/107 20130101; C12Q 2521/319 20130101; Y10S 435/81
20130101; C12Q 2535/131 20130101; C12Q 1/68 20130101; C12Q 1/6827
20130101; C12Q 1/6823 20130101; C12Q 1/6823 20130101; C12Q 1/6823
20130101 |
Class at
Publication: |
435/6 ;
536/23.1 |
International
Class: |
C12Q 001/68 |
Claims
What is claimed is:
1. A method of detecting deoxyribonucleic acid in a reaction
containing pyrophosphate, adenosine 5'-diphosphate, or a
combination thereof, the method comprising: depolymerizing a
deoxyribonucleic acid at a terminal nucleotide by enzymatically
cleaving the terminal internucleotide phosphodiester bond and
reforming same with a pyrophosphate molecule to form a free
deoxyribonucleoside triphosphate molecule according to the
reaction: dNA.sub.n+PP.sub.i.fwdarw.dNA.sub.n-1+dNTP; enzymatically
transferring terminal 5' phosphate groups from the
deoxyribonucleoside triphosphate molecules to adenosine
5'-diphosphate molecules to form adenosine 5'-triphosphate
according to the following general reaction
dNTP*+ADP.fwdarw.dNDP+ATP*, wherein P* is the terminal 5' phosphate
so transferred; and detecting the adenosine 5'-triphosphate formed
thereby.
2. The method of claim 1 wherein the depolymerizing step is
catalyzed by a DNA polymerase or reverse transcriptase selected
from the group consisting of T4 polymerase, Taq polymerase, AMV
reverse transcriptase and MMLV reverse transcriptase.
3. The method of claim 1, wherein the step of enzymatically
transferring terminal 5' phosphate groups from the nucleoside
triphosphate molecules to adenosine 5'-diphosphate molecules to
form adenosine 5'-triphosphate molecules is catalyzed by nucleoside
diphosphate kinase.
4. The method of claim 1, wherein the ATP detection method is a
luciferase detection system.
5. The method of claim 1, wherein the ATP detection method is a
NADH detection system.
6. The method of claim 1, wherein the depolymerizing step and
phosphate transferring steps are performed in a single pot
reaction.
7. The method of claim 1, together with the step of amplifying the
adenosine 5'-triphosphate molecules produced by the phosphate
transferring step.
8. The method of claim 7 wherein the amplification is performed
according to the method of claim 39.
9. A method of detecting polyadenylated mRNA in a reaction
containing pyrophosphate, the method comprising: depolymerizing the
polyadenylated mRNA at a terminal nucleotide by enzymatically
cleaving the terminal internucleotide phosphodiester bond and
reforming same with a pyrophosphate molecule to form a free
adenosine triphosphate molecule according to the reaction catalyzed
by poly(A) polymerase: NA.sub.n+PP.sub.i.fwdarw.NA.sub.n-1+ATP; and
detecting the adenosine 5'-triphosphate formed thereby.
10. The method of claim 9, wherein the ATP detection method is a
luciferase detection system.
11. The method of claim 9, wherein the ATP detection method is a
NADH detection system.
12. The method of claim 9, together with the step of amplifying the
adenosine 5'-triphosphate molecules produced by the depolymerizing
step.
13. The method of claim 12 wherein the amplification is performed
according to the method of claims 39.
14. A method of selectively detecting poly(A)-mRNA in a reaction
containing pyrophosphate, adenosine 5'-diphosphate, or a
combination thereof, the method comprising: hybridizing a
complimentary oligo(dT) probe to poly(A)-mRNA to form a RNA-DNA
hybrid, depolymerizing the oligo(dT) strand of the RNA-DNA hybrid
at the terminal nucleotide by enzymatically cleaving the terminal
internucleotide phosphodiester bond and reforming same with a
pyrophosphate molecule to form a deoxythymidine 5'-triphosphate
molecule according to the following general reaction catalyzed by a
reverse transcriptase: dTT.sub.n+PP.sub.i.fwdarw.dTT.sub.n-
-1+dTTP; enzymatically transferring terminal 5' phosphate groups
from the deoxythymidine 5'-triphosphate molecules to adenosine
5'-diphosphate molecules to form adenosine 5'-triphosphate
molecules according to the following general reaction:
dTTP*+ADP=dTDP+ATP*, wherein P* is the terminal 5' phosphate so
transferred; and detecting the adenosine 5'-triphosphate formed
thereby.
15. The method of claim 14, wherein the ATP detection method is a
luciferase detection system.
16. The method of claim 14, wherein the ATP detection method is a
NADH detection system.
17. The method of claim 14, together with the step of amplifying
the adenosine 5'-triphosphate molecules produced by the
depolymerizing step.
18. The method of claim 17 wherein the amplification is performed
according to the method of claim 39.
19. A method of detecting DNA in a reaction containing
phosphoribosylpyrophosphate, adenosine 5'-diphosphate, or a
combination thereof, the method comprising: producing free
deoxyadenosine monophosphate molecules from the nucleic acid by
digestion with a nuclease; enzymatically transferring the
pyrophosphate from phosphoribosylpyrophosphate molecules to the
deoxyadenosine monophosphate molecules to form deoxyadenosine
triphosphate molecules according to the following reaction
catalyzed by phosphoribosylpyrophosphate synthetase
dAMP+PRPP.fwdarw.dATP+ribose-5-PO.sub.4; enzymatically transferring
terminal 5' phosphate groups from the deoxyribonucleoside
triphosphate molecules to adenosine 5'-diphosphate molecules to
form adenosine 5'-triphosphate molecules according to the following
general reaction: dATP*+ADP.fwdarw.dADP+ATP*, wherein P* is the
terminal 5' phosphate so transferred; and detecting the ATP so
produced.
20. The method of claim 19, wherein the enzymatic transfer of
phosphate from the free deoxyadenosine triphosphate molecules to
adenosine 5'-diphosphate molecules is catalyzed by nucleoside
diphosphate kinase.
21. The method of claim 19, wherein the detection system is a
luciferase detection system.
22. The method of claim 19, wherein the detection system is a NADH
detection system.
23. The method of claim 19, wherein the pyrophosphate transferring
step and the phosphate transferring step are performed in a single
pot reaction.
24. The method of claim 19, together with the step of amplifying
the adenosine 5'-triphosphate molecules produced by the phosphate
transferring step.
25. The method of claim 24 wherein the amplification is performed
according to the method of claim 39.
26. A method of detecting RNA in a reaction containing
phosphoribosylpyrophosphate, the method comprising: producing free
ribonucleoside monophosphate molecules from the RNA by digestion
with a nuclease; enzymatically transferring the pyrophosphate from
phosphoribosylpyrophosphate molecules to the adenosine
monophosphate molecules to form adenosine triphosphate molecules
according to the following reaction catalyzed by
phosphoribosylpyrophosphate synthetase
AMP+PRPP.fwdarw.ATP+ribose-5-PO.sub.4; and detecting the ATP so
produced.
27. The method of claim 26, wherein the detection system is a
luciferase detection system.
28. The method of claim 26, wherein the detection system is a NADH
detection system.
29. The method of claim 26, together with the step of amplifying
the adenosine 5'-triphosphate molecules produced by the phosphate
transferring step.
30. The method of claim 29 wherein the amplification is performed
according to the method of claim 39.
31. A method for determining the presence and/or amount of cells
and cellular material, the method comprising: releasing the
contents of cells to form a cell lysate; adding phosphate donor
molecules and adenosine 5'-monophosphate molecules to the cell
lysate so that adenosine 5'-diphosphate molecules are produced by
the enzymatic transfer of a phosphate group from the donor to the
adenosine 5'-monophosphate according to the following general
reaction catalyzed by endogenous enzymes present in the cell
lysate: D--P+AMP.fwdarw.D+ADP; and producing adenosine
5'-triphosphate by the enzymatic transfer of a phosphate group from
the donor molecules to the adenosine 5'-diphosphate molecules
according to the following general reaction catalyzed by endogenous
enzymes present in the cell lysate sample: D--P+ADP.fwdarw.D+ATP;
and detecting the adenosine 5'-triphosphate produced thereby.
32. The method of claim 31 wherein the phosphate donor is selected
from the group consisting of deoxycytidine 5-triphosphate,
deoxyguanidine 5' triphosphate and deoxythymidine 5'
triphosphate.
33. A composition of matter for producing adenosine 5'-triphosphate
from DNA, pyrophosphate, and adenosine 5'-diphosphate, the
composition comprising a mixture of: a nucleoside diphosphate
kinase; and a nucleic acid polymerase, the nucleoside diphosphate
kinase and the nucleic acid polymerase provided in a sufficient
concentration to catalyze the production of the adenosine
triphosphate from DNA at about picogram to microgram amounts of
DNA.
34. A composition of matter for producing adenosine triphosphate
from DNA, phosphoribosylpyrophosphate and adenosine 5'-diphosphate,
the composition comprising a mixture of: a
phosphoribosylpyrophosphate synthetase; and a nucleoside
diphosphate kinase, the phosphoribosylpyrophosphate synthetase and
nucleoside diphosphate kinase in a sufficient concentration to
catalyze the production of the adenosine triphosphate from about
picogram to microgram amounts of DNA.
35. A kit containing reagents for the detection of DNA by
pyrophosphorolysis comprising: a vessel containing a nucleic acid
polymerase; and a vessel containing a nucleoside diphosphate
kinase.
36. The kit of claim 35 wherein the nucleic acid polymerase and
nucleoside diphosphate kinase are provided in the same
container.
37. A kit containing reagents for the detection of DNA by nuclease
digestion comprising: a vessel containing
phosphoribosylpyrophosphate synthetase; and a vessel containing a
nucleoside diphosphate kinase.
38. The kit of claim 37 wherein the phosphoribosylpyrophosphate
synthetase and nucleoside diphosphate kinase are provided in the
same container.
39. A method of producing a plurality of adenosine triphosphate
molecules from a nucleoside triphosphate molecule in a reaction
containing adenosine 5'-monophosphate molecules, high energy
phosphate donor molecules, or a combination thereof, the method
comprising the steps of: (1) enzymatically transferring the
terminal 5' phosphate group from a nucleoside triphosphate molecule
present in the sample to an adenosine 5'-monophosphate molecule
added to the sample to form an adenosine 5'-diphosphate molecule
and nucleoside diphosphate molecule according to the following
general reaction catalyzed by a first enzyme:
XTP+AMP.fwdarw.XDP+ADP; (2) enzymatically transferring a phosphate
group from a high energy phosphate donor molecule which may not be
utilized by the first enzyme to adenosine 5'-diphosphate molecules
to form adenosine 5'-triphosphate molecules according to the
following general reaction catalyzed by nucleoside diphosphate
kinase: ADP+D--P=ATP+D; and (3) amplifying the adenosine
triphosphate molecule so produced by repeating steps (1) and (2)
until the desired level of amplification is achieved.
40. The method of claim 39 wherein the first enzyme is selected
from the group consisting of nucleoside monophosphate kinase and
adenylate kinase.
41. The method of claim 39 wherein the high energy phosphate donor
is selected from the group of dCTP and AMP-CPP.
42. A method of detecting deoxyribonucleic or ribonucleic acid in a
reaction containing pyrophosphate, adenosine 5-monophosphate and a
high energy phosphate donor, or a combination thereof, the method
comprising the following steps performed in a single pot reaction:
depolymerizing a nucleic acid at a terminal nucleoside by
enzymatically cleaving the terminal internucleotide phosphodiester
bond and reforming same with a pyrophosphate molecule to form a
free ribonucleotide or deoxyribonucleoside triphosphate molecule
according to reaction 1: NA.sub.nPP.sub.i.fwdarw.NA.sub.n-1+XTP;
repeating the depolymerizing step to obtain at is least two
nucleoside triphosphate molecules; producing a plurality of
adenosine triphosphate molecules from the ribonucleoside
triphosphate or deoxyribonucleoside phosphate molecules by (i)
enzymatically transferring the terminal 5' phosphate group from the
ribonucleoside or deoxyribonucleoside triphosphate molecule formed
in reaction 1 to an adenosine 5-monophosphate molecule to produce
an adenosine 5-diphosphate molecule and a ribonucleoside or
deoxyribonucleoside diphosphate molecule according to reaction 2
catalyzed by a first enzyme XTP+AMP=XDP+ADP; (ii) enzymatically
transferring a phosphate group from a high energy phosphate donor
molecule which is not a substrate for the first enzyme to the
adenosine 5'-diphosphate molecules produced in reaction 2 to
produce an adenosine 5'-triphosphate molecules according to
reaction 3 catalyzed by a second enzyme: ADP+D--P=ATP+D; and (iii)
amplifying the adenosine triphosphate molecules so produced by
repeating steps (i) and (ii) until the desired level of
amplification is achieved; and detecting the ATP so produced.
43. The method of claim 42 wherein enzyme 1 is selected from the
group consisting of adenylate kinase and nucleoside monophosphate
kinase.
44. The method of claim 42 wherein enzyme 2 is selected from the
group consisting of pyruvate kinase and nucleoside diphosphate
kinase.
45. A kit containing reagents for the detection of nucleic acid by
nuclease digestion comprising: a vessel containing
phosphoribosylpyrophosphate synthetase; and a vessel containing a
nuclease.
46. A kit containing reagents for the detection of RNA by
pyrophosphorolysis comprising: a vessel containing
poly(A)-polymerase.
47. A kit containing reagents for the detection of cells and/or
cellular material in a sample comprising: a vessel containing
adenosine 5'-monophosphate; and a vessel containing a high energy
phosphate donor which may not be utilized by luciferase.
48. A method of assaying deoxyribonucleic acid in a reaction
containing pyrophosphate, adenosine 5'-diphosphate, or a
combination thereof, the method comprising: depolymerizing a
nucleic acid at a terminal nucleotide by enzymatically cleaving the
terminal internucleotide phosphodiester bond and reforming same
with a pyrophosphate molecule to form a free deoxyribonucleoside
triphosphate molecule according to the reaction:
dNA.sub.n+PP.sub.i.fwdarw.dNA.sub.n-1+dNTP; repeating the
depolymerizing step to obtain at least two deoxyribonucleoside
triphosphate molecules; enzymatically transferring terminal 5'
phosphate groups from the deoxyribonucleoside triphosphate
molecules to adenosine 5'-diphosphate molecules to form adenosine
5'-triphosphate according to the following general reaction
dNTP*+ADP.fwdarw.dNDP+ATP*, wherein P* is the terminal 5' phosphate
so transferred; and detecting the adenosine 5'-triphosphate formed
thereby.
49. The method of claim 48 wherein the depolymerizing step is
catalyzed by a DNA polymerase or reverse transcriptase selected
from the group consisting of T4 polymerase, Taq polymerase, AMV
reverse transcriptase and MMLV reverse transcriptase.
50. The method of claim 48, wherein the step of enzymatically
transferring terminal 5' phosphate groups from the nucleoside
triphosphate molecules to adenosine 5'-diphosphate molecules to
form adenosine 5'-triphosphate molecules is catalyzed by nucleoside
diphosphate kinase.
51. The method of claim 48, wherein the ATP detection method is a
luciferase detection system.
52. The method of claim 48, wherein the ATP detection method is a
NADH detection system.
53. The method of claim 48, wherein the depolymerizing step and
phosphate transferring steps are performed in a single pot
reaction.
54. The method of claim 48, together with the step of amplifying
the adenosine 5'-triphosphate molecules produced by the phosphate
transferring step.
55. The method of claim 54 wherein the amplification is performed
according to the method of claim 39.
56. A method of assaying polyadenylated mRNA in a reaction
containing pyrophosphate, the method comprising: depolymerizing the
polyadenylated mRNA at a terminal nucleotide by enzymatically
cleaving the terminal internucleotide phosphodiester bond and
reforming same with a pyrophosphate molecule to form a free
adenosine triphosphate molecule according to the reaction catalyzed
by poly(A) polymerase: NA.sub.n+PP.sub.i.fwdarw.NA.sub.n-1+ATP;
repeating the depolymerizing step to obtain at least 2 nucleoside
triphosphate molecules; and detecting the adenosine 5'-triphosphate
formed thereby.
57. The method of claim 56, wherein the ATP detection method is a
luciferase detection system.
58. The method of claim 56, wherein the ATP detection method is a
NADH detection system.
59. The method of claim 56, together with the step of amplifying
the adenosine 5-triphosphate molecules produced by the
depolymerizing step.
60. The method of claim 59 wherein the amplification is performed
according to the method of claims 39.
61. A method of selectively assaying poly(A)-mRNA in a reaction
containing pyrophosphate, adenosine 5'-diphosphate, or a
combination thereof, the method comprising: hybridizing a
complimentary oligo(dT) probe to poly(A)-mRNA to form a RNA-DNA
hybrid, depolymerizing the oligo(dT) strand of the RNA-DNA hybrid
at the terminal nucleotide by enzymatically cleaving the terminal
internucleotide phosphodiester bond and reforming same with a
pyrophosphate molecule to form a deoxythymidine 5'-triphosphate
molecule according to the following general reaction catalyzed by a
reverse transcriptase: dTT.sub.n*+PP.sub.i.fwdarw.dTT.sub.-
n-1+dTTP; repeating the depolymerizing step to obtain at least two
nucleoside triphosphate molecules; enzymatically transferring
terminal 5' phosphate groups from the deoxythymidine
5'-triphosphate molecules to adenosine 5'-diphosphate molecules to
form adenosine 5'-triphosphate molecules according to the following
general reaction: dTTP*+ADP=dTDP+ATP*, wherein P* is the terminal
5' phosphate so transferred; and detecting the adenosine
5'-triphosphate formed thereby.
62. The method of claim 61, wherein the ATP detection method is a
luciferase detection system.
63. The method of claim 61, wherein the ATP detection method is a
NADH detection system.
64. The method of claim 61, together with the step of amplifying
the adenosine 5-triphosphate molecules produced by the
depolymerizing step.
65. The method of claim 64 wherein the amplification is performed
according to the method of claim 39.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the field of molecular
biology, and in particular to the detection of nucleic acids and
cells. Methods and compositions for detection of extremely low
amounts of nucleic acids and cellular materials with ATP based
detection systems are described.
BACKGROUND
[0002] Methods for producing large amounts of recombinant protein
are well known. As the recombinant protein industry has developed,
the need for various quality control assays has arisen. An example
is the need for the quantitation of nucleic acids present in
recombinant protein preparations. Current guidelines require that
the amount of nucleic acid present in recombinant therapeutic
proteins be less than 10 pg of DNA per daily dose of recombinant
protein. Therefore, methods for detecting extremely low amounts of
nucleic acids are needed. Such methods would also find widespread
use for the quantitation of DNA in forensic samples.
[0003] Several methods of detecting low levels of nucleic acid have
been described. The first method is based on classical
hybridization techniques. This method utilizes radio-labeled
nucleic acid probes which bind to the DNA of interest. However,
this method has several disadvantages including poor
reproducibility, generation of large amounts of waste reagent, and
high background levels caused by nonspecific binding. Furthermore,
this technique is generally inappropriate for determining the
presence of low amounts of DNA of unknown sequence.
[0004] A second method of detecting nucleic acid utilizes
fluorescent dyes capable of intercalating into nucleic acids.
However, many interfering substances such as detergents, proteins,
and lipids affect the reproducibility of the signal generated by
this method.
[0005] A third method of detecting low levels of DNA utilizes
biotinylated single-stranded DNA binding protein (SSB),
streptavidin, an anti-DNA antibody fused to urease, and
biotinylated nitrocellulose as reagents. This assay is commercially
available from Molecular Devices and described in Kung et al.,
Picogram Quantitation of Total DNA Using SNA-Binding Proteins in a
Silicon Sensor-Based System, Anal. Biochem. 187: 220-27 (1990). The
assay is performed by incubating the streptavidin, biotin-SSB, and
the anti-DNA antibody together, allowing a complex to be formed.
The complex is then captured on the biotinylated filter, washed,
and the amount of captured urease is read. This method is highly
sensitive but has several disadvantages. These disadvantages
include costly reagents and the need for extensive controls.
[0006] A fourth method involves the depolymerization or degradation
of nucleic acids and detection of ATP by luciferase. Polynucleotide
polymerases are responsible for the synthesis of nucleic acids in
cells. These enzymes are also capable of catalyzing other reactions
as described in Deutscher and Kornberg, Enzymatic Synthesis of
Deoxyribonucleic Acid, J. Biol. Chem. 244(11):3019-28 (1969). Many,
but not all, polymerases are able to depolymerize nucleic acid in
the presence of either phosphate or pyrophosphate.
[0007] U.S. Pat. No. 4,735,897 describes a method of detecting
polyadenylated messenger RNA (poly(A)-mRNA). Depolymerization of
poly(A)-mRNA in the presence of phosphate has been shown to result
in the formation of ADP, which can be converted by pyruvate kinase
or creatine phosphokinase into ATP. RNA may also be digested by a
ribonuclease to AMP, converted to ADP by adenylate kinase, and then
converted to ATP by pyruvate kinase.
[0008] The ATP so produced is detected by a luciferase detection
system. In the presence of ATP and O2, luciferase catalyzes the
oxidation of luciferin, producing light which can then be
quantitated using a luminometer. Additional products of the
reaction are AMP, pyrophosphate and oxyluciferin.
[0009] The presence of ATP-generating enzymes in all organisms also
allows the use of a luciferase system for detecting the presence or
amounts of contaminating cells in a sample, as described in U.S.
Pat. 5,648,232. For example, ADP may be added to a sample suspected
of containing contaminating cells. The ADP is converted by enzymes
of the cell into ATP which is detected by a luciferase assay, as
described above. The disadvantage of this method is the relative
instability of the ADP substrate.
[0010] What is needed in the art are reliable, cost-effective
methods of detecting extremely low levels of nucleic acids, cells,
and cellular material in a wide variety of samples. The present
invention discloses novel methods for detecting low quantities of
DNA, RNA and of cells. These methods take advantage of novel
combinations of pyrophosphorolysis or enzymatic degradation of
nucleic acids, conversion of dNTPs to ATP, the conversion of AMP
directly to ATP, amplification of ATP to increase sensitivity,
depolymerization of oligonucleotide probes, and optimized reaction
conditions.
SUMMARY OF THE INVENTION
[0011] A need exists for quality control assays for proteins
produced by recombinant methods. Current guidelines suggest that
preparations of recombinant protein should contain less than 10
picograms of nucleic acid. There is also a need to be able to
quantitate extremely low levels of nucleic acids in forensics
samples. Therefore, it is an object of the present invention to
provide methods for detecting low amounts of nucleic acids and low
numbers of cells or cellular material. It is also an object of the
invention to provide compositions for the detection of nucleic
acids and kits for the detection of nucleic acids.
[0012] In one embodiment of the present invention a method is
provided for detecting and/or assaying deoxyribonucleic acid in a
reaction containing phosphate, adenosine 5-diphosphate, or a
combination thereof. The method comprises depolymerizing the
nucleic acid at a terminal nucleotide by enzymatically cleaving the
terminal internucleoside phosphodiester bond and reforming same
with a pyrophosphate molecule to form a deoxyribonucleoside
triphosphate molecule according to the following reaction:
NA.sub.n+PP.sub.i.fwdarw.NA.sub.n-31 1+dNTP
[0013] catalyzed by a DNA polymerase or reverse transcriptase
selected from the group consisting of T4 polymerase, Taq
polymerase, AMV reverse transcriptase, and MMLV reverse
transcriptase. In a quantitative assay for nucleic acids, the
depolymerizing step is repeated essentially to completion or
equilibrium to obtain at least two nucleoside triphosphate
molecules from a strand of minimally three nucleotides. For
detection of DNA, the depolymerizing step need not be repeated if
there are sufficient nucleic acid molecules present to generate a
signal. The next step involves enzymatically transferring terminal
5phosphate groups from the deoxyribonucleoside triphosphate
molecules to an adenosine 5'-diphosphate molecule to form adenosine
5'-triphosphate according to the following reaction:
DNTPI+ADP.fwdarw.NDP+ATP*
[0014] catalyzed by nucleoside diphosphate kinase and wherein P* is
the terminal 5' phosphate so transferred. The final step is the
detection of the ATP, either by a luciferase detection system or
NADH detection system. The depolymerizing step and phosphate
transferring step may optionally be performed in a single pot
reaction. If greater sensitivity is desired, the ATP molecules
produced by the phosphate transferring step or the NTPs produced by
the depolymerizing step may be amplified to form a plurality of ATP
molecules.
[0015] In another embodiment of the present invention, a method is
provided for detecting polyadenylated mRNA in a reaction containing
pyrophosphate. The polyadenylated mRNA is first depolymerized at a
terminal nucleotide by enzymatically cleaving the terminal
internucleoside phosphodiester bond and reforming same with a
pyrophosphate molecule to form a free ATP molecule according to the
following reaction:
NA.sub.n+PP.sub.i.fwdarw.NA.sub.n-1+ATP
[0016] catalyzed by poly(A) polymerase. In a quantitative assay for
RNA, the depolymerizing step is repeated essentially to completion
or equilibrium to obtain at least two nucleoside triphosphate
molecules from a strand of minimally three nucleotides. For
detection of DNA, the depolymerizing step need not be repeated if
there are sufficient nucleic acid molecules present to generate a
signal. The ATP molecules so formed are then detected with either a
luciferase detection system or a NADH detection system. The
sensitivity of the reaction may be increased by optionally
amplifying the ATP molecules.
[0017] In another embodiment of the present invention, a method is
provided for selectively detecting and/or assaying poly(A) mRNA in
a reaction containing pyrophosphate, adenosine 5'-diphosphate, or a
combination thereof. In this method, a complementary oligo (dT)
probe is hybridized to poly(A) mRNA to form an RNA-DNA hybrid. The
oligo (dT) strand of the RNA-DNA hybrid is then depolymerized at
the terminal nucleotide by enzymatically cleaving the terminal
internucleotide phosphodiester bond and reforming same with a
pyrophosphate molecule to form deoxythymidine 5'-triphosphate.
According to the following reaction:
[0018] TT.sub.n+PP.sub.i.fwdarw.TT.sub.n-1+dTTP
[0019]
[0020] catalyzed by a reverse transcriptase. In a quantitative
assay for nucleic acids, the depolymerizing step is repeated
essentially to completion or equilibrium to obtain at least two
nucleotide triphosphate molecules from a strand of minimally three
nucleotides. For detection of DNA, the depolymerizing step need not
be repeated if there are sufficient nucleic acid molecules present
to generate a signal. Next, the phosphate groups from the
deoxythymidine 5'-triphosphate are enzymatically transferred to
adenosine 5'-diphosphate molecules to form ATP molecules according
to the following reaction:
DTTPI+ADP.fwdarw.dTDP+ATP*
[0021] catalyzed by NDPK, wherein P* is the terminal 5' phosphate
so transferred. Finally, the ATP so formed is detected by a
luciferase detection system or an NADH detection system. If
increased sensitivity is desired, the terminal phosphate of the
dTTP may be transferred to ADP to form ATP as above followed by an
amplification of the resulting ATP.
[0022] In another embodiment of the present invention, a method is
provided of detecting DNA in a reaction containing
phosphoribosylpyrophosphate, adenosine 5'-diphosphate, or a
combination thereof. In this method, free deoxyribonucleoside
monophosphate molecules are produced from the nucleic acid by
digestion with a nuclease. A pyrophosphate group is then
enzymatically transferred from phosphoribosylpyrophosphate
molecules to the deoxyadenosine monophosphate molecules to form
deoxyadenosine triphosphate molecules according to the following
reaction:
dAMP+PRPP.fwdarw.dATP+ribose-5-PO.sub.4
[0023] catalyzed by phosphoribosylpyrophosphate synthetase. Next,
the terminal 5' phosphate groups from the deoxyadenosine
triphosphate molecules are enzymatically transferred to adenosine
5'-diphosphate molecules to form ATP molecules according to the
following reaction:
[0024] dATP*+ADP.fwdarw.dADP+ATP*
[0025]
[0026] catalyzed by NDPK wherein P* is a terminal 5' phosphate so
transferred. The ATP so produced may be detected by a luciferase
detection system or an NADH detection system. If desired, the
pyrophosphate transferring step and the phosphate transferring step
may be performed in a single pot reaction. If increased sensitivity
is required, the ATP molecules may be amplified.
[0027] Another embodiment of the present invention provides a
method of detecting RNA in a reaction containing
phosphoribosylpyrophosphate. Free ribonucleoside monophosphate
molecules are produced by digestion of RNA with a nuclease. Next, a
pyrophosphate molecule from phosphoribosylpyrophosphate molecules
is enzymatically transferred to the adenosine monophosphate
molecules to form adenosine triphosphate molecules according to the
following reaction:
[0028] NMP+PRPP.fwdarw.NTP+ribose--5--PO.sub.4
[0029]
[0030] catalyzed by phosphoribosylpyrophosphate synthetase. The ATP
so produced is then detected by a luciferase detection system or an
NADH detection system. If increased sensitivity is required, the
ATP so produced may be amplified.
[0031] Another embodiment of the present invention provides a
method for determining the presence and/or amount of cells and
cellular material present in the sample. In this method, the
contents of cells are released to form a cell lysate. Phosphate
donor molecules and adenosine 5'-monophosphate molecules are then
added to the cell lysate so that adenosine 5'-diphosphate molecules
are produced by the enzymatic transfer of an phosphate group from
the donor to the adenosine 5'-monophosphate according to the
following reaction:
D--P+AMP.fwdarw.D+ADP
[0032] catalyzed by endogenous enzymes present in thecell lysate.
ATP is then produced by the enzymatic transfer of a phosphate from
the donor molecules to adenosine 5'-diphosphate molecules according
to the following reaction:
D--P+ADP.fwdarw.D+ATP
[0033] also catalyzed by endogenous enzymes present in the cell
lysate sample. The adenosine 5'-triphosphate so produced is then
detected by either a luciferase detection system or an NADH
detection system. The phosphate donor of this embodiment may be
either deoxycytidine 5'-triphosphate, deoxyguanidine
5'-triphosphate, or deoxythymidine 5'-triphosphate.
[0034] The present invention further provides a composition of
matter for producing adenosine 5'-triphosphate from DNA,
pyrophosphate, and adenosine 5'-diphosphate. This composition
comprises a mixture of nucleoside diphosphate kinase and a nucleic
acid polymerase which are provided in a concentration sufficient to
catalyze the production of ATP from DNA at about picogram to
microgram amounts of DNA.
[0035] The present invention, also provides a composition of matter
for producing adenosine triphosphate from DNA,
phosphoribosylpyrophosphate, and adenosine 5'-diphosphate. This
composition comprises a mixture of a phosphoribosylpyrophosphate
synthetase and nucleoside diphosphate kinase in a sufficient
concentration to catalyze the production of adenosine triphosphate
from about picogram to microgram amounts of DNA.
[0036] The present invention provides various kits for nucleic acid
detection. First, a kit is provided which contains reagents for the
detection of DNA by pyrophosphorolysis. The kit contains a vessel
containing a nucleic acid polymerase and a vessel containing a
nucleoside disphosphate kinase. The nucleic acid polymerase and
nucleoside diphosphate kinase may be provided in the same
container. Second, a kit is provided which contains reagents for
the detection of nucleic acid by nuclease digestion. The kit
contains a vessel containing phosphoribosylpyrophosphate synthetase
and a vessel containing a nuclease. Third, a kit is provided which
contains reagents for the detection of RNA by pyrophosphorolysis.
The kit contains a vessel containing poly(A)-polymerase. Fourth, a
kit containing reagents for the detection of DNA by nuclease
digestion is provided. This kit contains a vessel containing
phosphoribosylpyrophosphate synthetase and a vessel containing
nucleoside disphosphate kinase. The phosphoribosylpyrophosphat- e
synthetase and nucleoside diphosphate kinase may optionally be
provided in the same container.
[0037] An embodiment of the present invention further provides a
kit containing reagents for the detection of cells and/or cellular
material in a sample. The kit contains a vessel containing
adenosine 5'-monophosphate and a vessel containing a high energy
phosphate donor which may not be utilized by luciferase.
[0038] The present invention also provides a method of amplifying a
nucleoside triphosphate molecule in a reaction containing adenosine
5'-monophosphate molecules, high energy phosphate donor molecules,
or a combination thereof. In this method, the terminal 5' phosphate
group from a nucleoside triphosphate molecule (XTP) present in the
sample is enzymatically transferred to an adenosine
5'-monophosphate molecule added to the sample to form adenosine
5'-diphosphate molecules and nucleoside diphosphate molecules (XTP,
either a ribonucleoside or deoxyribonucleoside triphosphate)
according to the following reaction:
XTP+AMP.fwdarw.XDP+ADP
[0039] catalyzed by a first enzyme which may be either nucleoside
monophosphate kinase or adenylate kinase. Next, a phosphate from a
high energy phosphate donor molecule which may not be utilized by
the first enzyme is enzymatically transferred to the adenosine
5'-diphosphate molecules to form adenosine 5'-triphosphate
molecules according to the following reaction:
[0040] ADP+D--P.fwdarw.ATP+D
[0041]
[0042] catalyzed by nucleoside diphosphate kinase or pyruvate
kinase. These two steps are then repeated until the desired level
of amplification is achieved. The high energy phosphate donors may
be either dCTP or AMP-CPP for NDPK and PEP for pyruvate kinase.
[0043] The present invention also provides a method for detecting
deoxyribonucleic or ribonucleic acid in a reaction containing
pyrophosphate, adenosine 5'-monophosphate, and a high energy
phosphate donor, or a combination thereof, in a single pot
reaction. First, nucleic acid is depolymerized at a terminal
nucleotide by enzymatically cleaving the terminal internucleotide
phosphodiester bond with a pyrophosphate molecule to form a free
ribonucleoside (XTP) or deoxynucleoside triphosphate molecule (XTP)
according to reaction 1:
NA.sub.n+PP.sub.i.fwdarw.NA.sub.n-1+XTP
[0044] catalyzed by a polymerase. The depolymerizing step is
repeated to obtain at least two nucleoside triphosphate molecules.
The ribonucleoside triphosphate molecules or deoxyribonucleoside
triphosphate molecules are then amplified by enzymatically
transferring the terminal 5' phosphate group from the nucleoside
triphosphate molecule formed in reaction 1 to an adenosine
5'-monophosphate to produce an adenosine 5'-diphosphate molecule
and a nucleoside 5'-diphosphate molecule (XDP) according to
reaction 2 catalyzed by a first enzyme:
XTP+AMP.fwdarw.XDP+ADP.
[0045] Next, a phosphate group from a high energy phosphate donor
molecule, which is not a substrate for the first enzyme, is
enzymatically transferred to the adenosine 5'-diphosphate molecules
produced in a reaction to produce adenosine 5'-triphosphate
molecules according to reaction 3 catalyzed by a second enzyme:
ADP+D--P.fwdarw.ATP+D.
[0046] The two amplification steps are repeated until the desired
level of amplification is achieved. Enzyme 1 in this method may be
either adenylate kinase or nucleoside monophosphate kinase, while
enzyme 2 may be either pyruvate kinase or nuceloside diphosphate
kinase.
DESCRIPTION OF THE PREFERRED EMBODIMENT
[0047] The present invention provides a method for detecting
extremely low levels of various nucleic acids including both
deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in
biological samples, especially samples of recombinant proteins. The
extreme sensitivity, reproducibility, ease of conducting the
reactions, and speed of conducting the reactions represent major
advantages over methods currently in use for low level detection of
nucleic acids.
[0048] The detection method may be divided into three general
steps. The first step is the production of the following
nucleosides: nucleoside monophosphates (XMPs) including the
ribonucleoside monophosphates (NMPs) adenosine 5'-monophosphate
(AMP), guanosine 5'-monophosphate (GMP), uridine 5'-monophosphate
(UMP), and cytidine 5'-monophosphate (CMP); deoxyribonucleoside
monophosphates (dNMPs) including deoxyadenosine 5'-monophosphate
(dAMP), deoxyguanosine 5'-monophosphate (dGMP), deoxythymidine
5'-monophosphate (dTMP), and deoxycytidine 5'-monophosphate (dCMP);
nucleoside triphosphates (XTPs) including the ribonucleoside
triphosphates (NTPs) adenosine 5'-triphosphate (ATP), guanosine
5-triphosphate (GTP), uridine 5'-triphosphate (UTP), and cytidine
5'-triphosphate (CTP); and the deoxyribonucleoside triphosphates
(dNTPs) including deoxyadenosine 5'-triphosphate (dATP),
deoxyguanosine 5'-triphosphate (dGTP), deoxythymidine
5'-triphosphate (dTTP), and cytidine 5'-triphosphate (dCTP). The
NMPs and dNMPs are produced by nuclease digestion and the NTPs and
dNTPs by depolymerization by pyrophosphorolysis. The second step,
used when the initial substrate is DNA, is the transfer of the
terminal phosphate from the dNTPs to ADP to form ATP. The optional
step of XTP amplification may be performed at this stage to
increase the sensitivity of the detection system especially when
measuring samples containing low levels of DNA in the range of 1-10
picograms of nucleic acid. The third step is detection of ATP by a
suitable detection method. Examples of such detection systems are
the luciferase detection system and NADH-based detection
system.
[0049] Nucleic acid polymerases generally catalyze the elongation
of nucleic acid chains. The reaction is driven by the cleavage of a
pyrophosphate released as each nucleoside is added. Each nucleoside
triphosphate has three phosphate groups linked to carbon 5 of
ribose or deoxyribose. The addition of a nucleoside to a growing
nucleic acid results in formation of a internucleoside
phosphodiester bond. This bond is characterized in having a 3'
linkage to carbon 3 of ribose or deoxyribose and a 5' linkage to
carbon 5 of ribose or deoxyribose. Each nucleoside is added through
formation of a new 3' linkage, so the nucleic acid strand grows in
a 5' to 3' direction. The 5' end of the nucleic acid is
characterized by a phosphate group attached to carbon 5.
[0050] Several polymerases are also known to catalyze the reverse
of the polymerization process. This reverse reaction is called
pyrophosphorolysis. The pyrophosphorolysis activity of DNA
polymerase was demonstrated by Deutscher and Kornberg, Enzymatic
Synthesis of Deoxyribonucleic Acids, J. Biol. Chem. 244: 3019-28
(1969). Other nucleic acid polymerases capable of
pyrophosphorolysis include DNA polymerase .alpha., DNA polymerase
.beta., T4 DNA polymerase, Taq polymerase, Klenow fragment, AMV
reverse transcriptase, and MMLV reverse transcriptase. However, not
all polymerases are known to possess pyrophosphorolysis activity.
For example, poly(A) polymerase has been reported to not catalyze
pyrophosphorylation. (See Sippel, Eur. J. Biochem. 37:31-40
(1973).)
[0051] A mechanism of pyrophosphorolysis has been suggested for RNA
polymerase. It is believed that the partial transfer of a Mg.sup.2+
ion from the attacking pyrophosphate to the phosphate of the
internucleoside phosphodiester bond of the RNA may increase the
nucleophilic reactivity of the pyrophosphate and the
electrophilicity of the diester as described in Rozovskaya et al.
Biochem. J. 224: 645-50 (1984). The internucleoside phosphodiester
bond is enzymatically cleaved by the addition of pyrophosphate to
the nucleoside 5phosphate and a new phosphodiester bond is formed
between the pyrophosphate and the nucleoside monophosphate.
[0052] The pyrophosphorolysis reaction can be summarized as
follows:
Reaction 1: NA.sub.n+PP.sub.i.fwdarw.NA.sub.n-1+XTP
[0053] wherein NA is a nucleic acid, PP.sub.i is pyrophosphate and
XTP is either a deoxyribonucleoside triphosphate molecule or a
ribonucleoside triphosphate molecule. The reaction may then be
repeated to obtain at least two XTP molecules. It should be noted
that the reaction may be repeated on the same nucleic acid molecule
or on a plurality of different nucleic acid molecules.
[0054] Preferred reaction mixes for depolymerization by
pyrophosphorolysis, including suitable buffers for each nucleic
acid polymerase analyzed are disclosed in the Examples. Under these
conditions, sufficient NTP or dNTP is produced to accurately detect
or assay extremely low amounts of nucleic acids (5-15
picograms.)
[0055] Even though the preferred reaction conditions for
polymerization and depolymerization by pyrophosphorolysis are
similar, the rates of these reactions vary greatly. For example,
AMV and RLV reverse transcriptases catalyze pyrophosphorolysis
under optimal conditions at a rate of about fifty- to one
hundred-fold less than polymerization as demonstrated in
Srivastavan and Modak, J. Biol. Chem. 255(5): 2000-04 (1980). Thus,
the high eficiency of the pyrophosphorolysis reaction was
unexpected and appears to be asscociated with extremely low levels
of DNA substrate in contrast to previous DNA pyrophosphorolysis
studies conducted on much higher amounts of DNA.
[0056] The pyrophosphorolysis activity of different nucleic acid
polymerases also varies. T4 polymerase appears to possess the
greatest pyrophosphorolysis activity as measured by a luciferase
assay for ATP produced by pyrophosphorolysis. Pyrophosphorolysis
using T4 polymerase resulted in about a 10 fold increase in light
production as compared to MMLV-RT and a 4 fold increase in light
production as compared to Taq polymerase.
[0057] The detection of nucleic acids at low picogram levels is
generally enhanced by fragmenting or partially digesting the
nucleic acid. Preferably, fragmentation is accomplished by
sonication or restriction enzyme digestion of the nucleic acid,
forming a plurality of smaller nucleic acid fragments. This step
probably enhances detection because the pyrophosphorolysis reaction
only proceeds from the DNA ends, as demonstrated in the Examples.
Providing a greater number of DNA ends means that more reactions
are occurring at any one time. It should be noted that DNA ends may
be present within a molecule as well as at the end of a linear DNA
fragment. For example, polymerases may catalyze pyrophosphorolysis
from a gap in a DNA segment or a nick in a DNA segment. The type of
enzyme used for pyrophosphorolysis and the type of substrate
determine whether fragmentation is necessary. For instance, the
data set forth in the Examples demonstrate that fragmenting greatly
increases detection of plasmid DNA when Tag polymerase is used, but
does not effect detection when T4 polymerase is used. However, when
chromosomal DNA is the substrate, fragmentation increases detection
from both enzymes.
[0058] The type of cut made by restriction enzyme digestion also
affects the pyrophosphorolysis activity of different nucleic acid
polymerases. For example, MMLV-RT and Taq polymerase can catalyze
pyrophosphorolysis of DNA fragments with 5' overhangs, but not 3'
overhangs. In contrast, T4 DNA polymerase catalyzes both 3' and 5'
end overhang and blunt end mediated pyrophosphorolysis. T4
polymerase is thus a preferred enzyme for pyrophosphorolysis. When
other nucleic acid polymerases are utilized for pyrophosphorolysis
of restriction enzyme treated DNA, care must be taken to match the
overhang specificity of the polymerase with the type of overhang
created by the restriction endonuclease.
[0059] It must be noted that sequence specificity of
pyrophosphorolysis for single stranded DNA has been previously
noted during sequencing by Tabor and Richardson, J. Biol. Chem. 265
(14): 8322-28 (1990). The sequence specificity of the
pyrophosphorolysis reaction was noted when some dideoxynucleoside
terminated sequence fragments were shown to be more susceptible to
degradation by phosphorolysis than other fragments.
[0060] Further, the type of polymerase used in the
pyrophosphorolysis reaction must be matched to the correct nucleic
acid substrate. In general, DNA polymerases and reverse
transcriptases are preferred for depolymerizing DNA, and RNA
polymerases are preferred for depolymerizing RNA. Reverse
transcriptases are preferred for depolymerizing RNA-DNA
hybrids.
[0061] Applicants have further demonstrated that poly(A) polymerase
may catalyze pyrophosphorolysis, even though no such reaction has
been previously reported. In fact, poly(A) polymerase has been
widely reported to not catalyze pyrophosphorolysis. See, for
example, Sippel, Eur. J. Biochem. 37:31-40 (1973) and Sano and
Feix, Eur. J. Biochem. 71:577-83 (1976). Surprisingly, the
applicants show that under the proper reaction conditions poly(A)
polymerase catalyzes phosphorolysis. Preferably, the manganese
chloride present in the previously reported buffers is omitted, the
concentration of sodium chloride is decreased, and the pH is
lowered from about 8.0 to about 7.5. Most preferably, poly(A)
polymerase pyrophosphorolysis reaction buffer contains about 50 mM
Tris-Cl pH 7.5, 10 mM MgCl.sub.2, 50 mM NaCl, and 2 mM NaPP.sub.i
(sodium pyrophosphate).
[0062] It is important to note that the depolymerization reaction
is the reverse of the polymerization reaction. Therefore, as
increasing amounts of free nucleoside triphosphates are produced by
depolymerization, a state of equilibrium may theoretically be
attained in which polymerization and depolymerization reactions are
balanced. Alternatively, where small amounts of nucleic acid are
detected, the reaction may go essentially to completion without
reaching equilibrium, i.e. the nucleic acid depolymerized into its
constituent subunits nucleotides by greater than 90%. This is
important in a quantitative test because the total amount of
nucleotides released is proprtional to the amount of signal
generated in the detection assay. When used for qualitative
detection of nucleic acid, it is not necessary that the reaction
reach equilibrium or go essentially to completion provided a
threshold level of nucleotides are produced. The mixture of
nucleoside triphosphate molecules produced by depolymerization will
preferably be converted to adenosine triphosphate as described
below. For either detection or assay, a detectable threshold level
of 6.times.10.sup.7 adenosine triphosphate molecules must be
provided for detection of light from a typical luciferase
assay.
[0063] In a preferred embodiment of the present invention for
detecting nucleic acids, nucleic acid polymerase and PP.sub.i are
added to a sample containing less than 1 .mu.g nucleic acid, down
to less than about 10 pg of nucleic acid. To increase the
sensitivity of the DNA detection, the DNA may be fragmented by
treatment with a restriction endonuclease or by sonication. Next,
the nucleic acid is degraded by pyrophosphorolysis releasing free
NTPs or dNTPs. Enzymes useful in the pyrophosphorolysis reaction
include AMV reverse transcriptase, MMLV reverse transcriptase, DNA
polymerase alpha and beta, Taq polymerase, T4 DNA polymerase,
Klenow fragment and poly(A) polymerase. Most preferably, T4
polymerase is utilized for DNA pyrophosphorolysis reactions because
of its recognition of 3' and 5' overhangs and blunt ends and high
processivity as noted above.
[0064] Luciferase, which is part of the preferred ATP detection
system, is inhibited by pyrophosphate (PP.sub.i). Therefore, care
must be taken not to transfer a highly inhibiting amount of
PP.sub.i to the ATP detection reaction. Preferably, the amount
PP.sub.i carried over to the ATP detection reaction results in a
concentration of PP.sub.i in the luciferase detection reaction of
less than about 100 .mu.M, although less than about 10 .mu.M is
desirable. Therefore, the amount of PP.sub.i utilized in the
pyrophosphorolysis reaction will be determined by the size of the
aliquot which is taken for use in the luciferase detection system.
The aliquot size may vary, but the amount of PP.sub.i transferred
or carried over to the luciferase detection reaction should
correspond to the PP.sub.i concentration parameters described above
so that the concentration of PP.sub.i is at least below about 100
.mu.M, and preferably below about 10 .mu.M.
[0065] In another embodiment, the nucleic acids may be first
degraded into NMP or dNMP by exonuclease digestion according to the
following reaction:
Reaction 2: NA.sub.n+H.sub.2O.fwdarw.NA.sub.n-1+XMP
[0066] wherein NA is a nucleic acid, XMP is either a
deoxyribonucleoside monophosphate or ribonucleoside monophosphate,
and n is the number of nucleosides in the nucleic acid.
[0067] Nuclease digestion may be accomplished by a variety of
nucleases including Si nuclease, nuclease BAL 31, mung bean
nuclease, exonuclease III and ribonuclease H. Nuclease digestion
conditions and buffers may be found in the Product Literature
available from commercial sources, or as disclosed in the
Examples.
[0068] After digestion with the nuclease, the NMPs or dNMPs are
converted to NTPs or dNTPs respectively. U.S. Pat. No. 4,375,897
describes the detection of RNA by digestion with nucleases followed
by conversion to NTP. This method utilizes a two-step scheme in
which adenylate kinase converts AMP to ADP, and pyruvate kinase
then converts ADP to ATP. This method is essentially limited to the
detection of poly(A) mRNA because no mechanism is suggested for
conversion of dNTPs to ATP, the preferred substrate for luciferase.
Nuclease digestion or phosphorolysis of DNA results in a mixture of
dNTPs which do not act as efficient substrates for luciferase.
[0069] In the biosynthesis of purine and pyrimidine
mononucleosides, phosphoribosylpyrophosphate (PRPP) is the
obligatory ribose-5'-phosphate donor. PRPP itself is formed in a
reaction catalyzed by PRPP synthetase through the transfer of
pyrophosphate from ATP to ribose-5-phosphate. This reaction is
known to be reversible as described in Sabina et al., Science 223:
1193-95 (1984).
[0070] In the present invention, the NMP or dNMP produced by
nuclease digestion is preferably converted directly to NTP or dNTP
by the enzyme PRPP synthetase in the following reactions:
Reaction 3: XMP+PRPP.fwdarw.XTP+ribose--5--PO.sub.4
[0071] wherein XMP is either adenosine monophosphate or
deoxyadenosine monophosphate and XTP is either a adenosine
triphosphate or deoxyadenosine triphosphate. Preferably, this
reaction produces a detectable threshold level of at least
6.times.10.sup.7 adenosine triphosphate molecules.
[0072] In this reaction, the pyrophosphate group of PRPP is
enzymatically transferred to XMP molecules, forming XTP molecules.
Reaction conditions and buffers are set forth in the Examples. When
RNA is the substrate, the ATP produced may be directly
detected.
[0073] Utilization of the PRPP reaction in the nucleic acid
detection system has several advantages over the prior art. First,
only one step is necessary to convert an AMP or dAMP to a ATP or
dATP, which simplifies the detection system. Second, contamination
of the detection reaction with exogenous ATP, ADP, or AMP is less
likely.
[0074] The dNTPs produced by pyrophosphorolysis or nuclease
digestion followed by pyrophosphorylation can theoretically be used
directly as substrates for luciferase, allowing detection of the
nucleic acid. However, the preferred substrate for luciferase is
ATP as demonstrated by Moyer and Henderson, Nucleoside Triphosphate
Specificity of Firefly Luciferase, Anal. Biochem. 131:187-89
(1983). When DNA is the initial substrate, a nucleoside diphosphate
kinase (NDPK) is conveniently utilized to catalyze the conversion
of dNTPs to ATP by the following general reaction:
Reaction 4: dNTP*+ADP.fwdarw.dNDP+ATP*
[0075] wherein dNTP is a mixture of deoxyribonucleoside
triphosphates and dNTP is the corresponding deoxyribonucleoside
triphosphate. In the reaction, the terminal 5'-triphosphate (P*) of
the dNTP is transferred to ADP to form ATP.
[0076] Enzymes catalyzing this reaction are generally known as
nucleoside diphosphate kinases (NDPK). NDPKs are ubiquitous,
relatively nonspecific enzymes. For a review of NDPK, see Parks and
Agarwal, in The Enzymes, Volume 8, P. Boyer Ed. (1973). The
conversion of NTPs or dNTPs to ATP by NDPK is preferably
accomplished by adding NDPK and a molar excess of ADP over the
amounts of NTPs or dNTPs expected to be produced by
pyrophosphorolysis, or nuclease digestion followed by
pyrophosphorylation by PRPP synthetase. Alternatively, if an
amplification scheme is used, a molar excess of AMP may be used as
the preferred substrate. The utilization of ADP requires
optimization of the amount of ADP added. Too much ADP results in
high background levels. A reaction containing NDPK contains about
0.01 to 0.50 .mu.M ADP, preferably about 0.05 .mu.M ADP.
Illustrative buffers and reaction components are set forth in the
Examples.
[0077] As an optional step, the NTP, dNTP, or ATP generated by the
pyrophosphorolysis or nuclease digestion schemes may be amplified
to give even greater sensitivity. Amplification may be required
when utilizing detection systems other than luciferase or when
increased levels of signal are needed for detection by a less
sensitive luminometer. Amplification of NTP means a continuous
reaction wherein 1 NTP gives rise to 2 NTPs, which can be cycled to
yield 4 NTPs and so on. When AMP is added to feed the amplification
reaction, ATP will accumulate while the amount of original NTP
remains the same. PCT publication WO 94/25619 and Chittock et al.,
Anal. Biochem., 255:120-6 (1998), incorporated herein by reference,
disclose amplification systems for ATP characterized by the
following coupled reactions:
[0078] Reaction 5:
C1+S1.fwdarw.2C2 and 2C2+2S2.fwdarw.2C1 2C1+2S1.fwdarw.4C2 and
4C2+4S2+E2.fwdarw.4C1 4C1+4S1.fwdarw.8C2 and
8C2+8S2+E2.fwdarw.8C1
[0079] wherein C1 is the target compound present in a sample to be
amplified, S1 is the amplification substrate, E1 is a catalytic
enzyme capable of utilizing C1 and S1 to produce C2, S2 is an
energy donating substrate, and E2 is a catalytic enzyme capable of
utilizing C2 and S2 to produce C1, which then recycles through the
reaction. According to this reaction scheme, each pass through the
coupled reaction doubles the amount of C1, which can be
subsequently detected. Patent Application GB 2,055,200 discloses an
amplification system utilizing adenylate kinase and pyruvate
kinase.
[0080] In designing a coupled ATP amplification reaction for use in
nucleic acid detection, two main requirements must be considered.
First, E1 must not be able to utilize the high energy phosphate
donor utilized by E2. If E1 can utilize the high energy phosphate
donor, the ATP amplification reaction would proceed in the absence
of NTP or dNTP produced as a result of pyrophosphorolysis or
nuclease digestion followed by pyrophosphorylation. This would
result in the undesirable occurrence of false positive results.
Second, a molar excess of the added high energy phosphate donor
must be provided as compared to the amount of dXTP or XTP expected
in the reaction. Third, E1 must be able to utilize either the NTP,
dNTP, or ATP produced in step 1 by pyrophosphorolysis or nuclease
digestion of the nucleic acid.
[0081] The amplification system of the present invention may be
characterized, as follows:
[0082] Reaction 6. XTP+AMP.fwdarw.XDP+ADP ADP+D--P.fwdarw.ATP
[0083]
[0084] wherein D-P is a high energy phosphate donor and E1 and E2
are enzymes capable of catalyzing the transfer of phosphates from
an XTP to AMP and from the D-P to ADP, respectively. The ATP so
produced may reenter the reaction (as XTP) and the reaction
repeated until the substrates are exhausted or equilibrium is
reached, resulting in the production of two ATPs for every ATP
supplied to or generated by the reaction. Note that when the target
XTP is any nucleoside triphosphate other than ATP the initial pass
through the cycle yields only 1 ATP which then reenters the cycle
to produce two ATP, which reenter the cycle to produce 4 ATP and so
on. Preferably, the amplification reaction produces a detectable
threshold level of 6.times.10.sup.7 adenosine triphosphate
molecules.
[0085] The XTP in reaction 6 is a ribonucleoside triphosphate or
deoxyribonucleoside triphosphate, which may preferably be ATP
provided by pyrophosphorolysis (Reaction 1) or created from XTP by
NDPK conversion of ADP to ATP (Reaction 5) or provided by nuclease
digestion coupled with pyrophosphorylation (Reaction 4) followed by
NDPK conversion to ATP (Reaction 5). It must be appreciated,
however, that when an amplification step is utilized for a DNA
substrate, the step of converting dNTP to ATP is inherent in the
amplification system. Therefore, a separate converting step is not
needed.
[0086] A nucleoside monophosphate kinase (NMPK) or adenylate kinase
is preferably utilized as enzyme 1 (E1). NMPKs occur as a family,
each of which is responsible for catalyzing the phosphorylation of
a particular NMP. Until recently, it was generally thought that ATP
and DATP were preferred phosphate donors. However, Shimofuruya and
Suzuki Biochem. Intl. 26(5):853-61 (1992) recently demonstrated
that at least some NMPKs can utilize other phosphate donors such as
CTP and UTP. Enzyme 2 (E2) is preferably nucleoside diphosphate
kinase (NDPK) or pyruvate kinase. NDPKs generally catalyze the
transfer of the terminal 5'-triphosphate of NTPs to NDPs to form
NTPs. Pyruvate kinase catalyzes the transfer of phosphate from
phosphoenolpyruvate to ADP to form ATP. These enzymatic activities
are utilized in the amplification reaction to transfer a phosphate
group from a high energy phosphate donor (D-P) to either ADP or an
NDP.
[0087] A high energy phosphate donor (D-P) that may be used by E2
but not by E1 is required. When E2 is NDPK, dCTP or .alpha., .beta.
methylene adenosine 5'-triphosphate (AMP-CPP) may be utilized as
D-P. When E2 is pyruvate kinase, phosphoenol pyruvate (PEP) is the
preferred high energy phosphate donor. The ability of NDPK to
utilize these substrates at efficiencies allowing production of
minute quantities of ATP was not known. It is surprising that these
high-energy phosphate donors utilized with NMPK and adenylate
kinase meet the requirements of the amplification reaction when the
recent literature suggests that NMPK (E1) may utilize phosphate
donors other than ATP or DATP. The nonspecificity of adenylate
kinase is also well known. The high energy phosphate donor and/or
AMP must be provided in a molar excess as compared to the amount of
ATP or dNTP expected present in the sample so that the high energy
phosphate donor is not recycled at an appreciable rate. Additional
buffers and reaction components are included in the Examples.
[0088] The third step of nucleic acid detection is detection of the
NTP, dNTP or amplified ATP. Two well known detection systems
include: the light emitting luciferase detection system, and the
NADH light adsorption detection system (NADH detection system).
[0089] The ATP so produced is detected by a luciferase detection
system. In the presence of ATP and O2, luciferase catalyzes the
oxidation of luciferin, producing light which can then be
quantitated using a luminometer. Additional products of the
reaction are AMP, pyrophosphate and oxyluciferin. The light can be
detected by a luminometer.
[0090] The preferred ATP detection buffer, which will be referred
to as LAR buffer, is formulated by mixing 19.1 ml of deionized
water; 800 gl of 0.5 M Tricine, pH 8.0; 70 .mu.l of 1M MgSO.sub.4;
4 .mu.l of 0.5 M EDTA; 0.108 g of DTT (dithiothreitol); 0.003 g of
Luciferin; and adjusting the pH to 7.8 if necessary. Preferably,
about 5 to 10 nanograms of recombinant luciferase (Promega Lot
6414002) is used in the reaction. Greater amounts of luciferase
have a tendency to increase non-specific background. Applicants
also have shown that deleting coenzyme A from the LAR reaction mix
decreases background.
[0091] In the NADH detection system, a combination of two enzymes,
phosphoglycerate kinase and glyceraldehyde phosphate dehydrogenase,
are used to catalyze the formation of NAD from NADH in the presence
of ATP. ATP is measured as a loss in fluorescence intensity because
NADH is fluorescent while NAD is not. Examples of NADH based ATP
assays are disclosed in U.S. Pat. Nos. 4,735,897, 4,595,655,
4,446,231 and 4,743,561, and UK Patent Application GB 2,055,200,
all incorporated herein by reference.
[0092] Certain of the above reactions may be performed as single
pot reactions. A single pot reaction is a reaction wherein at least
two enzymes (E1 and E2) with catalytic activity are present in the
same reaction mix and act on one or more substrate(s) (SI and S2).
The reactions catalyzed by the enzymes may occur simultaneously
where E1 acts on S1 and E2 acts on S2 successfully. Alternatively,
the reactions catalyzed by E1 and E2 may occur in a step-wise or
coupled manner where E1 acts on S1 to produce an intermediate
S2.sub.i and E2 then acts on S2.sub.i. Of course, such a coupled
reaction may also be essentially simultaneous.
[0093] The ability to utilize combinations or mixtures of the
enzymes of the present invention in single pot reactions is
surprising in light of the extremely low levels of nucleic acid
detection which are achieved. This low level detection is possible
even though some enzymes are used under less than optimal
conditions. As previously described, it was necessary to optimize
the concentration of PP.sub.i utilized in the pyrophosphorolysis
reactions so that luciferase would not be inhibited. Therefore,
aliquots from the NMP, dNMP, NTP, dNTP and ATP producing reactions
may be directly added to LAR buffer for luciferase detection
without any purification of the reaction products. The luciferase
reaction is not poisoned or otherwise quenched by the components of
the reactions. This desirable feature allows high throughput
screening with a minimal amount of time and effort, and also allows
great flexibility in the design of the overall detection
schemes.
[0094] Preferably, the pyrophosphorolysis reaction producing dNTP
and the NDPK catalyzed reaction in which the NTPs or dNTPs are
converted to ATP may be performed in a single pot reaction in the
nucleic acid polymerase buffer. NDPK activity is sufficient to
convert dNTP to ATP even though the polymerase buffer conditions
are suboptimal for NDPK activity. The polymerase enzyme and NDPK
may both be present initially in the reaction, or the NDPK may be
added directly to the reaction after an incubation period
sufficient for the production of NTP or dNTP. A nucleic acid
polymerase and NDPK may be provided in the same vessel or mixture
for use in the reactions described above. The mixture preferably
contains the nucleic acid polymerase and NDPK in a concentration
sufficient to catalyze the production of ATP when in the presence
of a nucleic acid, pyrophosphate and ADP. Preferably, the
polymerase is provided in a concentration of about 1 to 100
units/.mu.l, most preferably at about 5 units/.mu.l. Preferably,
the NDPK is provided in a concentration of 0.1 to 100 units/.mu.l,
most preferably at about 5 units/.mu.l. Preferably, the mixture is
greater than 99% pure.
[0095] Similarly, the PRPP synthetase and NDPK reactions can be
performed in a single pot reaction in the PRPP synthetase buffer.
Again, NDPK activity is sufficient even though conditions for NDPK
activity are suboptimal. The nuclease digested sample containing
free NMPs and dNMPs may be added to a reaction mix initially
containing PRPP synthetase and NDPK, or added to a PRPP synthetase
reaction followed by addition to a reaction mix containing NDPK and
the luciferase detection reaction components. The preferred buffers
and reaction components may be found in the Examples. PRPP
synthetase and NDPK may be provided in the same vessel or mixture
for use in the reactions described above. The mixture preferably
contains the PRPP synthetase and NDPK in a concentration sufficient
to catalyze the production of ATP when in the presence of
phosphoribosylpyrophosphate and ADP. Preferably, the NDPK is
provided in a concentration of 0.1 to 100 units/.mu.l, most
preferably at about 5 units/.mu.l. Preferably, the PRPP synthetase
is provided in a concentration of 0.001 to 10 units/.mu.l, most
preferably at about 0.01 units/.mu.l If amplification is desired,
the PRPP synthetase reaction must be heat inactivated, otherwise
the PRPP synthetase would convert the added AMP to ATP. Preferably
the mixture is greater than 99% pure.
[0096] The pyrophosphorolysis reaction and amplification reaction
may also be performed in a single pot reaction. In this single pot
reaction, the polymerases may be AMV reverse transcriptase, MMLV
reverse transcriptase, DNA polymerase alpha or beta, Taq
polymerase, T4 DNA polymerase, Klenow fragment or poly(a)
polymerase, a first enzyme for converting AMP to ADP may be
myokinase (adenylate kinase) or NMPK, and a second enzyme for
converting ADP to ATP may be pyruvate kinase or NDPK. The reaction
must be fed AMP, preferably Apyrase treated AMP so that background
due to contaminating ADP and ATP is minimized. Preferably 1 .mu.l
of 1U/.mu.l Apyrase may be added to 19 .mu.l of 10 mM AMP, followed
by incubation at room temperature for 30 minutes and heat
inactivation of the Apyrase by incubation at 70.degree. C. for 10
minutes. High energy phosphate donors must also be added to the
reaction. When pyruvate kinase is utilized phosphoenolpyruvate is
added, while when NDPK is utilized dCTP is added. Preferably, the
high energy phosphate donor is added about 15 minutes after a
preincubation with the polymerase, although this is not necessary.
These reactions may characterized as follows:
Reaction 7: NA.sub.n+PP.sub.i.fwdarw.NA.sub.n-1+XTP
XTP+AMP.fwdarw.ADP+XDP ADP+D--P.fwdarw.ATP+D
[0097] wherein NA is a nucleic acid, XTP is a nucleoside
triphosphate, either a deoxynucleoside or ribonucleoside
triphosphate, XDP is a nucleoside diphosphate, either a
deoxynucleoside or ribonucleoside diphosphate, and D-P is a high
energy phosphate donor. It should be appreciated that this reaction
produces ATP, the preferred substrate for luciferase, from dNTPs.
The amplification reaction proceeds as described in reaction 7 to
produce a detectable threshold level of 6.times.10.sup.7 adenosine
triphosphate molecules. Preferably, the polymerase is provided in a
concentration of about 1 to 100 units/.mu.l, most preferably at
about 5 units/.mu.l. Preferably, the NDPK is provided in a
concentration of 0.1 to 100 units/.mu.l, most preferably at about 1
unit/.mu.l. Preferably, the Preferably, the mixture is greater than
99% pure.
[0098] In another embodiment, the reactions described above may be
used to selectively detect poly(A) mRNA according to the following
scheme. First oligo(dT) primers are hybridized to the poly(A) tails
of the mRNA to form a DNA-RNA hybrid. Next, a pyrophosphorolysis
reaction is performed using reverse transcriptase (RT). Reverse
transcriptases which may be used in the present invention include
Mouse Mammary Leukemia Virus (MMLV) RT, Avian Myeloma Virus (AMV)
RT and Rous Sarcoma Virus (RSV) RT. An advantage of this detection
system is that these RTs catalyze pyrophosphorolysis of double
stranded nucleic acid and double stranded RNA-DNA hybrids, but not
single stranded nucleic acids. Thus, the amount of poly(A) RNA in a
total cellular RNA sample be determined. The pyrophosphorolysis
reaction produces dTTP according to the following reaction:
Reaction 8. TT.sub.n+PP.sub.i.fwdarw.TT.sub.n-1+dTTP;
[0099] wherein TT.sub.n is oligo(dT) and PP.sub.i is
pyrophosphate.
[0100] The dTTP can be converted to ATP by NDPK as described in
reaction 4 above, optionally amplified, and detected as described
above.
[0101] In another embodiment, the reactions described above may be
used to detect the presence of cells in a sample. U.S. Pat. No.
5,648,232, incorporated herein by reference, describes a method of
detecting cells in a sample. That method takes advantage of
adenylate kinase activity, which is present in all living
organisms. Briefly, a sample suspected of containing microorganisms
or other living cells is subjected to conditions causing cell
lysis. ADP is then added to the lysate, which is converted by
endogenous adenylate kinase activity to ATP by the following
reaction:
Reaction 9: ADP+ADP.fwdarw.ATP+AMP
[0102] The ATP produced by this reaction is then detected by the
luciferase assay system.
[0103] The present invention also provides a method of detecting
the presence of cells in a lysate of a sample suspected of
containing cellular material by using different substrates. This
system takes advantage of a coupled reaction catalyzed by
endogenous adenylate kinase activity (AK) and NDPK activity
according to the following reaction scheme:
Reaction 10: AMP+D--P.fwdarw.D+ADP and ADP+D--P.fwdarw.ATP+D.
[0104] wherein D-P is a high energy phosphate donor added to the
cell lysate and AMP is adenosine monophosphate added to the cell
lysate sample. In this reaction, adenosine 5'-diphosphate molecules
are produced by the enzymatic transfer of a phosphate group from
the high energy phosphate donor molecules (D-P) to the added
adenosine 5'-monophosphate (AMP) molecules. Then, adenosine
5'-triphosphate is produced by the enzymatic transfer of phosphate
from D-P molecules to the adenosine 5'-diphosphate molecules
according to the following general reaction catalyzed by endogenous
enzymes present in the cell lysate sample
[0105] Co-optimization of the concentrations of nucleosides added
to the samples was necessary to optimize light output from these
reactions. About 1 mM to 80 mM AMP and 1 mM to 100 mM dCTP may be
added to the test sample, and preferably about 10 mM AMP and 100 mM
dCTP may be added to the test sample. After addition of nucleosides
to the sample, the samples are preferably incubated at room
temperature for about 10 to 60 minutes, and light output from the
samples determined by a luminometer. Other preferred buffers and
reactions components may be found in the Examples.
[0106] This system has an important advantage over previously
described cell detections systems. The AMP and dCTP are much more
stable than ADP, so the results are more reproducible.
[0107] In another aspect of the present invention, a nucleic acid
detection test kit is provided for performing the
pyrophosphorolysis nucleic acid detection method. The nucleic acid
detection test kit comprises the essential reagents required for
the method of the nucleic acid detection invention. For nucleic
acid detection by pyrophosphorolysis, the kit includes a vessel
containing an enzyme capable of catalyzing pyrophosphorolysis such
as Taq polymerase, T4 polymerase, AMV reverse transcriptase, MMLV
reverse transcriptase, or poly(A) polymerase. The concentration of
polymerase is 0.1 to 100 units/.mu.l, preferably about 5
units/.mu.l. Kits for use in DNA detection also include a vessel
containing nucleoside diphosphokinase and a vessel containing ADP.
Preferably, these reagents are free of contaminating ATP and
adenylate kinase. The NDPK is provided in concentration of about
0.1 to 100 units/.mu.l, preferably about 1.0 units/.mu.l. The
contaminants may be removed by dialysis or Apyrase treatment.
Optionally, the kit may contain vessels with reagents for
amplification of dNTPs or NTP to ATP. Amplification reagents
include pyruvate kinase, adenylate kinase, NMPK, NDPK, AMP as the
amplification substrate, and dCTP or AMP-CPP as high-energy
phosphate donors. The kit may be packaged in a single enclosure
including instructions for performing the assay methods. The
reagents are provided in containers and are of a strength suitable
for direct use or use after dilution. A standard set may also be
provided to allow quantitation of results. Test buffers for optimal
enzyme activity may be included. Most preferably, the NDPK and
nucleic acid polymerase are provided in the same reaction mix so
that a single pot reaction may be performed consistently.
[0108] In another aspect of the present invention, a nucleic acid
detection kit is provided for performing the nuclease digestion
nucleic acid detection method of the present invention. This test
kit comprises the essential reagents required for this method.
These reagents include a nuclease, PRPP synthetase, PRPP, NDPK, and
ADP together with luciferase and luciferin. The nuclease is
provided in a concentration of about 1 to 500 units/.mu.l,
preferably about 20 units/.mu.l. The PRPP synthetase is provided in
concentration of about 0.01 units/.mu.l to 10 units/.mu.l,
preferably about 0.1 units/.mu.l. Preferably, the kit includes all
these reagents with luciferase and luciferin being provided as a
single reagent solution. Most preferably, the PRPP synthetase and
NDPK are provided in a single reaction mix so that a single pot
reaction containing these two enzymes may be performed, simplifying
the detection method. The kit is in the form of a single package
preferably including instructions to perform the method of the
invention. The reagents are provided in vessels and are of a
strength suitable for direct use or use after dilution. Preferably,
buffers which support the optimal enzyme activity are provided.
Optionally, reagents for amplification of the ATP signal may be
provided as described in the previous kit.
[0109] In another aspect of the present invention, a test kit is
provided for determining the presence of microorganisms or other
cells in a test sample. This test kit comprises the essential
reagents required for the method. These reagents include a
highenergy phosphate donor which may not be utilized by luciferase,
preferably dCTP, and AMP together with luciferase and luciferin.
Preferably, the kit includes all these reagents with luciferase and
luciferin being provided in the same solution. Preferably, the
reagents are free of contaminating components such as adenylate
kinase and ATP that would cause a false positive test. A cell lysis
cocktail may be provided for efficiently releasing the contents of
the target cells for each of the assays intended. For prokaryotic
microorganisms, only a cationic detergent is needed. For fungal
spores or eukaryotic cells assays, a further nonionic detergent
reagent is included. Reagents are provided in vessels and are of a
strength suitable for direct use or use after dilution. A buffer
solution for diluting the cell samples may also be provided.
[0110] Other aspects of the present invention will be made apparent
in the following examples. These Examples are intended to
illustrate the invention and in no way limit any aspect of the
invention.
EXAMPLES
Example 1
Detection of ATP using Luciferase
[0111] The ultimate sensitivity of detection using an enzyme based
detection system is related to the ability of the enzymatic
reaction to produce a measurable signal over background. This
example describes the detection of very low levels of ATP using
Luciferase.
[0112] Luciferase Assay Reagent buffer (LAR) was produced by
mixing: 19.1 ml of nanopure water; 800 .mu.l of 0.5 M Tricine
(Sigma T9784), pH 8.0; 70 .mu.l of 1 M MgSO.sub.4 (Promega AA319,
Lot #970931); 4 .mu.l of 0.5 M EDTA (Promega AA189, Lot #962131);
0.13 g of DTT (dithiothreitol, Promega V31SA); 0.003 g of Beetle
Luciferin (Promega E160C, Lot #79838); 0.0044 g of Coenzyme A, pH
7.8 (Pharmacia 28-3001-03 Lot #7053001031). A 0.1 M solution of ATP
was prepared by dissolving solid ATP (Sigma A9187) in Tris 10 mM pH
7.5. This stock solution was diluted into Tris 10 mM pH 7.5 to
produce solutions at 100 .mu.M, 1 .mu.M, 10 nM and 100 pM.
Recombinant Luciferase (Promega #1701, Lot #6414002) was diluted to
1 mg/ml, 100g/.mu.l, and 10 .mu.g/ml using nanopure water.
[0113] Reactions were assembled in duplicate containing the
following components in 1.5 ml polypropylene tubes as described in
Table 1.
1TABLE 1 Components Reaction LAR Luciferase Total Luciferase Added
1 50 .mu.l l ul of 1 mg/ml 1 .mu.g 2 50 .mu.l l ul of l00 .mu.g/ml
100 ng 3 50 .mu.l l ul of l0 .mu.g/ml 10 ng 4 50 .mu.l (none) 0
ng
[0114] Immediately upon addition of the Luciferase, the tube was
read in a Turner TD-20e Luminometer. The values obtained are listed
in Table 2.
2TABLE 2 Reaction Light Units Light 2 Avg. Filter* Total Light
Units 1 90.6 66.09 78.345 286 22406.67 2 2048 2096 2072 none 2072 3
148 122 135 none 135 4 0 0 0 none 0 *= light reduction filter used
to reduce signal, light units measured must be multiplied by filter
to obtain light output
[0115] Luciferase requires both ATP and Luciferin to produce a
light signal. The light produced in the reactions above is the
result of ATP contamination of either the LAR reagent or the
Luciferase added to the reactions. Ten and five nanogram levels of
Luciferase were chosen for further studies since they produced the
lowest level of background light without ATP addition, yet were
expected to give greatly increased light output upon addition of
ATP.
[0116] Reactions were assembled in duplicate containing 50 .mu.l of
LAR buffer, 1 .mu.l of stock Luciferase providing either 5 or 10 ng
of luciferase to the reaction, and ATP at the concentrations listed
below. Light output from reactions was then immediately determined
with a Turner TD-20e Luminometer. The results are described in
Table 3. These data indicate that Luciferase is capable of
detecting low levels of ATP if levels of Luciferase are used that
minimize the background resulting from ATP contamination of the
reagent.
3 TABLE 3 ATP Luciferase (ng) Conc. .mu.l Light Units Filter Avg.
Light Units 10 (none) na 131.7 119.6 (none) 125.7 10 100 uM 5 26.09
25.25 286 7221.5 10 l uM 5 249.5 226.1 (none) 237.8 10 l0 nM 5
131.7 232.6 (none) 182.2 10 100 pM 5 215.4 143.9 (none) 179.7 5
(none) na 52.76 50.04 (none) 51.4 5 100 uM 5 17.99 18.47 286 5213.8
5 l uM 5 156.4 174.4 (none) 167.9 5 l0 nM 5 46.13 34.37 (none)
40.3
Example 2
Limit of ATP Detection Using Luciferase
[0117] This example demonstrates that the light output values
obtained from reactions with very low levels of ATP are
statistically different from appropriate control reactions. The
limit of detection can be defined as the amount of the analyte that
generates a signal which has less than a 0.05 probability of
identity to the data from control reactions using the Student's
t-Test.
[0118] ATP (Sigma A9187, Lot #36H7808 Promega, stored overnight at
-20 degrees) in l0mM Tris-Cl pH 7.5 was diluted to 500 nM and 50
nM. Various amounts of ATP were added to 350 .mu.l LAR, with Tris
added to make up the difference in volume (385 total). Only Tris
was added to the control to determine the background signal. After
mixing, 6 aliquots of 50 .mu.l of the control and containing
samples were transferred to luminometer tubes. Luciferase (2 .mu.l
of 2.5 ng/.mu.l in 1X CCLR with 1 mg/ml BSA (1X CCLR Promega E153A,
Lot #7903201)) was added to the reaction, the tube was tapped to
mix the reagents and light output was immediately determined with
the Turner TD-20e luminometer. The data is presented in Table
4.
4TABLE 4 Reaction ATP (M) Light 1 0 2.744 2 0 2.606 3 0 2.849 4 0
2.834 5 0 2.801 6 0 2.778 7 4.5 .times. 10.sup.-10 4.883 8 4.5
.times. 10.sup.-10 5.192 9 4.5 .times. 10.sup.-10 4.945 10 4.5
.times. 10.sup.-10 4.220 11 4.5 .times. 10.sup.-10 5.282 12 4.5
.times. 10.sup.-10 5.216 13 9.1 .times. 10.sup.-10 7.167 14 9.1
.times. 10.sup.-10 8.100 15 9.1 .times. 10.sup.-10 7.774 16 9.1
.times. 10.sup.-10 8.047 17 9.1 .times. 10.sup.-10 8.010 18 9.1
.times. 10.sup.-10 7.677 19 1.82 .times. 10.sup.-9 10.70 20 1.82
.times. 10.sup.-9 11.02 21 1.82 .times. 10.sup.-9 11.93 22 1.82
.times. 10.sup.-9 11.91 23 1.82 .times. 10.sup.-9 12.27 24 1.82
.times. 10.sup.-9 11.92
[0119] The Student's t-Test (a 2-tailed test for 2 samples with
unequal variance) was used to analyze the data. The light output
from each ATP concentration was compared to the light output of the
background control, and a p-value determined for each comparison.
The results of the analysis are presented in Table 5. A p-value of
less than 0.05 indicates that the 2 sets of results being compared
are statistically different from each other. Each of the ATP
concentrations compared to background signal have a p-value of less
than 0.05. Therefore, this statistical test indicates that each of
the ATP concentrations analyzed is detectable over background.
5 TABLE 5 p -value 1.82 .times. 10.sup.-9M ATP 2.2 .times.
10.sup.-5 9.1 .times. 10.sup.-10M ATP 9.5 .times. 10.sup.-8 4.5
.times. 10.sup.-10M ATP 2.3 .times. 10.sup.-7
Example 3
Detection of DATP using Luciferase
[0120] Detection of polydeoxyribonucleosides using Luciferase can
in theory be performed through the measurement of DATP if the
enzyme used for detection can utilize DATP. In this example, the
ability of Luciferase to use deoxyadenosine triphosphate (dATP) as
compared to adenosine triphosphate (ATP) was tested.
[0121] Reactions were assembled containing 50 .mu.l of LAR, 2 or 4
.mu.l of luciferase stock (providing 5 or 10 ng of luciferase to
the reactions) and 0 or 5 .mu.l of 1 mM DATP Sigma (final
concentration of DATP approximately 100 .mu.M). Luciferase was the
last component added. Immediately upon enzyme addition, the light
output of the reactions was determined using a Turner TD-20e
Luminometer. The results are provided in Table 6. These data show
that Luciferase can be used to directly detect dATP.
6TABLE 6 Luciferase dATP + Level or - Light Units Avg. 5 ng - 423
295.7 359.4 5 ng + 1450 1621 1535.5 10 ng - 703 705.5 704.3 10 ng +
3684 3441 3562.5
Example 4
Pyrophosphate inhibition of Luciferase
[0122] The reaction of Luciferase produces pyrophosphate from ATP
or dATP and is inhibited by pyrophosphate. Some of the reaction
schemes described later use pyrophosphate as a substrate for other
enzymes. In order to use levels of pyrophosphate in these reactions
which do not inhibit detection of nucleoside using Luciferase, we
determined the levels of inhibition produced by various
concentrations of pyrophosphate on the production of light from
Luciferase.
[0123] A new buffer, LAR without Coenzyme A, was made as described
in Example 1. This buffer and the original LAR were then used to
formulate various reactions with the compositions shown below. The
reactions were assembled with Luciferase being the final component
added. Immediately upon enzyme addition, the light output of the
reactions were determined with a Turner TD-20e Luminometer. The
results are provided in Table 7. These data indicate that the light
output from Ltciferase can be measured in the presence of
pyrophosphate and that more than 50% of the activity can be seen
with pyrophosphate concentrations as high as 100 .mu.M. In
addition, these data indicate that removal of Coenzyme A from the
LAR greatly lowers the background light produced by the reactions
without greatly effecting the activity of Luciferase.
7TABLE 7 LAR with LAS minus ATP pp, Luciferase CoA (.mu.l) CoA
(.mu.l) (2 .mu.M) 1 mM 100 .mu.M (2.5 .mu.g/ml) Light Units Avg. 50
.mu.l -- -- - - 2 .mu.l 209.9 227 218.9 50 .mu.l -- 5 .mu.l - - 2
.mu.l 3462 3674 3568 50 .mu.l -- -- + - 2 .mu.l 9.73 9.54 9.6 50
.mu.l -- 5 .mu.l + - 2 .mu.l 169.5 180.9 175.2 50 .mu.l -- 5 .mu.l
- + 2 .mu.l 1452 1449 1450.5 -- 50 .mu.l -- - - 2 .mu.l 0.035 0.046
0.041 -- 50 .mu.l 5 .mu.l - - 2 .mu.l 3735 3289 3512 -- 50 .mu.l --
+ - 2 .mu.l 0.0003 0.0003 0.0003 -- 50 .mu.l 5 .mu.l + - 2 .mu.l
254.5 308 281.3 -- 50 .mu.l 5 .mu.l - + 2 .mu.l 2041 2069 2055
Example 5
Testing ADP as an inhibitor of Luciferase
[0124] Some of the reaction schemes described later use adenosine
diphosphate (ADP) as a substrate for other enzymes. ADP is a
possible inhibitor of Luciferase. Therefore, we determined the
levels of inhibition produced by various concentrations of ADP on
the production of light from Luciferase and ATP.
[0125] Stock solutions of ADP Sigma or ATP were dissolved in 10 mM
Tris-Cl pH 7.5 and diluted to produce various stock concentrations.
Reactions were assembled which contained 2 .mu.l of 2.5 .mu.g/ml
Luciferase, 50 .mu.l of LAR, 5 .mu.l of ADP or 5 .mu.l of 10 mM
Tris Cl pH 7.5 and 5 .mu.l of ATP or 5 .mu.l of 10 mM Tris Cl pH
7.5. The Luciferase was the final component added to these
reactions. Immediately upon enzyme addition, the light output of
the reactions was measured using a Turner TD-20e Luminometer. The
final nucleoside concentrations of the reactions and the light
output of the reactions are summarized in Table 8. These data
indicate that ADP does not greatly effect the ability of Luciferase
to produce light using ATP as a substrate. Thus, if low
concentrations of ADP are added to Luciferase reactions from
reactions performed using other enzymes, little effect on ATP
detection through the use of Luciferase is expected.
8 TABLE 8 ATP ADP Light output Average -- -- 485.8 423.5 454.7 2
.mu.M -- 4945 4930 4937.5 -- 100 .mu.M 4800 4418 4609 2 .mu.M 100
.mu.M 6834 7207 7020.5 -- 1 .mu.M 513 463 488 2 .mu.M 1 .mu.M 4303
4152 4227.5 -- 10 nM 419.6 419 419 2 .mu.M 10 nM 4534 4625
4579.5
Example 6
NDPK transformation of ADP to ATP, using deoxynucleosides
[0126] Luciferase can detect ATP at much lower concentrations than
dATP or other nucleosides. If deoxynucleoside triphosphates could
be used to generate ATP, an increase in sensitivity may result. For
this reason, we tested the ability of enzymes to transfer the
terminal phosphate of deoxynucleoside triphosphates to ADP, forming
ATP and deoxynucleoside diphosphates.
[0127] Reactions were assembled which contained 100 .mu.l of LAR,
10 ng of Luciferase in the presence or absence of deoxynucleoside
triphosphates (1 .mu.M final concentration when added), and 10
units of nucleoside diphosphate kinase (NDPK) (Sigma #N0379, Lot
#127F81802). The reactions were assembled with the exception of
luciferase and incubated for 15 min at room temperature. The
Luciferase was added and the light output of the reactions was
measured immediately using a Turner TD-20e Luminometer. The light
output values measured are provided in Table 9. These data confirm
that NDPK is capable of transferring the phosphate from nucleoside
triphosphates to ADP to form ATP which can be detected using
Luciferase.
9TABLE 9 Tube # dNTP ADP NDPK ATP Light Units 1 - + + 883 2 - - + +
15361 3 - + - 543 4 - - - + 21970 5 dATP + + 13356 6 dATP - + 151 7
dCTP + + 13007 8 dCTP - + 6.9 9 dGTP + + 13190 10 dGTP - + 7.3 11
TTP + + 19230 12 TTP - + 9.0
Example 7
NDPK transformation of ADP to ATP using NDPK and ATP Analogs
[0128] Some enzymes which may be used to transform nucleosides show
specificity for adenosine nucleosides as phosphate donors.
Adenosine nucleosides may not be used as high energy phosphate
donors for these converting enzymes if a Luciferase detection
system is to be utilized. This is because light would be generated
by Luciferase from the added adenosine nucleoside. However, the
converting enzymes may be utilized if an analog of adenosine is
identified that can be used by the converting enzymes but not by
Luciferase. This example indicates how such analogs can be tested
for their ability to be used by converting enzymes but not by
Luciferase.
[0129] Approximately 5 mg of ATP (Sigma A9187, Lot #36H7808),
.alpha.,.beta. methyleneadenosine 5'-Triphosphate (AMP-CPP) (Sigma
M6517, Lot #96H7813) and .beta., .gamma. methylene adenosine
5'-triphosphate (AMP-PCP) (Sigma M7510, Lot #34H7840) were diluted
in Tris-Cl, 10 mM, pH 7.5. The absorbance of a 1:100 dilution of
these solutions into 50 mM Tris-Cl, pH 7.5 was read at 259 nm using
a Beckman DU650 Spectrophotometer. The absorbances were used to
determine the concentration of these solutions using a molar
extinction coefficient of 15.4.times.10.sup.3 M. Recombinant
Luciferase was diluted into CCLR containing 1 mg/ml BSA to a
concentration of 2.5 ng/.mu.l. When the reactions were assembled, 2
.mu.l luciferase was added from the 2.5 ng/.mu.l stock solution and
the light emission of the solutions were immediately read using a
Turner TD-20e Luminometer. The data is provided in Table 10.
10TABLE 10 Reaction LAR ATP AMP-CPP* AMP-PCP* # rxn Avg. 1 50 .mu.l
-- -- -- 3 426.4 2 50 .mu.l 4 .mu.M -- -- 7 5762 3 50 .mu.l -- 552
.mu.M -- 2 349.2 4 50 .mu.l 4 .mu.M 552 .mu.M -- 2 5072.5 5 50
.mu.l -- 5.52 .mu.M -- 2 465.8 6 50 .mu.l 4 .mu.M 5.52 .mu.M -- 2
5843.5 7 50 .mu.l -- 55.2 nM -- 2 429.8 8 50 .mu.l 4 .mu.M 55.2 nM
-- 2 4152 9 50 .mu.l -- -- 1.14 mM 2 260.35 10 50 .mu.l 4 .mu.M --
1.14 mM 2 3735.5 11 50 .mu.l -- -- 11.4 .mu.M 2 431.25 12 50 .mu.l
4 .mu.M -- 11.4 .mu.M 2 5930 13 50 .mu.l -- -- 114 nM 2 389.35 14
50 .mu.l 4 .mu.M 114 nM 2 6093.5 *= final concentration in the
reaction, solution produced by concentrated stock solution
[0130] Micromolar solutions of these ATP analogs do not produce
light above reactions containing no added nucleoside and do not
greatly lower the light output of reactions containing low levels
of ATP from the values seen in the absence of these analogs. These
analogs do not inhibit Luciferase and are not utilized by
Luciferase. Thus, these data indicate that these analogs can be
tested for their ability to be used with enzymes for the
transformation of nucleosides.
[0131] The following reactions were performed to determine if
either AMP-CPP or AMP-PCP could be used by NDPK. All reactions were
assembled in duplicate and incubated at room temperature for 20
min. Ten nanograms of Luciferase was added and the light output of
the reactions immediately measured using a Turner TD-20e
Luminometer. The data is provided in Table 11. These data
demonstrate the analog AMP-CPP is utilized by the enzyme NDPK as a
substrate to generate ATP from ADP. The values seen with AMP-CPP,
ADP and NDPK present are substantially higher than those seen for
ADP alone, ADP and NDPK without AMP-CPP and NDPK alone. Analogous
experiments can be performed to test other enzymes for their
ability to use nucleoside substrates in a similar fashion.
11TABLE 11 ADP AMP-CPP AMP-PCP Reaction LAR-CoA (2 .times.
10.sup.-4M) NDPK (2 .times. 10.sup.-5M) (2 .times. 10.sup.-5M) Avg.
1 100 .mu.l -- -- -- -- 0.21 2 100 .mu.l 0.5 .mu.l -- -- -- 60.23 3
100 .mu.l 0.5 .mu.l 1 .mu.l -- -- 59.77 4 100 .mu.l 0.5 .mu.l 1
.mu.l 5 .mu.l -- 617.95 5 100 .mu.l -- 1 .mu.l 5 .mu.l -- 1.81 6
100 .mu.l 0.5 .mu.l 1 .mu.l -- 5 .mu.l 69.35 7 100 .mu.l -- 1 .mu.l
-- 5 .mu.l 0.03 8 100 .mu.l -- 1 .mu.l -- -- 0.05
Example 8
NMPK Transformations of ADP
[0132] This example demonstrates a method for testing the ability
of an enzyme to transform nucleoside diphosphates into nucleosides
which can be used by Luciferase for the generation of light. The
enzyme Nucleoside Monophosphate Kinase (NMPK, Sigma, N-4379) can
transfer the phosphate from ATP to UMP, forming UDP and ADP. This
experiment demonstrates that this enzyme preparation can also be
used to form ATP from ADP, probably through the reaction:
2ADP.fwdarw.ATP+AMP
[0133] The reactions were assembled in duplicate as prescribed in
Table 12 and incubated at room temperature for 30 min. At that
time, long of Luciferase was added and the light output of the
solutions was measured using a Turner TD-20e Luminometer. The data
is provided in Table 12. These data indicate that NMPK can
transform ADP into ATP. Similar experiments can be used to test the
ability of other enzymes to perform similar transformations.
12TABLE 12 Reaction LAR-CoA* NMPK** ADP(10.sup.bM) Avg. Light 1 100
.mu.l -- -- 0.58 2 100 .mu.l 10 .mu.l -- 0.23 3 100 .mu.l -- 5
.mu.l 14.81 4 100 .mu.l 10 .mu.l 5 .mu.l 211.4 * = Luciferase Assay
Reagent formulated without added Coenzyme A or ATP ** = Sigma
N-4379, Lot #96H0166, dissolved in ATP free water to 3.38 U/ml
Example 9
Combination of NMPK and NDPK, dCTP, and AMP
[0134] One potential method for amplifying an ATP signal requires
two enzymes and a phosphate donor. For this system to operate, the
first enzyme, E1, must be able to convert AMP to ADP but must be
unable to use the phosphate donor. The second enzyme, E2, must be
able to effectively use the phosphate donor to transform any ADP
formed to ATP. This example demonstrates a method to test the
ability of a combination of enzymes to be used in such a
combination reaction scheme. The Examples above demonstrate that
NDPK can transform ADP to ATP using dCTP as a phosphate donor.
[0135] The reactions were assembled as presented in Table 13 and
incubated for 30 min at room temperature. Then long of luciferase
in 2 .mu.l of 1X CCLR with 1 mg/ml BSA was added and the light
output of the reactions was measured using a Turner TD-20e
Luminometer. The data is presented in Table 13.
[0136] The reaction which could have produced significant ATP if
NMPK could transform AMP to ADP using dCTP as a substrate is
reaction 4. This reaction produced only minute amounts of ATP as
measured by Luciferase mediated light production. Reaction 5 (where
NDPK was used to transform ADP to ATP using added dCTP) and
reaction 6 (where NMPK was used to transform ADP to ATP) produced
much more ATP. Since all enzymes were shown to be active, these
data indicate that NMPK essentially cannot use dCTP to transform
AMP to ADP. This is the essential requirement for the ATP
amplification system described above. In this particular instance
E1, NMPK, cannot use the phosphate donor (dCTP) but can utilize AMP
and ATP to produce 2 ADP molecules (the reverse of the reaction 6).
The second enzyme, E2 (NDPK), can use the phosphate donor (dCTP) to
transform the ADP produced by the first enzyme to create 2ATP from
the 2ADP using 2dCTP. These ATPs can then re-enter the cycle. This
protocol can be used to test combinations of enzymes and phosphate
donors for their ability to act as the enzymes in our ATP
amplification schemes.
13TABLE 13 Reaction LAR-CoA AMP dCTP NMPK* NDPK** water Tris-Cl ADP
Light 1 100 .mu.l -- -- 10 .mu.l 1 .mu.l -- 15 .mu.l -- 0.0088 2
100 .mu.l 5 .mu.l -- -- -- 11 .mu.l 10 .mu.l -- 0.125 3 100 .mu.l
-- 10 .mu.l -- -- 11 .mu.l 5 .mu.l -- 0.159 4 100 .mu.l 5 .mu.l 10
.mu.l 10 .mu.l 1 .mu.l -- -- -- 10.93 5 100 .mu.l -- 5 .mu.l -- 1
.mu.l 10 .mu.l -- 10 .mu.l 4049 6 100 .mu.l -- -- 10 .mu.l 1 .mu.l
5 .mu.l 10 .mu.l 2309 7 100 .mu.l -- -- -- -- 11 .mu.l 5 .mu.l 10
.mu.l 115.9 * = NMPK concentration at 5 mg/ml o Sigma N4379, ** =
NDPK concentration of 10 units/.mu.l Sigma N0379, Nucleoside stocks
at: AMP (Sigma A2002, Lot #2OH7035), 2X10-5M; dCTP (Promega U122A,
Lot #6858402) , 2 .times. 10 .sup.-5M, ADP 1 .times.10.sup.-5M
(Sigma A2754 Lot #65H7880)
Example 10
Amplification of ATP using NMPK, NDPK, dCTP and AMP with ATP
Spikes
[0137] The enzyme combination presented in Example 9 should be
capable of greatly increasing the relative ATP concentration
through the cyclic amplification reaction scheme presented earlier.
This example demonstrates the amplification of different levels of
input ATP using these enzymes and nucleosides. The reactions were
assembled as presented in Table 14 and incubated at room
temperature. When the reactions reached incubation time 0 min, 20
min, 40 min, 60 min, 80 min, 100 min, 120 min, 180 min, and 240
min, 112 .mu.l samples of each reaction were transferred to
luminometer tubes and lOng of luciferase were added. The light
output of the reactions was immediately measured using a Turner
TD-20e Luminometer. The data is presented in Table 15.
[0138] The reactions with ATP added (Reactions 1, 2, and 3)
increased in ATP more rapidly than the reactions without added ATP
(Reaction 4). The rate of increase of the ATP was dependent upon
the amount of ATP first added to the reaction. Thus, this
combination of enzymes amplified the input ATP signal and the
amount of ATP produced at a particular time was dependent upon the
starting amount of ATP added.
[0139] In addition, this combination of reactions allows the user
to determine if any of the enzymes used are contaminated with
unexpected activities that may influence the system. For example,
removing the NMPK, dCTP or AMP from the system prevents any ATP
accumulation, as expected. However, eliminating the NDPK only has a
small influence on the rate of ATP accumulation. These data suggest
that the NMPK source used contains a small amount of activity which
can take the place of NDPK in this system.
[0140] Performing similar experiments should allow a user to
determine if other enzymes can be used in such amplifications
schemes, as shown in Example 11.
14TABLE 14 Reaction AMP dCTP NMPK NDPK ATP 1 10 .mu.l 10 .mu.l 100
.mu.l 10 .mu.l 1 pmol 2 10 .mu.l 10 .mu.l 100 .mu.l 10 .mu.l 100
fmol 3 10 .mu.l 10 .mu.l 100 .mu.l 10 .mu.l 10 fmol 4 10 .mu.l 10
.mu.l 100 .mu.l 10 .mu.l -- 5 10 .mu.l 10 .mu.l -- 10 .mu.l 1 pmol
6 10 .mu.l 10 .mu.l 100 .mu.l -- 1 pmol 7 -- 10 .mu.l 100 .mu.l 10
.mu.l 1 pmol 8 10 .mu.l -- 100 .mu.l 10 .mu.l 1 pmol
[0141]
15TABLE 15 Reaction 0 min 20 min 40 min 60 min 80 min 100 min 120
min 180 min 240 min 1 95 179.5 249.9 339.5 562 670.9 709.4 1157
1497 2 20.42 37.11 54.95 85.1 122.5 164.9 206.1 409.7 746 3 12.3
25.09 39.37 55.79 93.8 120.5 156.1 325.7 573.7 4 11.8 25.54 37.45
54.64 87.8 113.1 143.8 299.1 548.7 5 96.4 75 63.63 56.75 51.72
54.72 56.61 58.97 60.97 6 110 183.5 247.8 285.9 426.1 503.9 624.5
958 1301 7 91.7 99 98.6 85.2 93.2 94.2 95.4 91.5 90.4 8 3.521 2.755
2.31 2.058 2.092 2.173 1.682 1.088 0.73
Example 11
Amplification of ATP using Adenylate Kinase and Pyruvate Kinase
[0142] This example demonstrates a second ATP amplification system
using a non-nucleoside based phosphate donor. The enzymes used are:
adenylate kinase (an enzyme which produced 2ADP from one ATP and
one AMP but which cannot use phosphoenol pyruvate (PEP) as a
phosphate donor and Pyruvate Kinase (an enzyme which phosphorylates
ADP to form ATP using PEP as a phosphate donor). The reactions were
assembled as presented in Table 16. These reactions were incubated
at room temperature and 109 .mu.l of the reactions was removed at
0, 30, 60, and 120 min. Luciferase (2 .mu.l, long in 1X CCLR with 1
mg/ml BSA) was added and the light output of the reaction was
immediately measured using a Turner TD-20e Luminometer. The data is
presented in Table 17.
[0143] After 30 minutes of incubation the reaction containing ATP
(reaction 1) increased much more rapidly than the reaction with no
ATP added (reaction 2). Thus, the ATP sample was amplified. Also
note that in this set of reactions, the ATP content of reactions 1
and 2 reached a final ATP level. This indicates that the reactions
had reach an equilibrium value.
[0144] Finally, note that the reaction with no added AMP also
increased over time. This suggests that one of the components was
contaminated with either AMP or ADP. Further experiments
demonstrated that the contaminating nucleoside was present in the
pyruvate kinase solution used in this study. The following example
demonstrates a method for removing this contaminating
nucleoside.
16TABLE 16 Reaction ATP AMP AK PEP PK Tris Buffer LAR-CoA 1 12.5
.mu.l 5 .mu.l 10 .mu.l 5 .mu.l 12.5 .mu.l -- -- 500 .mu.l 2 -- 5
.mu.l 10 .mu.l 5 .mu.l 12.5 .mu.l 12.5 .mu.l -- 500 .mu.l 3 12.5
.mu.l -- 10 .mu.l 5 .mu.l 12.5 .mu.l 5 .mu.l -- 500 .mu.l 4 12.5
.mu.l 5 .mu.l -- 5 .mu.l 12.5 .mu.l -- 10 .mu.l 500 .mu.l 5 12.5
.mu.l 5 .mu.l 10 .mu.l -- 12.5 .mu.l -- 5 .mu.l 500 .mu.l 6 12.5
.mu.l 5 .mu.l 10 .mu.l 5 .mu.l -- -- 12.5 .mu.l 500 .mu.l * The
concentrations of these components were: ATP, 1 .times.10.sup.-6M;
AMP, 1 .times.10.sup.-4M; Adenylate Kinase (AK) (Sigma M5520, lot
#16H9558), 7 U/.mu.l in 50 mM KPO.sub.4, 15 mM MgCl.sub.2, pH 7.5
(Buffer A); PEP, (Phosphoenolpyruvate, Sigma P-7002, Lot #46H3777,
100 mM in deionized water; PK (pyruvate kinase, (Sigma P-7286, Lot
#45H9504), 0.1 U/.mu.l in Buffer A), and; Tris Cl, 10 mM Tris Cl,
pH 7 .5.
[0145]
17TABLE 17 Time (Min) Reaction 0 30 60 120 1 93.6 536.4 683.8 670.4
2 14.98 120.6 594.8 639.3 3 105.5 219.4 321 384.7 4 112.5 97.2 98.8
94.1 5 83.1 16.84 16.03 15.02 6 90.6 21.61 22.79 21.2
Example 12
Removal of interfering substances in Pyruvate Kinase using
Dialysis
[0146] This example demonstrates methods for detecting
contaminating nucleosides in enzymes used in the various
technologies discussed in the other examples and removing the
contaminating material.
[0147] Additionally, another amplification scheme is described.
This scheme utilizes: Adenylate Kinase (E1); NDPK (E2); AMP; and
the ATP analog AMP-CPP as the high energy phosphate donor. If AMP
is left out of this reaction, no increase in an initial ATP signal
should take place unless one of the other materials is contaminated
with AMP (or ADP).
[0148] Reactions performed as described in Example 11 suggested
that one of the components may have adenosine nucleoside
contamination. One of the components suspected of contamination is
pyruvate kinase. A sample of this enzyme was dialyzed against 50 mM
KPO4.sub.1 15 mM MgCl.sub.2 pH 7.6 in Spectra Por Dialysis tubing
with a molecular weight cut off of 3,500 da. The dialysis was
performed twice against 1000X amount of buffer for several hours at
4.degree. C. to remove free adenosine. The reactions were assembled
according to Table 18. These data indicate that following dialysis
the enzyme solution was slightly more dilute than prior to
dialysis. By adding 5.3 .mu.l of the post dialysis enzyme and 5.0
.mu.l of the pre-dialysis enzyme, equal amounts of PK were added to
the reactions.
[0149] 500 microliters of LAR-CoA was added to the assembled
reactions and the final reactions incubated at room temperature. At
0, 10, 20, and 30 min, 114 .mu.l of these reactions were added to
long of luciferase in 5 .mu.l of 1X CCLR with lmg/ml BSA and the
light output of the solutions was immediately measured using a
Turner TD-20e Luminometer. The data is presented in Table 19.
[0150] Two main observations can be derived from this data. First,
the AK, NDPK, AMP, AMP-CPP enzyme-substrate combination can be used
to amplify an ATP signal. However, production of ATP from some
contamination source allows reactions not given ATP added to
achieve a final ATP concentration similar to those given an ATP
spike.
[0151] The reaction to which no AMP or PK were added (reaction 2)
does not increase over time. However, the reactions to which the
undialized PK was added and no AMP was added give high light output
over time (reaction 5). Addition of dialyzed PK to reactions
lacking AMP (reaction 8) demonstrate increased light output over
time, but thle rate of increase is dramatically reduced from that
seen without dialysis.
[0152] This Example demonstrates that yet another ATP amplification
system can be used to generate higher ATP levels from a starting
ATP spike. In addition, this Example shows that these systems can
be used to determine if solutions contain contaminating nucleosides
and that dialysis can be used to fractionate contaminating
nucleosides from enzymes utilized in ATP amplification
reactions.
18TABLE 18 Reaction AMP ATP NDPK AMP-CPP AK PK Pi Buffer Tris 1 10
.mu.l 10 .mu.l 5 .mu.l 10 .mu.l 10 .mu.l -- 5 .mu.l -- 2 -- 10
.mu.l 5 .mu.l 10 .mu.l 10 .mu.l -- 5 .mu.l 10 .mu.l 3 10 .mu.l -- 5
.mu.l 10 .mu.l 10 .mu.l -- 5 .mu.l 10 .mu.l 4 10 .mu.l 10 .mu.l 5
.mu.l 10 .mu.l 10 .mu.l 25 .mu.l Sample 1 -- -- 5 -- 10 .mu.l 5
.mu.l 10 .mu.l 10 .mu.l 25 .mu.l Sample 1 -- 10 .mu.l 6 10 .mu.l --
5 .mu.l 10 .mu.l 10 .mu.l 25 .mu.l Sample 1 -- 10 .mu.l 7 10 .mu.l
10 .mu.l 5 .mu.l 10 .mu.l 10 .mu.l 26.5 .mu.l Sample 2 -- -- 8 --
10 .mu.l 5 .mu.l 10 .mu.l 10 .mu.l 26.5 .mu.l Sample 2 -- 10 .mu.l
9 10 .mu.l -- 5 .mu.l 10 .mu.l 10 .mu.l 26.5 .mu.l Sample 2 -- 10
.mu.l The compositions of these solutions were: AMP, 1 .times.
10.sup.-4M; ATP, 2 .times. 10.sup.-6M; NDPK, 0.1 U/.mu.l; AMP-CPP,
1 .times. 10.sup.-3M; AK, 0.75 units/.mu.l; PK, 5 .mu.l of pyruvate
kinase pre-dialysis (sample 1) or post dialysis (sample 2), Pi
buffer (described above); and, Tris, 10 mM Tris - Cl, pH 7.5.
[0153]
19TABLE 19 Time (Min) Reaction 0 10 20 30 1 60.35 349.7 529 563.1 2
54.73 50.74 49.79 52.79 3 51.62 59.11 279 420.7 4 87.4 666.6 754.2
779 5 73.1 213.9 354.6 412.4 6 24.41 479.1 701.7 707.6 7 69.9 449.3
577.3 595.2 8 50.92 76.03 118.4 148.9 9 12.92 229.3 541.3 569.4
Example 13
PRPP Synthetase, Reactions with Adenosine
[0154] The enzyme 5' phopshorylribose 1' pyrophosphate synthetase
(PRPP Synthetase) transfers a pyrophosphate from ATP to D-ribose 5'
phosphate. This experiment was performed to determine if this
enzyme could be used with AMP and 5'phosphoribose 1' pyrophosphate
to generate ATP and D-ribose 5' phosphate.
[0155] ATP, AMP and PRPP were diluted in 10 mM Tris, pH 7.3. PRPP
Synthetase (Sigma #P0287) was diluted in PRPP Synthetase reaction
buffer (see below). 2 .mu.l of ATP, 2 .mu.l of AMP, 2 .mu.l of
PRPP, and 2 .mu.l of PRPP Synthetase (or appropriate buffers) were
added as indicated in Table 20 to 20 .mu.l of PRPP Synthetase
reaction buffer.
[0156] The reactions were incubated in the 37.degree. C. water bath
for 30 min. The tubes were removed from the water bath and 100
.mu.l of LAR (without CoA) was added. Then, 126 .mu.l was
transferred to a luminometer tube. long of Luciferase was added in
5 .mu.l of 1X CCLR containing 1 mg/ml BSA and light output measured
with Turner TD-20e Luminometer. The data presented in Table XXX.
This data demonstrates that PRPP Synthetase can transfer
pyrophosphate from non-nucleoside substrates to AMP to form
ATP.
[0157] The nucleoside concentrations in the reaction were: ATP
(when added) 1.2.times.10.sup.5M; AMP (when added)
2.9.times.10.sup.-5M, and; PRPP (when added) 2.6.times.10.sup.-5M.
6.times.10.sup.-4 units of the enzyme (PRPP Synthetase) was added
per reaction. PRPP Synthetase buffer is 50 mM triethanolamine, 50
mM potassium phosphate, pH 7, 0.37 mM EDTA, 10 mM MgCl.sub.2, 1
mg/ml BSA.
20TABLE 20 Tube # AMP PRPP PRPP Syn Light Units 1 + + + 3440 3424 2
+ - + 0.522 0.501 3 + + - 6.649 4.619 4 - + + 7.096 7.139 5 + - -
1.874 0.430 6 - - - 5.203 4.794 7 - + + 96.0 0.361 8 - - - 462.8
0.603
Example 14
PRPP Synthetase, Reactions with deoxyadenosine
[0158] Some schemes for the detection of DNA require the conversion
of dAMP, generated by nuclease digestion of DNA, to DATP. This
example demonstrates that the enzyme PRPP Synthetase can perform
the transformation of dAMP to DATP using PRPP as a cosubstrate. In
addition, this transformation can be monitored by Luciferase
detection at much higher sensitivities if the DATP formed is used
to transform ADP to ATP through the action of NDPK added to the
reaction.
[0159] The reactions were assembled in duplicate as shown in Table
21. The concentrations of the reaction components were: dAMP
2.9.times.10.sup.-4 M in 10 mM Tris pH 7.3; AMP 2.9.times.10.sup.-4
M in 10 mM Tris pH 7.3; PRPP 2.6 X10.sup.-4 M in 10 mM Tris pH 7.3;
PRPP Syn (PRPP Synthetase) (Sigma #P0287) 100X dilution of stock
enzyme which is at 0.03 units/gl. The components were added to
twenty microliters of PRPP Synthetase Buffer (see Example 13).
After incubating for 47 min at 37.degree. C., After incubating, 100
.mu.l of LAR was added to all reactions along with long of
luciferase and the light output of the reactions was immediately
measured. The data is presented in Table 22. PRPP was able to
utilize dAMP as a substrate (comparing reaction 1 to 2, 3, 4 and
5). However, the amount of light produced by reaction was not very
great, probably due to the fact that luciferase uses DATP at a much
lower efficiency than ATP as presented earlier.
21 TABLE 21 Reaction dAMP PRPP PRPP Syn 1 2 .mu.l 2 .mu.l 2 .mu.l 2
2 .mu.l -- 2 .mu.l 3 2 .mu.l 2 .mu.l -- 4 2 .mu.l -- -- 5 -- 2
.mu.l 2 .mu.l
[0160]
22 TABLE 22 Reaction Tube A Tube B Avg. 1 18.2 22.1 20.15 2 1.4 1.4
1.4 3 4.2 3.8 4 4 2.1 1.8 1.95 5 13.1 15.8 14.45
[0161] In order to demonstrate the transfer of phosphate from DATP
to ADP to form ATP, the reactions presented in Table 23 were
assembled in duplicate in twenty microliters of PRPP Synthetase
Buffer (for solution compositions, see tables above). They were
then incubated at 37.degree. C. for 34 min. The added components
had the following formulations: ADP 2.3.times.10.sup.-2 M in 10 mM
Tris pH 7.3; NDPK-1000X dilution of Sigma #N0379 at 10 units/.mu.l
(final concentration 0.01 units/.mu.l). The tubes were then
incubated for an additional 60 min at 37.degree. C., long of
Luciferase were added, and the light output was measured using a
Turner TD-20e Luminometer. The data is presented in Table 24. These
data indicate that the dATP produced by the PRPP Synthetase
reaction can be transferred to ADP by the action of NDPK to produce
ATP.
23TABLE 23 Reaction dAMP PRPP PRPP Syn ADP NDPK 1 2 .mu.l 2 .mu.l 2
.mu.l 2 .mu.l 2 .mu.l 2 2 .mu.l 2 .mu.l 2 .mu.l -- -- 3 2 .mu.l 2
.mu.l 2 .mu.l 2 .mu.l -- 4 2 .mu.l 2 .mu.l -- 2 .mu.l 2 .mu.l 5 --
2 .mu.l 2 .mu.l 2 .mu.l 2 .mu.l
[0162]
24TABLE 24 Light Units Reaction Tube A Tube B 1 812.1 839.3 2 19.2
37.5 3 53.6 52.6 4 168.4 173.1 5 43.6 38.9
Example 15
Digestion of PolydA using Nucleases
[0163] One potential method for detecting DNA would be to digest
the polymer to deoxynucleoside 5' monophosphate, transform the
deoxynucleoside monophosphates to deoxynucleoside triphosphates,
form ATP from the deoxynucleoside triphosphates using ADP and NDPK,
and then detect the ATP using Luciferase. This example demonstrates
the digestion of a deoxyadenosine polymer.
[0164] A solution of deoxyadenosine 5' monophosphate was made by
adding 990 .mu.l of water and 10 .mu.l of 1X TE buffer (10 mM Tris
Cl, 1 mM EDTA pH 8.0) to 25 units of polyadenylic acid (Pharmacia
27-786, Lot #5017836021). A reaction was assembled with the
following materials: 450 .mu.l of nanopure water, 50 .mu.l of 10 X
S1 Nuclease buffer (Promega Corp. M577A, Lot #6748605) and 10 .mu.l
of the polydeoxyadenylic acid solution above. The absorbance change
at 260 nm was monitored on a Beckman DU650 Spectrophotometer. The
rate of change in the absorbance of the solution was 0.0020
Abs/min. At this point, 1 .mu.l of S1 Nuclease (Promega Corp.
E576B, Lot #6800810) was added and the absorbance change of the
solution redetermined and found to be 0.0156 abs/min. Since small
oligonucleosides and mononucleosides display absorbance values
higher than a corresponding amount of polynucleoside, this
indicates that this enzyme can digest the polymer.
[0165] The reaction conditions given below are those used to digest
the polydeoxyadenylic acid polymer samples that are used in later
examples.
[0166] Three reactions were assembled which contained:
25 Reaction 1: 90 .mu.l of the polydeoxyadenylic acid solution
described above, 10 .mu.l of 10X S1 nuclease reaction buffer.
Reaction 2: As Reaction 1 above. Reaction 3: 9O .mu.l of nanopure
water, 10 .mu.l of S1 nuclease reaction buffer.
[0167] At time equals zero minutes of digestion, 10 .mu.l of each
of these was removed and added to 490 .mu.l of 50 mM Tris Cl pH
8.0. Immediately, 1 .mu.l of S1 nuclease was added to the remaining
reaction mixtures 1 and 3 but not 2, and the mixtures were allowed
to incubate at room temperature. Additional 10 .mu.l samples of the
reactions were removed after 20, 50 and 140 min of reaction and
diluted into 490 .mu.l of 50 mM Tris Cl pH 8.0. The data is
presented in Table 25. The absorbance of the solution in Reaction
#1 increased, again indicating that the polymer in this reaction
was digested over time. A second set of reactions was produced as
described above. The only difference with these reactions was that
50 units of Sigma Poly(dA)(Sigma P-0887, Lot #67H0226) was
dissolved in 1.5 ml of TE buffer and used in the reactions. After
the 140 minutes of digestion, these reactions were used as
described in Example 16.
26 TABLE 25 Net Absorbance at 260 nm of Samples From Reaction Time
#1 #2 #3 0 0 0 0 20 0.0726 -0.0088 -0.0025 50 0.1425 0.0291 -0.0041
140 0.1445 -0.003 -0.0044
Example 16
Detection of Poly (dA) using nucleases and PRPP Synthetase
[0168] In this example, the digested polynucleoside described in
Example 15 is detected by two different methods. Both methods begin
with transformation of the deoxynucleosides to deoxynucleoside
triphosphates using PRPP Synthetase and PRPP. In the first method,
ADP is converted to ATP using the deoxynucleoside triphosphates
formed in the PRPP synthetase reaction and the resulting ATP
detected using Luciferase. In the second method, AMP is converted
to ATP using the deoxynucleoside triphosphates formed by the PRPP
Synthetase reaction, simultaneously amplified and detected using
Luciferase.
[0169] Table 26 presents the components of the PRPP Synthetase
reaction. The concentrations of the components were: PRPP,
2.6.times.10.sup.-4 M in 10 mM Tris-Cl pH 7.5; PRPP Synthetase,
6.times.10.sup.31 4 Units of Sigma P0287 per 2 .mu.l in PRPP
Synthetase Buffer. For composition of Buffer, refer to PRPP
Synthetase Buffer in Example 13. The nucleoside digests containing
S1 were diluted in deionized water to yield the amount of polymer
listed in the Table in 8 .mu.l of solution and added to the
appropriate reactions. The digest containing no polymer was diluted
identically to those with polymer. Eight microliters of this
solution contained all the components in the samples containing
720ng of polymer except the Poly(dA). All the reactions were
incubated 32 min in a 37.degree. C. water bath. At this point all
the reactions were heated at 95.degree. C. for 5 min to inactivate
the PRPP Synthetase and cooled in an ice bath for 5 min.
27TABLE 26 Reac- PRPP tion Digest Buffer PRPP Synthetase poly(dA)
S1 1 720 ng 80 .mu.l 2 .mu.l 2 .mu.l -- -- 2 72 ng 80 .mu.l 2 .mu.l
2 .mu.l -- -- 3 7.2 ng 80 .mu.l 2 .mu.l 2 .mu.l -- -- 4 0.72 ng 80
.mu.l 2 .mu.l 2 .mu.l -- -- 5 -- 80 .mu.l 2 .mu.l 2 .mu.l 720 ng --
6 -- 80 .mu.l 2 .mu.l 2 .mu.l -- (720 ng
[0170] A. First Detection Method
[0171] Twenty microliters of each reaction was added to 100 .mu.l
of LAR minus CoA. Ten nanograms of Luciferase was immediately added
and the light production of the reactions was measured. A second 20
.mu.l sample was added to 100 .mu.l of LAR minus CoA, followed by
addition of ADP (2 .mu.l of 2 .mu.g/ml stock) and NDPK (2 .mu.l of
1.times.10.sup.-2 U/.mu.l), and allowed to incubate 20 min at room
temperature. After the incubation, long of luciferase was added to
the reactions and the light production of the reactions was
measured using a Turner TD-20/20 luminometer at 52.1- sensitivity.
The data obtained for these measurements are presented in Table
27.
[0172] These data show that direct measurement of the
deoxynucleoside triphosphates is possible using luciferase if
relatively high amounts of digested DNA are to be detected (see
reaction 1 vs. 5 and 6 in the no NDPK column). However, much more
sensitive detection is provided when the deoxynucleoside
triphosphates are used to convert ADP to ATP using NDPK.
28TABLE 27 Light Units Reaction DNA no NDPK w/NDPK 1 180 ng 43 711
2 18 ng 15 227 3 1.8 ng 13 77 4 0.18 ng 11 37 5 no S1 13 161 6 no
poly 11 28
[0173] B. Second Detection Method
[0174] Twenty microliters of the reaction mixtures from the heat
inactivated PRPP Synthetase reactions were added to ATP
amplification reactions in an attempt to use the initial
deoxynucleoside triphosphates to produce of ATP. This would allow
easier detection of the DATP produced by the PRPP synthetase
reaction.
[0175] The reactions were assembled as demonstrated in Table 28.
The reactions were mixed and the first aliquot of 109.3 .mu.l
({fraction (1/7)} of the reaction) was removed immediately after
adenylate kinase was added. The aliquot was placed in a luminometer
tube, long luciferase was added, the tube tapped to mix, and then
the light output was measured with a Turner TD-20/20 luminometer at
52.1% sensitivity. Subsequent aliquots were removed at 20 minute
intervals and measured immediately. The reactions were incubated at
room temperature. The data obtained is presented in Table 29.
[0176] These results show that it is possible to amplify the dATP
produced from digested DNA after conversion to nucleoside
triphosphates. Note that the light output obtained by this method
is greater than the light output of then on-amplified PRPP
Synthetase method.
29TABLE 28 (Reaction Components**) Reaction DNA LAR-CoA AMP PEP AK
PK Tris PRPP Butter 1 poly(dA) 180 ng, 20 .mu.l* + + + + + -- -- 2
poly(dA) 18 ng, 20 .mu.l* + + + + + -- -- 3 poly(dA) 1.8 ng, 20
.mu.l* + + + + + -- -- 4 poly(dA) + + + + + -- -- 5 poly(dA) no S1
+ + + + + -- -- 6 Si nuclease, no + + + + + -- -- 7 ATP 14 .mu.l 2
mM + + + + + -- 6 .mu.l 8 dATP 14 .mu.l 2 nM + + + + + -- 6 .mu.l 9
dATP 14 .mu.l 200 nM + + + + + -- 6 .mu.l 10 dATP 14 .mu.l 20 nM +
+ + + + -- 6 .mu.l 11 none + + + + + 14 .mu.l 6 .mu.l *These
reactions used 20 .mu.l of the heat-inactivated PRPP Synthetase
reactions from the first part of this example. **The components
were: ATP (Sigma A9187) in 10 mM Tris pH 7.5, dATP (Sigma D6500) in
10 mM Tris pH 7.5, AMP 7 .mu.l of 2 .times. 10.sup.-4M in 10 mM
Tris pH 7.5, LAR-CoA (LAR without CoA) 700 .mu.l per reaction tube,
PEP (phosphoenol pyruvate-ammonium salt) (synthesized) 7 .mu.l of
100 mM, AK (adenylate kinase/myokinase) (Sigma M5520) 14 .mu.l of
0.75 units/.mu.l in Buffer A, PK (pyruvate kinase) (Sigma P7286,
dialyzed 48 hours) 17.5 .mu.l of 0.13 units/.mu.l, Tris 10 mM pH
7.5, PRPP Synthetase Buf #fer-see example 13.
[0177]
30 TABLE 29 0 minutes* 20 minutes* 40 minutes* 1 11.56 187.30
5860.0 2 1.89 25.26 1598.0 3 2.09 12.52 671.3 4 1.34 20.21 1638.0 5
1.56 17.04 1009.0 6 1.11 9.44 691.1 7 27.21 315.30 7426.0 8 8.84
186.20 7177.0 9 1.52 9.76 295.6 10 1.14 5.18 184.1 11 0.72 4.10
169.7 *Light output in Relative Light Units
Example 17
Digestion of PhiX 174 HinF1 fragments
[0178] Polynucleoside encountered in nature is often double
stranded. The DNA fragments generated by digestion of Phi X 174 DNA
using endonuclease HinF I are double stranded DNA fragments of
various sizes. In order to test whether double stranded DNA could
be detected, the Phi X 174 was directed used as a test substrate or
digested with nucleases to produce nucleosides which could be
converted to nucleoside triphosphates as in previous Examples.
[0179] The following conditions were used to digest DNA fragments
from bacteriophage Phi X 174. These materials were placed in three
1.5 ml polypropylene tubes: 50 .mu.l of PhiX174 Hin FI fragments
(Promega G175A, Lot #773603); 40 .mu.l of 5 mM Mg SO.sub.4; 5 .mu.l
of Exo III buffer (10X) (Promega E577B, 4853216), and 5 .mu.l of
Nanopure water. Fifty microliters of TE buffer and 40 .mu.l of 5 mM
MgSO.sub.4; 5 .mu.l of ExoIII buffer (10X) and 5 .mu.l of Nanopure
water were added to one sample. Two of the samples containing Phi X
174 DNA were further treated with 2 .mu.l of Exo III (Promega
M181A, 5512708) and the tubes placed in a 37.degree. C. water bath
for 60 min. ExoIII was also deleted to the sample without DNA and
the sample incubated at 37.degree. C. for 60 min.
[0180] At this time, 800 .mu.l of nanopure water and 100 .mu.l of
S1 Nuclease Buffer (10X) (Promega M577A, Lot #6748605) were added
to all samples. Three microliters of S1 nuclease (Promega E576B,
Lot #789881) were then added to all samples. All samples were
incubated at 37.degree. C. for 30 min.
[0181] Two hundred microliters from each of the three tubes
containing DNA were diluted with 300 .mu.l of 1X TE Buffer and the
absorbance read at 260 nm using a Beckman DU 650 spectrophotometer.
The readings recorded were: tube one (no nuclease addition),
0.3073; tube two (treatment with Exo III), 0.5495; tube three
(treatment with Exo III and S1), 0.5190. The increased absorbance
values of the tubes treated with nuclease indicates that the
polymer was digested. These digests were subsequently used in other
studies (see Example 18).
Example 18
Detection of PhiX 174 Hin F1 fragments using Nucleases, PRPP
Synthetase, NDPK
[0182] This example demonstrates the detection of DNA by digestion
of the polymer to nucleoside monophosphates using nucleases,
transformation of the nucleoside monophosphates to nucleoside
triphosphates using PRPP Synthetase and PRPP along with
transformation of ADP to ATP using the nucleoside triphosphates
generated by the action of PRPP Synthetase, and detection of the
ATP using Luciferase. A sample of deoxynucleoside (Poly (dA)) was
prepared as described in example 17. Different amounts of
deoxynucleoside were used in the reactions as presented in Table
30.
[0183] The following additions were made to each reaction: 2 .mu.l
of PRPP, 2 .mu.l of PRPP Synthetase, and 20 .mu.l PRPP Synthetase
buffer. The reactions proceeded at 37.degree. C. for 28 minutes at
which time the reactions were transferred to 100 .mu.l LAR
containing 2 .mu.l of ADP and 2 .mu.l of NDPK. This second reaction
was allowed to proceed at room temperature for 20 min. The amount
of ATP produced was measured by the addition of 10 ng of Luciferase
followed by measuring light output with a luminometer. The data is
presented in Table 30.
[0184] These data show that this combination of enzymes allows
detection of DNA.
31TABLE 30 Reac- tion Nucleoside Amount in Rxn Light Units 1 dAMP
200 ng, 600 pmoles 1018 2 dAMP 20 ng, 60 pmoles 636 3 dAMP 2 ng, 6
pmoles 178 4 dAMP 200 pg, 600 fmoles 83 5 none 0 ng 69 6 Phi X174
only 100 ng 46 (= 300 pmoles dNMP; approx. 75 pmoles dAMP) 7 Phi
X174 + ExoIII 100 ng 472 8 Phi X174 + Exo + S1 100 ng 448 9 No DNA
+ Exo + S1 0 ng 55
Example 19
Detection of PhiX 174 Hin Fl fragments using reverse transcriptase
and NDPK
[0185] The following example demonstrates the detection of DNA
fragments having nucleoside overhangs on their ends using reverse
transcriptase. The reactions were assembled as demonstrated in
Table 31.
[0186] The components were: Buffer, 5X MMLV-RT Buffer, (Promega
Part #M531A, Lot #7090101); DNA, PhiX 174 Hin F1 Fragments (Promega
Part #Gl75A, Lot #7733602); NaPP.sub.i, 10 mM Sodium Pyrophosphate
(Promega Part #C113A, Lot# 6675705); ADP, 1 .mu.M ADP (Sigma
A-5285, Lot #56H7815); NDPK, nucleoside diphosphate kinase, (Sigma
N-0379, Lot #127F81802) 1U/.mu.l in 25 mM sodium citrate; MMLV-RT
(Promega M170A, Lot #6980019, 1U/.mu.l), incubated for 30 min at
370C, then 2 .mu.l of the reactions was added to 100 .parallel.l of
L/L (Promega FF2021, Enliten Luciferase/Luciferin Reagant). The
light production by the reactions was immediately measured with a
Turner TD-20e Luminometer. The data is presented in Table 31.
[0187] These data show that MMLV-RT can be used to
pyrophosphorylate DNA and that the resulting nucleosides can be
used to transform ADP to ATP and the ATP formed detected using
Luciferase. Other enzymes can be tested for their ability to
perform this reaction in a similar fashion.
32TABLE 31 Rx Buffer DNA NaPPi ADP NDPK water MMLV-RT Light 1 4
.mu.l 1 .mu.l of 100 ng/.mu.l 1 .mu.l 2 .mu.l 1 .mu.l 10 .mu.l 1
.mu.l 165. 2 4 .mu.l 1 .mu.l of 20 ng/.mu.l 1 .mu.l 2 .mu.l 1 .mu.l
10 .mu.l 1 .mu.l 155. 3 4 .mu.l 1 .mu.l of 4 ng/.mu.l 1 .mu.l 2
.mu.l 1 .mu.l 10 .mu.l 1 .mu.l 58.9 4 4 .mu.l 1 .mu.l of 800
pg/.mu.l 1 .mu.l 2 .mu.l 1 .mu.l 10 .mu.l 1 .mu.l 18.0 5 4 .mu.l 1
.mu.l of 160 ng/.mu.l 1 .mu.l 2 .mu.l 1 .mu.l 10 .mu.l 1 .mu.l 4.54
6 4 .mu.l 1 .mu.l of 32 ng/.mu.l 1 .mu.l 2 .mu.l 1 .mu.l 10 .mu.l 1
.mu.l 1.70 7 4 .mu.l -- 1 .mu.l 2 .mu.l 1 .mu.l 11 .mu.l 1 .mu.l
0.95 8 4 .mu.l 1 .mu.l of 1 .mu.l 2 .mu.l 1 .mu.l 11 .mu.l 0.62
Example 20
Limit of DNA Fragment Detection Using Reverse Transcriptase, NDPK,
and Luciferase
[0188] As shown in Example 18, DNA can be detected using luciferase
when the DNA is fragmented and pyrophosphorylated using a reverse
transcriptase to produce dNTPs and the terminal phosphate is
transferred from the dNTPs to ADPs to form ATP. This Example
demonstrates that the light units produced in reaction containing
very low levels of DNA are statistically significant compared to
values for the appropriate control reactions. As with Example 2,
the limit of detection is statistically determined using Student's
t-Test. The reactions presented in Table 32 were assembled in
duplicate. The components were: Buffer 5X MMLV-RT Buffer (Promega
M531A); DNA, PhiX 174 Hin F1 fragments diluted in TE (Promega
G175A); TE, Tris EDTA (Promega AA641); NaPPi, 40 mM Sodium
Pyrophosphate (Promega C113A); ADP, ADP 2 uM in Tris 10 mM pH 7.3
(Sigma A5285); NDPK, 1 unit/.mu.l in water (Sigma N0379); water,
nanopure water; MMLV-RT, MMLV reverse transcriptase 200 units/.mu.l
(Promega M170A).
[0189] All reagents except DNA and MMLV-RT were added to a 1.5 ml
polypropylene tube and mixed. Then duplicate 16.5 .mu.l aliquots
were transferred to new polypropylene tubes. One microliter of
MMLV-RT was added to each tube, followed by 2.5 .mu.l of DNA at
varying concentrations or 2.5 .mu.l of TE. The reactions were
incubated at 37.degree. C. for 10 min, then 2 .mu.l of the 20 .mu.l
reaction was added to 100 .mu.l of L/L reagant (which includes
luciferase, Promega F202A and F180A, mixed) in a luminometer tube.
The tubes were tapped to mix, and then light output levels were
immediately measured using a Turner TD-20e luminometer at 52.1%
sensitivity (this sensitivity is comparable to the Turner TD-20/20
readings). The data is presented in Table 33.
[0190] The light output for each DNA concentration (6 readings
each) was compared to the light output of the background control
(no DNA), and a p-value determined for each comparison. The results
of the analysis are presented in the following Table 34. As in
Experiment 2, the p-values were less than 0.05 for each sample
tested. Therefore, less than 10 pg of DNA can be reliably
detected.
33TABLE 32 Reaction Buffer DNA TE NaPPi ADP NDP water MMLV-RT 1 4
.mu.l -- 2.5 .mu.l 0.5 .mu.l 1 .mu.l 1 .mu.l 10 .mu.l 1 .mu.l 2 4
.mu.l 2.5 .mu.l of -- 0.5 .mu.l 1 .mu.l 1 .mu.l 10 .mu.l 1 .mu.l 40
pg/.mu.l 3 4 .mu.l 2.5 .mu.l of -- 0.5 .mu.l 1 .mu.l 1 .mu.l 10
.mu.l 1 .mu.l 100 pg/ .mu.l 4 4 .mu.l 2.5 .mu.l of -- 0.5 .mu.l 1
.mu.l 1 .mu.l 10 .mu.l 1 .mu.l 200 pg/.mu.l
[0191]
34TABLE 33 Reaction Amount of DNA Light Units 1 no DNA tube 1A
1.166 2 no DNA tube 1A 1.189 3 no DNA tube 1A 1.190 4 no DNA tube
1B 1.071 5 no DNA tube 1B 1.124 6 no DNA tube 1B 1.159 7 10 pg DNA
tube 2A 1.355 8 10 pg DNA tube 2A 1.498 9 10 pg DNA tube 2A 1.464
10 10 pg DNA tube 2B 1.485 11 10 pg DNA tube 2B 1.519 12 10 pg DNA
tube 2B 1.189 13 25 pg DNA tube 3A 2.360 14 25 pg DNA tube 3A 2.159
15 25 pg DNA tube 3A 2.344 16 25 pg DNA tube 3B 2.126 17 25 pg DNA
tube 3B 2.087 18 25 pg DNA tube 3B 2.148 19 50 pg DNA tube 4A 4.501
20 50 pg DNA tube 4A 4.920 21 50 pg DNA tube 4A 4.751 22 50 pg DNA
tube 4B 4.721 23 50 pg DNA tube 4B 4.809 24 50 pg DNA tube 4B
4.929
[0192]
35TABLE 34 Student's t-Test for DNA detection p-value l0 pg
0.002377925 25 pg 3.9211E-07 50 pg 4.2734E-09
Example 21
Detection of Blunt End DNA Fragments using Reverse Transcriptase
and NDPK
[0193] The following example demonstrates the detection of DNA
fragments having blunt ends using reverse transcriptase. A reaction
master mix was made containing: 80 .mu.l of 5X MMLV-RT Buffer
(Promega Part #MS31A, Lot #7090101); 10 .mu.l of 40 mM Sodium
Pyrophosphate (Promega Part #C113A, Lot #6675705); 10 .mu.l of 1
.mu.M ADP (Sigma A-5285, Lot #56H7815); 20 .mu.l of NDPK (Sigma
N-0379, Lot #127F81802 1U/.mu.l); and 210 .mu.l of deionized
water.
[0194] DNA samples consisted of ladders of DNA fragments in
multiples of 25bp (Promega G451, Lot #84791) and 50 bp (Promega
G452, Lot #84796) in 1X TE buffer. These materials were diluted
into 1X TE buffer to produce a series of solutions at different DNA
concentrations. The reactions were assembled as demonstrated in
Table 35. The composition of these components was: MM, Master Mix
(described above), and 200U/.mu.l MMLV-RT (Promega Part
#M531A).
[0195] These reactions were incubated for 30 min at 37.degree. C.
After incubation, 2 .mu.l of the solution was added to 100 .mu.l of
L/L reagent and the light production of the reaction was measured
using a Turner TD-20e Luminometer. The data is presented in Table
35. These data demonstrate that sensitive DNA detection of blunt
ended fragments can be achieved through pyrophosphorolysis of the
DNA followed by conversion of ADP to ATP.
36TABLE 35 Rx MM DNA MMLV-RT Light 1 18 .mu.l 100 ng 25 bp ladder 1
.mu.l 142.5 2 18 .mu.l 20 ng 25 bp ladder 1 .mu.l 66.28 3 18 .mu.l
4 ng 25 bp ladder 1 .mu.l 20.33 4 18 .mu.l 800 pg 25 bp ladder 1
.mu.l 5.216 5 18 .mu.l 160 pg 25 bp ladder 1 .mu.l 1.606 6 18 .mu.l
32 pg 25 bp ladder 1 .mu.l 0.902 7 18 .mu.l -- 1 .mu.l 0.717 8 18
.mu.l 100 ng 25 bp ladder -- 0.571 9 18 .mu.l 100 ng 50 bp ladder 1
.mu.l 149.2 10 18 .mu.l 20 ng 50 bp ladder 1 .mu.l 84.43 11 18
.mu.l 4 ng 50 bp ladder 1 .mu.l 27.56 12 18 .mu.l 800 pg 50 bp
ladder 1 .mu.l 6.694 13 18 .mu.l 160 pg 50 bp ladder 1 .mu.l 2.829
14 18 .mu.l 32 pg 50 bp ladder 1 .mu.l 1.323 15 18 .mu.l -- 1 .mu.l
0.951 16 18 .mu.l 100 ng 50 bp ladder -- 0.751
Example 22
Detection of PolyA RNA using Poly A Polymerase
[0196] This example demonstrates the detection Poly A mRNA by the
pyrophosphorylation of the poly A segment. The reactions were
assembled as demonstrated in Table 36. The compositions of the
reaction materials was: 10X Buffer-0.5M Tris-HCl, pH 7.5, 0.1 M
MgCl.sub.2, 0.5 M NaCl; Globin mRNA GibcoBRL cat#18103-028
(dissolved in H.sub.2O); NaPP.sub.I, 20 mM sodium pyrophosphate
(Promega C113A, in deionized water); Poly A Polymerase, (Sigma
P4058, 1U/.mu.l). These reactions were incubated at 37.degree. C.
for 30 min, then 2 .mu.l of the reaction was added to 100 .mu.l of
L/L Reagent and the light output of the reaction immediately
measured using a Turner TD-20e Luminometer. The data is presented
in Table 37. These data demonstrate that Poly A Polymerase is
capable of pyrophosphorylating the RNA and that the resulting
nucleoside triphosphates can be detected using luciferase, even if
only very low levels of RNA are present.
37TABLE 36 Reaction 10X Globin NaPPi Poly A Water 1 2 .mu.l 1 .mu.l
of 50 ng/.mu.l 1 .mu.l 1 .mu.l 15 .mu.l 2 2 .mu.l 1 .mu.l of 10
ng/.mu.l 1 .mu.l 1 .mu.l 15 .mu.l 3 2 .mu.l 1 .mu.l of 2 ng/.mu.l 1
.mu.l 1 .mu.l 15 .mu.l 4 2 .mu.l 1 .mu.l of 400 pg/.mu.l 1 .mu.l 1
.mu.l 15 .mu.l 5 2 .mu.l 1 .mu.l of 80 pg/.mu.l 1 .mu.l 1 .mu.l 15
.mu.l 6 2 .mu.l 1 .mu.l of 16 pg/.mu.l 1 .mu.l 1 .mu.l 15 .mu.l 7 2
.mu.l -- 1 .mu.l 1 .mu.l 15 .mu.l
[0197]
38TABLE 37 Reaction Light RNA Present in L/L 1 772.2 5000 pg 2
172.3 1000 pg 3 33.53 200 pg 4 7.727 40 pg 5 1.85 8 pg 6 0.743
.sup. 1. 6pg 7 0.594 --
Example 23
Detection of Poly A RNA using Reverse Transcriptase and NDPK
[0198] This example demonstrates another method for the detection
of mRNA, particularly polyA mRNA. In this method, a DNA segment is
hybridized to the mRNA and the probe is pyrophosphorylated using a
reverse transcriptase and pyrophosphate. As the pyrophosphorylation
occurs, the deoxynucleoside triphosphates are used to convert ADP
to ATP using the enzyme NDPK. The ATP of the final solution is then
measured using luciferase.
[0199] The reactions were assembled as presented in Table 38. The
reaction components were: Buffer, 5X MMLV-RT Buffer (Promega Part
#M531A, Lot #7090101); mRNA, Globin mRNA (GibcoBRL cat# 18103-028
dissolved in H.sub.2O); Poly (dT), 0.2 .mu.M oligo dT(50), NaPPi,
2OmM Sodium Pyrophosphate, (Promega C113A in deionized water); ADP,
10 mM ADP ( Sigma A-5285 Lot #56H7815); NDKP, nucleoside
diphosphate kinase, 1U/.mu.l, (Sigma N-0379 Lot #127F81802);
MMLV-RT, (Promega Part #M531A, Lot #7090101) 200U/.mu.l; and
200U/.mu.l Superscript II GibcoBRL cat# 18064-014).
[0200] These were incubated at 37.degree. C. for 30 min and 2 .mu.l
of the reactions was added to 100 .mu.l of L/L reagent. The light
production of the reactions was immediately measured using a Turner
TD-20e Luminometer. The data is presented in Table 39.
39TABLE 38 Rx Suffer mRNA Poly (dT) NaPPi APP NDPK MMLV-RT
Superscript water 1 4 .mu.l 1 .mu.l of 50 ng/.mu.l 1 .mu.l 1 .mu.l
2 .mu.l 1 .mu.l 1 .mu.l -- 9 .mu.l 2 4 .mu.l 1 .mu.l of 10 ng/.mu.l
1 .mu.l 1 .mu.l 2 .mu.l 1 .mu.l 1 .mu.l -- 9 .mu.l 3 4 .mu.l 1
.mu.l of 2 ng/.mu.l 1 .mu.l 1 .mu.l 2 .mu.l 1 .mu.l 1 .mu.l -- 9
.mu.l 4 4 .mu.l 1 .mu.l of 400 pg/.mu.l 1 .mu.l 1 .mu.l 2 .mu.l 1
.mu.l 1 .mu.l -- 9 .mu.l 5 4 .mu.l 1 .mu.l of 80 pg/.mu.l 1 .mu.l 1
.mu.l 2 .mu.l 1 .mu.l 1 .mu.l -- 9 .mu.l 6 4 .mu.l -- 1 .mu.l 1
.mu.l 2 .mu.l 1 .mu.l 1 .mu.l -- 9 .mu.l 7 4 .mu.l 1 .mu.l of 50
ng/.mu.l 1 .mu.l 1 .mu.l 2 .mu.l 1 .mu.l -- 1 .mu.l 9 .mu.l 8 4
.mu.l 1 .mu.l of 10 ng/.mu.l 1 .mu.l 1 .mu.l 2 .mu.l 1 .mu.l -- 1
.mu.l 9 .mu.l 9 4 .mu.l 1 .mu.l of 2 ng/.mu.l 1 .mu.l 1 .mu.l 2
.mu.l 1 .mu.l -- 1 .mu.l 9 .mu.l 10 4 .mu.l 1 .mu.l of 400 pg/.mu.l
1 .mu.l 1 .mu.l 2 .mu.l 1 .mu.l -- 1 .mu.l 9 .mu.l 11 4 .mu.l 1
.mu.l of 80 pg/.mu.l 1 .mu.l 1 .mu.l 2 .mu.l 1 .mu.l -- 1 .mu.l 9
.mu.l 12 4 .mu.l -- 1 .mu.l 1 .mu.l 2 .mu.l 1 .mu.l -- 1 .mu.l 9
.mu.l
[0201]
40TABLE 39 Rx mRNA Light Units 1 5 ng 647.2 2 1 ng 425.4 3 0.2 ng
113.9 4 40 pg 43.56 5 8 pg 23.66 6 -- 21.52 7 5 ng 648.5 8 1 ng
500.4 9 0.2 ng 144.2 10 40 pg 45.85 11 8 pg 28.17 12 -- 19.71
Example 24
Detecton of RNA using nucleases, PRPP synthetase
[0202] this example demonstrates the detection of RNA by digestion
of RNA by nucleases, transformation of the AMP produced to ATP by
PRPP Synthetase, and detection of the ATP produced using
Luciferase. Three reactions were assembled. These were: Digest 1
(250 ng Globin mRNA and S1 in a 10 .mu.l reaction); Digest 2 (same
as Digest 1, however no S1 nuclease was added); Digest 3 (same as
Digest 1, but without Globin mRNA). After these digests had
incubated 30 min at 37.degree. C., they were used to compose the
reactions presented in Table 40. [For the concentrations of these
solutions, see descriptions under reaction composition table in
Example 16.]
[0203] The reactions were incubated for 30 min at 37.degree. C.,
100 .mu.l of LAR-CoA and long of Luciferase were added to each
tube, and the light output of the reactions were measured using a
Turner TD-20e Luminometer. The data is presented in Table 40. These
data show that this combination of enzymes can be used to detect
relatively low levels of RNA.
41TABLE 40 PRPP Digest Polymer Synthetase PRPP Light Reaction AMP #
and .mu.l Added Buffer Synt Units 1 2 .mu.l of -- -- 20 .mu.l 2
.mu.l 3869 2.9 e-4 M (200 ng) 2 2 .mu.l of -- -- 20 .mu.l 2 .mu.l
1287 2.9 e-5 M (20 ng) 3 2 .mu.l of -- -- 20 .mu.l 2 .mu.l 192.9
2.9 e-6 M (2 ng) 4 2 .mu.l of -- -- 20 .mu.l 2 .mu.l 118.6 2.9 e-7
M (200 pg) 5 -- 1, 0.2 .mu.l 5 ng 20 .mu.l 2 .mu.l 48.4 6 -- 1,
0.02 .mu.l 0.5 ng 20 .mu.l 2 .mu.l 14.8 7 -- 2, 0.2 .mu.l 5 ng 20
.mu.l 2 .mu.l 10.9 8 -- 2, 0.02 .mu.l 0.5 ng 20 .mu.l 2 .mu.l 10.6
9 -- 3, 0.2 .mu.l -- 20 .mu.l 2 .mu.l 10.3 10 -- 3, 0.02 .mu.l --
20 .mu.l 2 .mu.l 11
Example 25
Improved Detection of Cells through the addition of Materials that
Allow ATP to be Produced from the Enzymes in the Cells.
[0204] One common way to detect the presence of cells is to assay
the ATP content of materials which may contain cells. However, such
detection methods are limited by the very small concentration of
ATP that is present in samples which may contain very few cells.
Several types of enzymatic activities are required in every living
cell. These activities are involved in the transformation of
nucleosides into nucleoside triphosphates for use in cellular
metabolism. In particular, the activities known as adenylate kinase
and nucleoside diphosphokinase are widely found in cells. Since
these enzymes are expected to exist in all cell lysates, addition
of AMP and dCTP should result in the formation of ATP in cell
lysates through the reactions:
AMP+dCTP+adenylate kinase .fwdarw.ADP+dCDP
ADP+dCTP+NDPK.fwdarw.ATP+dCDP
[0205] If, however, the enzymes which remove ATP from such extracts
are active enough to remove the ATP as it is formed, no build-up of
ATP will result.
[0206] This example demonstrates that ATP can be detected in cell
lysate samples to which AMP and dCTP are added. The nucleoside
transformations such as those presented above probably increase ATP
concentration. Therefore, lower amounts of cells and cellular
materials can be detected by taking advantage of the transformation
activity of these enzymes to produce ATP from AMP thereby detecting
ATP directly.
[0207] A sample of E. coli JM109 was grown in Luria broth for 2
hours. The cells were harvested by centrifugation at 7240 g for 10
min and then resuspended in 1.times.TBS. The cells were spun down
again the same way and resuspended in 1.times.TBS. A sample of the
resuspended cells was removed, diluted into sterile Luria broth and
plated onto Luria Agar plates to determine the number of cells per
microliter of resuspended cell culture. The cell culture was then
lysed by sonication and the lysate was used in the reactions
presented in Table 41. After an incubation of 40 min at room
temperature, long of luciferase was added to the reactions and the
light output of the reactions was immediately measured using a
Turner TD 20-e luminometer. The data is presented in Table 41.
42TABLE 41 Reaction Lysate AMP (0.1 mM) dCTP (1 mM) Buffer* Light 1
10 .mu.l** -- 2 .mu.l 100 .mu.l 142 2 10 .mu.l** 2 .mu.l -- 100
.mu.l 69 3 10 .mu.l** -- -- 100 .mu.l 67 4 -- 2 .mu.l 2 .mu.l 100
.mu.l 4 5 10 .mu.l** 2 .mu.l 2 .mu.l 100 .mu.l 175 6 1 .mu.l** 2
.mu.l 2 .mu.l 100 .mu.l 18.85 7 0.1 .mu.l 2 .mu.l 2 .mu.l 100 .mu.l
4.6 8 0.01 .mu.l 2 .mu.l 2 .mu.l 100 .mu.l 3.5 *buffer = 100 .mu.l
LAR **Lysate made from resuspended cells at a concentration of 1.8
.times. 10.sup.5 cells/.mu.l. Dilutions of this lysate were made in
1 .times. TBS and 10 .mu.l added to 5, 6, 7, and 8, the amount of
initial lysate equivalent to that added is above.
[0208] This data shows that addition of dCTP (reaction 1) allows
more light to be measured from a cell sample than can be measured
if no addition was made to the lysate (reaction 3) or if AMP alone
was added to the lysate (reaction 2). However, even more light can
be found if both AMP and dCTP is added to the lysate (reaction 5).
Dilution of the lysate results in a reduction of the light produced
by reactions given AMP and dCTP (reactions 6 and 7). However, the
amount of light found is still above the amount expected to present
by simple dilution of the lysate. These data then, show that
improved cell detection can be demonstrated if additions can be
made to the lysate which should result in an increased ATP level in
the sample.
Example 26
Optimization of the Concentrations of Nucleotides Added to form
Additional ATP in Cell Lysates
[0209] Example 25 demonstrates that additional ATP can be detected
in cell lysates if materials are added to the lysate. This example
demonstrates that the concentration of the material added can be
adjusted to produce a substantially higher level of ATP than is
originally present in the lysates. A cell lysate produced from a
known amount of cells was produced as in Example 23. This new
lysate was used in the reactions presented in Table 42. The
reactions were incubated for 40 min at room temperature. After
incubation, long of luciferase was added to the reactions and the
light output of the reactions was immediately read using a Turner
TD-20e luminometer. The data is presented in Table 42.
[0210] These data indicate that the concentration of both
nucleotides can be co-optimized to obtain light values far superior
to those seen in either the lysate without additions or with
non-optimal additions.
43TABLE 42 Stock AMP Stock dCTP Reaction conc. conc. Lysate LAR-CoA
Light 1 -- 100 mM 10 .mu.l* 100 .mu.l 144.8 2 80 mM -- 10 .mu.l*
100 .mu.l 1.515 3 -- -- 10 .mu.l* 100 .mu.l 38.4 4 80 mM 100 mM --
100 .mu.l 1.743 5 0.1 mM 1 mM 10 .mu.l* 100 .mu.l 139.4 6 1 mM 1 mM
10 .mu.l* 100 .mu.l 247.3 7 10 mM 1 mM 10 .mu.l* 100 .mu.l 569.2 8
80 mM 1 mM 10 .mu.l* 100 .mu.l 336.7 9 80 mM 1 mM 10 .mu.l* 100
.mu.l 273.9 10 1 mM 1 mM 10 .mu.l* 100 .mu.l 283.3 11 1 mM 10 mM 10
.mu.l* 100 .mu.l 239.6 12 1 mM 100 mM 10 .mu.l* 100 .mu.l 358.8 13
10 mM 1 mM 10 .mu.l* 100 .mu.l 666.7 14 10 mM 10 mM 10 .mu.l* 100
.mu.l 1236 15 10 mM 100 mM 10 .mu.l* 100 .mu.l 2320 16 80 mM 1 mM
10 .mu.l* 100 .mu.l 339.8 17 80 mM 10 mM 10 .mu.l* 100 .mu.l 761.9
18 80 mM 100 mM 10 .mu.l* 100 .mu.l 1970 *lysate was made from
resuspended cells at a concentration of 3 .times. 10.sup.4 cells/ul
**2 .mu.l of stock used
Example 27
Time Course of ATP Increase in Lysates Following Addition of Enzyme
Substrates
[0211] The examples above indicate cell detection sensitivity
increased by addition of dCTP and/or AMP followed by an incubation
period prior to ATP detection using luciferase. This example
demonstrates that detection reaction may be teporally optimized as
well.
[0212] The cell lysate was made as in Example 25 and was frozen.
This lysate was thawed and used to compose the reactions presented
in Table 43. Samples were removed from reaction 5 at 1, 5, 15, 30,
60, 90, 120, 150, and 180 min and at 5 min for the other reactions.
These samples were added to 10 ng of luciferase and the light
output of the reaction was measured immediately using a Turner
TD-20e luminometer. The results from the samples taken at Smin are
presented in Table 43. The results from the samples of reaction 5
taken over time are presented in Table 44.
[0213] Note that the light output for the reaction rises
dramatically over time and reaches final values far above those
reported in the previous example. These data indicate that the most
sensitive detection of cells will require optimization of the
reaction time used for detection in addition to optimization of the
added materials.
44TABLE 43 Reaction Lysate AMP dCTP Light 1 10 .mu.l -- 100 mM 329
2 10 .mu.l 10 mM -- 80.4 3 10 .mu.l -- -- 241.8 4 -- 10 mM 100 mM
1.129 5 10 .mu.l 10 mM 100 mM 347.5
[0214]
45 TABLE 44 Time Light 1 136.5 5 347.5 15 1325 30 6379 60 21078 90
33204 120 41470 150 43844 180 36579
Example 28
Determination of the effect of increasing the number of DNA ends on
detection of DNA through pyrophosphorylation.
[0215] Reverse transcriptases and DNA polymerases usually bind to
DNA segments which can be used as substrates in polymerization
reactions. Plasmid DNAs have no DNA ends since they are a
covalently closed circular molecule. In general, such a molecule
would not be expected to undergo pyrophosphorylation unless the DNA
is first modified to transform it into a substrate for the reverse
transcriptase or polymerase. In this example, an experiment is
described that confirms that plasmid DNA is not as good a substrate
for pyrophosphorylation as digested fragments. In addition, using
an enzyme to cleave the DNA which generates more new DNA ends than
one that generates fewer ends may improve the detection of the
DNA.
[0216] The reactions were assembled as presented in Table 45. The
components were: Plasmid, pGEM 3ZF(+) (1 mg/ml, Promega
corporation, Part #P227A); Buffer, 10 X Buffer B (Promega
Corporation, Part #R002A); Sau 3AI, Endonuclease Sau 3AI, (Promega
Corporation, 8U/.mu.l, Part #R619E); Bam H1, Endonuclease Bam Hi
(Promega Corporation, 10U/.mu.l, Part #R602A). The solutions were
incubated at 37.degree. C. for 1 hr, then heated at 70.degree. C.
for 10 min, and allowed to cool to room temperature. The solutions
were then added to the reactions presented in Table 46. The
reactions were incubated at 37.degree. C. for 20 min. After
incubation, 2 .mu.l of the reaction solution was added to 100 .mu.l
of L/L reagent and the light output of the reactions was
immediately measured using a Turner TD-20e luminometer. The data is
presented in Table 46.
[0217] These data again demonstrate that detection of DNA by
pyrophosphorylation is possible. In addition, these data
demonstrate that digestion of plasmid DNA is needed prior to
treatment using reverse transcriptase. Bam Hi produces only one DNA
fragment from the plasmid while Sau 3A produces over 10 fragments
from this plasmid. These data demonstrate that light production
increases with increasing fragment end number.
46TABLE 45 Solution Plasmid Buffer Water Sau 3A Bam H1 1 1 .mu.l 5
.mu.l 44 .mu.l -- -- 2 -- 5 .mu.l 45 .mu.l -- -- 3 -- 5 .mu.l 44
.mu.l -- 1 .mu.l 4 -- 5 .mu.l 44 .mu.l 1 .mu.l -- 5 1 .mu.l 5 .mu.l
43 .mu.l -- 1 .mu.l 6 1 .mu.l 5 .mu.l 43 .mu.l 1 .mu.l --
[0218]
47TABLE 46 Reaction MM MMLV-RT Solution Light Rx1- 18 .mu.l -- 1
.mu.l #1 0.87 Rx1+ 18 .mu.l 1 .mu.l 1 .mu.l #1 0.787 Rx2- 18 .mu.l
-- 1 .mu.l #2 0.906 Rx2+ 18 .mu.l 1 .mu.l 1 .mu.l #2 0.75 Rx3- 18
.mu.l -- 1 .mu.l #3 0.932 Rx3+ 18 .mu.l 1 .mu.l 1 .mu.l #3 0.714
Rx4- 18 .mu.l -- 1 .mu.l #4 0.856 Rx4+ 18 .mu.l 1 .mu.l 1 .mu.l #4
0.713 Rx5- 18 .mu.l -- 1 .mu.l #5 0.837 Rx5+ 18 .mu.l 1 .mu.l 1
.mu.l #5 2.909 Rx6- 18 .mu.l -- 1 .mu.l #6 0.811 Rx6+ 18 .mu.l 1
.mu.l 1 .mu.l #6 8.757
Example 29
Demonstration of DNA Detection Using Pyrophosphorylation Catalyzed
by a Thermostable DNA Polymerase.
[0219] Both reverse transcriptases and DNA polymerases catalyze the
addition of nucleotides to a DNA strand. As shown in the earlier
examples, reverse transcriptases can be used to catalyze the
pyrophosphorylation of DNA thereby allowing its detection using
coupled enzymatic reactions. In this example, we demonstrate that
DNA polymerases also can be used to catalyze this reaction and that
the DNA polymerase from Thermus aquaticus (Taq) in fact produces
more light from a set amount of input DNA than does the reverse
transcriptase.
[0220] A master mix (MM) was made which comprised: 10 X buffer
(Promega Part #M19OG, Lot #7675526), 20 .mu.l, 25mM MgCl.sub.2,
40.mu.l, 40 mM Sodium Pyrophosphate, 5 .mu.l; Taq DNA Polymerase
(Promega Part #M166B, Lot #7474623) [storage buffer b], 5U/.mu.l,
10 .mu.l; water, 100 .mu.l. This solution was mixed by vortex and
then used to compose the following reactions: Reactions 1-3 (17.5
.mu.l of master mix, 2.5 .mu.l of 1 X TE); Reactions 4-6 (17.5 gl
of master mix, 1 .mu.l of 100 pg DNA/.mu.L [PhiX 174 HinF1
Fragments, Promega G175A diluted to the concentration listed using
1 X TE buffer], 1.5 .mu.l of 1 X TE); and Reactions 7-9 (17.5 .mu.l
of master mix, 2.5 .mu.l of 100 pg DNA/.mu.l). The solutions were
mixed and 30 .mu.l of mineral oil was used to cover the aqueous
solution. The solutions were incubated at 70.degree. C. for 30 min.
Fifteen microliters were removed to which 1 .mu.l of 1U/gl NDPK and
1.5 .mu.l of 1 uM ADP were added. After an additional 15 min at
room temperature, 2.3 .mu.l of each sample was added to 100 .mu.l
of L/L reagent. The light output of the reactions were immediately
measured using a luminometer. The data is presented in Table
47.
[0221] These results demonstrate the pyrophosphorylation reaction
can be catalyzed by DNA polymerases and that low amounts of DNA may
be detected. The values obtained from reactions with 10 and 25 pg
of DNA are statistically different from the no DNA addition
values.
48TABLE 47 Reactions DNA* Light Units Measured Mean Sd. p-value**
1-3 0 pg 0.915 0.653 0.837 0.802 0.135 4-6 10 pg 5.718 7.718 7.397
6.958 1.089 <.0094 7-9 25 pg 11.8 11.18 14.79 12.59 1.93
<.0086 *amount of DNA present in the luciferase assay tube **p
value for comparison of the results from no DNA addition to this
Mentioned in earlier examples, any value <0.05 is usually
considered a significant difference
Example 30
Additional DNA detecton experiments
[0222] This example is a direct comparison of the detection of DNA
by a reverse transcriptase (MMLV-RT) a thermostable DNA polymerase
(Taq Polymerase) and a non-thermostable DNA Polymerase (T4 DNA
Polymerase). Also shown is another example of how the particular
structure of the DNA fragments utilized in the reaction must be
matched to the properties of the DNA modifying enzyme. The enzymes
generally fail to produce a signal from supercoiled plasmid DNA
since all these enzymes require a DNA end to start their reactions.
MMLV-RT and Taq DNA Polymerase utilize DNA species having a 5'
overhang but cannot use a DNA having a 3' overhang as a substrate.
In contrast, T4 DNA Polymerase utilizes DNA substrates with both 5'
overhangs and 3' overhangs. This ability may be due to its 3'
exonuclease activity. In addition, this Example shows that
reactions using T4 DNA polymerase produce more light than from
equivalent reactions with either of the other two enzymes.
[0223] The reactions were assembled as presented in Table 48. The
solutions were incubated at 37.degree. C. for 1 hr then at
70.degree. C. for 10 min. At this point, 1 .mu.l of each reaction
was diluted to 20 .mu.l with water to give a concentration of 1000
pg DNA/.mu.l. Solution MM was made as follows: 40 .mu.l 5X MMLV-RT
Reaction Buffer (Promega Part M531A); 5 .mu.l 40 mM Sodium
pyrophosphate; 20 .mu.l 1 .mu.M ADP; 5 .mu.l 1U/.mu.l NDPK; and 180
.mu.l water. The reactions were mixed and 18 .mu.l were transferred
into 8 tubes. One microliter of reaction 1 above and 1 .mu.l of
MMLV-RT (200U/.mu.l) were added to tubes 1 and 2; 1 .mu.l of
reaction 2 above and 1 .mu.l of MMLV-RT were added to tubes 3 and
4; 1 .mu.l of reaction 3 above and 1 .mu.l of MMLV-RT were added to
tubes 5 and 6; and 1 .mu.l of reaction 3 was added to tubes 7 and
8. The tubes were incubated 20 min at 37.degree. C. and then 2
.mu.l of the solutions were added to 100 .mu.l of L/L and the light
output of the resulting mixture was immediately measured using a
Turner TD-20e luminometer. The data is presented in Table 49.
[0224] A second MM Mix was made for use with T4 DNA Polymerase as
follows: 20 .mu.l 10X Buffer C (Promega Part #R003A); 5 .mu.l 40 mM
sodium pyrophosphate; 20 .mu.l 1 .mu.M ADP; 5 .mu.l 1U/.mu.l NDPK;
and 130 .mu.l water. This solution was mixed by vortex and then
used to compose the 8 reaction mixtures described in the paragraph
above. Incubations were performed at 37.degree. C. for 20 min and
then 2Al of the reaction mixtures were added to 100 .mu.l of L/L
with luciferase. The light output was immediately measured and the
data presented in Table 50 was obtained.
[0225] These data show that both of these enzymes can
pyrophosphorylate DNA having 5' overhangs. However the T4 DNA
polymerase can also pyrophosphorylate DNA having 3' overhangs
(produced by Sph I digestion of DNA) while the reverse
transcriptase cannot utilize this form of DNA.
[0226] A final MM Mix was made containing: 20 .mu.l 10 X Taq Buffer
(Promega Part #M190G); 40 .mu.l 25 mM MgCl.sub.2; 5 .mu.l 40 mM
sodium pyrophosphate, and 105 .mu.l of water. This new MM Mix was
used to produce 8 mixtures. Mixture 1 and 2 contained 17 .mu.l of
this new MM Mix, 1 .mu.l of diluted DNA reaction 1, and 2 .mu.l of
Taq DNA Polymerase (Promega Part #M166B); mixtures 3 and 4
contained 17 .mu.l MM, 1 .mu.l from reaction 2, and 2 .mu.l Taq;
mixtures 5 and 6 contained 17 .mu.l MM, 1 .mu.l from reaction 3,
and 2 .mu.l Taq; and mixture 7 and 8 contained 17 .mu.l MM and 1
.mu.l from reaction 3. The mixtures were mixed by vortex action, 30
.mu.l of mineral oil was placed over the tubes and they were
incubated at 70.degree. C. for 20 min. Fifteen microliters of each
tube was removed and 1 .mu.l of 1U/.mu.l of NDPK and 1.5 .mu.l of 1
.mu.M ADP was added to each tube. The tubes were incubated at room
temperature for 15 min, 2.3 .mu.l were removed from each reaction
and added to .sup.100 .mu.l of L/L reagent containing luciferase
and the light output of the reactions measured immediately using a
luminometer. The data is presented in Table 51.
[0227] These data show that Taq DNA Polymerase can utilize DNA
having a 5' overhang. However, very little light output results
when the DNA has a 3' overhang. Thus, Taq polymerase appears to be
similar to MMLV-RT in that it will catalyze the pyrophosphorylation
of a DNA if it has a 5' overhang but not if it has a 3' overhang.
T4 DNA Polymerase will catalyze pyrophosphorylation with either
form of DNA overhang. In addition, by comparing all the data it is
clear that much more light is produced if the reactions are
performed using T4 polymerase than using either of the other
enzymes.
49 TABLE 48 Reaction Buffer DNA Bam HI Sph I Water 1 5 .mu.l 1
.mu.l -- -- 44 .mu.l 2 5 .mu.l 1 .mu.l 2 .mu.l -- 42 .mu.l 3 5
.mu.l 1 .mu.l -- 2 .mu.l 42 .mu.l The solutions used were: Buffer,
Promega Buffer B (Part #R002A); DNA, pGEM 3ZF+ (1 mg/ml), Promega
Part #P227A; Bam HI, Promega Bam HI, Part #R602A, Sph I, Promega
Sph I, Part #R626A.
[0228]
50TABLE 49 Reaction Light Units DNA Condition MMLV-RT Added 1 1.472
supercoiled + 2 1.445 supercoiled + 3 5.156 linear, 5' overhang + 4
4.699 linear, 5' overhang + 5 1.504 linear, 3' overhang + 6 1.494
linear, 3' overhang + 7 1.412 linear, 3' overhang - 8 1.378 linear,
3' overhang -
[0229]
51TABLE 50 Reaction Light Units DNA condition T4 DNAP added 1 2.214
supercoiled + 2 1.946 supercoiled + 3 44.46 linear, 5' overhang + 4
32.53 linear, 5' overhang + 5 37.29 linear, 3' overhang + 6 32.11
linear, 3' overhang + 7 1.446 linear, 3' overhang - 8 1.361 linear,
3' overhang -
[0230]
52TABLE 51 Reaction Light Units DNA Condition Taq DNAP added 1
1.125 supercoiled + 2 1.174 supercoiled + 3 8.110 linear, 5'
overhang + 4 9.687 linear, 5' overhang + 5 1.623 linear, 3'
overhang + 6 1.515 linear, 3' overhang + 7 1.004 linear, 3'
overhang - 8 1.046 linear, 3' overhang -
Example 31
Detection of Genomic DNA
[0231] In this example, high molecular weight DNA is measured using
pyrophosphorylation of the DNA, transfer of the terminal phosphate
from the dNTPs to ADP to form ATP and measurement of the ATP using
Luciferase. High molecular weight DNA can be detected at a higher
sensitivity if it is first cleaved using endonucleases.
[0232] The reactions were assembled as in Table 52. The materials
used in the reactions were: Buffer; 10X Multicore Buffer (Promega
Part #R999A); Yeast DNA, S. cerevisiae DNA (380 .mu.g/ml) (Promega
Part #G301A); Mouse DNA (300 .mu.g/ml) (Promega Part #G309A); Eco
RI, Endonuclease Eco RI, 12u/.mu.l, (Promega Part #R601A). The
reactions were heated at 37.degree. C. for 60 min then at
70.degree. C. for 10 min. At that point, 1 .mu.l of each of these
reactions were diluted to 20 .mu.l by the addition of 19 .mu.l of
water.
[0233] A solution (MM) was made which contained: 24 .mu.l of 10X
Buffer C (Promega Part #R003A); 6 .mu.l of 40 nmM Sodium
Pyrophosphate, 24 .mu.l of 1 .mu.M ADP, 6 .mu.l of 1u/.mu.l NDPK,
and 156 .mu.l of water. The reactions presented in Table 53 were
assembled using this mix.
[0234] The added DNAs in the reactions 1A through 10A above refer
to the diluted materials from reactions 1-4 described in Table 53.
The T4 DNA Pol is T4 DNA Polymerase (Promega Part #M421F). These
reactions were incubated at 37.degree. C. for 20 min, then 2 .mu.l
of the reactions was added to 100 .mu.l of L/L reagent. The light
produced by the reactions was immediately measured using a Turner
TD-20e luminometer. The data is presented in Table 54. Note that
the reactions demonstrate that the system can detect genomic DNA.
In addition, Eco RI treatment prior to pyrophosphorylation results
in higher light values than are seen without Eco RI
pretreatment.
53TABLE 52 Reaction Buffer Yeast DNA Mouse DNA Water Eco RI 1 5
.mu.l 2.6 .mu.l -- 42.4 .mu.l -- 2 5 .mu.l -- 3.3 .mu.l 41.7 .mu.l
-- 3 5 .mu.l 2.6 .mu.l -- 40.4 .mu.l 2 .mu.l 4 5 .mu.l -- 3.3 .mu.l
39.7 .mu.l 2 .mu.l
[0235]
54 TABLE 53 Reaction MM DNA Added T4 DNA Pol lA and 2A 18 .mu.l 1
.mu.l#1 1 .mu.l 3A and 4A 18 .mu.l 1 .mu.l#2 1 .mu.l 5A and 6A 18
.mu.l 1 .mu.l#3 1 .mu.l 7A and 8A 18 .mu.l 1 .mu.l#4 1 .mu.l 9A 18
.mu.l 1 .mu.l#3 -- 10A 18 .mu.l 1 .mu.l#4 --
[0236]
55 TABLE 54 Reaction Light Eco R1 T4 DNA Pol Sampled Units
Treatment Treatment lA 2.424 - + 2A 1.94 - + 3A 1.989 - + 4A 1.665
- + 5A 12.27 + + 6A 11.9 + + 7A 23.23 + + 8A 20.26 + + 9A 0.651 + -
10A 0.724 + -
Example 32
Optimization of ADP Concentrations Used in DNA Detection by
Pyrophosphorylation.
[0237] In this example, we examine the effect of varying the ADP
concentration on the detection of DNA by the T4 DNA Polymerase
catalyzed pyrophosphorylation of the DNA and transfer of the
terminal phosphates of the dNTPs to ADP using NDPK. Increasing the
concentration of ADP increases the background seen without ATP
addition. Increasing the ADP concentration also can increase the
signal seen upon DNA pyrophosphorylation. An optimal amount of
added ADP can be determined by selecting the concentration of ADP
which results in the best fold increase in signal over
background.
[0238] ADP ( Sigma potassium ADP, A-5285, Lot #56H7815) was
dissolved in distilled water to various concentrations ranging from
0.2 to 20 .mu.M. The Bam HI digest of PGEM 3ZF+ described in
example 26 above was used to form a reaction solution (solution MM)
composed of: 40 .mu.l 10X Buffer C (Promega Part #R003A), 10 .mu.l
of 40 mM sodium pyrophosphate; 10 .mu.l of 1u/.mu.l of NDPK; 1
.mu.l of 20 ng/.mu.l Bam HI digested pGEM 3ZF+; and 299 .mu.l of
water. These solutions were used to compose the reactions presented
in Table 55.
[0239] As ADP concentration increases, the total light value
increases for both the reactions containing polymerase and those
without polymerase as demonstrated in Table 56. In this example the
best fold increase in the signal, as defined as fold increase in
signal over background, is seen with 0.05 .mu.M ADP in the
pyrophosphorylation reaction.
56TABLE 55 Reaction RM T4 DNA Pol ADP 1,2 18 .mu.l 1 .mu.l 1 .mu.l
of 0.2 uM 3 18 .mu.l -- 1 .mu.l of 0.2 .mu.M 4,5 18 .mu.l 1 .mu.l 1
.mu.l of 1 .mu.M 6 18 .mu.l -- 1 .mu.l of 1 .mu.M 7,8 18 .mu.l 1
.mu.l 1 .mu.l of 2 .mu.M 9 18 .mu.l -- 1 .mu.l of 2 .mu.M 10,11 18
.mu.l 1 .mu.l 1 .mu.l of 10 .mu.M 12 18 .mu.l -- 1 .mu.l of 10
.mu.M 13,14 18 .mu.l 1 .mu.l 1 .mu.l of 20 .mu.M 15 18 .mu.l -- 1
.mu.l of 20 .mu.M The T4 DNA Pol used was Promega T4 DNA Polymerase
(10 u/ul) (Pt# M421F).
[0240]
57TABLE 56 Reac- Fold Above tion Light ADP DN Avg. Blank Avg. no
polymerase 1 9.19 0.01 .mu.M + 8.81 8.317 16.9 2 8.43 0.01 .mu.M +
3 0.43 0.01 .mu.M - 4 28.36 0.05 .mu.M + 29.05 28.39 42.9 5 29.74
0.05 .mu.M + 6 0.662 0.05 .mu.M - 7 43.29 0.10 .mu.M + 41.54 40.18
29.6 8 39.78 0.10 .mu.M + 9 1.359 0.10 .mu.M - 10 77.4 0.50 .mu.M +
74.9 68.9 11.5 11 72.49 0.50 .mu.M + 12 5.969 0.50 .mu.M - 13 82.4
1.0 .mu.M + 80.1 69.38 6.42 14 77.98 1.0 .mu.M + 15 10.81 1.0 .mu.M
-
Example 33
Detection of ATP using Fluorescence-based Methods
[0241] In addition to detecting ATP by luciferase-based methods,
ATP can be detected using fluorescence-based systems. For the
fluorescence-based measurements, an ATP determination kit was used
(Sigma #366-A Lot#117H6017). This kit uses a combination of 2
enzymes, phosphoglycerate kinase and glyceraldehyde phosphate
dehydrogenase, to catalyze the formation of NAD from NADH in the
presence of ATP. Since the NADH is fluorescent, but the NAD is not,
ATP can be measured as a loss in fluorescence intensity. The
reaction buffer was prepared from kit components as follows: 3 ml
of the supplied buffer solution was diluted in 5.25 ml of nanopure
water, and 0.75 ml of 12% trichloroacetic acid was added. One vial
of the supplied NADH was reconstituted in 1 ml of nanopure water;
the enzyme mix was used as supplied. For each measurement, 10 .mu.l
of enzyme mix and 20 .mu.l of NADH were added to 1.5 ml of reaction
buffer in a clear plastic 10 mm cuvette. Fluorescence was read in a
SPEX Fluorolog Fluorimeter using SPEX dm3000 Software, with
absorbance and emission wavelengths set at 340 nm and 460 nm,
respectively.
[0242] ATP samples at various concentrations were prepared by
serially diluting ATP tenfold into 10 mM Tris, pH 7.3. Varying
amounts of each dilution was added to the cuvette and the decrease
in fluorescence was recorded (Table 57). For comparison ATP was
also quantitated using luciferase. 20 .mu.l of each ATP dilution
was added to 100 .mu.l LAR with 10 ng of luciferase and light
output was measured using a TD-20e luminometer. Each dilution was
measured in duplicate (Table 58).
[0243] This example indicates that ATP can be detected by at least
two separate methods. In the fluorescence-based system, changes of
approximately 200,000 fluorescent light units were significant,
which corresponds to 1 nanomole of ATP. The luciferase assay was
sensitive to lower levels of ATP.
58TABLE 57 Decrease in ATP Volume Mass Fluorescence Units in
Concentration Added Added 10,000's 10 mM 20 .mu.l 200 nmoles 135
nd.sup.a nd.sup.a 1 mM 20 .mu.l 20 nmoles 84.3 132 nd.sup.a 1 mM 10
.mu.l 10 nmoles 89.3 nd.sup.a nd.sup.a 1 mM 5 .mu.l 5 nmoles 76.4
nd.sup.a nd.sup.a 100 .mu.M 40 .mu.l 4 nmoles 66.7 60.2 nd.sup.a
100 .mu.M 20 .mu.l 2 nmoles 23.9 21.9 20.8 100 .mu.M 10 .mu.l 1
nmole 19.1 22.0 18.9 100 .mu.M 5 .mu.l 500 pmoles 7.6 6.9 6.8 10
.mu.M 20 .mu.l 200 pmoles 11.6 10.0 11.1 10 .mu.M 10 .mu.l 100
pmoles 10.4 6.9 6.6 1 .mu.M 20 .mu.l 20 pmoles 8.2 8.4 5.2 1 .mu.M
10 .mu.l 10 pmoles 8.0 8.1 5.3 0.1 .mu.M 20 .mu.l 2 pmoles 3.2 5.6
3.6 0.01 .mu.M 20 .mu.l 200 fmoles 8.1 9.7 6.8 Tris 20 .mu.l -- 4.3
3.7 3.8 Tris 10 .mu.l -- 4.0 3.3 3.5 nd.sup.a, not done
[0244]
59TABLE 58 APT, 20 .mu.l of Light Units 10 mN 102,417 102,731 1 mM
117,718 98,842 100 .mu.M 47,676 44,101 10 .mu.M 7690 6996 1 .mu.M
812 798 0.1 .mu.M 76.8 67.8 0.01 .mu.M 7.0 4.5 Tris 0.06 0.06
Example 34
Detection of ATP using Fluorescence; PRPP Synthetase, Reactions
with Adenosine
[0245] ATP was synthesized by the enzyme PRPP Synthetase from the
substrates AMP and PRPP as in Example 13, except the reactions were
done in larger volumes and the substrates were at higher
concentrations. 20 .mu.l of AMP (29 mM) and 20 .mu.l of PRPP (26
mM) were incubated with 20 .mu.l of PRPP Synthetase
(6.times.10.sup.-3 units) in 200 .mu.l of PRPP Synthetase buffer.
The reactions are summarized in Table 59. After a 30 minute
incubation at 37.degree. C., the PRPP Synthetase was
heat-inactivated at 94.degree. C. for 10 minutes. The ATP was then
quantitated using both a fluorescence-based system and a
luciferase-based system. For the fluorescence-based measurements,
an ATP determination kit was used (Sigma #366-A Lot#117H6017) as
described in Example 33. Twenty microliter aliquots of the PRPP
reactions were then added to cuvettes containing 1.5 ml of buffer,
10 .mu.l of enzyme mix and 20 .mu.l of NADH. The decrease in
fluorescence was monitored. Four to six measurements were made for
each reaction (Table 60). For the luciferase-based assay, 20 .mu.l
was added to 100 .mu.l of LAR and 10 ng of luciferase. Each
reaction was determined in triplicate. Light output was measured
using a Turner TD-20e luminometer (Table 61). This example
demonstrates that ATP production by PRPP Synthetase can be measured
using fluorescence or luciferase.
60TABLE 59 PRPP Syn PRPP Reaction Buffer AMP PRPP Synthetase 1 200
.mu.l 20 .mu.l 20 .mu.l 20 .mu.l 2 200 .mu.l 20 .mu.l -- 20 .mu.l 3
200 .mu.l -- 20 .mu.l 20 .mu.l 4 200 .mu.l 20 .mu.l 20 .mu.l --
[0246]
61TABLE 60 Decrease in Fluorescence Units Reaction (in 10,000's)
Average 1 49.1 48.0 47.3 49.0 nd.sup.a nd.sup.a 48.4 2 2.48 3.30
2.37 10.9 7.06 9.57 5.95 3 3.36 2.30 11.06 7.63 10.5 nd.sup.a 6.97
4 3.48 1.68 4.83 0.62 5.74 3.37 3.29 nd.sup.a, not done
[0247]
62 TABLE 61 Reaction Light Units 1 8923 9995 9562 2 0.001 0.000
0.013 3 1939 1760 1770 4 27.9 23.7 23.0
Example 35
Detection of ATP using Fluorescence; Cell Lysates
[0248] ATP can also be generated by incubating cell lysates with
AMP and dCTP as described in Examples 25, 26 and 27. The Sigma ATP
determination kit described in Example 33 was also used to detect
ATP in this system. Reactions were assembled as described below
(Table 62) and incubated at room temperature. ATP concentrations
were quantitated at 80 minutes and 140 minutes using luciferase. In
this assay 15 .mu.l of each reaction was added to 100 .mu.l of LAR
and 10 ng of luciferase. Light output was measured using a Turner
Luminometer TD-20e (Table 63). During the time course, ATP was also
measured by fluorescence. The procedure was as described in Example
33, except that 15 gl of each reaction was added per reading,
instead of 20 gl. The first set of time points began at 80 minutes;
the second set of readings began at 140 minutes. Each reaction was
assayed in duplicate or triplicate (Table 64). This example
demonstrates that ATP synthesized in cell lysates can be detected
using a luciferase or a fluorescence assay.
63TABLE 62 E. coli 0.05 M 100 mM Reaction Lysate MgSO.sub.4 10 mM
AMP dCTP 1 100 .mu.l 20 .mu.l 20 .mu.l 10.5 .mu.l 2 -- 20 .mu.l 20
.mu.l 10.5 .mu.l 3 100 .mu.l -- 20 .mu.l 10.5 .mu.l 4 100 .mu.l 20
.mu.l -- 10.5 .mu.l 5 100 .mu.l -- -- 10.5 .mu.l
[0249]
64TABLE 63 Light Units Reactions T = 80 minutes T = 140 minutes 1
33,519 65,522 2 2.158 2.086 3 362.7 370.6 4 0.5 0.561 5 1.898
1.057
[0250]
65 TABLE 64 Decrease in Fluorescence Units (in 10,000's) Reaction
First Time Point Second Time Point 1 27.1 29.4 83.8 87.3 2 11.9 8.2
1.3 1.2 3 12.2 8.2 4.1 4.7 4 5.0 4.1 4.2 2.8 5 nd.sup.a nd.sup.a
4.8 7.3 nd.sup.a, not done
Example 36
Extremely Sensitive DNA Measurement by Amplification of
Pyrophosphorylation Reaction Products
[0251] This Example demonstrates that AMP can be a source of
extraneous nucleotides that result in unwanted background
amplification in reactions spiked with a nucleoside triphosphate
and that the detection limit for DNA measured through the
pyrophosphorylation of the sample can be lowered if the products
are amplified.
[0252] Two reactions were assembled. Reaction 1 consisted of: 2
.mu.l of 10X Buffer C (Promega Corp. R003A, Lot 7544205); 0.5 .mu.L
of 40 mM sodium pyrophosphate; 2 .mu.l of 1 mM AMP; 1 ul of
0.25U/.mu.l Myokinase (Sigma M3003, Lot 116H9516); 1 .mu.l of
0.17U/ul Pyruvate Kinase (Sigma N 0379, Lot 127F81802); 1 .mu.l of
10U/.mu.l T4 DNA Polymerase (Promega M421F Lot 617506) and 11.5
.mu.l of water. Reaction 2 was identical to Reaction 1 except that
the AMP was treated with Apyrase in a reaction consisting of 20
.mu.l of 10 mM AMP and 1 .mu.l of 1U/.mu.l Apyrase (Sigma A 6535
lot 127H7010) for 30 min at room temperature, followed by a heat
inactivation step to eliminate the Apyrase activity by treatment at
70.degree. C. for 10 Min.
[0253] At time 0, 1 .mu.l of 10 mM PEP was added to each reaction
and the reaction was mixed and incubated at room temperature. At 2
min, 2 .mu.l of the reaction was removed and added to 100 .mu.l of
L/L reagent and the light output measured using a Turner TD-20e
Luminometer as described above. The following data were collected:
Reaction 1; 817.4 light units, Reaction 2; 7.3 light units. Since
there should be no ATP produced by this reaction unless extraneous
nucleoside di- or triphosphate is added as a contaminant in a
reagent, this demonstrates that the AMP probably contained some
level of contaminating nucleotide which was eliminated by Apyrase
treatment.
[0254] The following reactions were assembled: Reaction A contained
the components described in Reaction 2 above except that 1 .mu.l of
1 ng Hin F1 Fragments (Promega Corp, G175A Lot 7733602) diluted to
this concentration with 1X TE Buffer )/.mu.l was added and the T4
DNA Polymerase was not added to the initial reaction mix; Reaction
B, same as Reaction A but the DNA added was at a concentration of
100 pg DNA/.mu.l; Reaction C, same as Reaction A but the DNA added
was at a concentration of 10 pg DNA/.mu.l; Reaction D, same as
Reaction A but the DNA added was at a concentration of 1 pg
DNA/.mu.l; and, Reaction E, same as Reaction A but with 1 .mu.l of
1X TE Buffer added and no DNA added.
[0255] One microliter of T4 DNA Polymerase was added to each
reaction and the reactions were incubated at 37C for 15 min. After
this incubation, 1 .mu.l of 10 mM PEP was added to each reaction
and incubated again at room temperature for 10 min. At that time, 2
.mu.l of each reaction was added to 100 .mu.l of L/L reagent and
the light output of the reaction was measured using a Turner
Luminometer as described above. The data are presented in Table 65.
This Example demonstrates that the products of the
pyrophosphorylation reaction can be coupled to an ATP amplification
system to increase the sensitivity of DNA measurement.
66 TABLE 65 Light Measured From Samples Incubated at Room
Temperature pg DNA* 10 min 20 min A 100 pg 917.3 1156 B 10 pg 112.1
1119 C 1 pg 4.68 919 D 0.1 pg 2.61 873 E 0 1.52 650 *The DNA
reported in this column is the actual DNA equivalent Luciferase
reaction. The amount is approximately 10% of the total
pyrophosphorylated.
* * * * *