Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-01-09
2002-04-30
Campbell, Eggerton A. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100
Reexamination Certificate
active
06379898
ABSTRACT:
FIELD OF THE INVENTION
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
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.
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.
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.
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.
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.
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.
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 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. No. 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.
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
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.
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
n
+PP
i
→NA
n−1
+dNTP
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 5′ phosphate groups from the deoxyribonucleoside triphosphate molecules to an adenosine 5′-diphosphate molecule to form adenosine 5′-triphosphate according to the following reaction:
dNTP*+ADP→NDP+ATP*
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.
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
n
+PP
i
→NA
n−1
+ATP
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 nucleotid
Leippe Donna M.
Lewis Martin K.
Nelson Lisa S.
Nelson Michelle A.
Shultz John W.
Campbell Eggerton A.
Welsh & Katz Ltd.
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