Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2001-08-08
2003-03-18
Horlick, Kenneth R. (Department: 1656)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C536S023500, C536S024320, C536S024330, C435S006120, C435S091100, C435S091200, C435S810000
Reexamination Certificate
active
06534645
ABSTRACT:
BACKGROUND OF THE INVENTION
Methods for amplifying nucleic acids provide useful tools for the detection of human pathogens, detection of human genetic polymorphisms, detection of RNA and DNA sequences, for molecular cloning, sequencing of nucleic acids, and the like. In particular, the polymerase chain reaction (PCR) has become an important tool in the cloning of DNA sequences, forensics, paternity testing, pathogen identification, disease diagnosis, and other useful methods where the amplification of a nucleic acid sequence is desired. See e.g.,
PCR Technology: Principles and Applications for DNA Amplification
(Erlich, ed., 1992);
PCR Protocols: A Guide to Methods and Applications
(Innis et al., eds, 1990).
PCR permits the copying, and resulting amplification, of a target nucleic acid. Briefly, a target nucleic acid, e.g. DNA, is combined with a sense and antisense primers, dNTPs, DNA polymerase and other reaction components. See Innis et al. The sense primer can anneal to the antisense strand of a DNA sequence of interest. The antisense primer can anneal to the sense strand of the DNA sequence, downstream of the location where the sense primer anneals to the DNA target. In the first round of amplification, the DNA polymerase extends the antisense and sense primers that are annealed to the target nucleic acid. The first strands are synthesized as long strands of indiscriminate length. In the second round of amplification, the antisense and sense primers anneal to the parent target nucleic acid and to the complementary sequences on the long strands. The DNA polymerase then extends the annealed primers to form strands of discrete length that are complementary to each other. The subsequent rounds serve to predominantly amplify the DNA molecules of the discrete length.
A variety of factors can lead to non-functional PCR or other amplification reactions. One drawback of PCR is that artifacts can be generated from mis-priming and primer dimerization. Those artifacts can be exacerbated in traditional multiplex PCR. Multiple sets of primers increase the possibility of primer complementarity at the 3′-ends, leading to primer-dimer formation. These artifacts deplete the reaction of dNTPs and primers and outcompete the multiplex templates for DNA polymerase. Such artifacts can be reduced by careful primer design and the use of “hot start” PCR. See Chou, Q. et al. (1992)
Nucleic Acids Research,
20: 1717-1723. It is increasingly difficult, however, to eliminate all interactions which promote the mis-priming and primer dimerization in a multiplex amplification as the reaction may contain many primers at high concentration.
Additionally, multiplex PCR has been observed to suppress the amplification of one template in preference for another template. A number of factors are involved in this suppression. For example, when a multiplex PCR reaction involves different priming events for different target sequences, the relative efficiency of these events may vary for different targets. This can be due to the differences in thermodynamic structure, stability, and hybridization kinetics among the various primers used.
Simple user error, of course, can also result in a nonfunctional amplification reaction. For instance, the absence of nucleotides or enzyme due to negligence or degradation will lead to a nonfunctional reaction. Similarly, where probes are used to monitor a particular reaction, a nonfunctional probe will lead to a false negative reaction. This can occur, for instance, when there is an absence of probe or the probe does not bind to its hybridization site efficiently. Use of probes, particularly fluorescent probes, are commonly used for monitoring the accumulation of reaction products in real time, i.e. while that amplification reaction is progressing.
Several schemes for controlling for failure of an amplification reaction have been described. See, e.g., Edwards, M., et al. PCR PRIMER, A LABORATORY MANUAL (Dieffenbach, C., et al., eds. 1995) pages 157-171. For example, it is common to run positive and negative control reactions in separate reaction tubes. Simple positive controls include a known amount of template, while negative controls do not have any template in the reaction. These controls are run under the same conditions as a test sample and provide the tester with information about the quality of the enzymes and nucleotides, etc., as well as whether the test solutions are contaminated.
More recently, internal controls for PCR have been developed. Internal controls are advantageous because they are run in the exact same reaction mixture as the test sample and therefore there is no question about the activity of the reagents in the test sample itself. Moreover, internal controls are more efficient by allowing for the use of fewer reactions and less reaction solution and reagents.
Internal controls typically involve multiple reactions performed in the same reaction tube (e.g., multiplex PCR). In such reactions, the presence of at least one amplification product indicates that some variables, such as the enzyme and nucleotides, were functional during the reaction. See, e.g. Levinson, G. et al.
Human Reprod.
7(9):1304-1313 (1992).
In addition, internal controls to verify the presence of the target template have also been described. For example, in multiplex assays where closely related templates such as pathogen strains are distinguished by amplifying differing sequences, primers for a sequence common to all templates provides a positive control for amplification. See, e.g., Kaltenboeck, B., et al.
J. Clin. Microbiol.
30(5):1098-1104 (1992); Way, J., et al.
App. Environ. Microbiol.
59(5):1473-1479 (1993); Wilton, S. et al.
PCR Methods Appl.
1:269-273 (1992). Rosenstraus et al. (
J. Clin. Microbiol.
36(1):191-197 (1998)) have described an internal control containing primer binding regions identical to those of the target sequence and that contain a unique probe-binding region that differentiates the control from the amplified target sequence.
As discussed above, it is often desirable to quantify PCR products using various fluorescent probes. Examples of useful fluorescent probes include, e.g., fluorescence resonance energy transfer (FRET), molecular beacon, and TaqMan® probes. Currently, however, there is no internal control method that validates the activity of a target specific probe in the same reaction mixture as the test sample. Therefore, to have a fully validated amplification reaction, a positive control must be run in a separate reaction tube to insure that the target specific probe is functioning properly.
Accordingly, there is a need for internal control compositions and methods useful for measuring these and other amplification variables. The present invention meets this need and provides useful methods and compositions for performing a totally internally controlled amplification reaction.
SUMMARY OF THE INVENTION
The present invention provides methods of performing an amplification reaction. The steps of the reaction comprise:
(a) combining in an aqueous solution,
(i) a target probe, a first control probe and a second control probe;
(ii) a first 5′ primer, a first 3′ primer and a target template, the target template comprising a hybridization site for the first 5′ primer, the first 3′ primer and the target probe;
(iii) a first control template, the first control template comprising a hybridization site for the first 5′ primer, the first 3′ primer and the first control probe; and
(iv) a second 5′ primer, a second 3′ primer and a second control template, the second control template comprising a hybridization site for the second 5′ primer, the second 3′ primer, the target probe and a second control probe;
(b) performing an amplification reaction; and
(c) quantifying binding of the target probe, first control probe and second control probe.
In some embodiments, the quantifying step is performed during the amplification reaction. In some embodiments, the quantifying step is performed after the a
Cepheid
Horlick Kenneth R.
Spiegler Alexander H.
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