Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-01-19
2001-03-13
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S023100, C536S024330, C536S024300, C536S025300, C536S025320, C536S026710
Reexamination Certificate
active
06200757
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
Significant morbidity and mortality are associated with infectious diseases. More rapid and accurate diagnostic methods are required for better monitoring and treatment of disease. Molecular methods using DNA probes, nucleic acid hybridizations and in vitro amplification techniques are promising methods offering advantages to conventional methods used for patient diagnoses.
A method for the enzymatic amplification of specific segments of DNA known as the polymerase chain reaction (PCR) method has been described. This in vitro amplification procedure is based on repeated cycles of denaturation, oligonucleotide primer annealing, and primer extension by thermophilic polymerase, resulting in the exponential increase in copies of the region flanked by the primers. The different PCR primers, which anneal to opposite strands of the DNA, are positioned so that the polymerase catalyzed extension product of one primer can serve as a template strand for the other, leading to the accumulation of a discrete fragment whose length is defined by the distance between the 5′-ends of the oligonucleotide primers.
Another method has also been described for amplifying nucleic acid sequences. This method is referred to as single primer amplification. The method provides for the amplification of a target sequence that possesses a stem-loop or inverted repeat structure where the target sequence is flanked by relatively short complementary sequences. Various methods for creating such a target sequence in relation to the presence of a polynucleotide analyte to be detected have also been described.
The amplification methods described above require that samples suspected of having a specific nucleotide sequence be heated at about 95° C. and then be repetitively thermally cycled between one or two lower temperatures and about 95° C. The higher temperatures denature duplexes and the lower temperatures permit hybridization of the primer and chain extension.
The above methods are extremely powerful techniques for high sensitivity detection of target DNA molecules present in very small amounts. The correlation between the number of original target DNA molecules and the number of specifically amplified products is influenced by a number of variables. Minor variations in buffer or temperature conditions can greatly influence reaction-to-reaction amplification efficiencies. Further, clinical samples of DNA targets can contain inhibitory factors that can suppress enzymatic amplification. In addition, such clinical samples also contain irrelevant DNA, which can be present in very large amounts relative to the target DNA molecules.
The above amplification methods suffer from interference caused by random partial hybridization of primers used in such amplification to irrelevant DNA, i.e., DNA that is not target DNA and to which the primers bind non-specifically or non-selectively. A competition between target DNA and irrelevant DNA for the enzyme and the primer thus is created. As a result the efficiency of the amplification of the target DNA molecules is decreased. At best this leads to difficulty in distinguishing amplified target DNA from amplified irrelevant DNA. The amplification of irrelevant DNA to any substantial degree can interfere with specific amplification of target DNA to prevent detection of the target DNA completely.
One approach for this problem is to avoid chain extension of low temperature non-specifically hybridized primers by heating the reaction mixture to 95° C. prior to adding a critical reagent such as a polymerase enzyme or magnesium that is required to activate the polymerase. This can be accomplished by using a wax layer to separate the various reaction components until a high temperature is reached. Alternatively, an inhibitory antibody against the polymerase can be added at low temperature. The antibody denatures at elevated temperature and allows the enzyme to become reactivated. Another approach involves the use of AmpliTaq Gold enzyme as the polymerase in PCR reactions. Another method involving chain extension of an oligonucleotide primer is a method for the detection of differences in nucleic acids described in U.S. patent application Ser. No. 08/771,623, the disclosure of which is incorporated herein by reference. Briefly, the branch migration method detects a difference between two related nucleic acid sequences. In the method, if there is a difference between the two related nucleic acid sequences, a stable quadramolecular complex is formed comprising both of the nucleic acid sequences in double stranded form. Usually, the complex comprises a Holliday junction. Both members of at least one pair of non-complementary strands within the complex have labels. The association of the labels as part of the complex is determined as an indication of the presence of the difference between the two related sequences. The method may be employed for detecting the presence of a mutation in a target nucleic acid sequence.
In the above method for the detection of differences between two related DNA sequences, non-specific priming can be a problem for mutation detection by inhibition of DNA branch migration. All amplification products incorporate the “tail” sequences of the tailed primers and hence are able to participate in the formation of four-stranded DNA complexes with both specific PCR products and with each other. Since the sequences on both sides of the junction are completely different from each other, such complexes never undergo strand separation by branch migration and thus generate non-specific signal. One approach to alleviate this problem is to use a two-step PCR procedure or nested PCR. It is highly desirable, however, to perform the above method using a single PCR reaction with just one set of primers.
A method for avoiding the above problems that is inexpensive and more controllable than the approaches mentioned above is desirable.
2. Description of the Related Art
U.S. Pat. No. 5,338,671 (Scalice, et al.) discusses DNA amplification with thermostable DNA polymerase and polymerase inhibiting antibody.
Compositions and methods for inhibiting dimerization of primers during storage of polymerase chain reaction reagents is disclosed in U.S. Pat. No. 5,565,339 (Bloch, et al.) (Bloch I).
Use of grease or wax in the polymerase chain reaction is discussed in U.S. Pat. No. 5,411,876 (Bloch, et al.) (Bloch II).
U.S. Pat. No. 5,599,660 (Ramanujam, et al.) discloses a method and preparation for sequential delivery of wax-embedded, inactivated biological and chemical reagents.
A method for reducing non-specific priming in DNA amplification is disclosed in U.S. Pat. No. 5,348,853 (Wang, et al.).
TaqStart Antibody™ used in hot start PCR facilitated by a neutralizing monoclonal antibody directed against Taq DNA polymerase is described by Kellogg, et al.,
BioTechniques
(1994) 16(6): 1134-1137.
Co-amplification of target nucleic acid using volume exclusion agent in reaction composition and a test kit and test device useful therefor is discussed in U.S. Pat. No. 5,705,366 (Backus).
Heat-mediated activation of affinity-immobilized Taq DNA polymerase is described by Nilsson, et al., in
BioTechniques
(1997) 22(4):744-751.
Oligonucleotide inhibitors of Taq polymerase facilitate detection of low copy number targets by PCR are discussed by Dang, et al.,
J. Mol. Biol.
(1996) 24:268-278.
A simple procedure for enhancing PCR specificity is described by Weighardt, et al.,
PCR Methods and Applications
(1993) 3:77-80.
A simplified hot start PCR using AmpliTaq Gold enzyme is discussed by Birch, etal., Nature (1996) 381:445-446.
A hot start procedure using wax beads is disclosed by Chou, et al.,
Nucleic Acids Research
(1992) 20:1717-1723.
W. B. Barnes discusses PCR amplification of up to 35-kb DNA with high fidelity and high yield from &lgr;-bacteriophage templates in
Proc. Nat. Acad. Sci.
USA (1994) 91:2216-2220.
PCT application WO 96/03526A1 (Niveleau) discusses nucleic acid amplification method using a modified nucleoside and detection of
Kurn Nurith
Lishanski Alla
Liu Yen Ping
Taylor Marc
Dade Behring Inc.
Gattari Patrick G.
Horlick Kenneth R.
Leitereg Theodore J
Taylor Janell E.
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