Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-07-30
2001-10-02
Myers, Carla J. (Department: 1655)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S006120, C435S069100
Reexamination Certificate
active
06297365
ABSTRACT:
FIELD OF THE INVENTION
The present invention features compositions, reagents and methods for enhancing an amplification protocol. Preferably, the compositions, reagents and methods are used in conjunction with an RNA polymerase driven transcription-associated amplification protocol.
BACKGROUND OF THE INVENTION
None of the references described herein are admitted to be prior art to the claimed invention.
Nucleic acid amplification involves the enzymatic synthesis of nucleic acid amplicons that contain a sequence complementary to a nucleic acid sequence being amplified. Nucleic acid amplification can be performed using different techniques such as those involving transcription-associated amplification, the polymerase chain reaction (PCR), ligase chain reaction (LCR) and strand displacement amplification (SDA).
Uses of nucleic acid amplification include diagnostic and synthetic applications. Diagnostic applications of nucleic acid amplification typically involve screening for whether amplicons are produced, the amount of amplicon produced, and/or determining whether produced amplicons contain a particular sequence.
Transcription-associated amplification of a nucleic acid sequence generally employs an RNA polymerase, a DNA polymerase, deoxyribonuclcoside triphosphates, ribonucleoside triphosphates, and a promoter-template complementary oligonucleotide. The promoter-template complementary oligonucleotide contains a 5′ sequence recognized by an RNA polymerase and a 3′ sequence that hybridizes to a template nucleic acid in a location 3′ of a target sequence that is sought to be amplified. After hybridization of the promoter-template complementary oligonucleotide to the template, a double-stranded promoter is formed upstream from the target sequence. Double-stranded promoter formation generally involves DNA polymerase activity.
RNA polymerase-associated amplification is initiated by the binding of an RNA polymerase to a promoter region that is usually double-stranded. The RNA polymerase proceeds downstream from the promoter region and synthesizes ribonucleic acid in a 5′ to 3′ direction. Multiple copies, generally in the range of 100-3,000 RNA transcripts, can be produced by RNA polymerase-associated amplification using a single template.
Different formats can be employed for performing transcription-associated amplification. Examples of different formats are provided in publications such as Burg et al., U.S. Pat. No. 5,437,990; Kacian et al., U.S. Pat. No. 5,399,491; Kacian et al., U.S. Pat. No. 5,554,516; Kacian et al., International Application No. PCT/US93/04015, International Publication No. WO 93/22461; Gingeras et al., International Application No. PCT/US87/01966, International Publication No. WO 88/01302; Gingeras et al., International Application No. PCT/US88/02108, International Publication No. WO 88/10315; Davey and Malek, European Application No. 88113948.9, European Publication No. 0 329 822 A2; Malek el al., U.S. Pat. No. 5,130,238; Urdea, International Application No. PCT/US91/00213, International Publication No. WO 91/10746; McDonough et al., International Application No. PCT/US93/07138, International Publication No. WO 94/03472; and Ryder et al., International Application No. PCT/US94/08307, International Publication No. WO 95/03430. (Each of these references is hereby incorporated by reference herein.)
PCR amplification is described by Mullis et al., U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159, and in
Methods in Enzymology
, 155:335-350 (1987). (Each of these references is hereby incorporated by reference herein.)
An example of LCR is described in European Patent Publication No. 320 308 (hereby incorporated by reference herein). LCR uses at least four separate oligonucleotides. Two of the oligonucleotides hybridize to a nucleic acid template so that the 3′ end of one oligonucleotide and the 5′ end of the other oligonucleotide are positioned for ligation. The hybridized oligonucleotides are then ligated forming a full-length complement to the target sequence in the nucleic acid template. The double-stranded nucleic acid is then denatured, and third and fourth oligonucleotides are hybridized to the complementary strand and joined together. Amplification is achieved by further cycles of hybridization, ligation, and denaturation, producing multiple copies of the target sequence and the sequence complementary to the target sequence.
SDA is an isothermal amplification reaction based on the ability of a restriction enzyme to nick the unmodified strand of a hemiphosphorothioate form of its recognition site, and on the ability of a DNA polymerase to initiate replication at the nick and displace a downstream non-template strand. (See, e.g., Walker,
PCR Methods and Applications
, 3:25-30 (1993), Walker et al.,
Nucleic Acids Res
., 20:1691-1996 (1992), and Walker et al.,
Proc. Natl. Acad. Sci
., 89:392-396 (1991). Each of these references is hereby incorporated by reference herein.) The steps used in generating fragments for carrying out autocatalytic SDA amplification are indicated to be adaptable for generating fragments for transcription-associated amplification or amplification carried out using Q-beta technology. (Walker et al.,
Nucleic Acids Res
., 20:1691-1696 (1992).)
SUMMARY OF THE INVENTION
The present invention features inhibitors of target-independent amplification and the use of such inhibitors for enhancing an amplification protocol. The inhibitors are believed to enhance an amplification protocol by inhibiting the ability of one or more nucleic acid polymerases to use nucleic acid in a polymerase reaction in the absence of target nucleic acid.
“Target-independent amplification” refers to the amplification of a nucleic acid sequence that is not a target nucleic acid sequence. The target nucleic acid sequence is present on a target nucleic acid and is a nucleotide base sequence, or region, sought to be amplified.
It is believed that the present invention benefits nucleic acid amplification by using competitors of amplification oligonucleotides to sequester amplification enzymes in solution from non-target nucleic acid such as amplification oligonucleotides. The competitors appear to compete with amplification oligonucleotides for binding to one or more amplification enzymes, and may be added in an excess amount relative to the amplification oligonucleotides.
In the absence of target nucleic acid, affected amplification enzymes are occupied by the competitors, and the ability of the enzymes to participate in a polymerase reaction involving non-target nucleic acid is inhibited. Amplification oligonucleotides hybridized to target nucleic acid favorably compete with the competitors for amplification enzyme binding. Thus, the competitors function as reversible inhibitors of amplification enzymes.
Amplification enzymes are nucleic acid polymerases that catalyze the synthesis of polynucleotides by polymerizing nucleoside triphosphates. Reversible inhibition of amplification enzymes is carried out to prevent the formation of undesirable side-products, such as one or more of the following: (1) a primer-dimer; (2) an RNA replicating nucleic acid; (3) a single-stranded primer extended RNA or DNA; and (4) a modification to a primer rendering the primer unable to participate in the amplification of a target nucleic acid sequence. The types of undesirable side-products that can be formed will depend upon the particular amplification protocol that is performed.
Amplification oligonucleotides hybridize to target nucleic acid and participate in an amplification reaction. Examples of amplification oligonucleotides include template-complementary probes, such as primers, and promoter-template complementary probes, such as promoter-primers.
While inhibitors of target-independent amplification described by the present invention are expected to function by competing with amplification oligonucleotides for binding to an amplification enzyme, unless otherwise specified in the claims, the claims are not limited to a particular mechanism.
Adams Christopher C.
Brentano Steven T.
Schroth Gary P.
Cappellari Charles B.
Chakrabarti Arun Kr.
Gen-Probe Incorporated
Heber Sheldon O.
Myers Carla J.
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