Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-02-11
2001-06-12
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S024300
Reexamination Certificate
active
06245519
ABSTRACT:
FIELD OF INVENTION
The present invention relates to compositions for protecting detectable labels from chemical or enzymatic alteration and to their use in amplification and detection methods.
BACKGROUND OF THE INVENTION
None of the references described herein are admitted to be prior art to the claimed invention.
A target nucleic acid sequence can be detected by various methods using detection probes designed to preferentially hybridize to the target sequence over other sequences that may be present in a sample. Examples of target sequences include sequences initially present in a sample or produced as part of an amplification procedure.
Examples of detection probes include oligonucleotides and derivatives thereof able to preferentially hybridize to a target nucleic acid containing a target nucleic acid sequence over other nucleic acids that may be present in a sample. Hybridization of detection probes to target nucleic acid sequences results in the formation of detectable probe:target hybridization complexes under appropriate conditions.
Detecting detectable probe:target hybridization complexes is facilitated using a labeled detection probe. Different labels and assay formats can be used to detect the presence or amount of an analyte in a sample. Examples of detectable labels include radioisotopes, fluorescent molecules, chemiluminescent molecules, chromophors, enzymes, enzyme substrates and ligands. Examples of references describing the detection of nucleic acid using fluorescent and chemiluminescent molecules include Arnold et al., U.S. Pat. Nos. 5,283,174 and Becker et al. 5,731,148, both of which are hereby incorporated by reference herein.
To facilitate detection of a target nucleic acid sequence, the number of target sequences in a sample can be increased using nucleic acid amplification techniques. Nucleic acid amplification involves the enzymatic synthesis of nucleic acid containing a sequence complementary to a nucleic acid sequence being amplified. Nucleic acid amplification can be performed using different techniques such as those involving transcription-based amplification, the polymerase chain reaction (PCR), ligase chain reaction (LCR) and strand displacement amplification (SDA).
Transcription-based amplification of a nucleic acid sequence generally employs an RNA polymerase, a DNA polymerase, deoxyribonucleoside 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 sequence 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 nucleic acid sequence. Double-stranded promoter formation generally involves DNA polymerase activity. Generally, a second oligonucleotide primer is employed to facilitate double-stranded promoter formation.
Transcription-based amplification involves 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 RNA transcripts are produced by transcription-based amplification using a single template.
Different formats can be employed for performing transcription-based amplification. Examples of different formats are provided in publications such as Burg et al., U.S. Pat. Nos. 5,437,990; Kacian et al., 5,399,491; Kacian et al., 5,554,516; McDonough et al., 5,766,849; Ryder et al., 5,786,183; Malek et al., 5,130,238; 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; and Urdea, International Application No. PCT/US91/00213, International Publication No. WO 91/10746. (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, which is 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 nucleic acid sequence. 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 nucleic acid sequence and the sequence complementary to the target nucleic acid 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-based amplification or amplification carried out using Q-beta technology (Walker et al.,
Nucleic Acids Res.,
20:1691-1696 (1992), which is hereby incorporated by reference herein.
SUMMARY OF INVENTION
The present invention features compositions and methods that are useful for storing labeled detection probes and detecting whether a target nucleic acid sequence is present in a sample. Preferred compositions are made up of a detection probe containing a label susceptible to a chemical or enzymatic alteration and a protection probe that protects the label from alteration and/or decreases the ability of the detection probe to inhibit nucleic acid amplification. Such compositions can be used, for example, to stabilize a detection probe label and to prevent a detection probe from hybridizing prematurely to amplified or target nucleic acid.
Chemical and enzymatic alterations of a detection probe label are changes in chemical identity or bonding effecting a signal produced from the altered label compared to a signal produced from an unaltered label. Examples of chemical and enzymatic alterations include oxidation, reduction, acid hydrolysis, base hydrolysis, alkylation and enzymatic cleavage or hydrolysis. Preferably, the chemical or enzymatic alteration causes a loss of signal detectability from the label.
A label susceptible to a chemical or enzymatic alteration, also referred to herein as a “susceptible label”, contains a labile group that undergoes such alteration in an aqueous solution containing an agent normally able to act on the labile group. Preferably, a labile group is subject to hydrolysis in an aqueous solution having a pH between about pH 4 and about pH 9. Examples of labile groups include an ester linkage and a thioester linkage.
A protection probe protects the label from alteration when the label is altered to a lesser extent in the presence of the protection probe than in the absence of the protection probe. In preferred embodiments, the differ
Brentano Steven T.
McDonough Sherrol H.
Nelson Norman C.
Cappellari Charles B.
Gen-Probe Incorporated
Heber Sheldon O.
Horlick Kenneth R.
Strzelecka Teresa
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