Measuring analytes using competitive interference and recuperation of enzyme activity
A technique for analyzing analytes and solutions, applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., which can solve the problems of low sensitivity and resolution
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Embodiment 1
[0054] Interfering with Polymerization Using Anti-Analyte Antibodies in Polymerase Reactions
[0055] This example illustrates how anti-analyte antibodies can be used as binding molecules to intentionally interfere with polymerization in a polymerase reaction in a dose-dependent manner. The selected binding molecule binds to the analog, the analog binds to the DNA template, the primer, or both, and the binding of the binding molecule to the analog interferes with the polymerase reaction.
[0056] Select the DNA template for the polymerase reaction. The DNA template chosen was the following 51mer single-stranded oligonucleotide:
[0057] 5'-ACCAACACCACACAACCAACACACACCAACCACAACACCACCATGTCTC-3' (SEQ ID NO: 7)
[0058] Primer sequences for this reaction can also be chosen. The primer sequence is the following oligonucleotide: 5'-GAGACAUGG-3' (SEQ ID NO: 4)
[0059] Analogs were combined with the primers in this example. Uracil (U) is conjugated to the analog using methods kno...
Embodiment 2
[0071] Detection of analyte concentration in a sample using anti-analyte antibodies to interfere with polymerization in a polymerase reaction
[0072] The conditions and materials taught in Example 1 were used for this example unless otherwise indicated. Example 1 describes how, in a polymerase reaction, anti-analyte antibodies interfere with polymerization and in a dose-dependent manner. This example illustrates how interference can be used to detect the concentration of an analyte in a sample in the presence of the analyte.
[0073] Prepare 50 µL of a reaction mixture containing 10 mM Tris-HCl (pH 7.9), 50 mM NaCl, 10 mM MgCl 2, 2.5nmol of each dNTP, PICOGREEN (Invitrogen, San Diego) diluted 1:200 and anti-Dig antibody Fab fragment (Roche Diagnostics, Germany) 33nM. Analytes are chosen to test detection techniques that competitively bind the analyte. Dig was chosen as the analyte, and samples with different Dig analyte concentrations were added to the reaction mixture to ...
Embodiment 3
[0077] Interfering with cleavage using anti-analyte antibodies in an exonuclease reaction
[0078] Examples 1 and 2 describe how to use a competitive binding assay to determine the concentration of analyte by interfering with the polymerization reaction using anti-analyte antibodies. In this embodiment, anti-analyte antibodies are used to interfere with the exonuclease cleavage reaction to determine the content of the analyte in the sample.
[0079] The substrate to be cleaved in the exonuclease reaction is the following double-stranded DNA (dsDNA) fragment: the sense strand sequence is
[0080] 5'-ACCAACACCACACACAACCAACACACACCAACCACAACACCACCATGTCTCTCTGAC-3' (SEQ ID NO: 5)
[0081] An analogue of the analyte is labeled with Dig at the 3' end of the sequence, where the analogue competes with the analyte for binding to the binding molecule.
[0082] Antisense strand sequence is
[0083] 5'-GTCAGAGAGACATGGTGGTGTTGTGGTTGGTGTGTGTTGGTTGTGTGTGGTGTTGGT-3' (SEQ ID NO: 1)
[0084] P...
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