System for simultaneous detection of multiple binding events in the same reaction
First Claim
1. A system for the simultaneous multiplexed determination of a plurality of events employing electrophoresis to distinguish the events, comprising an electrophoretic device for electrophoretic separation and detection, a container containing a first set of first agents comprising differing mobility regions defining differing target-binding regions and a second reagent comprising at least one active agent under conditions where said active agent modifies members of said first set bound to a target resulting in a change of electrophoretic mobility of said first agents bound to target to provide a modified member retaining said mobility region, and transfer of said at least one modified member to said electrophoretic device for separation and detection of said at least one modified member, with the proviso that when said first and second reagents comprise oligonucleotides, said mobility region is other than an oligomer.
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Abstract
Methods and compositions are provided for detecting target molecules, e.g. DNA sequences, particularly single nucleotide polymorphisms, using a pair of nucleotide sequences, a primer and a snp detection sequence, where the snp detection sequence binds downstream from the primer to the target DNA in the direction of primer extension, or ligands and receptors. The methods employ e-tags comprising a mobility-identifying region joined to a detectable label and a target-binding region. The result of the binding of the target-binding region to the target is to have a bond cleaved in the starting material with the production of a detectable product with a different mobility from the starting material, where the different e-tags can be separated and detected
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Citations
26 Claims
- 1. A system for the simultaneous multiplexed determination of a plurality of events employing electrophoresis to distinguish the events, comprising an electrophoretic device for electrophoretic separation and detection, a container containing a first set of first agents comprising differing mobility regions defining differing target-binding regions and a second reagent comprising at least one active agent under conditions where said active agent modifies members of said first set bound to a target resulting in a change of electrophoretic mobility of said first agents bound to target to provide a modified member retaining said mobility region, and transfer of said at least one modified member to said electrophoretic device for separation and detection of said at least one modified member, with the proviso that when said first and second reagents comprise oligonucleotides, said mobility region is other than an oligomer.
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2. A system for the simultaneous multiplexed determination of a plurality of events employing electrophoresis to distinguish the events, employing e-tags comprising a region having two functionalities, a first functionality for binding to or bound to a detectable label and a second functionality bonded to a target-binding region, said system comprising a vessel containing a plurality of e-tag moieties with each e-tag bonded to a different target-binding region, and a sample, whereby the presence in the sample of a target for each of said e-tag moieties results in a modification of said c-tag to produce modified e-tags with a change in mobility of said e-tag, and an electrophoresis device for separating and detecting said modified e-tags, means for moving said modified e-tags to said electrophoresis device and a data processor for receiving and processing data from said electrophoresis device, with the proviso that in the event that said first functionality is not bound to said detectable label, said system further comprising joining said modified e-tag to said detectable label and when said first and second reagents comprise oligonucleotides, said mobility region is other than an oligomer
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8. A method for performing multiplexed determinations in a nucleic acid sample, employing a reagent having a plurality of first members, each first member having an oligonucleotide sequence homologous to a target sequence and a non-oligomeric e-tag bonded to a nucleotide, and a second reagent having a nucleic acid sequence homologous to a target sequence proximal to said first member homologous sequence, with the proviso that when said first member comprises a DNA sequence interrupted by an RNA sequence a ribonuclease specific for a chimeric double stranded nucleic acid is substituted for said second reagent, said method comprising:
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combining a target nucleic acid sample with said first and second reagents under conditions where said first and second reagents comprising nucleic acid sequences bind to homologous target sequences and said first and/or second reagent is modified to change the mobility of said e-tag by joining any first reagent and second reagent bound to target nucleic acid, releasing said e-tag from at least a portion of said sequence of said first reagent or cleaving said RNA interrupting said DNA to produce modified e-tags; and
separating said modified e-tags by electrophoresis, whereby the presence of target nucleic acid is determined. - View Dependent Claims (9, 10, 11, 12)
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13. A method for detecting at least one target nucleic acid sequence in a nucleic acid sample, said method comprising:
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combining under bond cleavage conditions;
a reagent system capable of cleaving a cleavable bond of an e-tag linked target-binding sequence, said nucleic acid sample and a reagent pair consisting of a primer and said e-tag linked target-binding sequence, each reagent pair having sequences homologous for each nucleic acid sequence to be determined, wherein each said primer specifically binds to said target nucleic acid and said target-binding sequence binds to said target nucleic acid downstream from said primer, wherein each said target-binding sequence is characterized by being linked to a non-oligomeric e-tag specific for each said nucleic acid sequence;
executing at least one cycle of cleavage of said cleavable bond, whereby said e-tag is released substantially free of said target-binding sequence;
separating released e-tags into individual fractions; and
detecting said e-tag fractions, whereby the presence in said target nucleic acid sample of said at least one nucleic acid sequence is detected;
with the proviso that, when separation is performed solely by means of differences in mass, the e-tags that are separated all have the same number of nucleotides bonded to the e-tag. - View Dependent Claims (14, 15, 16, 17, 18, 19)
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20. A method for performing a plurality of simultaneous determinations in a vessel in relation to a plurality of targets, each target having at least one epitope, employing a first composition with different e-tags specific for each of said targets linked to a target-binding region that comprises a reciprocal binding member to said epitopes, or said first composition is different candidate enzyme substrates and said e-tags are specific for each enzyme substrate, said method comprising:
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combining said first composition with a second composition, wherein said second composition is suspected of containing at least one target or contains at least one enzyme for said candidate substrates, and additional reagents, so that binding of said target-binding sequence to a target results in a change in the mass or electrophoretic mobility of said e-tag linked target-binding sequence to produce a product;
separating said products into discrete packets for identification;
whereby said determinations are made as to each of said e-tags. - View Dependent Claims (21, 22)
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23. A library comprising a plurality of non-oligomeric e-tags, said e-tags having a molecular weight in the range of about 150 to 5,000, each e-tag, with the entities to which it is attached, providing a different mobility in electrophoresis, wherein at least 5 of the e-tags comprise:
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a mobility-identifying region comprising at least one negative or positive charge, a target-binding region comprising at least one nucleotide or nucleotide analog, an amino acid or poly(amino acid), an enzyme substrate or a first functionality for bonding to any of them;
a detectable label or a second functionality for binding to a detectable label, wherein when said mobility-modifying region is joined to or for joining to said poly(amino acid), said first functionality is a cleavable bond. - View Dependent Claims (24, 25, 26)
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Specification