Bio-rad Laboratories, Inc.

Do, Duc

Litterst, Claudia

Maar, Dianna

Dianna Maar

John Frederick Regan

Claudia Litterst

Samantha Cooper

Eli Hefner

Wei Yang

Duc Do

Niels Klitgord

Tina C Legler

Dawne N Shelton

Nicholas Heredia

Yann Jouvenot

Svilen Tzonev

Geoff McDermott

Eli A Hefner

Svilen S Tzonev

Digital assay system, including methods, apparatus, and compositions, for assay of one or more targets in a set of partitions containing a generic reporter of target amplification.

Digital assays with a generic reporter

Method of analysis for alleles of a target. In the method, a plurality of fluid volumes may be formed. Each fluid volume may contain a first primer pair to amplify a first allele of a target, a second primer pair to amplify a second allele of the target, a first reporter including a first photoluminophore and providing a primer of the first primer pair, and a second reporter including a second photoluminophore and providing a primer of the second primer pair. Each of the first and second reporters may include an oligomer having a quencher, and the oligomer may be configured to base-pair with the primer of the first primer pair and the primer of the second primer pair. The first and second alleles may be amplified using the first and second primer pairs. Photoluminescence may be detected. A level of each allele may be determined.

Amplification reporter with base-pairing oligomers

Methods and compositions are provided for improved nucleic acid amplification assays. In some embodiments, the nucleic acid amplification assay is a tagged amplicon primer extension (TAPE) nucleic acid amplification reaction.

Split-cycle and tape amplification

Methods and compositions for detecting genetic instability using digital amplification assays. The methods may be performed in a set of isolated volumes and generally may involve competitive hybridization of a competitor and a probe/primer with a normal allele and one or more mutant alleles of a microsatellite locus. The competitor may be configured to compete similarly with, or to outcompete, the primer/probe for hybridization with the normal allele. The primer/probe may be configured to outcompete the competitor for hybridization with various mutant alleles of the locus that alter the length of the repetitive sequence by different amounts. Isolated volumes in which the primer/probe outcompetes the competitor may be enumerated, and represent one or more of the mutant alleles. The methods may enable diagnosing microsatellite instability and treating a subject based on the diagnosis.

Digital amplification assays for genetic instability

System, including methods, apparatus, and compositions, for performing amplification assays with an amplification reporter including a first oligomer and a second oligomer capable of base-pairing with one another below a melting temperature of the reporter. The reporter may have a detectable photoluminescence that is affected, such as reduced, by base-pairing of the first and second oligomers with one another. A target, such as a nucleic acid target sequence, may be amplified in at least one volume, such as a plurality of partitions, above the melting temperature, and photoluminescence of the reporter may be detected from the at least one volume below the melting temperature. A property of the target, such as a concentration of the target, may be determined based on the photoluminescence detected.

Methods of, and compositions for, analysis using digital amplification assays with unconventional/inverse changes in photoluminescence to indicate the presence of one or more targets. In an exemplary method, isolated volumes may be formed, only a subset of which contain a target. Each volume may include a probe having a label, a sink having a quencher and configured to hybridize with the probe to quench the label, and a separator configured to hybridize with the probe and/or the sink to block hybridization of the sink with the probe. An amplification reaction may be performed in the volumes to generate an amplicon corresponding to the target. The separator may hybridize with the amplicon, and may be extended or degraded by the amplification reaction. Photoluminescence of the label may be detected from the volumes, and the photoluminescence of target-positive volumes may be less than that of target-negative volumes.

Digital amplification assays with unconventional and/or inverse changes in photoluminescence

Methods of detecting or quantifying short RNA or DNA molecules using split cycle amplification are provided.

Small nucleic acid quantification using split cycle amplification

Methods and compositions are provided for improved nucleic acid amplification assays.

Digital assay system, including methods, apparatus, and compositions, for performing target assays in partitions each containing a generic reporter and a specific reporter for target amplification.

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Mountain House, CA, United States of America

Dianna Maar