Brandeis University

Other

John E Rice

Lawrence J Wangh

Kenneth E Pierce

J Aquiles Sanchez

Cristina Hartshorn

Arthur Henry Reis, Jr

Jesse J Salk

Yanwei Jia

Arthur Reis

Adam E Osborne

Nicholas Rice

Skye Fishbein

Stephen Van Hooser

James M Rice

Robert W Rice

Alexandra Isabel King Over

J Acquiles Sanchez

Jesus A Sanchez

Aquiles J Sanchez

Jr Arthur H Reis

Kenneth Pierce

Provided herein are reagents and kits for detection of multiple target sequences in a single-tube, single-color assay, and methods of use thereof. In particular, multiplex assays are provided for the detection ofcomplex target sequences (e.g., katG, rpoB, inhA promotor, pncA, etc.).

Multiplex target detection assay

Provided herein are reagents for improving PCR accuracy.

Reagents for improving PCR accuracy

Provided herein are kits for performing for nucleic acid sequences.

Kits and reaction mixtures for analyzing single-stranded nucleic acid sequences

Provided herein are methods for detecting and identifying strains of mycobacteria, and compositions and kits for performing such methods. In particular, nucleic acid amplification and fluorescence detection methods are provided for the detection and differentiation of mycobacteria based on, for example, pathogenicity, species, and antibiotic resistance or sensitivity. Compositions and methods are provided herein to identify and differentiate mycobacteria in mixtures of different mycobacteria and mycobacteria and non-mycobacteria.

Compositions, methods, and kits for detecting and identifying mycobacteria

Provided herein are mispriming prevention reagents, compositions and kits comprising such reagents and methods of use thereof.

Mispriming prevention reagents

The present invention relates to compositions and methods for nucleic acid based diagnostic assays. In particular, the present invention provides primers for asymmetric PCR and other amplification modalities. In some embodiments, the present invention provides multiple primers for amplification of related nucleic acid targets in a single reaction.

Compositions and methods for nucleic acid based diagnostic assays for variable sequence targets

Modified double-stranded oligonucleotides that have terminal regions on each of their strands, that have a hybrid length of 6-50 nucleotides long, that have a melting temperature Tm of at least 32&#xb0; C., and that include 2-4 modifying groups, each covalently attached to a different terminal region, preferably to a terminal nucleotide, said modifying groups being polycyclic substituents that do not have bulky portions that are non-planar, said modified oligonucleotide being capable of binding to the 5' exonuclease domains of DNA polymerases and, when included in a PCR or other primer-dependent DNA amplification reaction at a concentration, generally not more than 2000 nM, that is effective for at least one of the functions of suppressing mispriming, increasing polymerase selectivity against 3&#x2032; terminal mismatches. Increasing polymerase selectivity against AT-rich 3&#x2032; ends, reducing scatter among replicates, suppressing polymerase 5&#x2032; exonuclease activity, and inhibiting polymerase activity; as well as amplification reaction mixtures containing such modified double-stranded oligonucleotides, and amplification reactions, amplification assays and kits that include such modified double-stranded oligonucleotides.

Reagents and methods of PCR

Homogenous detection during or following PCR amplification, preferably LATE-PCR, utilizing fluorescent DNA dye and indirectly excitable labeled primers and probes, improves reproducibility and quantification. Low-temperature homogeneous detection during or following non-symmetric PCR amplification, preferably LATE-PCR, utilizing fluorescent DNA dye and indirectly excitable labeled mismatch-tolerant probes permits analysis of complex targets. Sequencing sample preparation methods following LATE-PCR amplifications reduce complexity and permit 'single-tube' processing.

Methods for sequential DNA amplification and sequencing

Provided herein are methods for detecting mutations in nucleic acid, and compositions and kits for performing such methods. In particular, nucleic acid amplification and fluorescence detection methods are provided to detect mutations and assess the mutational load. The methods are based on a set of adjacently binding probes wherein one probe is labelled with a quencher and the other is a self-indicating probe labelled with fluorophore and quencher. The binding of the probes is analyzed by melting curve analysis.

Detecting mutations in DNA

A non-symmetric polymerize chain reaction (PCR) amplification method employing a limiting primer in low concentration whose concentration-adjusted melting point at least equals, and preferably exceeds, that of the excess primer, the latter in turn not being more than 25&#xb0; C. below the melting temperature of the amplicon. Assays employing such amplification and labeled hybridization probes, including assays that include a detection step following primer extension or a low-temperature probe, or both. Kits for performing such assays and primer or primer-and-probe sets for performing the foregoing amplifications and assays.

Late-PCR

Provided herein are fluorescence detection methods for nucleic acid sequences and to kits for performing such methods.

Methods, kits and reaction mixtures for analyzing single-stranded nucleic acid sequences

This disclosure relates to amplification and detection of a rare variant or variants of a DNA sequence in an abundant variant of the sequence, such as detection of a low-level somatic mutations and minority alleles in an excess of normal nucleic acid target sequences.

Detecting nucleic acid variations within populations of genomes

Modified double-stranded oligonucleotides that have terminal regions on each of their strands, that have a hybrid length of 6-50 nucleotides long, that have a melting temperature Tm of at least 32&#xb0; C., and that include 2-4 modifying groups, each covalently attached to a different terminal region, preferably to a terminal nucleotide, said modifying groups being polycyclic substituents that do not have bulky portions that are non-planar, said modified oligonucleotide being capable of binding to the 5' ex-nuclease domains of DNA polymerases and, when included in a PCR or other primer-dependent DNA amplification reaction at a concentration, generally not more than 2000 nM, that is effective for at least one of the functions of suppressing mispriming, increasing polymerase selectivity against 3&#x2032; terminal mismatches, increasing polymerase selectivity against AT-rich 3&#x2032; ends, reducing scatter among replicates, suppressing polymerase 5&#x2032; exonuclease activity, and inhibiting polymerase activity; as well as amplification reaction mixtures containing such modified double-stranded oligonucleotides, and amplification reactions, amplification assays and kits that include such modified double-stranded oligonucleotides.

Growing community of inventors

Natick, MA, United States of America

John E Rice