Brandeis University

Other

Cristina Hartshorn

Lawrence J Wangh

John E Rice

Kenneth E Pierce

J Aquiles Sanchez

Arthur Henry Reis, Jr

Jesse J Salk

Arthur Reis

Stephen Van Hooser

Skye Fishbein

J Acquiles Sanchez

Aquiles J Sanchez

Jr Arthur H Reis

Kenneth Pierce

Provided herein are reagents for improving PCR accuracy.

Reagents for improving PCR accuracy

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

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

Biological samples containing nucleic acids, RNA and DNA, are freed from bound proteins by incubation with a chaotropic agent such as a guanidium salt, and the mixture is readied for further processing by dilution of such agent to a concentration below 0.05 M without physical isolation of RNA and DNA from one another or from other components of the reaction mixture. Methods include such preparation and further processing, such as amplification and detection, which may be performed in a single container. Chaotropic agent may be supplied as a dried reagent adhered to a container. Kits may include such reagents, alone or with amplification reagents.

Nucleic acid processing kits and devices

A non-symmetric polymerise 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.

An assay comprising more than one primer pair and more than one detection probe, a low copy number synthetic amplicon corresponding to each of the primer pairs. The assay can detect and distinguish between various sub-types and strains of an influenza virus using any suitable nucleic acid amplification technique. Related kits and methods are also described.

Detection and analysis of influenza virus

Primers, probes and methods for nucleic acid amplification

An additive for preventing mispriming in polymerase chain reaction (PCR) amplifications and assays comprising a hairpin oligonucleotide having a stem duplex greater than six nucleotides in length and a stabilized stem terminus. The additive improves PCR amplifications, including LATE-PCR amplifications when added to initial amplification reaction mixtures. It can be included in oligonucleotide sets and in kits for PCR amplification and assays.

Reagents and methods for improving reproducibility and reducing mispriming in PCR amplification

Nucleic acid processing methods, kits and devices

A non-symmetric polymerase 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.

Growing community of inventors

Needham, MA, United States of America

Cristina Hartshorn