Methods of monitoring hybridization during polymerase chain reaction are disclosed. These methods are achieved with rapid thermal cycling and use of double stranded DNA dyes or specific hybridization probes. A fluorescence resonance energy transfer pair comprises fluorescein and Cy5 or Cy5.5. Methods for quantitating amplified DNA and determining its purity are carried out by analysis of melting and reannealing curves.

Monitoring amplification of DNA during PCR

Systems, including apparatus and methods, for performing assays. These systems may involve separating sample components by partitioning them into droplets or other partitions, amplifying or otherwise reacting the components within the droplets, detecting the amplified components, or characteristics thereof, and/or analyzing the resulting data, among others.

Droplet-based assay system

The present invention provides multi-well plates and column arrays in which samples (eg, cell lysates containing nucleic acids of interest, such as RNA) can be analyzed and/or processed In one embodiment, the microfiltration arrangement is a multilayer structure, including: (i) a column plate having an array of minicolumns into which samples can be placed; (ii) a discrete filter element disposed in each minicolumn; (iii) a drip-director plate having a corresponding array of drip directors through which filtrate may egress; and (iv) a receiving-well plate having a corresponding array of receiving wells into which filtrate can flow The invention provides multi-well microfiltration arrangements that are relatively simple to manufacture and that overcome many of the problems associated with the prior arrangements relating to: (i) cross-contamination due to wicking across a common filter sheet or (ii) individual filter elements entrapping sample constituents within substantial dead volumes Further, the invention provides multi-well microfiltration arrangements that adequately support discrete filter elements disposed in the wells without creating substantial preferential flow Additionally, the invention provides multi-well microfiltration arrangements that avoid cross-contamination due to aerosol formation, pendant drops and/or splattering Other disclosed features of the invention provide for the automated covering or heat-sealing of filtrate samples separately collected in an array of wells

Multi-well microfiltration apparatus

An apparatus and method employing a plurality of light emitting devices which each can get light through a respective optical fiber toward a respective sample of a plurality of samples in a time-staggered manner. Light is generated in each of the samples at different times consistent with the times at which light is irradiated onto the sample. A single detector is used to detect the lights emitted from the plurality of samples at these different times. A plurality of bifurcated optical cable are coupled to the light emitting devices and single light detector, and the integrated end of each bifurcated cable acts as the light emitting port and light detecting port. Multiple targets can be detected from each of the plurality of samples in the same manner by providing an apparatus and method employing a different plurality of light emitting devices and single detector for each target to be detected.

Automated Optical Reader for Nucleic Acid Assays

A system for nucleic acid amplification of a sample comprises partitioning the sample into partitioned sections and performing PCR on the partitioned sections of the sample. Another embodiment of the invention provides a system for nucleic acid amplification and detection of a sample comprising partitioning the sample into partitioned sections, performing PCR on the partitioned sections of the sample, and detecting and analyzing the partitioned sections of the sample.

Chemical amplification based on fluid partitioning

A system is provided for carrying out real time fluorescence-based measurements of nucleic acid amplification products. In a preferred embodiment of the invention, an excitation beam is focused into a reaction mixture through a surface, the reaction mixture containing: (i) a first fluorescent indicator capable of generating a first fluorescent signal whose intensity is proportional to the amount of an amplification product in the volume of the reaction mixture illuminated by the excitation beam and (ii) a second fluorescent indicator homogeneously distributed throughout the reaction mixture capable of generating a second fluorescent signal proportional to the volume of reaction mixture illuminated by the excitation beam. Preferably, the excitation beam is focused into the reaction mixture by a lens through a portion of a wall of a closed reaction chamber containing the reaction mixture. The same lens is used to collect the first and second fluorescent signals generated by the first and second fluorescent indicators, respectively, in response to the excitation beam. The ratio of the fluorescent intensities of the first and second fluorescent signals provides a stable quantitative indicator of the amount of amplification product synthesized in the course of the amplification reaction.

Linda G. Lee

Papers

System for real time detection of nucleic acid amplification products

Verfahren und vorrichtung zur echtzeiterfassung der produkte von nukleinsäureamplifikation