Leland Stanford Junior University

Robert D Chambers

Juan G Santiago

Mostafa Ronaghi

Alexandre Persat

Reto B Schoch

Moran Bercovici

Supreet Singh Bahga

Govind V Kaigala

A method and system are presented for fast and efficient isolation, purification and quantitation of nucleic acids from complex biological samples using isotachophoresis in microchannels. In an embodiment, a sieving medium may be used to enhance selectivity. In another embodiment, PCR-friendly chemistries are used to purify nucleic acids from complex biological samples and yield nucleic acids ready for further analysis including for PCR. In another embodiment, small RNAs from biological samples are extracted, isolated, preconcentrated and quantitated using on-chip ITP with a high efficiency sieving medium. The invention enables fast concentration and separation (takes 10s to 100s of seconds) of nucleic acids with high selectivity and using lower volumes of reagents (order of 10s of &#x3bc;L to focus less than 1 &#x3bc;g/&#x3bc;L of nucleic acid).

Isotachophoretic focusing of nucleic acids

Improved electrophoretic analysis is provided by interaction of anionic and cationic isotachophoresis (ITP) shock waves that propagate toward each other, and analysis of the resulting interaction zones. These shock wave interactions can provide qualitatively different capabilities from conventional ITP methods. Shock wave interaction can enable a single assay to identify analyte and quantify its concentration via isotachophoretic focusing followed by separation of the concentrated analytes via electrophoresis, without any mid-assay alteration of the externally imposed experimental conditions (i.e., no switching, valve operation, etc. during the measurement). As another example, shock wave interaction can enable a single assay to provide coupled ITP processes with different electrolyte concentrations (as in cascade-ITP) in a single simple system (again, without any mid-assay alteration of the externally imposed experimental conditions).

Isotachophoresis having interacting anionic and cationic shock waves

A method and system are presented for fast and efficient isolation, purification and quantitation of nucleic acids from complex biological samples using isotachophoresis in microchannels. In an embodiment, a sieving medium may be used to enhance selectivity. In another embodiment, PCR-friendly chemistries are used to purify nucleic acids from complex biological samples and yield nucleic acids ready for further analysis including for PCR. In another embodiment, small RNAs from biological samples are extracted, isolated, preconcentrated and quantitated using on-chip ITP with a high efficiency sieving medium. The invention enables fast concentration and separation (takes 10s to 100s of seconds) of nucleic acids with high selectivity and using lower volumes of reagents (order of 10s of &#x3bc;L to focus less than 1 pg/&#x3bc;L of nucleic acid).

A novel method for visualizing electrokinetic process zones (e.g., for isotachophoresis (ITP)) is provided. We introduce negligibly small concentrations of a fluorophore that is not focused by isotachophoresis. This non-focusing tracer (NFT) migrates through multiple isotachophoresis zones. As it enters each zone, the NFT concentration adapts to the local electric field in each zone. ITP zones can then be visualized with a point detector or camera. The method can be used to detect, identify, and quantify unknown analyte zones, and can visualize complex and even transient electrophoresis processes. This visualization technique is particularly suited to microfluidic and lab-on-a-chip applications, as typical fluorescence microscopes and CCD cameras can provide high-resolution spatiotemporal data.

Non-focusing tracers for indirect detection in electrophoretic displacement techniques

Indirect detection and/or identification of analytes by ITP can be enhanced by adding a mixture of labeled carrier ampholytes (CAs) to the sample to provide a continuous range of mobility markers. Each analyte can be detected and quantified by corresponding gaps in the CA signal. This approach does not require a priori choice of fluorophores and can be readily applied (without extensive and specific design) to a wide range of analytes. Analyte identification can be expedited by computing a normalized signal integral (NSI) from the CA signals. Empirical calibrations can relate the NSI to effective mobility. Effective mobility results under two or more different pH conditions can be used to determine analyte pKa and fully ionized mobility, which are analyte properties that can facilitate analyte identification.

Growing community of inventors

Enfield, NH, United States of America

Robert D Chambers