US 9,753,007 B1Granted

Isotachophoretic focusing of nucleic acids

AssigneeThe Board of Trustees of the Leland Stanford Junior University

InventorsRobert D. Chambers, Juan G. Santiago, Alexandre Persat, Reto B. Schoch, Mostafa Ronaghi

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Abstract

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 μL to focus less than 1 μg/μL of nucleic acid).

Claims41

Dependent claims

An isotachophoretic method for concentrating a target nucleic acid and for separating said target nucleic acid from at least one other macromolecule in a sample, said method comprising the steps of:

(a) applying a sample comprising a mixture of a target nucleic acid and ionic macromolecules that are not target nucleic acids to a sample well connected to at least one liquid channel;
(b) causing said sample comprising said mixture of a target nucleic acid and ionic macromolecules that are not target nucleic acids to be in contact with a trailing electrolyte (TE) and a leading electrolyte (LE) in said at least one liquid channel, said TE having an effective-mobility magnitude greater than at least one of said ionic macromolecules that are not target nucleic acids and less than said target nucleic acid, said LE having an effective-mobility magnitude greater than said target nucleic acid, and wherein said LE and TE contacting said sample are in free solution, and are at a pH of from 4 to 10;
and
(c) applying a voltage difference along said at least one liquid channel, said at least one liquid channel containing said sample, said LE, and said TE, to cause concentration of said target nucleic acid in an isotachophoresis interface between LE and TE and separation of said at least one of said ionic macromolecules that are not target nucleic acids from said isotachophoresis interface between LE and TE.

1 more element

Dependencies are inferred from the claim text.

Facts

Application: 14/497,050Filed 25 Sep 2014 · continuation
Priority: 3 Mar 2009
Granted: 5 Sep 2017
Term ends: Not estimatedThe source records don’t support an estimate.

Classification6 codes

G01N 27/447
Systems: using electrophoresis
B01D 57/02
Separation, other than separation of solids, not fully covered by a single other group or subclass, e.g. B03C: by electrophoresis
C12N 15/101
By means of a solid support carrier, e.g. particles, polymers: by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
G01N
Investigating or analysing materials by determining their chemical or physical properties

Family

Application61/209,199Us provisional application · filed 3 Mar 2009
Application12/716,142Parent of this continuation · filed 2 Mar 2010
US 8,846,314 B2Parent of this continuation
US 2010/0224494 A1Published application
US 9,753,007 B1This patent

From the US filing history. Foreign family members aren’t listed.

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