Microfluidic device-based nucleic acid purification method
Inventors
Young, Lincoln C. • Zhou, Peng • Spizz, Gwendolyn • Yasmin, Rubina
Assignees
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Publication Number
US-9169479-B2
Publication Date
2015-10-27
Expiration Date
Abstract
A method is provided for purifying nucleic acid from a sample in a microfluidic device. The method can be used to purify nucleic acids from any source known in the art that comprises nucleic acids, such as prokaryotic or eukaryotic organisms, viruses, cell, tissues, organs, etc. In a specific example, the tissue is whole blood. The method for purifying nucleic acid may run fully automated in the microfluidic device.
Core Innovation
The invention provides a microfluidic device-based method for purifying nucleic acid from a lysed liquid sample solution. The method performs a first purification step using a first purification matrix, where an organic solvent is added to the lysed solution and a first capture medium is added to capture the nucleic acid. The nucleic acid-bound first capture medium is separated, washed, separated again, and then eluted to obtain an eluted nucleic acid.
After the first purification step, the method performs a second purification step using a second purification matrix. The second purification step includes re-establishing an appropriate molecular charge on the eluted nucleic acid, re-capturing the nucleic acid from the solution using a second capture medium, washing the re-captured nucleic acid, and eluting the re-captured nucleic acid to obtain a purified nucleic acid. This two-stage purification workflow is implemented within the microfluidic device using first and second purification matrices.
The second-stage molecular charge re-establishment includes adding a suitable buffer and an organic solvent to the eluted nucleic acid. Additional refinements include providing a microfluidic device that contains all necessary reagents for automatic purification, or manually adding at least some reagents to a microfluidic device. Further refinements include repeating washing and separating steps a desired number of times and performing RNase treatment or DNase treatment after elution to remove RNA or DNA to obtain DNA only or RNA only, respectively.
Claims Coverage
The independent claims are clm-00001 and clm-00009, each covering a microfluidic device-based purification workflow that uses two purification matrices: a first capture/elution stage followed by a second charge re-establishment and re-capture stage.
Microfluidic device with first and second purification matrices
providing a suitable microfluidic device comprising a first purification matrix and a second purification matrix
First purification step on first purification matrix using organic solvent and first capture medium
performing a first purification step using the first purification matrix, the first purification step comprising adding an organic solvent to the lysed solution; adding a first capture medium to the lysed solution; capturing the nucleic acid to the first capture medium; separating the nucleic acid-bound first capture medium from the solution; washing the nucleic acid-bound first capture medium with a wash solution; separating the washed first capture medium from the wash solution; and eluting the nucleic acid from the washed first capture medium
Second purification step with molecular charge re-establishment and second capture medium
performing a second purification step using the second purification matrix, the second purification step comprising reestablishing an appropriate molecular charge on the eluted nucleic acid; re-capturing the nucleic acid from the solution using a second capture medium; washing the re-captured nucleic acid with a wash solution; and eluting the re-captured nucleic acid to obtain a purified nucleic acid
Across both independent claims, the coverage centers on a microfluidic device-based, two-matrix nucleic acid purification workflow: first capturing nucleic acids on a first capture medium after organic solvent addition with wash/separation and elution, then re-establishing an appropriate molecular charge to re-capture nucleic acids on a second capture medium followed by washing and elution to obtain purified nucleic acid.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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