Methods for microbial DNA analysis
AssigneePathogenDx, Inc.
InventorsMichael Edward Hogan, Benjamin Alan Katchman, Candy Mavis Rivas, Yasmine Eve Baca
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Abstract
Provided herein are methods for isolating nucleic acids from intact cells in a sample of intact cells and cell free nucleic acids. The intact cells and cell free nucleic acids are captured and concentrated within the pores of a silicate matrix in a microporous silicate filter. Within the silicate matrix the cell free nucleic acids are degraded with a nuclease, the intact cells are lysed with the released DNA binding to the silicate, the nuclease treated cell free nucleic acids and contaminants from the lysed are washed from the silicate matrix and the DNA bound to the silicate is eluted therefrom to form an isolated DNA product.
Claims12
A method for isolating nucleic acids from intact cells, comprising the steps of:
- obtaining a sample comprising intact cells and cell free nucleic acids;
- capturing the intact cells and the cell free nucleic acids within a macroporous silicate matrix with pores of 10 μm to 50 μm in size;
- degrading in situ the captured cell free nucleic acids with a micrococcal nuclease;
- releasing cellular nucleic acids from the captured intact cells;
- and
- eluting the cellular nucleic acids from the macroporous silicate matrix with an elution buffer to produce an isolated nucleic acid product.
A method for isolating nucleic acids from intact cells, comprising the steps of:
- obtaining a sample comprising intact cells and cell free nucleic acids;
- suspending the sample in a suspension buffer to form a cellular suspension;
- flowing directionally the cellular suspension through a macroporous silicate matrix with pores size of 10 μm to 50 μm in size to capture the intact cells and cell free nucleic acids within pores therein;
- degrading in situ within the macroporous silicate matrix the cell free nucleic acids with a micrococcal nuclease;
- adding a lysing/binding buffer to the macroporous silicate matrix to lyse the captured intact cells to release cellular nucleic acids therefrom, said cellular nucleic acids binding to the macroporous silicate matrix;
- and
- adding an elution buffer to the macroporous silicate matrix to elute the cellular nucleic acids therefrom to produce an isolated nucleic acid product.
Dependencies are inferred from the claim text.