Methods for microbial DNA analysis
Inventors
Hogan, Michael Edward • Katchman, Benjamin Alan • Rivas, Candy Mavis • Baca, Yasmine Eve
Assignees
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Publication Number
US-11618917-B2
Publication Date
2023-04-04
Expiration Date
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.
Core Innovation
The invention relates to methods for isolating nucleic acids from intact cells by addressing both intact cells and cell free nucleic acids. A sample comprising intact cells and cell free nucleic acids is obtained, and the method captures the intact cells and the cell free nucleic acids within a macroporous silicate matrix having pores of 10 µm to 50 µm in size. The cell free nucleic acids captured in the matrix are degraded in situ with micrococcal nuclease.
After in situ degradation of the captured cell free nucleic acids, cellular nucleic acids are released from the captured intact cells. The cellular nucleic acids are then eluted from the macroporous silicate matrix with an elution buffer to produce an isolated nucleic acid product. The isolated product comprises cellular nucleic acids, including DNA and/or RNA as defined in dependent claims.
In related workflow language, the sample is suspended in a suspension buffer to form a cellular suspension, which is flowed directionally through the macroporous silicate matrix to capture the intact cells and cell free nucleic acids within pores. A lysing/binding buffer is added to lyse the captured intact cells, and cellular nucleic acids bind to the macroporous silicate matrix. Optionally, a washing solution is flowed through the macroporous silicate matrix to remove nuclease treated cell free nucleic acids and contaminants prior to elution. The macroporous silicate matrix is defined as a glass made from silicon oxide alone or combined with boron oxide or aluminum oxide.
Claims Coverage
The document provides two independent claims. Across the independent claims, there are six main inventive features: (i) obtaining a sample with intact cells and cell free nucleic acids, (ii) capturing both within a macroporous silicate matrix with 10 µm to 50 µm pores, (iii) degrading captured cell free nucleic acids in situ using micrococcal nuclease, (iv) lysing captured intact cells to release cellular nucleic acids and binding them to the matrix in one claim, (v) eluting cellular nucleic acids with an elution buffer to produce an isolated nucleic acid product, and (vi) using directional flow via a suspension buffer in the second independent claim.
Macroporous silicate matrix capture with defined pore size
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
In situ micrococcal nuclease degradation of cell free nucleic acids
degrading in situ the captured cell free nucleic acids with micrococcal nuclease within the macroporous silicate matrix
Release and elution of cellular nucleic acids to produce an isolated nucleic acid product
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
Directional flow capture using a cellular suspension
suspending the sample in a suspension buffer to form a cellular suspension and flowing directionally the cellular suspension through a macroporous silicate matrix with pores of 10 µm to 50 µm in size to capture the intact cells and cell free nucleic acids within pores therein
Lysing/binding and matrix binding of cellular nucleic acids
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
Optional washing to remove nuclease treated cell free nucleic acids and contaminants
adding a washing solution through the macroporous silicate matrix to remove nuclease treated cell free nucleic acids and contaminants
Together, the independent claims focus on capturing intact cells and cell free nucleic acids in a macroporous silicate matrix of 10 µm to 50 µm pores, degrading the captured cell free nucleic acids in situ with micrococcal nuclease, and then releasing or lysing intact cells followed by elution to produce an isolated nucleic acid product, including workflows that use directional suspension flow and lysing/binding with matrix binding.
Stated Advantages
Improved discrimination of live vs dead microbes.
Documented Applications
Microbial DNA analysis intended to distinguish intact-cell (live) nucleic acids from extracellular/cell-free nucleic acids associated with dead cells and biofilm.
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