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-11319582-B2
Publication Date
2022-05-03
Expiration Date
Abstract
Provided herein are methods for isolating nucleic acids from intact cells in a sample of intact cells, contamination dead cells, cell debris, and biofilm using two separation steps, either by centrifugation or filtration, performed in sequentially. Also provided is a method for isolating nucleic acids from intact cells using a first separation step followed by treatment with a nuclease and then a second separating step. Provided herein is a related method for isolating DNA from intact cells using a nuclease that produces DNA cuts on double stranded DNA, followed by a second separating step.
Core Innovation
The disclosure provides methods for isolating nucleic acids from intact cells while reducing nucleic acids originating from contaminating dead cells, cell debris, and biofilm. A sample comprising intact cells, contaminating dead cells, cell debris, and biofilm is subjected to a first separation to obtain a first fraction comprising intact cells together with the contaminating dead cells, cell debris, and biofilm.
The first fraction is suspended in a nuclease reaction buffer and incubated with a nuclease to degrade nucleic acids in the contaminating dead cells, the cell debris, and the biofilm to obtain a partially nuclease-degraded sample. A second separation is then performed on the partially nuclease-degraded sample to remove the contaminating dead cells, the cell debris, and the biofilm to obtain a second fraction consisting of the intact cells, and nucleic acids are isolated from the intact cells in the second fraction.
The approach is described for nucleic acids generally and specifically for isolating DNA from intact cells using a double stranded DNA specific deoxyribonuclease to degrade DNA in the contaminating dead cells, the cell debris, and the biofilm. Dependent aspects further define the workflow by specifying that the first and second separations are performed independently by centrifuging, filtering, or a combination of both, and that the nuclease can be an endonuclease, an exonuclease, or an endo-exonuclease.
Claims Coverage
The provided independent claims are clm-00001 and clm-00010, covering nucleic acids generally from intact cells and DNA specifically using a double stranded DNA specific deoxyribonuclease. In total, the independent claims recite two inventive features organized around a first separation, a nuclease incubation targeted at contaminants, and a second separation before recovery of intact-cell fractions.
Two sequential separations around contaminant nuclease degradation
A sample comprising intact cells, contaminating dead cells, cell debris, and biofilm is subjected to a first separation to obtain a first fraction; the first fraction is incubated with a nuclease to degrade nucleic acids in the contaminating dead cells, the cell debris, and the biofilm to obtain a partially nuclease-degraded sample; a second separation is performed on the partially nuclease-degraded sample to remove the contaminating dead cells, the cell debris, and the biofilm to obtain a second fraction consisting of the intact cells; nucleic acids are isolated from the intact cells in the second fraction.
Double stranded DNA specific deoxyribonuclease targeting contaminant DNA
A sample comprising intact cells, contaminating dead cells, cell debris, and biofilm is subjected to a first separation to obtain a first fraction; the first fraction is suspended in a deoxyribonuclease reaction buffer and incubated with a double stranded DNA specific deoxyribonuclease to degrade DNA in the contaminating dead cells, the cell debris, and the biofilm to obtain a partially deoxyribonuclease-degraded sample; a second separation is performed to remove the contaminating dead cells, the cell debris, and the biofilm to obtain a second fraction consisting of the intact cells; and DNA is isolated from the intact cells in the second fraction.
Across the independent claims, the core claim coverage is the combination of a first separation that enriches a fraction containing intact cells and contaminants, a nuclease incubation that degrades nucleic acids or contaminant DNA in the contaminants, and a second separation that removes the contaminating dead cells, cell debris, and biofilm so that nucleic acids or DNA can be isolated from the recovered intact cells.
Stated Advantages
Reduces nucleic acids from contaminating dead cells, cell debris, and biofilm by degrading nucleic acids in those contaminants prior to isolating nucleic acids from intact cells.
Provides nucleic-acid isolation from intact-cell fractions after removing contaminating dead cells, cell debris, and biofilm.
Documented Applications
Microbial DNA/RNA analysis intended to distinguish nucleic acids from intact cells versus nucleic acids from contaminating dead cells, cell debris, and biofilm.
Analysis using micrococcal nuclease and DNase I/DNase with comparative results indicating reduced false positives versus prior dye-based methods, including microarray/quantification and qPCR outcomes as described in the provided content.
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