Methods for microbial DNA analysis
AssigneePathogenDx, Inc.
InventorsMichael Edward Hogan, Benjamin Alan Katchman, Candy Mavis Rivas, Yasmine Eve Baca
Life of this patent
Estimating the term…
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.
Claims14
A method for isolating nucleic acids from intact cells, consisting of the steps of:
- obtaining a sample comprising intact cells, contaminating dead cells, cell debris, and biofilm;
- performing a first separation on the sample to obtain a first fraction comprising the intact cells, the contaminating dead cells, the cell debris, and the biofilm;
- suspending the first fraction in a nuclease reaction buffer to obtain a suspension;
- incubating the suspension 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;
- performing a second separation 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
- isolating the nucleic acids from the intact cells in the second fraction.
A method for isolating DNA from intact cells, consisting of the steps of:
- obtaining a sample comprising intact cells, contaminating dead cells, cell debris and biofilm;
- performing a first separation on the sample to obtain a first fraction comprising the intact cells, the contaminating dead cells, the cell debris and the biofilm;
- suspending the first fraction in a deoxyribonuclease reaction buffer to obtain a suspension;
- incubating the suspension 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;
- performing a second separation on the partially deoxyribonuclease-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
- isolating the DNA from the intact cells in the second fraction.
Dependencies are inferred from the claim text.