COMBINED LYSIS PROTOCOL FOR COMPREHENSIVE CELL LYSIS
Disclosed are methods for lysis of cells, such as bacteria present in microbiomes, that combine three lysis steps—(1) heat, (2) detergent and (3) base—into a single step and that can be completed in a short period of time, e.g., a few minutes. The methods combine a normally incompatible detergent and base lysis, allows for simplified removal of detergent after lysis, and importantly, yields improved quantities of genomic DNA (gDNA) from difficult to lyse bacteria.
1 .- 17 . (canceled)
18 . A method for releasing genomic DNA from biologic cells in a sample by lysing the cells, comprising the sequential steps of:
a) producing a mixture solution by mixing an aqueous solution containing the biologic cells from the sample with (i) an amount of an ionic detergent, and (ii) an amount of a base such that the ionic detergent and the base in the mixture solution are at concentrations effective for releasing the genomic DNA from the cells by lysing the cells after the ionic detergent is dissolved in the mixture solution;
b) heating the mixture solution to at least about 50° C. for a time such that the ionic detergent is dissolved in the mixture;
c) cooling the mixture solution to 40° C. or less for a time effective to precipitate the ionic detergent, thereby producing a precipitate comprising the ionic detergent; and
d) separating the precipitate comprising the ionic detergent from the mixture solution,
wherein the genomic DNA released from the biologic cells is present in a solution generated by said separating the precipitate comprising the ionic detergent from the mixture solution; and wherein the genomic DNA released from the biologic cells is ready for analysis, PCR amplification, sequencing, purification, or storage.
19 . The method of claim 18 , wherein the ionic detergent is selected from the group consisting of: sodium dodecyl sulfate (SDS), N-Lauroylsarcosine sodium salt, and sodium deoxycholate.
20 . The method of claim 18 , wherein the ionic detergent of the mixture solution in step b) is from about 0.1% by weight to about 10% by weight.
21 . The method of claim 18 , wherein the ionic detergent of the mixture solution in step b) is about 1% by weight.
22 . The method of claim 18 , wherein the base is selected form the group consisting of:
potassium hydroxide (KOH), lithium hydroxide (LiOH), sodium hydroxide (NaOH), rubidium hydroxide (RbOH), cesium hydroxide (CsOH), calcium hydroxide (Ca(OH) 2 ), strontium hydroxide (SR(OH) 2 ), and barium hydroxide (Ba(OH) 2 ).
23 . The method of claim 18 , wherein the base of the mixture solution in step b) is from about 0.05 molar to about 1 molar.
24 . The method of claim 18 , wherein the base of the mixture solution in step b) is about 0.2 molar.
25 . The method of claim 18 , wherein the ionic detergent of the mixture solution in step b) is 1% by weight and the base of the mixture solution in step b) is 0.2 molar.
26 . The method of claim 18 , wherein the at least about 50° C. is a temperature range from at least about 50° C. to about 100° C.
27 . The method of claim 26 , wherein the heating step is conducted at a temperature about 95° C.
28 . The method of claim 27 , wherein the time conducted in the heating step is at least 0.25 minutes.
29 . The method of claim 18 , wherein the 40° C. or less is at a temperature range from about 4° C. to 40° C.
30 . The method of claim 29 , wherein the cooling temperature range from about 4° C. to 40° C. is about 20° C. to about 25° C.
31 . The method of claim 30 , wherein the time conducted in the cooling step is for at least 0.25 minutes.
32 . The method of claim 18 , wherein the separating step is conducted by a method selected from the group consisting of: centrifugation of the mixture solution, filtration of the mixture solution and gravity settling of the mixture solution.
33 . The method of claim 18 , wherein the sample is selected from the group consisting of: feces, cell lysate, tissue, blood, tumor, tongue, tooth, buccal swab, phlegm, mucous, wound swab, skin swab, vaginal swab, or any other biological material or biological fluid originally obtained from a human, animal, plant, or environmental samples, raw samples, complex samples, mixtures of samples, and microbiome samples.
34 . The method of claim 18 , wherein the sample is from an organism selected from the group consisting of: multicellular organisms, unicellular organisms, prokaryotes, eukaryotes, microbes, bacteria, archaea, protozoa, algae, fungi and viruses.
35 . The method of claim 18 , wherein the DNA released from the biologic cells is in relative abundance with, or has the correct relative abundance compared to, the relative abundance of the biologic cells in the sample.
36 . The method of claim 18 , wherein the DNA released from the biologic cells is proportional to, or in proportion with, the biological cells in the sample.