Patent Application
20190024076
Disclosed are a method and device for lysing cells to release or extract genomic DNA (gDNA) from inside cells. The disclosed method uses a mixture of microscopic glass beads and cells (for example, spores) that form a semi-dry cake that clings to a larger metal ball and the sides of the tube during bead beating lysis, greatly improving the efficiency of the bead beating process. Since the cells are caked onto the glass beads and metal ball, each time the ball strikes the side of the container, cells are impacted. The device is designed to produce a chaotic motion with a dual purpose, first ensuring that sufficient force is generated to open the cells, and second that the metal ball impacts are distributed across the interior surface of the container so that all of the cell mixture is subjected to sufficient impacts to break the cells. The result is that spores and other microbes that typically need to be boiled or beaten for up to 30 minutes to achieve adequate lysis, can be opened in seconds using the disclosed semi-dry bead beating method. The method reduces the number of steps and hands-on time by rapidly opening difficult to lyse cells, while preserving the integrity of the DNA.
Many cell-based and DNA-based analytical methods require releasing cell contents including DNA from inside a cell to facilitate analysis. Opening the cells to release the contents is called ‘lysis.’ For example, methods used to investigate the microbiome using DNA sequencing techniques first require lysis of microbes so the DNA can be extracted. Most microbiomes are communities of bacteria, archaea and fungi that vary tremendously in their susceptibility to lysis techniques. Differential susceptibility presents a significant problem to microbial population researchers, who want to ensure that the toughest (usually Gram-positive) and the easiest (usually Gram-negative) to lyse bacteria are represented in proportion to their population in the original sample. Unfortunately, most microbial lysis protocols work well for some microbes, but poorly for others. (Comparison of lysis techniques for microbiome—Sanqing Yuan, Dora B. Cohen, Jacques Ravel, Zaid Abdo, Larry J. Forney. Evaluation of Methods for the Extraction and Purification of DNA from the Human Microbiome. PLoS ONE 7(3): e33865. doi:10.1371/journal.pone.0033865; Additionally, rapid and simple alkaline lysis techniques used to recover plasmid DNA typically also remove the microbial genomic DNA, which is the target for microbiome screening (Alkaline Lysis opens cells but removes gDNA—Birnboim, H C. and Doly, J., A rapid alkaline extraction procedure for screening recombinant plasmid DNA, Nucleic Acids Res. 7(6), 1979, 1513-1524; in contrast, KOH lysis can be used to recover bacterial genomic DNA Raghunathan, Arumugham et al. “Genomic DNA Amplification from a Single Bacterium.” Applied and Environmental Microbiology 71.6 (2005): 3342-3347. PMC. Web. 29 September 2016). There are multiple lysis techniques known in the art that attack cellular integrity based on different biochemical methods, including lysozyme (enzymatic attack on the peptidoglycan cell wall), strong base (chemical attack), detergent (solubilizes cell membranes), bead beating or shaking (mechanical disruption), and heat DNA extraction methods affect microbiome profiling results: Wagner Mackenzie B, Waite D W, Taylor M W Evaluating variation in human gut microbiota profiles due to DNA extraction method and inter-subject differences. Frontiers in Microbiology. 2015; 6:130. doi:10.3389/fmicb.2015.00130). Most published or commercially available DNA preparation methods use one or more of these methods to lyse cells, usually in sequential steps that can take a significant amount of time, especially when handling many samples at once. While individual lysis methods are usually sufficient for applications where incomplete or partial lysis yields sufficient DNA for the protocol being performed, they often do not yield DNA from microbiome samples in proportion to the original community, and may fail to lyse certain microbes altogether. For example, a detergent-based lysis may disrupt a subset of cells with weak cell walls and strong cell membranes, but not open detergent-resistant microbes with strong cell walls, leading to under-representation or absence of DNA from detergent resistant cells in the resulting DNA preparation. In another example, bead beating of microbes sufficient to lyse cells with strong cell membranes may shear or destroy DNA released early in the process from easily lysed cells. Additionally, the various methods of lysis tend to be incompatible with each other, and need to be performed sequentially if used in combination. For example, lysozyme will not work in the presence of detergents or strong base. Certain detergents precipitate in the presence of strong base. Bead beating is difficult to combine with a heating process. While individual shortcomings may be overcome by running separate lysis protocols in series, this increases the complexity, time, and cost involved. Importantly, detergents such as sodium dodecyl sulfate (SDS) must be removed after lysis, because SDS interferes with downstream DNA manipulation. Additionally, certain microbes may be resistant to lysis protocols run sequentially, depending on protocol order. For example, certain microbes with tough peptidoglycan cell walls may have an outer envelope of lipid bi-layer that protects from an initial treatment with strong base or lysozyme. A simultaneous combination of multiple methods may be effective, or a long sequence of multiple steps, to yield DNA from all microbes in a sample, but the use of a single method that opens all cells would be a significant improvement.
Devices and shakers for conducting bead beating are known in the art. For examples, see Table 1. Bead beaters and shakers capable of processing large numbers of samples tend to be large and heavy with powerful motors and fast moving parts that can be dangerous to users. The devices disclosed herein are small, efficient and safe. Smaller units known in the art typically process a few samples at a time. The devices disclosed herein can process 1-48 samples at once. The devices known in the art use regular or simple motions to process samples, whereas the disclosed devices use a random motion that results in more forceful impacts and wide range of motion that results in more uniform lysis in less time at slower (less dangerous) speeds.
The methods and devices disclosed herein utilize a bead beating procedure that works rapidly to lyse cells, including even the most difficult microbial spores (Bacillus subtilis spores are described herein as an example), yet preserves DNA of sufficient size to generate the large amplicons needed for applications and techniques where proportional lysis is desired or necessary, such as high resolution microbiome characterization. The result is a simple, rapid protocol that uniformly opens all cells in a sample, yielding a more representative DNA profile across a sample containing different cellular constituents, such as the microbiome.
Disclosed are methods and devices for lysis of cells, such as bacteria present in microbiomes, that can be completed in a short period of time, e.g., less than a minute, and importantly, yield improved quality and quantities of genomic DNA (gDNA) from difficult to lyse samples, such as bacterial spores.
The subject matter that is regarded as the invention is particularly pointed out and distinctly claimed in the claims at the conclusion of the specification. The foregoing and other objects, features, and advantages of the invention will be apparent from the following detailed description taken in conjunction with the accompanying drawings.
DNA-based analytical methods, for example, the methods described in U.S. patent application Ser. No. 15/372,588 titled “Methods for DNA Preparation for Multiplex High Throughput Targeted Screening” by Mark Driscoll and Thomas Jarvie, that is incorporated herein by reference in its entirety, generate DNA by lysing the cells in the target microbiome, after which the resulting DNA is used as template in PCR amplification targeting the 16S, 23S or other rRNA (ribosomal RNA) gene sequences, present in all bacteria and archaea. Microbes can be identified using their rRNA gene sequence, which varies slightly in most, if not all, bacteria and archaea. The variation in rRNA gene sequence means that individual species of bacteria and archaea have characteristic DNA variations in the rRNA gene that serve as identifiers, or fingerprints, for that species. Kits, protocols and software enable comprehensive fingerprinting of the microbes in a sample, and permit simultaneous rRNA fingerprinting of many samples at once, at high resolution, using rRNA gene sequences. Known microbes can be identified after sequencing by mapping the rRNA gene DNA sequences to a microbial genomic database. Unknown microbes will contain rRNA sequences that are different from any of the microbes in the database, but can be tracked using their unique rRNA sequence. In addition, the number of reads obtained for each microbe in a sample can reveal the relative abundances of each microbe in a sample. The relative abundance can be an important indicator of the state of each individual microbiome. Lysis techniques that change relative abundances of microbes, or leave out certain microbes altogether, can lead to sequencing results that incorrectly characterize the state of the microbiomes being studied. The invention as described is an improved lysis method for achieving the correct relative abundances of microbes from a sample.
As used herein, “non-periodic motion” means any motion that does not have a regular period of repetition or orbital motion and repeats itself at non regular intervals of time.
As used herein, “random motion” and “chaotic motion” are used interchangeably to mean any motion in which the speed and three-dimensional direction continually change.
As used herein “semi-dry cake” means a sample of biologic cells having 1% to 30% weight of sample (for example, spores or microbes) per weight of moisture.
As used herein, “micron” and “micrometer” are used interchangeably to mean a unit of length equal to one millionth of a meter.
Disclosed herein is a device for shaking samples in a non-periodic motion and random motion simultaneously, having a stroke length of from about 1 cm to about 2.5 cm and a speed of from about 5 Hz to about 50 Hz. In some embodiments, the device is configured to hold samples in microfuge tubes or microtiter plates or other vessels suitable for high-throughput analysis. In some embodiments, the number of tubes that can be processed simultaneously is between one and 96. In some embodiments, the microtiter plate is selected from the group consisting of: 8, 16, 24, 46, 96, or 384 well plate. In some embodiments the volume of the tubes or wells in the microtiter plates is between 200 microliters to 2 ml.
Disclosed herein is a method for lysing biologic cells in a sample to release DNA from the cells, comprising the sequential steps of: (a) mixing a first aqueous solution or semi-solid sample containing one or more biologic cells with (i) a 4.5 mm steel (or other biocompatible material) ball; and (ii) ˜100 micron beads (glass or other material 4 or greater on the Mohs hardness scale) to form a semi-dry cake and (b) shaking the mixture of (a) in a non-periodic and random motion for a period of time effective to release DNA from the cells; (c) resuspending the semi-dry cake in a second aqueous solution and allowing the beads to settle out; (d) recovering the second aqueous solution from the settled solid components in the mixture, wherein DNA released from the biologic cells is present in the recovered second aqueous solution.
In some embodiments, the first aqueous solution is water. In some embodiments, the first aqueous solution contains Tris or other buffers, salts or detergents to control pH or limit biological activity. In some embodiments, the first solution is a non-aqueous liquid sufficient to form a semi-dry cake when added to the beads. In some embodiments, the first aqueous solution contains one or more biologic cells.
In some embodiments, the steel ball has a diameter of from about 2 millimeters to about 10 millimeters. In some embodiments, the steel ball has a diameter of about 4.5 millimeters. In some embodiments, the glass beads have a diameter of from about 10 micrometers to about 300 micrometers. In some embodiments, the glass beads have a diameter of about 100 micrometers. In some embodiments, the glass beads are added to the first aqueous solution at a concentration of about 83% glass beads by weight (for example, 10 ul (10 mg) sample to 50 mg beads).
In some embodiments, the glass beads are added to the aqueous solution at a concentration of from 40% to 99% by weight.
In some embodiments, the shaking is conducted for a period of from about 5 seconds to about 10 minutes.
In some embodiments, the shaking is conducted for 2 minutes.
In some embodiments, cell lysis is measured by terbium fluorescence of dipicolinic acid, which is co-located with DNA inside spores
In some embodiments, the release of DNA during lysis is followed by UV spectroscopy (OD260).
In some embodiments, the release of DNA during lysis is determined by DNA fluorescence assays.
In some embodiments, the separation of steel and glass beads from the second aqueous solution is conducted by a method selected from the group consisting of: centrifugation, filtration and gravity settling.
In some embodiments, the biologic cells originate from a sample 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 sample, including raw samples, complex samples, mixtures, and microbiome samples.
In some embodiments, the biologic cells originate from an organism selected from the group consisting of: spores, biofilms, multicellular organisms, unicellular organisms, prokaryotes, eukaryotes, microbes, bacteria, archaea, protozoa, algae and fungi.
In some embodiments, the shaker unit has a rigid, flat tube or plate support element (free element) into which multiple plates or tubes can be loaded (
The following is an illustrative embodiment of the cell lysis methods disclosed herein:
Step 1: 2 microliters of B. subtilis spores (1 OD/ml) were suspended in 8 ul water to form a 10 μl mixture.
Step 2: A 2 ml microcentrifuge tube (Dot Scientific #RN2000-GMT) was prepared, containing a single 4.5 mm steel ball and 0.05 g glass beads (acid washed, 100 um, Sigma G4649) (see
Step 3: 10 μl of spore suspension was added to steel bead/glass bead mixture.
Step 4: Tube was placed in shaker. Semi-dry cake forms on outside of bead (see
Step 5: The lysis of spores releases dipicolinic acid (DPA), which can be measured via terbium fluorescence.
Step 6: DNA quality was assessed by PCR of the 16S rRNA gene (see
As a comparison, spore lysis and DNA quantity were followed during boiling lysis of spores as shown in
Bead beating parameters were also explored using the novel shaker device disclosed herein. As shown in
Shaker intensity was tested up to 55 beats per second with similar results as shown in
The methods and devices disclosed here may be useful for any applications that require lysis of cells and spores, such as microbiome sequencing (for example, 16S rRNA or other targeted gene profiling), DNA applications (for example, whole genome shotgun profiling), non-DNA applications (for example, protein, RNA, metabolites, and organelles), and diagnostic applications for difficult to lyse pathogens.
One or more embodiments of the present invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
This application claims priority to patent application No. 62/533,821, filed Jul. 18, 2017, the content of which is incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 62533821 | Jul 2017 | US |
