KR20240003845A - Increase the therapeutic effect of HIV virus through CD4+ immune cells - Google Patents

Abstract

A method and device for disrupting cells to release or extract genomic DNA (gDNA) are disclosed. The method greatly improves the efficiency of the bead beating process by using a mixture of microscopic glass beads and cells (e.g., spores) that form a semi-dry cake that sticks to the larger metal balls and sides of the tube during bead beating disruption. . The device generates a chaotic movement where sufficient force is generated to open the cells and the metal ball impacts are distributed across the inner surface of the vessel to ensure that all cell mixtures receive sufficient impact to destroy the cells. As a result, spores and other difficult-to-disrupt microorganisms can be opened within seconds. This method reduces the number of steps and operating time by quickly opening difficult-to-disrupt cells while maintaining the integrity of the DNA.

Description

{Increase the therapeutic effect of HIV virus through CD4+ immune cells}

Methods and devices for disrupting cells to release or extract genomic DNA (gDNA) from internal cells are disclosed. The disclosed method uses a mixture of microscopic glass beads and cells (e.g., spores) to form a semi-dry cake that sticks to the larger metal balls and the sides of the tube during bead beating lysis. Greatly improves efficiency. Because the cells cake on the glass beads and metal balls, the cells are shocked each time the balls hit the side of the container. The device is designed to create chaotic movement with a dual purpose: firstly, to ensure that sufficient force is generated to open the cells, and secondly, to ensure that the impact of the metal balls is distributed throughout the inner surface of the vessel, so that all cell mixture is destroyed. The goal is to ensure that you receive sufficient shock. As a result, spores and other microorganisms that would normally require boiling or beating for up to 30 minutes to achieve proper disruption can be opened within seconds using the disclosed semi-dry bead beating method. This method reduces the number of steps and operating time by quickly opening difficult-to-disrupt cells while maintaining the integrity of the DNA.

Many cell-based and DNA-based assays require the release of cellular contents, including DNA, from inside the cell to facilitate analysis. Opening the cell to release its contents is called 'disruption'. For example, methods used to investigate the microbiome using DNA sequencing technology first allow the DNA to be extracted.

Requires destruction of microorganisms. Most microbiomes are communities of bacteria, archaea, and fungi that vary widely in susceptibility to fracture techniques. Differential susceptibility is a major problem for microbial population researchers who want to ensure that the hardest (usually Gram-positive) and easiest (usually Gram-negative) bacteria to disrupt are represented in proportion to their populations in the original sample. Unfortunately, most microbial disruption protocols work well for some microorganisms but not others. (Comparison of Disruption Technologies for Microbiome - Sanqing Yuan, Dora BCohen, 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): e33865doi:101371/journalpone0033865]) Additionally, the rapid and simple alkaline-based disruption technique used to recover plasmid DNA is generally used to isolate the genome, which is the target of microbiome screening. DNA is also removed (alkaline disruption opens the cells, but removes gDNA - Birnboim, HC and Doly, J, A rapid alkaline extraction procedure for

screening recombinant plasmid DNA, Nucleic Acids Res 7(6), 1979, 1513-1524]) In contrast, KOH disruption can be used to recover bacterial genomic DNA [Raghunathan, Arumugham et al "Genomic DNA Amplification from a Single Bacterium" Applied and Environmental Microbiology 716 (2005): 3342-3347

PMC Web 29 Sept 2016] Various biochemical methods, including lysozyme (enzymatic attack on peptidoglycan cell walls), strong bases (chemical attack), detergents (solubilize cell membranes), and bead beating or shaking (mechanical disruption). There are a number of disruption techniques known in the art that attack cellular integrity based on heat DNA extraction and impact microbiome profiling results: Wagner Mackenzie B, Waite DW, Taylor MW Evaluating variation in human gutmicrobiota profiles due to DNA extraction method and inter-subject differences Frontiers in Microbiology 2015;6:130 doi:103389/fmicb201500130] Most published or commercially available

DNA preparation methods utilize one or more of the above methods to disrupt cells in sequential steps that can generally take considerable time, especially when handling multiple samples at once. Although individual disruption methods are generally sufficient for applications where incomplete or partial disruption yields sufficient DNA for the protocol being performed, individual disruption methods often produce DNA from microbiome samples in proportion to the original population. It may not completely destroy certain microorganisms. For example, detergent-based disruption can destroy a subset of cells with weak cell walls and strong cell membranes, but does not open up detergent-resistant microorganisms with strong cell walls, so that DNA from detergent-resistant cells is included in the resulting DNA preparation. may appear in small amounts or may not be present. In another example, microbial bead beating sufficient to disrupt cells with strong cell membranes may shear or destroy DNA released early in the process from cells that are easily disrupted. Additionally, various crushing methods tend to be non-commercial and, when used in combination, must be performed sequentially. For example, lysozyme will not work in the presence of detergents or strong bases. Certain detergents precipitate in the presence of strong bases. Bead beating is difficult to combine with heating processes. Executing separate shredding protocols sequentially can overcome their individual drawbacks, but this increases the complexity, time, and cost involved. Importantly, detergents such as sodium dodecyl sulfate (SDS) must be removed after disruption, as SDS interferes with downstream DNA manipulation. Additionally, certain microorganisms may be resistant to disruption protocols performed sequentially, depending on the protocol order. For example, certain microorganisms with tough peptidoglycan cell walls may have an envelope of lipid bilayers that protect them from initial processing by strong bases or lysozyme.

A method and device for disrupting cells, such as bacteria, present in the microbiome, which can be completed in a short period of time, e.g., less than one minute, and importantly, with improved quality and quantity from samples that are difficult to disrupt, such as bacterial spores. A method and device for calculating genomic DNA (gDNA) are disclosed.

The methods and devices disclosed herein utilize a bead beating procedure that acts rapidly to disrupt cells, including even the most difficult microbial spores (Bacillus subtilis spores are described herein as an example), while providing proportional Applications and technologies where fragmentation is desirable or inevitable, such as high-resolution microbiome characterization

Preserve DNA of sufficient size to generate the large amplicons required. The result is a simple, rapid protocol that uniformly opens all cells within a sample to generate a more typical DNA profile from a sample containing various cellular components, such as the microbiome.

Figure 5 is a graph showing DPA release and corresponding DNA release from spores after disruption by the methods described herein. Spore disruption was measured using a terbium fluorescence assay as shown in Figure 3. The amount of DNA was measured by qPCR quantification as shown in Figure 4. Spores were bead beaten on the X-axis for up to 10 minutes as described in detail.

Figure 1 shows a 2 milliliter microcentrifuge tube containing a single 45 millimeter steel ball and 005 grams of 100 micrometer glass beads.

The subject matter regarded as the present invention is specifically stated and explicitly claimed in the claims at the end of the specification. The above and other objects, features and advantages of the present invention will become apparent from the following detailed description together with the accompanying drawings.

In an embodiment, the glass beads are added to the aqueous solution at a concentration of 40 to 99 weight percent.

Claims (1)

The release of DPA was measured for each test as an indicator of spore disruption.
Shaking at 10 and 55 BPS (beats per second) resulted in significant spore disruption. Agitation at approximately 6 BPS was variable with significant crushing for either test, demonstrating that speeds above 6 BPS are preferred. As a positive control, spores were boiled for 30 minutes.