Cell Lysis Method Research Articles

Optimizing the indirect extraction of prokaryotic DNA from soils

Abstract The objective of this work was to develop protocols to selectively extract prokaryotic DNA from soils, representative of the whole community, amenable to high-throughput whole genome shotgun sequencing. Prokaryotic cells were extracted from soils by blending, followed by gradient centrifugation. Detergent (sodium deoxycholate) was required for complete dispersion of soil aggregates and detachment of prokaryotic cells from a broad range of soil types. Repeated extractions of a given soil sample were critical to maximize cell yield. Furthermore, cells obtained through repeated extractions captured unique prokaryotic assemblages that would otherwise have been missed in single-pass extractions. DNA was isolated from extracted cells using one of the following treatments: i) lysozyme–SDS–proteinase K (enzymatic) digestion; ii) potassium ethyl xanthogenate digestion; or iii) enzymatic digestion of cells embedded in agarose plugs. In addition, these methods were compared to a commercial bead-beating extraction kit (MoBio UltraClean). Of the indirect DNA extraction methods, plug digestion generated the largest yields (up to 70% of yields obtained by direct DNA extraction) of high-molecular weight DNA (>400 kb). Thus, plug digestion is amenable to large-insert metagenomic library construction and analysis. Comparisons of banding patterns generated by RAPD-PCR and DGGE indicated that sequence composition and inferred community composition of a given extract varied greatly with DNA isolation method. While overall diversity did not change significantly with the cell lysis method, analysis of 16S rRNA gene clone libraries revealed that each extraction procedure produced unique distributions of prokaryotic phyla within the sample population.

A method for extracting tissue proteins for use in lymphocyte function assays

Currently human T-cell responses to unpurified tissue extracts cannot be easily measured because current cell lysis methods yield a lysate that is toxic. Here we describe the optimization of a protocol for extracting proteins from human tissues in a format that is compatible with the functional analysis of human T cells. The tissue was homogenized in a mixture of butan-1-ol, acetonitrile and water and then lyophilized. Lyophilized protein extracts were dissolved in 8 M urea because urea did not affect T-cell function when present at 0.08 M or less. Using this method cytokine production and proliferation responses were detected from islet, acinar and spleen extracts. Hence, our method allows the rapid preparation of human tissue lysates in a format that is compatible with the analysis T-cell responses. We suggest that this method will facilitate the analysis of adaptive immune responses to tissues in transplantation, cancer and autoimmunity.

Evaluation of Methods for Cyanobacterial Cell Lysis and Toxin (Microcystin-LR) Extraction Using Chromatographic and Mass Spectrometric Analyses

Contamination of microcystins, a family of heptapeptide hepatotoxins, in eutrophic water bodies is a worldwide problem. Due to their poisoning effects on animals and humans, there is a requirement to characterize and quantify all microcystins present in a sample. As microcystins are, for most part, intracellular toxins produced by some genera of cyanobacteria, lysing cyanobacterial cells to release all microcystins is considered an important step. To date, although many cell lysis methods have been used, little work has been conducted comparing the results of those different methods. In this study, various methods for cell lysis and toxin extraction from the cell lysates were investigated, including sonication, bead beating, freeze/thaw, lyophilization and lysing with TritonX-100 surfactant. It was found that lyophilization, followed by extraction with 75% methanol, was the most effective for extracting toxins from Microcystis aeruginosa cells. Another important step prior to the analysis is removing impurities and concentrating the target analyte. For these purposes, a C18 Sep-Pak solid phase extraction cartridge was used, with the percentage of the eluent methanol also evaluated. As a result, methanol percentages higher than 75% appeared to be the best eluting solvent in terms of microcystin-leucine-arginine recovery efficiency for the further chromatographic and mass spectrometric analyses.

Continuous cell cross over and lysis in a microfluidic device

Complex sample preparation processes are major stumbling blocks for the development of lab-on-a-chip (LOC). We herein advance a microfluidic device for chemical cell lysis using a cell cross over (CCO) technology for the purpose of minimizing the sample preparation steps. The proposed device allows cells to continuously cross over from a cell carrier to a cell lysis solution in a CCO region and to be automatically lysed. For the successful CCO and cell lysis, microflow patterns and cell movements in the CCO region are investigated by experimental as well as numerical studies. EL-4 mammalian cells are used for the demonstration of the performance of the proposed device. The DNA sample obtained from the developed device is quantitatively and qualitatively compared with the one obtained from a conventional chemical cell lysis method by using a UV–Vis spectrophotometer and gel electrophoresis. The quantitative analysis shows that the recovered DNA is 86% compared to the one obtained from the conventional chemical cell lysis.

Successful Identification of Glycine Transporter Inhibitors Using an Adaptation of a Functional Cell-Based Assay

Glycine transporter (GlyT1) function is typically measured by radiolabeled glycine uptake using lysis methods or scintillation proximity assays (SPAs), which have limited throughput. This study shows the adaptation of the standard cell lysis method to a screening assay with improved throughput and assay characteristics. The assay takes advantage of the 384-well format, standard laboratory automation, and cryopreserved CHO-K1 cells stably overexpressing human GlyT1a transporter (CHO-K1/hGlyT1a) that were validated and banked in advance of screening. The assay was evaluated for the time course of glycine uptake, K(m), V(max), Z' factor analysis, and IC(50) value determination with reference GlyT1 inhibitors. Screening of 118,000 compounds at 10 microM identified 4556 compounds (3.9%) as inhibitors. Positive compounds (>50% inhibition) were retested in the assay at 4 inhibitor concentrations. Compounds demonstrating greater than 40% inhibition at 10 microM were considered as confirmed positives, yielding a 68% confirmation rate from the original screen. To eliminate compounds that nonspecifically inhibited glycine uptake, IC(50) values were determined in both GlyT1 and GlyT2 assays, and those compounds that inhibited GlyT2 were removed from consideration. The screening campaign identified 300 small molecules as selective GlyT1 inhibitors for lead optimization, demonstrating the utility of this cost-effective method.

Evaluation of cell lysis methods for platinum metallomic studies of human malignant cells

Three cell lysis methods—freeze–thaw, osmosis, and a chemical detergent-based method—were evaluated as sample treatment procedures for platinum metallomic studies of in vitro grown human malignant cells exposed to cisplatin. The lysis methods are relatively mild, resemble those commonly used in proteomic studies, and were selected because of the proven reactivity of platinum drug metabolites and indications that platinum in exposed cells and plasma is mainly associated with proteins. The chemical method gave an absolute lysis efficiency of greater than 80%, whereas the freeze–thaw and osmosis methods gave approximately 30% lower efficiency. The within- and between-batch lysis reproducibilities were, for all methods, better than 20 and 24% relative standard deviations, respectively. Total platinum concentration normalized to lysate protein content was statistically the same for all lysis methods. Reagents in the chemical lysis buffer did, however, react with platinum analyte compounds, making this method unsuitable for analysis of reactive compounds or for metallome profiling encompassing analytes with unknown reactivity. Of the lysis methods evaluated here, osmosis gave the highest cisplatin recovery, likely because this protocol is chemically inert and can be carried out at a constant low temperature. Therefore, it is the recommended cell lysis method for the determination of reactive and unknown intracellular platinum compounds.

A Survey of Polymerase Chain Reaction (PCR) Amplification Studies of Unicellular Protists Using Single‐Cell PCR

We surveyed a variety of studies that have used single-cell polymerase chain reaction (SC-PCR) to examine the gene sequences of a diversity of unicellular protists. Representatives of all the Super-Groups of eukaryotes have been subjected to SC-PCR with ciliates and dinoflagellates being most commonly examined. The SC-PCR was carried out either by directly amplifying a single lysed cell or by first extracting DNA and following this with amplification of the DNA extract. Cell lysis methods included heating, freezing, mechanical rupture, and enzyme digestion. Cells fixed or preserved with ethanol, methanol, and Lugol's have also been used successfully. Heminested or seminested PCR might follow the initial PCR, whose products were then directly sequenced or cloned and then sequenced. The methods are not complicated. This should encourage protistologists to use SC-PCR in the description of new or revised taxa, especially rare and unculturable forms, and it should also enable the probing of gene expression in relation to life history stages.

A method for obtaining DNA from compost

An effective cell lysis method for extraction of bacterial genomic DNA from compost was developed in this study. Enzymatic disruption method, physical-chemical combination method, and commercial kit method were used to extract DNA from compost samples and were compared by analyzing DNA yield and efficient cell lysis. The results showed that all the three methods can be used to extract high-quality DNA from compost, but the enzymatic method had better cell lysis efficiency and DNA yields than others without the use of special equipment and expensive spending. Comparison of different methods for lysing gram-positive bacteria Bacillus subtilis indicated that the enzymatic cell lysis is superior for destroying the gram-positive cell wall. Spin-bind DNA column was used for DNA purification, and the purity of the purified sample was checked by polymerase chain reaction to amplify a region of the 16S rRNA. Results indicated that the part of 16S rRNA were amplified from all the purified DNA samples, and all the amplification products could be digested by the restriction enzyme HhaI.

Proteomic detection of proteins involved in perchlorate and chlorate metabolism

Mass spectrometry and a time-course cell lysis method were used to study proteins involved in perchlorate and chlorate metabolism in pure bacterial cultures and environmental samples. The bacterial cultures used included Dechlorosoma sp. KJ, Dechloromonas hortensis, Pseudomonas chloritidismutans ASK-1, and Pseudomonas stutzeri. The environmental samples included an anaerobic sludge enrichment culture from a sewage treatment plant, a sample of a biomass-covered activated carbon matrix from a bioreactor used for treating perchlorate-contaminated drinking water, and a waste water effluent sample from a paper mill. The approach focused on detection of perchlorate (and chlorate) reductase and chlorite dismutase proteins, which are the two central enzymes in the perchlorate (or chlorate) reduction pathways. In addition, acetate-metabolizing enzymes in pure bacterial samples and housekeeping proteins from perchlorate (or chlorate)-reducing microorganisms in environmental samples were also identified.

Cell lysis methods for high‐throughput screening or miniaturized assays

The widely and routinely used cell lysis techniques are still being evolved to address the technical needs of micro-scale sample processing, for example in high-throughput screening or miniaturized assays. This review focuses on several recently emerged mechanical, physical and biological methods designed for lysing cells on small scales for screening or analysis.

All-in-One Tube Method for Quantitative Gene Expression Analysis in Oligo-dT30 Immobilized PCR Tube Coated with MPC Polymer

In this report, we have developed a novel quantitative RT-PCR protocol in which the procedure including mRNA purification can be performed in an all-in-one tube. To simplify gene expression analysis, oligo-dT(30) immobilized PCR tubes were used serially to capture mRNA, synthesize solid-phase cDNA, and amplify specific genes. The immobilized oligo-dT(30) can efficiently capture mRNA directly from crude human cell lysates. The captured mRNA is then amplified by one-step reverse transcription PCR (RT-PCR) with initial cDNA synthesis followed by PCR. In RT-PCR, this new reusable PCR tube device can be employed for multiple PCR amplifications with different primer sets from a solid-phase oligo-dT(30) primed cDNA library. This paper introduces a novel and highly reliable all-in-one tube method for rapid cell lysis, followed by quantitative preparation and expression analysis of target mRNA molecules with small amounts of sample. This procedure allows all steps to be carried out by sequential dilution in a single tube, without chemical extraction. We demonstrate the utility of this novel method by quantification of two housekeeping genes, beta-actin and GAPDH, in HeLa cells. We believe this new PCR device can be useful as a platform for various mRNA expression analyses, including basic research, drug screening, and molecular toxicology, as well as for molecular pathological diagnostics.

Method to Screen Substrates of Apical Sodium-Dependent Bile Acid Transporter

Human apical sodium-dependent bile acid transporter (hASBT) is a potential prodrug target under study. Development of prodrugs that target hASBT may yield compounds with low solubility and/or susceptibility to hydrolysis. A transport uptake method is needed that increases compound solubility and avoids NaOH for cell lysis during postexperimental cell sample preparation. The overall goal was to develop an assay method to screen for hASBT uptake of novel compounds. The first objective was to determine the maximum cosolvent concentrations that are compatible with an hASBT active transport assay. The second objective was to develop a NaOH-free cell lysis method to process cell samples from these uptake studies. The following cosolvents were studied: dimethylacetamide (DMAC), dimethylformamide (DMF), dimethylsulfoxide (DMSO), ethanol, methanol, polyethylene glycol-400, propylene glycol, and dioxane. Initial studies included taurocholate flux studies across hASBT-Madin-Darby canine kidney monolayers using up to 10% cosolvent, as well as cytotoxicity studies. The effect of selected cosolvent concentrations on the hASBT Michaelis-Menten kinetic parameters was evaluated. Additionally, two acetonitrile-based cell lysis methods that do not use NaOH were evaluated in terms of percent sample recovery and hASBT kinetic parameters. Results showed that the maximum permissible cosolvent concentrations for hASBT uptake studies, without compromising assay results or causing cytotoxicity, are 1% DMAC, 1% DMF, 2.5% DMSO, 2.5% methanol, and 2.5% ethanol. Additionally, both NaOH-free, acetonitrile-based cell lysis methods provided similar recovery and hASBT results, compared to NaOH method. Hence, an assay method was developed to screen for active transport, allowing for cosolvents that can solubilize compounds and avoid NaOH sample treatment, which can otherwise degrade compound.

Facile, reagentless and in situ release of Escherichia coli intracellular enzymes by heat-inducible autolytic vector for high-throughput screening

In an effect to broaden the application of the heat-inducible autolytic vector pUC18-cI857/p(R)-SRRz-rrnB previously developed, a new vector pUC18-cI857/p(R)(T41C)-SRRz-rrnB (pEAS-1b) was quantitatively characterized under various growth temperatures, heat induction temperatures and durations, and IPTG (isopropyl beta-d-thiogalactoside) induction times, after resolving its erratic lysis profile found previously. Escherichia coli BL21 cells harboring this vector grew well at temperatures <36 degrees C, and lysed efficiently (97.0 +/- 0.8%) just 0.5 h after heat induction at 42 degrees C for 30 min when cell growth was performed at 35 degrees C. Application of this autolytic vector either in 96-well plates, or on nitrocellulose membranes, or on agar plates led to facile, efficient and consistent release of intracellular recombinant enzymes (e.g., a lysis efficiency of 91.8 +/- 1.1% was obtained in 96-well plates). Further application in directed evolution was illustrated by improving the thermostability of amadoriase using this vector. This reagentless and in situ cell lysis method has the potentials for lysis of miniaturized samples in clinical diagnosis and bioanalytical detection, and even for lysis of cells in the microarray format.

An efficient and reliable DNA extraction method for preimplantation genetic diagnosis: a comparison of allele drop out and amplification rates using different single cell lysis methods

To evaluate methods of DNA extraction from single cells for their suitability to amplify and provide a correct diagnosis of target disease genes. Experimental study. University hospital laboratory. Two normal adult male and female blood donors. Exon 51 of the dystrophin gene and the ZFX/ZFY gene were amplified from single lymphocytes using nested PCR. Five different methods of DNA extraction were tested (lysis in distilled water with freezing and thawing using liquid nitrogen, lysis in distilled water, alkaline lysis buffer, Proteinase K/sodium dodecyl sulfate (SDS) buffer, and N-lauroylsarcosine salt solution). Allele drop out and amplification rate. The amplification efficiency from single unaffected lymphocytes was 89.0% using the liquid nitrogen method, 88.1% with the distilled water lysis method, 97.5% with the alkaline lysis buffer method, 91.5% with the Proteinase K/SDS lysis buffer method, and 84.8% using the N-lauroylsarcosine salt solution method. The mean allele drop out rate was 16.7%, 43.9%, 2.0%, 9.8%, and 18.9%, respectively, for each lysis method using single male lymphocytes as a template. Based on these results, DNA extraction using an alkaline lysis buffer results in more efficient rates of DNA amplification and less allele drop out than the other methods of DNA extraction tested. This method is suitable for the lysis of single cells in clinical preimplantation genetic diagnosis.

Proteinase K challenged by a novel protease

Proteinase K is used in forensic DNA extraction methods for cell lysis and degradation of proteins. Here we compare Proteinase K with a novel protease. We conclude that there is no need to exchange Proteinase K in our methods.

Cell Lysis Method Affects Assessment of Microbial Diversity Based on Ribotyping Analysis

The microbial community in Kawah Hujan, Kamojang, West Java, Indonesia, was analyzed using 16S-rRNA-gene-sequencing combining with denaturing-gradient-gel electrophoresis (DGGE) technique. Two different cell lysis methods, enzymatic-based, and physical treatment-based DNA extraction, were used to isolate chromosomal DNA for 16S rDNA gene-fragment amplification. The DGGE profiles showed some differences in banding pattern that were obtained from both cell lysis methods. The DNA sequence analysis of the individual DGGE bands revealed that most of the band sequences obtained by physical treatment were close to 16S rRNA gene fragments from the bacterial domain, while most of band sequences performed by enzymatic method had high homology with 16S rRNA gene fragments from archaeal domain. Further analysis of the sequences from both methods performed by comparisons with the Ribosomal Database Project showed that some of DGGE sequences from Kawah Hujan consisted unique 16S rDNA sequences.

A High Through-Put Protocol for Quantifying Nucleic Acids in Individual Microcrustaceans Using New Generation RNA and DNA Specific Dyes

Quantifying growth rate in microcrustacea is important to plankton biologists when determining primary productivity. RNA:DNA ratios have been widely used as an indicator of instantaneous growth rate in microcrustaceans in nutritional and environmental studies. It is important when measuring nucleic acids in these small organisms that it is done efficiently and with high sensitivity, so that small differences in nucleic acid content can be accurately detected. A technique is presented for the accurate, high-throughput determination of RNA and DNA content in individual microcrustacea, based on two new generation fluorescent dyes that bind preferentially to either RNA (Quant-iT™ RNA) or DNA (Quant-iT™ DNA). Contaminant interference (either from detergents or enzymes used in the chemical cell lysis process, or cell based contaminants such as proteins) can significantly affect fluorescence readings of these dyes. Through the development of our protocol, we show that proteinase K was an ineffective cell lysis method, whereas sarcosyl efficiently lysed cells and could be used when its final assay concentration was diluted to avoid interference with dye fluorescence. Organic solvents were also utilized to remove cell based contaminants that caused an interference with the Quant-iT™ DNA dye fluorescence. Through the use of specific RNA and DNA dyes and by achieving low levels of technical variation, this study developed a rapid and reliable method for nucleic acid extraction and quantification in Artemia.

Lysis efficiency of standard DNA extraction methods for Halococcus spp. in an organic rich environment

The extraction of nucleic acids from a given environment marks a crucial and essential starting point in any molecular investigation. Members of Halococcus spp. are known for their rigid cell walls, and are thus difficult to lyse and could potentially be overlooked in an environment. Furthermore, the lack of a suitable lysis method hinders subsequent molecular analysis. The effects of six different DNA extraction methods were tested on Halococcus hamelinensis, Halococcus saccharolyticus and Halobacterium salinarum NRC-1 as well as on an organic rich, highly carbonated sediment from stromatolites spiked with Halococcus hamelinensis. The methods tested were based on physical disruption (boiling and freeze/thawing), chemical lysis (Triton X-100, potassium ethyl xanthogenate (XS) buffer and CTAB) and on enzymatic lysis (lysozyme). Results showed that boiling and freeze/thawing had little effect on the lysis of both Halococcus strains. Methods based on chemical lysis (Triton X-100, XS-buffer, and CTAB) showed the best results, however, Triton X-100 treatment failed to produce visible DNA fragments. Using a combination of bead beating, chemical lysis with lysozyme, and thermal shock, lysis of cells was achieved however DNA was badly sheared. Lysis of cells and DNA extraction of samples from spiked sediment proved to be difficult, with the XS-buffer method indicating the best results. This study provides an evaluation of six commonly used methods of cell lysis and DNA extraction of Halococcus spp., and the suitability of the resulting DNA for molecular analysis.

Phosphoproteomics

AbstractProtein phosphorylation is one of the most important mechanisms of regulating protein function in cells, and it plays an important role in controlling diverse biological processes, including cellular proliferation, migration, and metabolism. The term “phosphoproteome” refers to the complement of proteins that undergoes phosphorylation, the extent of their phosphorylation status at the level of individual residues, as well as the dynamics of the phosphorylation events in response to various stimuli. This unit provides methods for enrichment of phosphorylated proteins and peptides using anti‐phosphotyrosine antibodies or titanium dioxide, respectively. Support protocols are provided for two detergent‐free cell lysis methods, fractionation of proteins prior to enrichment, and use of stable isotope labeling by amino acids in cell culture (SILAC) method for studying dynamics of phosphorylation events. Curr. Protoc. Protein Sci. 50:24.4.1‐24.4.21. © 2007 by John Wiley &amp; Sons, Inc.

An efficient and reliable DNA extraction method for preimplantation genetic diagnosis: a comparison of allele drop-out and amplification rate using different single cell lysis methods

Single cell polymerase chain reaction (PCR) for preimplantation genetic diagnosis (PGD) requires highly efficient cell lysis system. We compared of allele drop-out (ADO) and amplification rate in different DNA extraction methods from single lymphocytes.