Electricity-free, sequential nucleic acid and protein isolation

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern for governments, industries and medical professionals worldwide. For example, EHECs, combined with Shigella, are responsible for the deaths of approximately 325,000 children each year and are particularly prevalent in the developing world where laboratory-based identification, common in the United States, is unavailable (1). The development and distribution of low cost, field-based, point-of-care tools to aid in the rapid identification and/or diagnosis of pathogens or disease markers could dramatically alter disease progression and patient prognosis. We have developed a tool to isolate nucleic acids and proteins from a sample by solid-phase extraction (SPE) without electricity or associated laboratory equipment (2). The isolated macromolecules can be used for diagnosis either in a forward lab or using field-based point-of-care platforms. Importantly, this method provides for the direct comparison of nucleic acid and protein data from an un-split sample, offering a confidence through corroboration of genomic and proteomic analysis. Our isolation tool utilizes the industry standard for solid-phase nucleic acid isolation, the BOOM technology, which isolates nucleic acids from a chaotropic salt solution, usually guanidine isothiocyanate, through binding to silica-based particles or filters (3). CUBRC's proprietary solid-phase extraction chemistry is used to purify protein from chaotropic salt solutions, in this case, from the waste or flow-thru following nucleic acid isolation(4). By packaging well-characterized chemistries into a small, inexpensive and simple platform, we have generated a portable system for nucleic acid and protein extraction that can be performed under a variety of conditions. The isolated nucleic acids are stable and can be transported to a position where power is available for PCR amplification while the protein content can immediately be analyzed by hand held or other immunological-based assays. The rapid identification of disease markers in the field could significantly alter the patient's outcome by directing the proper course of treatment at an earlier stage of disease progression. The tool and method described are suitable for use with virtually any infectious agent and offer the user the redundancy of multi-macromolecule type analyses while simultaneously reducing their logistical burden.     

Perfectly complementary nucleic acid enzymes

The ability to maximize the use of available nucleic acid sequence space would have been crucial during the presumed RNA world and confers selective advantage in many contemporary organisms. One way to access sequence space at a higher density would be to make use of both strands of a duplex nucleic acid for the production of functional molecules. As a demonstration of this possibility, two pairs of nucleic acid enzymes were engineered to be perfect complements, each with the capacity to adopt a distinct structure and catalyze a particular chemical transformation. Both members of each pair of enzymes exhibited nearly the same level of activity as the canonical form of the corresponding catalytic motif. The ability to generate functional nucleic acids encoded by both strands of a duplex has implications for the evolution of catalytic nucleic acids and the prospects for realizing maximum functionality from a given genetic sequence. Present address (Scott T. Kuhns): CancerVax Corp., 9393 Towne Center Drive, San Diego, CA 92121, USA     

Use of aurintricarboxylic acid as an inhibitor of nucleases during nucleic acid isolation.

Aurintricarboxylic acid (ATA) is a general inhibitor of nucleases. ATA has been shown to inhibit the following enzymes in vitro: DNAse I, RNAse A, S1 nuclease, exonuclease III, and restriction endonucleases Sal I, Bam HI, Pst I and Sma I. The observed inhibition is consistent with the proposal by Blumenthal and Landers (BBRC 55, 680, 1973) that most nucleic acid binding proteins will be sensitive to ATA. The action of ATA as a nuclease inhibitor can be used to advantage in the isolation of cellular nucleic acids.     

A modified Escherichia coli protein production strain expressing staphylococcal nuclease,capable of auto-hydrolysing host nucleic acid

The large-scale production of recombinant biotherapeutics, particularly recombinant proteins, provides significant process and regulatory challenges to the biotechnology industry in order to meet the regulatory agencies stringent requirements in a cost-effective manner. Host cell derived nucleic acid causes problems from both a process and a regulatory perspective, as high molecular weight chromosomal DNA is responsible both for the viscosity of cell lysates, and it is a source of heterologous DNA sequences whose inclusion in the final product must be prevented. We have constructed a modified Escherichia coli JM107 expression host (JMN), containing a staphylococcal nuclease expression cassette, integrated into the host chromosome at the dif locus. The nuclease is expressed as a fusion to the ompA signal peptide, and is translocated to the periplasm of the cell, protecting the cytoplasmic nucleic acid from any toxic activity. The nuclease is released during cell lysis, where it subsequently acts to hydrolyse host nucleic acid present in the lysate. Results with this strain show that sufficient levels of nuclease activity are produced to completely auto-hydrolyse the host's chromosomal DNA to a size non-visible on 1% agarose gel, generating a markedly lower lysate viscosity. This provides a suitable methodology to remove heterologous DNA sequences early in the product stream and decrease lysate viscosity, improving the efficiency of downstream processing and product yield, whilst avoiding the addition of exogenous nuclease and its prohibitive costs at large-scale.     

Binding preferential of chickpea cold shock protein during nucleic acid interactions

Cold shock proteins (CSPs) have a widespread occurrence from prokaryotes to eukaryotes including plants. These proteins are known to possess nucleic acid binding properties. CSPs have a single cold shock domain in prokaryotes while N-terminal and C-terminal flanking regions are present in eukaryotic CSPs. The objective of this study was to investigate nucleic acid binding preferential for the chickpea CSP. Full cDNA of chickpea CSP was cloned and sequenced. The sequence was submitted to GenBank (accession no. KM036036) at NCBI. Multiple sequence alignment and phylogenetic analysis further revealed that the inferred amino acid sequence belongs to CSP family. Molecular docking was performed between the CSP and variety of nucleic acids entities. These results suggest that CSPs of chickpea possess preferential binding affinity for single stranded nucleic acids. Docking results suggest that homo-polymer entities of RNA polyU RNA (20mer) form most stable complex.     

The problem of nuclease activity in nucleic acid hybridization reactions: Theoretical considerations

The presence of nucleuses during DNA-DNA or DNA-RNA hybridization reactions alters the kinetics of hybrid formation. Unfortunately, while the effect (even of small amounts of nuclease) on the C₀t curve may be large, it may not be readily detectable. The effect of various types of nuclease is shown. As many nucleuses cause a shift in the position rather than a change in shape of the curve, all studies involving nucleic acid hybridization should assay for the presence of nucleases and care must be taken to avoid their presence as contaminants.     

Improved RNA isolation from cells in tissue culture using a commercial nucleic acid extractor.

The Applied Biosystems 340A Nucleic Acid Extractor automates isolation of either DNA or RNA from tissue or cells in culture. We have found that several modifications to the manufacturer's recommended protocol greatly improve the quality of RNA that can be routinely isolated from cells in culture. These modifications include lysis of monolayer cells directly on plates, centrifuging samples after homogenization to remove precipitable RNase contaminants and purging the instrument's reagent lines with 0.1% diethyl pyrocarbonate. These simple modifications enhance both RNA quality and reproducibility of yield.     

A microwave-based method for nucleic acid isolation from environmental samples

AIMS: A simple and rapid method was described for DNA isolation directly from activated sludge or other environmental sources, including soil and sediments. METHODS AND RESULTS: The present method is based on microwave thermal shock and provides DNA suitable for further analysis. It is also effective for RNA extraction. CONCLUSION: The protocol is effective, easy, fast and does not require the use of expensive equipment or reagents. SIGNIFICANCE AND IMPACT OF THE STUDY: The described method can be applied to difficult substrates in environmental microbiology studies.     

Efficient protoplast isolation from fungi using commercial enzymes

Several commercial polysaccharases have been compared for their ability to liberate protoplasts from fungi. These enzymes were found to contain side activities capable of hydrolysing fungal cell walls. Protoplasts have been commonly isolated from fungi using enzyme systems prepared by workers in their own laboratories. However, these procedures are time consuming and considerable variation may be found between different batches of enzyme. The present study shows that high yields of protoplasts can be prepared from a variety of fungi using relatively cheap commercial enzymes. The yields obtained were normally as good as or better than those previously produced.     

Multiple roles for ATP hydrolysis in nucleic acid modifying enzymes

Enzymes that operate on nucleic acid substrates are faced with the unusual situation where the substrate is much larger than themselves. Despite the potential to promote catalysis by utilizing the significant binding energy available through their interaction with substrate, ATP hydrolysis is frequently a part of the mechanism of these enzymes. The reasons for this have become clearer in recent years, and a surprising range of ways that these enzymes utilize the free energy of hydrolysis of ATP has been revealed. This review describes these different mechanisms in the context of the biochemical reactions that they support.