Rapid preparation of denatured double-stranded DNA templates for sequencing

This article proposes protocol for rapid preparation (ds) DNA templates for sequencing based on double-stranded DNA denaturation and its recovery by extraction with Wizard DNA purification resin (Promega). This method is an alternative to commonly used procedure employing denatured-DNA recovery by ethanol precipitation.     

Rapid and reliable dideoxy sequencing of double-stranded DNA

We report a simple and reliable protocol for nucleotide sequencing using the Sanger dideoxy technique on linearized double-stranded DNA molecules with specific oligonucleotide primers. The method is demonstrated for restriction fragments cloned into the plasmid vectors pSP64 and pSP65 using two vector-specific primers, the M 13 reverse primer and a new SP6 primer, flanking the multiple cloning site. Template DNA may be prepared by a rapid alkaline lysis procedure. Mild linearization conditions with the appropriate restriction endonuclease avoid the appearance of artifact bands.     

Rapid isolation and sequencing of purified plasmid DNA from Bacillus subtilis.

We report two methods for isolation of plasmid DNA from the gram-positive bacterium Bacillus subtilis. The protoplast alkaline lysis procedure was developed for general use, and the protoplast alkaline lysis magic procedure was developed for isolation of DNA for sequencing. Both procedures yielded large amounts of high-quality DNA in less than 1 h, while current protocols require 4 to 7 h to perform and give lower yields and quality. Plasmid DNA was obtained from strains containing either high- or low-copy-number plasmids. In addition, the procedures were easily adapted to yield large amounts of plasmid DNA suitable for sequencing from another gram-positive organism, Staphylococcus aureus. Further, we demonstrated that neither chloramphenicol, used for plasmid selection, nor the mutation recE4 reduced plasmid DNA yield from the strains we examined.     

Rapid screening of complex DNA samples by single-molecule amplification and sequencing

Microbial cloning makes Sanger sequencing of complex DNA samples possible but is labor intensive. We present a simple, rapid and robust method that enables laboratories without special equipment to perform single-molecule amplicon sequencing, although in a low-throughput manner, from sub-picogram quantities of DNA. The method can also be used for quick quality control of next-generation sequencing libraries, as was demonstrated for a metagenomic sample.     

Rapid and simple removal of contaminating RNA from plasmid DNA without the use of RNase

A procedure for the removal of RNA and RNA fragments from large quantities of pBR322 plasmid DNA without the use of RNase is described. Sephacryl S-300 is employed for the separation of low-molecular-weight RNA from plasmid DNA molecules on the basis of gel filtration. The technique thus circumvents many of the dangers associated with treating plasmid DNA preparations with RNase. The procedure should be generally applicable to the purification of virtually any type of plasmid DNA isolated from a bacterial host.     

Rapid removal of acetimidoyl groups from proteins and peptides. Applications to primary structure determination.

Methylamine buffers can be used for the rapid quantitative removal of acetimidoyl groups from proteins and peptides modified by treatment with ethyl or methyl acetimidate. The half-life for displacement of acetimidoyl groups from fully amidinated proteins incubated in 3.44 M-methylamine/HCl buffer at pH 11.5 and 25 degrees C was approx. 26 min; this half life is 29 times less than that observed in ammonia/HCl buffer under the same conditions of pH and amine concentration. Incubation of acetimidated proteins with methylamine for 4 h resulted in greater than 95% removal of acetimidoyl groups. No deleterious effects on primary structure were detected by amino acid analysis or by automated Edman degradation. Reversible amidination of lysine residues, in conjunction with tryptic digestion, has been successfully applied to the determination of the amino acid sequence of an acetimidated mouse immunoglobulin heavy chain peptide. The regeneration of amino groups in amidinated proteins and peptides by methylaminolysis makes amidination a valuable alternative to citraconoylation and maleoylation in structural studies.     

Rapid incorporation of label from ribonucleoside disphosphates into DNA by a cell-free high molecular weight fraction from animal cell nuclei

A readily sedimentable nuclear fraction from Chinese hamster embryo fibroblast (CHEF/18) cells catalyzes incorporation of 14C-rCDP into DNA. Data indicated that this incorporation is made possible by the conversion of rCDP into a small and functionally compartmentalized, rather than a large and freely diffusible, pool of dCTP. This catalytically active sedimentable fraction from S phase CHEF/18 cells or actively replicating calf thymus cells contains nascent and template DNA, and numerous enzymes required for DNA biosynthesis including ribonucleoside diphosphate reductase, thymidylate synthetase, dihydrofolate reductase, DNA methylase, topoisomerase and DNA polymerase. We have named this catalytically active macromolecule the replitase. The replitase fraction contained spherical particles with a diameter of approximately 24 to 30 nm and had an estimated molecular weight on the order of 5 X 10(6).     

Rapid identification of multi-strain HBV infection in patient by high-throughput DNA sequencing

Hepatitis B is a well-known risk factor for the development of liver cancer and is closely associated with patient morbidity and mortality. Viral mutants and variants have the potential to evade immune response and prolong infection, and thus it is crucial to develop a methodology for the rapid identification of multi-strain hepatitis infections in patients. Here we describe a method based on selective region amplification of viral genome and deep sequencing, which may be used for rapid identification of multi-strain hepatitis B virus (HBV) infection in patients. The method works even with significantly low amounts of patients’ serum samples, where the wet-lab procedures take about 1.5 days, followed by a quick bioinformatic analysis to reveal the final results. Our method can potentially be applied to the rapid and reliable identification of multi-strain HBV infection and help improve treatment regiments.     

Two-step cycle sequencing improves base ambiguities and signal dropouts in DNA sequencing reactions using energy-transfer-based fluorescent Dye terminators

The use of automated fluorescent DNA sequencer systems and PCR-based DNA sequencing methods plays an important role in the actual effort to improve the efficiency of large-scale DNA analysis. While dideoxy-terminators labeled with energy-transfer dyes (BigDyes) provide the most versatile method of automated DNA sequencing, premature terminations result in a substantially reduced reading length of the DNA sequence. Premature terminations are usually evidenced by base ambiguities and are often accompanied by diminished signal intensity from that point on in the sequence. I studied a two-step protocol for Taq cycle sequencing using the ABI BigDye terminator for reducing premature terminations in DNA sequences. I demonstrate that combining the annealing step with the extension step at one temperature (60°C) reduces premature terminations in DNA sequences that regularly contain premature terminations when the three temperature steps are used. This modification significantly increases the number of accurately read bases in DNA sequences.     

Genomic-scale capture and sequencing of endogenous DNA from feces

Genomic-level analyses of DNA from non-invasive sources would facilitate powerful conservation and evolutionary studies in natural populations of endangered and otherwise elusive species. However, the typical low quantity and poor quality of DNA that is extracted from non-invasive samples have generally precluded such work. Here we apply a modified DNA capture protocol that, when used in combination with massively-parallel sequencing technology, facilitates efficient and highly-accurate resequencing of megabases of specified nuclear genomic regions from fecal DNA samples. We validated our approach by comparing genetic variants identified from corresponding fecal and blood DNA samples of six western chimpanzees (Pan troglodytes verus) across more than 1.5 megabases of chromosome 21, chromosome X, and the complete mitochondrial genome. Our results suggest that it is now feasible to conduct genomic studies in natural populations for which constraints on invasive sampling have otherwise long been a barrier. The data we collected also provided an opportunity to examine western chimpanzee genetic diversity at unprecedented scale. Despite high mitochondrial genome diversity (π = 0.585%), western chimpanzees have a low ratio (0.42) of X chromosomal (π = 0.034%) to autosomal (chromosome 21 π = 0.081%) sequence diversity, a pattern that may reflect an unusual demographic history of this subspecies.     

Rapid and simple preparation of plasmids suitable for dideoxy DNA sequencing and other purposes.

Plasmid DNA released from bacteria by boiling in the presence of lysozyme and Triton x-100 and without further purification can be sequenced by the dideoxy method using T7 DNA polymerase, when conditions during alkali denaturation and subsequent ethanol precipitation are adjusted to remove contaminants. The samples remain in the same microcentrifuge tubes from the harvesting of the bacteria until the splitting of the sample into four aliquots for the termination reactions. Less background label is observed with end-labelled primers (radioactivity or fluorescence), but even when radioactive nucleotides are incorporated during the sequencing reactions, 250 bases or more can be read from template prepared from 1.5 ml bacterial culture. The DNA can also be cut by restriction enzymes; the purification procedure described thus provides the rapid preparation of plasmids for a variety of purposes.     

Hijacked DNA repair proteins and unchained DNA polymerases

Somatic hypermutation of immunoglobulin (Ig) genes occurs at a frequency that is a million times greater than the mutation in other genes. Mutations occur in variable genes to increase antibody affinity, and in switch regions before constant genes to cause switching from IgM to IgG. Hypermutation is initiated in activated B cells when the activation-induced deaminase protein deaminates cytosine in DNA to uracil. Uracils can be processed by either a mutagenic pathway to produce mutations or a non-mutagenic pathway to remove mutations. In the mutagenic pathway, we first studied the role of mismatch repair proteins, MSH2, MSH3, MSH6, PMS2 and MLH1, since they would recognize mismatches. The MSH2–MSH6 heterodimer is involved in hypermutation by binding to U:G and other mismatches generated during repair synthesis, but the other proteins are not necessary. Second, we analysed the role of low-fidelity DNA polymerases η, ι and θ in synthesizing mutations, and conclude that polymerase η is the dominant participant by generating mutations at A:T base pairs. In the non-mutagenic pathway, we examined the role of the Cockayne syndrome B protein that interacts with other repair proteins. Mice deficient in this protein had normal hypermutation and class switch recombination, showing that it is not involved.     

Rapid and effective removal of perfluorooctanoic acid from proteomics samples

We recently demonstrated that perfluorooctanoic acid (PFOA), a volatile surfactant, is as effective as sodium dodecyl sulfate at solubilizing the membrane proteins. PFOA can be removed by repeated evaporation prior to mass spectrometry analysis. However, the removal of PFOA by evaporation is a lengthy process that takes approximately 6 h. Toward the goal of decreasing the length of time required to remove PFOA from protein digests, we tested the efficiency of PFOA removal and subsequent peptide recovery using strong cation exchange (SCX) chromatography, hydrophilic interaction chromatography (HILIC), fluorous solid phase extraction (FSPE), and anion exchange (ANX) chromatography. We found that all these chromatographic techniques except ANX chromatography remove PFOA thoroughly from protein digest. Peptide recovery rates from the SCX chromatography varied widely; nonacidic peptides were recovered at a rate of up to 95%, while acidic peptides were recovered at a rate of less than 10%. On the other hand, acidic peptides were recovered well from HILIC, while peptides whose pIs are greater than 6 were recovered poorly. Peptide recovery using FSPE was considerably lower, less than 10% for most of the peptides. These results indicate that the SCX and HILIC chromatography provide a more rapid alternative to the evaporation method for applications in which recovery of entire set of peptides is not required.     

Rapid identification of bacterial isolates from aqueous kava (Piper methysticum) extracts by polymerase chain reaction and DNA sequencing

Aim:  Fresh kava beverages have a limited shelf life under refrigerated conditions. The objective of this study was to isolate and identify bacteria in aqueous extracts of kava rhizome.
Methods and Results:  The internal part of kava rhizome was used to minimize soil contamination. Three kava extracts were prepared, serially diluted and plated on nutrient agar. Isolated colonies were identified by sequencing polymerase chain reaction amplicons targeting the eubacterial 16S rDNA and the tuf gene of Staphylococcus . Seventy-five bacterial isolates belonged to 16 genera. Bacillus , Cellulomonas , Enterococcus , Pectobacterium and Staphylococcus were identified in all kava extracts.
Conclusions:  Kava rhizome contains large amounts of starch and fibre, which justify the presence of polysaccharide-degrading bacteria in the extracts. Bacillus cereus group and Staphylococcus species may produce toxins and cause foodborne illness.
Significance and Impact of the Study:  The results of this study provide fundamental information that may be used to enhance the microbial quality and safety of kava beverages.     

Kinetics of nonspecific binding reactions of proteins with DNA flexible coils: site-based and molecule-based association reactions

Domain effects on the pseudo-first-order kinetics of the reversible and irreversible association of proteins or other ligands with nucleic acids containing multiple binding sites are treated using the classical reaction-diffusion equation applied to a spherical cell model of the nucleic acid solution and a diffuse-sphere model for the nucleic acid chain molecule. Both uniform and Gaussian distributions of chain segments are analyzed. In general, the details of the segment distribution do not have a major effect on the kinetics of association. Domain effects are best examined experimentally by determining the effect of the molecular weight of the nucleic acid on the kinetics of the association reaction. A theoretical framework is presented that permits such data to be analyzed simply. Kinetic studies over a wide range of nucleic acid molecular weights are required in order to separate the contributions of diffusion and reaction to the observed kinetics, and to determine the contributions of site-based and molecule-based elements to the rate constants.     

Rapid determination of bacterial ribosomal RNA sequences by direct sequencing of enzymatically amplified DNA

Ribosomal RNA sequences are an appealing target for bacterial classification as well as for development of group- or species-specific DNA probes. Using the polymerase chain reaction and synthetic primers, the feasibility of this gene amplification technique for rapid sequence determination of the major 16S ribosomal RNA domains from small amounts of input DNA is demonstrated. Information useful for phylogenetic classification as well as for construction of specific DNA probes may be obtained by comparison with known sequences.