Oligonucleotide purification in milligram quantities

An inexpensive and simple procedure for the purification of synthetic oligonucleotides is described. Hands-on time is only 30 to 40 minutes and multiple samples can be prepared simultaneously. One- to two-milligram quantities can easily be handled by a single column with no further purification required for the DNA to be used in a wide variety of molecular biological uses.     

Isolation and random amplified polymorphic DNA analysis of genomic DNA from soybean embryos

Summary An isolation procedure was developed for the extraction of genomic DNA and Random Amplified Polymorphic DNA (RAPD) analysis using individual soybean embryos. This procedure can be used to quickly and efficiently isolate DNA from a large number of individuals. DNA isolations were analyzed for total yield, integrity, and usefulness as a template in RAPD analysis.     

PCR amplification of streptococcal DNA using crude cell lysates

Gram-positive organisms such as streptococci and enterococci are often difficult to lyse. Obtaining DNA for procedures such as PCR amplification usually requires a large scale isolation for each strain under investigation. We describe a simple procedure for small volumes of whole cells, involving pretreatment with detergent and proteinase that allows for efficient release of DNA for PCR amplification. This procedure is fast, reproducible, can be used with a large number of samples, and has been successfully applied to a variety of streptococcal and enterococcal strains.     

A biotinylated DNA probe to detect bacterial cells in artificially contaminated foodstuffs

A biotin-labelled DNA probe was used in a dot-blot hybridization test to demonstrate the presence of Escherichia coli in a variety of artificially contaminated foodstuffs. Positive hybridization was detected by using a streptavidine/polyalkaline phosphatase conjugate to generate an insoluble coloured precipitate in the presence of an appropriate dye. The colour intensity was measured with a computer-controlled image analysis system which assessed objectively the hybridization signal produced by each sample. The method was capable of distinguishing positive hybridization at cell concentrations exceeding 10(4) cells/dot-blot, equivalent to 2 x 10(7) cells/g food, and had none of the drawbacks normally associated with the use of radioactively labelled DNA in hybridization techniques. The procedure is highly specific and takes less than 30 h. Many samples can be screened simultaneously and the procedure can be used to detect any species for which a suitable DNA probe is available.     

Identification of the most significant amphipathic helix with application to HIV and MHV envelope proteins

Amphipathic helices, which play important roles in protein structure,occur in a wide variety of lengths. Yet existing methods employfixed window lengths. We present a hierarchical procedure thatidentifies the Q most significant amphipathic helices regardlessof length. Since the observed hydrophobicities are not normallydistributed, test statistics usually employed for least-squaresregression are inappropriate for assessing statistical significanceof amphipathic helices. We show that an adjusted F statisticprovides a good test. An application to the envelope proteinof HIV finds an unexpected long amphipathic helix in gp41. Received on July 12, 1989; accepted on February 28, 1990     

Rapid generation of directed and unmarked deletions in Xanthomonas

We have devised a rapid four-step procedure for the generation of directed and unmarked chromosomal deletions in bacteria, based on the use of a novel cloning vector containing the Bacillus subtilis sacB gene that encodes levansucrase and confers sucrose sensitivity, which can be used for counter-selection. Using this technique, we describe the construction of a 6.5 kb directed and unmarked deletion in a phytopathogenicity region of the chromosome in Xanthomonas campestris. This procedure allows rapid and easy transfer of a wide variety of mutant allelic DNA to the bacterial chromosome, and should be adaptable to various bacteria besides Xanthomonas spp.     

A biotinylated DNA probe to detect bacterial cells in artificially contaminated foodstuffs

A biotin-labelled DNA probe was used in a dot-blot hybridization test to demonstrate the presence of Escherichia coli in a variety of artificially contaminated foodstuffs. Positive hybridization was detected by using a streptavidine/polyalkaline phosphatase conjugate to generate an insoluble coloured precipitate in the presence of an appropriate dye. The colour intensity was measured with a computer-controlled image analysis system which assessed objectively the hybridization signal produced by each sample. The method was capable of distinguishing positive hybridization at cell concentrations exceeding 10⁴ cells/dot-blot, equivalent to 2×10⁷ cells/g food, and had none of the drawbacks normally associated with the use of radioactively labelled DNA in hybridization techniques. The procedure is highly specific and takes less than 30 h. Many samples can be screened simultaneously and the procedure can be used to detect any species for which a suitable DNA probe is available     

Southern blot analysis with synthetic oligonucleotides. Application to prostatic protein genes.

1. An ethanol precipitation procedure was developed to purify radiolabeled DNA and oligonucleotide probes to be used in Southern blots. 2. The radiolabeled probes produced strong hybridization signals on a clear background on Southern blot analysis of single gene copies even after 5 days of exposure on X-ray films. 3. An oligonucleotide probe complementary to human glandular kallikrein-1 coding region (amino acids 161-167) detected a single DNA fragment after digestion with Bam H1, Hind III or Pst 1. 4. Another oligonucleotide probe coding for the same region of human prostate-specific antigen detected 3 DNA fragments on Southern blots by contrast to a 1.5 kb full length cDNA probe which detected the presence of only one strong hybridization signal. 5. Oligonucleotide probes appear to be excellent tools for gene mapping. Their sensitivity, specificity and limitations can be compared to the one of monoclonal antibodies used in epitope mapping of proteins.     

Multiplex polymerase chain reaction amplification and direct sequencing of homologous sequences: point mutation analysis of the ras genes.

ras proto-oncogenes are activated by point mutation in a wide variety of human and animal tumors, making ras gene analysis a major area of clinical and basic cancer research. Activating point mutations, in each of the three ras genes (Ha-, Ki-, or N-ras), usually occur in one of three specific codons (12, 13, or 61). Thus, an adequate assessment of activating ras gene mutations should include the analysis of at least nine codons. We have developed a rapid method for point mutation analysis of the ras genes, which involves simultaneous (multiplex) PCR amplification of all three homologous ras genes (in the regions surrounding codons 12-13 and codon 61) in a single reaction starting with only 1 microgram of genomic DNA. Although multiplex PCR has been previously used for unrelated sequences, we demonstrate here that multiplex PCR can also be used for highly homologous sequences. Importantly, after coamplification, each of the homologous ras genes can be individually and specifically sequenced even though the other two closely related genes are present in the same template mixture, by using high-stringency conditions permitted by Taq DNA polymerase. An automated multicycle DNA sequencing procedure is used to allow the double-stranded PCR products to be sequenced directly without the need to generate single-stranded templates, further simplifying the protocol. Our multiplex PCR amplification and direct DNA sequencing procedures should greatly facilitate more complete analyses of activating ras gene point mutations, particularly in studies involving many tumor samples.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of technology for purification of plasmid DNAs for pharmaceutical purposes

Optimization of the stage of fractioning of nucleotide material has been performed, and a technology for purification and obtaining of plasmid DNAs for pharmaceutical purposes has been developed. Plasmid DNAs can be used for construction of DNA vaccines to urgent viral infections. Conditions for extraction and preliminary purification of DNA by salt and alcohol precipitations were determined. An effective gel-filtration procedure of separation of plasmid DNA from contaminants was suggested.     

An improved method for single step purification of metagenomic DNA

An improved method for purification of intact metagenomic DNA from soil has been developed using Q-Sepharose, which purified the DNA from phenolic and humic acid contaminants in a single step. The entire procedure for purification took only 45 min. A total of 81% of DNA was recovered after purification and there was 84% reduction in humic acid contents. The purified DNA was readily digested with restriction enzymes and can be further used for molecular applications.     

A new method for the rapid identification of genes encoding restriction and modification enzymes.

We have constructed derivatives of Escherichia coli that can be used for the rapid identification of recombinant plasmids encoding DNA restriction enzymes and methyltransferases. The induction of the DNA-damage inducible SOS response by the Mcr and Mrr systems, in the presence of methylated DNA, is used to select plasmids encoding DNA methyltransferases. The strains of E. coli that we have constructed are temperature-sensitive for the Mcr and Mrr systems and have been further modified to include a lacZ gene fused to the damage-inducible dinD locus of E. coli. The detection of recombinant plasmids encoding DNA methyltransferases and restriction enzymes is a simple, one step procedure that is based on the induction at the restrictive temperature of the lacZ gene. Transformants encoding DNA methyltransferase genes are detected on LB agar plates supplemented with X-gal as blue colonies. Using this method, we have cloned a variety of DNA methyltransferase genes from diverse species such as Neisseria, Haemophilus, Treponema, Pseudomonas, Xanthomonas and Saccharopolyspora.     

Non-destructive genetic sampling in fish. An improved method for DNA extraction from fish fins and scales

DNA-based studies have been one of the major interests in conservation biology of endangered species and in population genetics. As species and population genetic assessment requires a source of biological material, the sampling strategy can be overcome by non-destructive procedures for DNA isolation. An improved method for obtaining DNA from fish fins and scales with the use of an extraction buffer containing urea and further DNA purification with phenol-chloroform is described. The methodology combines the benefits of a non-destructive DNA sampling and its high efficiency. In addition, comparisons with other methodologies for isolating DNA from fish demonstrated that the present procedure also becomes a very attractive alternative to obtain large amounts of high-quality DNA for use in different molecular analyses. The DNA samples, isolated from different fish species, have been successfully used on random amplified polymorphic DNA (RAPD) experiments, as well as on amplification of specific ribosomal and mitochondrial DNA sequences. The present DNA extraction procedure represents an alternative for population approaches and genetic studies on rare or endangered taxa.     

A PCR-based method for manipulation of the vaccinia virus genome that eliminates the need for cloning.

A general method is described for altering specific genes of vaccinia virus (VV). We demonstrate and evaluate the procedure by gene inactivation, using a dominant selectable marker in conjunction with recombinant polymerase chain reaction (PCR). Primers based on the sequence of the target gene enable amplification of flanking arms and their subsequent attachment to the gpt cassette that confers resistance to mycophenolic acid. Linear PCR constructs are transfected into cells infected with wild-type vaccinia virus. Mutant viruses with gpt inserted into the target gene by homologous recombination are then selected by growth in the presence of MPA. This technique was applied to the vaccinia virus thymidine kinase gene and compared to the traditional method of constructing gpt-containing plasmids by cloning. The PCR scheme was found to be highly efficient and could theoretically be used to insert any foreign DNA element into any nonessential target gene for which partial or complete sequence information is available. The procedure can potentially be used for a wide variety of genetic modifications, including the insertion of foreign genes, with poxviruses and other DNA viruses. Genomes of microorganisms, such as bacteria and yeast that can be transformed with linear DNA, are also candidates for manipulation by this methodology.     

Deproteination of nucleic acids by filtration through a hydrophobic membrane.

To remove or inactivate an enzyme from DNA in multistep procedures in molecular biology, it is often necessary to phenol extract the solution, followed by chloroform extraction and ethanol precipitation. In addition to being time-consuming and hazardous, there can be significant loss of DNA with this procedure, especially when small volumes or amounts of DNA are being used. We have found that filtering analytical reaction mixtures through a hydrophobic membrane specifically to remove protein is a rapid alternative to phenol extraction. Within broad limits commonly encountered in molecular biology, filtration through a polyvinylidene difluoride membrane quantitatively removes a variety of enzymes without significant loss of double-stranded nucleic acid.     

Blunt-end and single-strand ligations by Escherichia coli ligase: influence on an in vitro amplification scheme

A ligase-based, in vitro DNA amplification system (LAR) has been described by Wu and Wallace [Genomics 4 (1989) 560-569]. This strategy is based on the ability of a DNA ligase to join the 5' phosphate of one DNA molecule to the 3' hydroxyl of a second during a nick-closing reaction. Escherichia coli DNA ligase has been used in place of the T4 DNA ligase in our study in order to limit template-independent ligation activities, which lower the sensitivity of this amplification procedure. The results of this study indicate that E. coli ligase also joins blunt-ended DNA molecules and some single-stranded oligodeoxyribonucleotides, in the absence of a complementary template, with an efficiency which is sensitive to both the concentrations of DNA substrate and enzyme.     

Isothermal strand-displacement amplification applications for high-throughput genomics

Amplification of source DNA is a nearly universal requirement for molecular biology applications. The primary methods currently available to researchers are limited to in vivo amplification in Escherichia coli hosts and the polymerase chain reaction. Rolling-circle DNA replication is a well-known method for synthesis of phage genomes and recently has been applied as rolling circle amplification (RCA) of specific target sequences as well as circular vectors used in cloning. Here, we demonstrate that RCA using random hexamer primers with 29 DNA polymerase can be used for strand-displacement amplification of different vector constructs containing a variety of insert sizes to produce consistently uniform template for end-sequencing reactions. We show this procedure to be especially effective in a high-throughput plasmid production sequencing process. In addition, we demonstrate that whole bacterial genomes can be effectively amplified from cells or small amounts of purified genomic DNA without apparent bias for use in downstream applications, including whole genome shotgun sequencing.     

Biochemistry and structural DNA nanotechnology: an evolving symbiotic relationship

Structural DNA nanotechnology is derived from naturally occurring structures and phenomena in cellular biochemistry. Motifs based on branched DNA molecules are linked together by sticky ends to produce objects, periodic arrays, and nanomechanical devices. The motifs include Holliday junction analogues, double and triple crossover molecules, knots, and parallelograms. Polyhedral catenanes, such as a cube or a truncated octahedron, have been assembled from branched junctions. Stiff motifs have been used to produce periodic arrays, containing topographic features visible in atomic force microscopy; these include deliberately striped patterns and cavities whose sizes can be tuned by design. Deliberately knotted molecules have been assembled. Aperiodic arrangements of DNA tiles can be used to produce assemblies corresponding to logical computation. Both DNA structural transitions and branch migration have been used as the basis for the operation of DNA nanomechanical devices. Structural DNA nanotechnology has been used in a number of applications in biochemistry. An RNA knot has been used to establish the existence of RNA topoisomerase activity. The sequence dependence of crossover isomerization and branch migration at symmetric sites has been established through the use of symmetric immobile junctions. DNA parallelogram arrays have been used to determine the interhelical angles for a variety of DNA branched junctions. The relationship between biochemistry and structural DNA nanotechnology continues to grow.