Electrophoresis of polyester-backed polyacrylamide gels.
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Acrylamides↗
Biomedical subjects
B J Bassam
Publications and source records attributed to B J Bassam.
Buffer components tailor DNA amplification with arbitrary primers.
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Animals↗
DNA amplification fingerprinting of the Azolla-Anabaena symbiosis.
The Azolla-Anabaena symbiosis has been used for centuries as a nitrogen biofertilizer in rice paddies. Genetic improvement of the symbiosis has been limited by the difficulty in identifying Azolla-Anabaena accessions and Anabaena azollae strains. The recently developed technique of DNA amplification fingerprinting (DAF) was applied to this problem. DAF uses single, short, oligonucleotide primers of arbitrary sequence to direct amplification of a characteristic set of DNA products by a thermostable DNA polymerase in a thermocycling reaction. The products are separated in polyacrylamide gels and detected by silver staining. DAF could easily distinguish and positively identify accessions of Azolla-Anabaena with DNA extracted from the intact symbiosis. The contribution of prokaryotic Anabaena sequences to the fingerprint of the intact symbiosis, however, ranged from 0 to 77%, depending on the primer sequence. Therefore, DNA extracted from the intact symbiosis would not be suitable for Azolla taxonomy studies. The fingerprints of Anabaena strains isolated by sucrose gradient centrifugation from different species of Azolla could be easily distinguished, and DAF patterns were used to confirm the maternal pattern of transmission of Anabaena in a sexual hybrid. Template DNA extracted from roots was used to produce fingerprints for Azolla without interference from the microsymbiont. Comparison of the patterns from the parents and a hybrid gave strong evidence confirming sexual hybridization.
Anabaena↗
Enhanced detection of polymorphic DNA by multiple arbitrary amplicon profiling of endonuclease-digested DNA: identification of markers tightly linked to the supernodulation locus in soybean.
Multiple endonuclease digestion of template DNA or amplification products can increase significantly the detection of polymorphic DNA in fingerprints generated by multiple arbitrary amplicon profiling (MAAP). This coupling of endonuclease cleavage and amplification of arbitrary stretches of DNA, directed by short oligonucleotide primers, readily allowed distinction of closely related fungal and bacterial isolates and plant cultivars. MAAP analysis of cleaved template DNA enabled the identification of molecular markers linked to a developmental locus of soybean (Glycine max L. Merrill). Ethyl methane sulfonate (EMS)-induced supernodulating, near-isogenic lines altered in the nts locus, which controls nodule formation, could be distinguished from each other and from the parent cultivar by amplification of template pre-digested with 2-3 restriction enzymes. A total of 42 DNA polymorphisms were detected using only 19 octamer primers. In the absence of digestion, 25 primers failed to differentiate these soybean genotypes. Several polymorphic products co-segregated tightly with the nts locus in F2 families from crosses between the allelic mutants nts382 and nts1007 and the ancestral G. soja Sieb. & Succ. PI468.397. Our results suggest that EMS is capable of inducing extensive DNA alterations, probably around discrete mutational hot-spots. EMS-induced DNA polymorphisms may constitute sequence-tagged markers diagnostic of specific genomic regions.
DNA↗
Automated "hot start" PCR using mineral oil and paraffin wax.
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Base Sequence↗
DNA amplification fingerprinting of bacteria.
We have amplified short arbitrary stretches of total bacterial DNA to produce highly characteristic and complex DNA fingerprints. This DNA amplification fingerprinting (DAF) strategy involves enzymatic amplification of DNA directed by a single arbitrary oligonucleotide primer. Amplification produces a characteristic spectrum of products that is adequately resolved by polyacrylamide gel electrophoresis and visualized by silver staining. Although DAF is simple in concept, we found that amplification parameters must be within an optimal range for reproducibility. We establish a safe window for these parameters, which include magnesium, primer and enzyme concentration as well as cycle number. The refined procedure was used to distinguish between clinical isolates of Streptococcus uberis, Klebsiella pneumoniae, and Escherichia coli. The use of template DNA concentrations higher than 1 ng.microliters-1 and high MgCl2 levels was especially important for reproducibility when amplifying small bacterial genomes. We tested a truncated Thermus aquaticus DNA polymerase, the Stoffel fragment, and found it more tolerant of reaction conditions, more efficient in the amplification of short products, and able to produce more informative fingerprints when compared to the normal thermostable polymerase from which it was derived. Because DAF produces representative fingerprints quickly and reliably from bacteria regardless of prior genetic or biochemical knowledge, we anticipate the general use of this diagnostic tool for bacterial identification and taxonomy.
Animals↗
Primer-template interactions during DNA amplification fingerprinting with single arbitrary oligonucleotides.
DNA amplification fingerprinting (DAF) is the enzymatic amplification of arbitrary stretches of DNA which is directed by very short oligonucleotide primers of arbitrary sequence to generate complex but characteristic DNA fingerprints. To determine the contribution of primer sequence and length to the fingerprint pattern and the effect of primer-template mismatches, DNA was amplified from several sources using sequence-related primers. Primers of varying length, constructed by removing nucleotides from the 5' terminus, produced unique patterns only when primers were 8 nucleotides or fewer in length. Larger primers produced either identical or related fingerprints, depending on the sequence. Single base changes within this first 8-nucleotide region of the primer significantly altered the spectrum of amplification products, especially at the 3' terminus. Increasing annealing temperatures from 15 degrees to 70 degrees C during amplification did not shift the boundary of the 8-nucleotide region, but reduced the amplification ability of shorter primers. Our observations define a 3'-terminal oligonucleotide domain that is at least 8 bases in length and largely conditions amplification, but that is modulated by sequences beyond it. Our results indicate that only a fraction of template annealing sites are efficiently amplified during DAF. A model is proposed in which a single primer preferentially amplifies certain products due to competition for annealing sites between primer and terminal hairpin loop structures of the template.
Animals↗
Subtyping of Streptococcus uberis by DNA amplification fingerprinting.
Total DNA of Streptococcus uberis from cows with mastitis was analyzed by DNA amplification fingerprinting (DAF) and compared with restriction endonuclease fingerprinting (REF). DAF grouped 22 strains into 15 distinct patterns, while REF grouped them into 12 patterns. These results suggest that DAF is a useful technique for subtyping strains of S. uberis.
Base Sequence↗
Fast and sensitive silver staining of DNA in polyacrylamide gels.
The photochemically derived silver stain of nucleic acids in polyacrylamide gels originally described by Merril et al. (1981, Science 211, 1437-1438) was modified to reduce unspecific background staining and increase sensitivity (down to 1 pg/mm2 band cross-section). Detection limits for double-stranded DNA fragments from HaeIII endonuclease digests of phage phi X174 were maintained despite eliminating oxidation pretreatment of fixed gels and reducing silver nitrate concentration. Preexposure to formaldehyde during silver impregnation enhanced sensitivity and the inclusion of the silver-complexing agent sodium thiosulphate in the image developer decreased background staining. Higher formaldehyde concentration during image development resulted in darker bands with good contrast. The procedure almost halves the number of steps, solutions and experimental time required and can be used for the staining of DNA fragments in polyacrylamide gels bound to a polyester backing film by controlling temperature during image development. We have applied this improved staining procedure for the routine analysis of complex DNA profiles generated by DNA amplification fingerprinting (DAF).
Bacteriophages↗
DNA amplification fingerprinting using very short arbitrary oligonucleotide primers.
The surprising finding that amplification of genomic DNA can be directed by only one oligonucleotide primer of arbitrary sequence to produce a characteristic spectrum of short DNA products of varying complexity, was applied as a strategy to detect genetic differences between organisms. This approach, DNA amplification fingerprinting (DAF), does not depend on cloning or DNA sequence information and can generate fingerprints from DNA of viral, bacterial, fungal, plant and animal origins. Primers as short as 5 nucleotides in length can produce complex banding patterns that are resolved by polyacrylamide gel electrophoresis and silver staining. Amplification fragment length polymorphisms (AFLPs) were detected between different human individuals as well as between soybean cultivars. It is anticipated that DAF will have wide application for DNA analysis.
Base Sequence↗
Identification of a nodD-dependent locus in the Rhizobium strain NGR234 activated by phenolic factors secreted by soybeans and other legumes.
Transfer of the strain NGR234nodD 1 gene into the narrow host range R. trifolii strain ANU843 on either a 6.7-kb HindIII or 17-kb XhoI fragment broadens the host range of this bacterium to include the tropical legumes Vigna unguiculata, Glycine ussuriensis, Leucaena leucocephala, and siratro (Macroptilium atropurpureum). Contrary to previous data (Bassam et al. 1986), mutagenesis of the 17-kb XhoI fragment with a mini-Mu lac transposon (Mu dII1734) showed that a functional nodD 1 gene was essential for extended host range. Gene expression studies using both Mu dII1734 fusions and a promoter-cloning vector indicated that several loci, including the nodD 1 gene, are constitutively expressed. No evidence was found for regulation of the strain NGR234 nodD 1 gene by its product. Another locus nod-81, was induced only in the presence of exudates from various plant species, including soybean (Glycine max). Whereas the expression of nod-81 was dependent on the presence of a functional nodD 1 gene product, a regulatory nod-box DNA sequence was not detected 5' to this gene by using available oligonucleotide hybridization probes. The nod-81 locus was induced by genistein, daidzein, naringenin, and coumestrol from both cotyledon and root tissue of freshly germinated soybean seedlings. A broad spectrum of commercially available phenolic compounds stimulated induction of the nod-81 locus, including some that antagonize nod gene induction in other Rhizobium species. The nodD 1 gene product from strain NGR234 was shown to determine the spectrum of compounds that induce nod-81 expression.
Base Sequence↗
DNA amplification fingerprinting using arbitrary oligonucleotide primers.
DNA amplification fingerprinting (DAF) is a strategy for genetic typing and mapping that uses one or more very short (> or = 5 nt) arbitrary oligonucleotides to direct the enzymatic amplification of discrete portions of a DNA template resulting in a spectrum of products characteristic of the DNA starting material. Polymorphisms from simple banding patterns are useful as genetic markers while more complex and informative patterns are suitable for DNA fingerprinting. The use of polyacrylamide gel electrophoresis and silver staining can adequately resolve the spectrum of DAF products into detailed and reproducible patterns.
Base Sequence↗
DNA fingerprinting using arbitrary primer technology (APT): a tool or a torment.
A recent variation of the PCR has allowed DNA fingerprints to be obtained independently of prior sequence information and with unparalleled ease. Several approaches, conveniently grouped under the general term of arbitrary primer technology (APT), include the popular RAPD, DAF, and AP-PCR methods. A great deal of attention has been focussed on these methods and questions have arisen regarding reproducibility, DNA fingerprint resolution, and even the future of the technology itself. Here we discuss these issues and examine some of the unique properties of DNA amplification using a single short arbitrary primer.
DNA Fingerprinting↗