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Current Issues in Molecular Biology is published by MDPI from Volume 43 Issue 1 (2021). Previous articles were published by another publisher in Open Access under a CC-BY (or CC-BY-NC-ND) licence, and they are hosted by MDPI on mdpi.com as a courtesy and upon agreement with Caister Press.

Curr. Issues Mol. Biol., Volume 1, Issue 1 (July 1999) – 6 articles

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994 KiB

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Identification of Lactobacilli and Bifidobacteria

by Gerald W. Tannock

Curr. Issues Mol. Biol. 1999, 1(1), 53-64; https://doi.org/10.21775/cimb.001.053 - 31 Jul 1999

Cited by 2 | Viewed by 1390

Abstract

Selective culture media and phenotypic tests enable lactobacilli to be differentiated from morphologically similar bacteria. The accurate identification of Lactobacillus species can be accomplished by reference to 16S rRNA gene sequences. Species-specific, PCR primers that target the 16S-23S rRNA spacer region are available for a limited number of Lactobacillus species. Molecular methods for the comprehensive identification of Bifidobacterium species are not yet available. Only DNA-DNA reassociation provides a reliable means of species identification for this genus at present. Bifidobacteria can be differentiated from morphologically similar bacteria by the use of genus-specific, PCR primers or oligonucleotide probes. Full article

903 KiB

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A PCR-Based Method for Isolation of Genomic DNA Flanking a Known DNA Sequence

by Catherine A. Boulter and Dipa Natarajan

Curr. Issues Mol. Biol. 1999, 1(1), 47-52; https://doi.org/10.21775/cimb.001.047 - 31 Jul 1999

Viewed by 457

Abstract

We describe a simple PCR-based method for the isolation of genomic DNA that lies adjacent to a known DNA sequence. The method is based on the directional cloning of digested genomic DNA into the multiple cloning site of a pUC-based plasmid to generate a limited genomic library. The library is plated onto a number of selective LA plates which are incubated overnight, and recombinant plasmid DNA is then isolated from resistant colonies pooled from each plate. PCR amplification is performed on the pooled recombinant plasmid DNAs using primers specific for the pUC vector and the known genomic sequence. The combination of efficient directional cloning and bacterial transformation gives relative enrichment for the genomic sequence of interest and generates a simple DNA template, enabling easy amplification by PCR. Full article

980 KiB

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The Yeast Two-Hybrid System: Criteria for Detecting Physiologically Significant Protein-Protein Interactions

by Erica A. Golemis, Ilya Serebriiskii and Susan F. Law

Curr. Issues Mol. Biol. 1999, 1(1), 31-46; https://doi.org/10.21775/cimb.001.031 - 31 Jul 1999

Cited by 2 | Viewed by 490

Abstract

In vivo transcription-based assays for protein-protein interactions such as the two-hybrid system are powerful methods for identifying novel proteins based on their physical association with known proteins of biological interest, or for characterizing the degree and nature of interactions between sets of proteins. Because of the complexity inherent in assays taking place within a living organism, a key issue for the effective use of two-hybrid approaches is the ability to determine whether apparent interactions are likely to be physiologically relevant. In this article, a number of the different two-hybrid systems currently available for use will be reviewed. Then, taking as a model one such system, the Interaction Trap, examples of different reagents for use in varying the affinity range of detectable interactions will be outlined. Also set forth are a number of protocols to establish an appropriate set of conditions for either screening a library or analysing the interaction phenotype between protein sets. Finally, a number of general guidelines are suggested for trouble-shooting two-hybrid results, and for eliminating falsely positive interactions. Full article

961 KiB

Open AccessReview

DNA Splicing by Directed Ligation (SDL)

by Yuri A. Berlin

Curr. Issues Mol. Biol. 1999, 1(1), 21-30; https://doi.org/10.21775/cimb.001.021 - 31 Jul 1999

Viewed by 532

Abstract

Splicing by directed ligation (SDL) is a method of in-phase joining of PCR-generated DNA fragments that is based on a pre-designed combination of class IIS restriction endonuclease recognition and cleavage sites. Since these enzymes cleave outside of their recognition sites, the resulting sticky end can have any desired sequence, and the site itself can be removed and does not appear in the final spliced DNA product. SDL is based on the addition of class IIS recognition sites onto primers used to amplify DNA sequences. Cleavage of the PCR products results in elimination of the recognition site-containing flanking sequences and leaves the DNA fragments crowned with protruding ends. With careful design of the sticky ends, several segments can be ligated together in a predetermined order in a single reaction. SDL requires fewer rounds of amplification than overlap extension methods, and is particularly useful for creating a series of recombinants that differ in one segment. Full article

607 KiB

Open AccessReview

Synthesis of Infectious Viroids and Other Circular RNAs

by M. A. Rezaian

Curr. Issues Mol. Biol. 1999, 1(1), 13-20; https://doi.org/10.21775/cimb.001.013 - 31 Jul 1999

Viewed by 483

Abstract

Viroids are small autonomously replicating RNAs that share structural features with other subviral circular single-stranded RNAs of plants. Viroids and other circular single-stranded RNAs can be synthesised in vitro by a PCR-based procedure using a simple set of reactions. Two end-to-end primers are selected from a desired region of the viroid, one for the synthesis of the first strand cDNA and another for the production of the second strand DNA. The second primer contains an 18 nucleotide T7 promoter at its 5' end, and is selected such that the G nucleotide at the transcription start site represents a G in the viroid. Linked reverse transcription-PCR results in linear double-stranded DNA consisting of the viroid sequence and the T7 promoter. Run-off transcription of the PCR product allows the synthesis of exact-length linear viroid RNA which can be circularised by T4 RNA ligase following an enzymic modification of the 5' triphosphate to a monophosphate. This procedure results in authentic viroid molecules and obviates the need for construction and cloning of DNA in the form of tandem repeats for infectivity tests. It also allows PCR-based manipulation of circular RNAs, thus greatly simplifying structure-function analyses of viroid molecules. Full article

982 KiB

Open AccessReview

The Application of Molecular Biology

by Robert C. Tait

Curr. Issues Mol. Biol. 1999, 1(1), 1-12; https://doi.org/10.21775/cimb.001.001 - 31 Jul 1999

Viewed by 4173

Abstract

Molecular biology methods have tremendous value not only in the investigation of basic scientific questions, but also in application to a wide variety of problems affecting the overall human condition. Disease prevention and treatment, generation of new protein products, and manipulation of plants and animals for desired phenotypic traits are all applications that are routinely addressed by the application of molecular biology methods. Because of the wide applicability of these methods, they are rapidly becoming a pervasive - some would argue invasive - aspect of our technologically based society. The public concerns that address the application of these methods should be addressed by informed public discussion and debate. While scientists can be extremely critical of the quality, interpretation, and significance of experimental results, they have a rather remarkable tendency to be non-judgmental of the relative social merits of many applications of scientific research. It remains a public responsibility to be sufficiently well-informed to critically assess the merits of applied science research and participate in a communal decision-making process regarding the extent to which a new technology will be allowed to affect society. Full article

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Curr. Issues Mol. Biol., Volume 1, Issue 1 (July 1999) – 6 articles

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