Current Issues in Molecular Biology

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Duplex DNA Capture

by Vadim V. Demidov, Nikolay O. Bukanov and Maxim D. Frank-Kamenetskii

Curr. Issues Mol. Biol. 2000, 2(1), 31-35; https://doi.org/10.21775/cimb.002.031 - 01 Jan 2000

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This article describes the sequence-specific isolation and purification of intact double-stranded DNA (dsDNA) by oligonucleotide/PNA-assisted affinity capture (OPAC). The OPAC assay is based on selective tagging of a DNA duplex by biotinylated oligodeoxyribonucleotide (ODN) through formation of a so-called PD-loop. The PD-loop is assembled with the aid of a pair of PNA "openers", which allow sequence-specific targeting with a Watson-Crick complementary ODN probe in the exposed region of the dsDNA. The protocol involves three steps. First, two cationic bis-PNAs locally pry the DNA duplex apart at a predetermined site. Then, the exposed DNA single strand is targeted by a complementary biotinylated ODN to selectively form a stable PD-loop complex. Finally, the capture of dsDNA is performed using streptavidin covered magnetic beads. The OPAC procedure has many advantages in the isolation of highly purified native DNA over other affinity capture and amplification techniques. Full article

570 KiB

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PCR Clamping

by Henrik Ørum

Curr. Issues Mol. Biol. 2000, 2(1), 27-30; https://doi.org/10.21775/cimb.002.027 - 01 Jan 2000

Cited by 1 | Viewed by 644

Abstract

An efficient, PCR based method for the selective amplification of DNA target sequences that differs by a single base pair is described. The method utilises the high affinity and specificity of PNA for their complementary nucleic acids and that PNA cannot function as [...] Read more.

579 KiB

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Lactic Acid Bacteria as Live Vaccines

by Annick Mercenier, H. Müller-Alouf and Corinne Grangette

Curr. Issues Mol. Biol. 2000, 2(1), 17-25; https://doi.org/10.21775/cimb.002.017 - 01 Jan 2000

Cited by 5 | Viewed by 806

Abstract

Mucosal routes for vaccine delivery offer several advantages over systemic inoculation from both immunological and practical points of view. The development of efficient mucosal vaccines therefore represents a top prority in modern vaccinology. One way to deliver protective antigens at the mucosal surfaces is to use live bacterial vectors. Until recently most of these were derived from attenuated pathogenic microorganisms. As an alternative to this strategy, non-pathogenic food grade bacteria such as lactic acid bacteria (LAB) are being tested for their efficacy as live antigen carriers. The LABVAC european research network is presently comparing the vaccine potential of Lactococcus lactis, Streptococcus gordonii and Lactobacillus spp. To date, it has been shown that systemic and mucosal antigen-specific immune responses can be elicited in mice through the nasal route using the three LAB systems under study. Data on successful oral and vaginal immunisations are also accumulating for L. lactis and S. gordonii, respectively. Moreover, the immune responses can be potentiated by co-expression of interleukins. Future areas of research include improvement of local immunisation efficiency, analysis of in vivo antigen production, unravelling of the Lactobacillus colonisation mechanisms and construction of biologically contained strains. Full article

575 KiB

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In Vitro Selection of Functional Nucleic Acid Sequences

by Dan Nieuwlandt

Curr. Issues Mol. Biol. 2000, 2(1), 9-16; https://doi.org/10.21775/cimb.002.009 - 01 Jan 2000

Cited by 1 | Viewed by 492

Abstract

The power of in vitro selection methods for the isolation of nucleic acids that display a desired property derives from the enormous number of sequence variants that can be surveyed with relative ease using controlled in vitro biochemistry. This methodology has found a variety of applications, ranging from the study of nucleic acid-protein interactions and natural ribozymes to the isolation of nucleic acids with potential as diagnostic or therapeutic reagents or with new catalytic activities. The number of reported applications is growing exponentially, and each application presents new variables and challenges. The goal of this article is to guide prospective users through the myriad decisions that must be made in the design and execution of a successful in vitro selection experiment. Full article

587 KiB

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Universal TA Cloning

by Ming-Yi Zhou and Celso E. Gomez-Sanchez

Curr. Issues Mol. Biol. 2000, 2(1), 1-7; https://doi.org/10.21775/cimb.002.001 - 01 Jan 2000

Cited by 2 | Viewed by 6012

Abstract

TA cloning is one of the simplest and most efficient methods for the cloning of PCR products. The procedure exploits the terminal transferase activity of certain thermophilic DNA polymerases, including Thermus aquaticus (Taq) polymerase. Taq polymerase has non-template dependent activity which preferentially adds a single adenosine to the 3'-ends of a double stranded DNA molecule, and thus most of the molecules PCR amplified by Taq polymerase possess single 3'-A overhangs. The use of a linearized "T-vector" which has single 3'-T overhangs on both ends allows direct, high-efficiency cloning of PCR products, facilitated by complementarity between the PCR product 3'-A overhangs and vector 3'-T overhangs. The TA cloning method can be easily modified so that the same T-vector can be used to clone any double-stranded DNA fragment, including PCR products amplified by any DNA polymerase, as well as all blunt- and sticky-ended DNA species. This technique is especially useful when compatible restriction sites are not available for the subcloning of DNA fragments from one vector to another. Directional cloning is made possible by appropriate hemi-phosphorylation of both the T-vectors and the inserts. With a single T-vector at hand, any DNA fragment can be cloned without compromising the cloning efficiency. The universal TA cloning method is thus both convenient and labor-saving. Full article

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Curr. Issues Mol. Biol., Volume 2, Issue 1 (January 2000) – 5 articles

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