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  • If the factors needed for A NHEJ are


    If the factors needed for A-NHEJ are not completely defined and the A-NHEJ reaction mechanism nebulous, it is also fair to say that the biological role(s) of A-NHEJ is even more poorly understood. Most of the current interest in A-NHEJ, however, stems from its implicated use in the chromosomal translocations that are present in cancer cells. Sequencing of human cancer genomes has revealed that many [25], [26], [27], albeit not all [28] chromosomal translocations have microhomology at their breakpoint junctions, which implicates A-NHEJ in their genesis. This hypothesis gained support from work in which LIGIII conditionally-null murine AUY-922 sale showed decreased translocation frequencies and reduced microhomology usage [29], [30], [31], [32]. An additional biological process where A-NHEJ has been implicated is in the random insertion events associated with rAAV (recombinant adeno-associated virus)-mediated gene targeting. Although rAAV can mediate high frequency gene targeting, the majority of the viral integration events still occur randomly [33]. Moreover, our laboratory has reported that a reduction in the C-NHEJ genes Ku70 [34] and LIGIV [35] had almost no impact on the random rAAV integration rate – implying that these events may be mediated instead by A-NHEJ. In summary, although A-NHEJ was a neglected subject for many years, in the past decade it has proven itself to be an increasingly interesting and biologically relevant topic. A key feature of A-NHEJ is its dependence on LIGIII [16]. Unlike the other ligases, LIGIII is molecularly heterogeneous [12], [36], [37]. Thus, alternative translation initiation generates mitochondrial and nuclear forms of LIGIII, which either contain or lack a MLS (mitochondrial localizing sequence), respectively [36]. The existence of LIGIII isoforms implies diverse functional roles for LIGIII. One experimental approach to unraveling the complexity of LIGIII is to generate a LIGIII-deficient model system, which has already been accomplished in the chicken cell line, DT40 [38], and in the mouse [30], [32], [39]. In these systems the gene is essential due to its presumed requirement for mitochondrial DNA replication. Moreover, in LIGIII conditionally-null mice no obvious nuclear DNA repair phenotypes could be detected [30], [32]. The extrapolation of these studies to humans is unfulfilled as neither LIGIII patients nor LIGIII-deficient human cell systems have been described.
    Materials and methods
    Conflict of interest statement
    Funding This work was supported by grants from the National Institutes of Health (GM088351), the National Cancer Institute (CA154461) and by a research contract provided by Horizon Discovery, Ltd.
    Introduction The design of the electrochemical biosensor described in this paper could equally have been applied to any genomic DNA but the white rot fungus, Ganoderma boninense was selected in this study. This is a pathogen, which causes both basal and upper stem rot in oil palms and presents a major threat to a highly lucrative industry in the south East Asia. G. boninense has a devastating effect on a plantation; it causes direct loss of the stands and reduces the yield of the palms, creating the need for earlier replanting [1]. As soon as young palms show symptoms of the disease they inevitably die within a year or two while older trees survive only a few years longer [2]. As well as trying a multitude of methods of control, extensive work has already been carried out on the early detection of G. boninense[3]. After experimenting with PCR [4], researchers tried polyclonal antibody for ELISA technique [5]. Finally, a multiplex PCR-DNA kit [6] was designed for the Malaysian Palm Oil Board although it is far from offering a complete solution. The PCR technique is sensitive to contamination, and because it can be inhibited by secondary metabolites, it is critical to purify the DNA before the reaction [7], [8].