sn38 australia Experimental section br Results and discussio

Experimental section
Results and discussion
Conclusion In summary, we have developed a fluoresence method to sensitively detect hAAG on the basis of TdT-activated Endo IV-assisted hyperbranched amplification. This method shows the following advantages: (1) the combination of TdT-mediated extension with Endo IV-mediated cleavage of signal probes can induce efficient hyperbranched amplification, ensuring the high sensitivity of hAAG assay (0.090 U/mL). (2) Although the sensitivity of this method is almost the same as the magnetic bead-based fluorescent assay (0.1 U/mL) [15], the method can be performed in a simple ‘‘mix and detection’’ manner, without the involvement of complicated immobilization, separation and washing steps. (3) Compared with the highly sensitive single-molecule detection method [14], the fluorescence instrument in the developed method is low cost and it can be used prevalently. (4) The reaction scheme is relatively simple, and only two DNA oligonucleotides are used. Moreover, the hAAG activity detection can be finished in a short time (∽100 min). (5) This method can be further applied for quantification of hAAG activity in complex HeLa nuclear extract, providing a broad spectrum of therapeutic applications such as clinical diagnostics.
Introduction DNA glycosylases are the catalysts of the first step of the sn38 australia excision repair (BER), the primary repair mechanism for small base lesions arising from deamination, oxidation, methylation and replication-associated misincorporation. Their role in the control of genetic integrity is inferred from in vitro and in vivo studies and from human diseases, although rare, associated with germline mutations in their coding genes. Aside this evidence there is a plethora of studies where the effects of single nucleotide polymorphisms (SNPs) in DNA glycosylases have been evaluated in population studies and analysed for function, although not systematically. SNPs are the most common type of genetic variation among people (there are many millions) and usually occur in non-coding regions. Only when they are located within genes or their regulatory regions they may affect the gene function and thus play a more direct role in disease. In this review we will briefly introduce the structure, function and biological importance of DNA glycosylases as inferred from knock-out (ko) model mice and human diseases with germline mutations and then we will focus on the impact of SNPs on gene function and disease.
DNA glycosylases: structure, function and biological importance
SNPs: function and disease In this section the studies currently available in the literature on the function of SNPs are reported in conjunction with their association with disease (Table 2). The description of the SNP-disease association studies is focused on large size epidemiological studies and/or meta-analysis of published studies when available. It should be taken into consideration that the conclusions of all the other studies summarized below are hampered by several factors including limited sample size.
Concluding remarks: present and future perspectives What emerges from this overview is that there is a clear association between SNPs in DNA glycosylases that are mostly involved in the repair of oxidatively-induced DNA lesions and susceptibility to diseases which all present as major risk factor oxidative stress. Notwithstanding the limits of the epidemiological studies, the scarce information on function of genetic variants and the limitation of studies exploring one or a few SNPs, the overall disease spectrum includes cancer (mostly lung, breast, GI tract), ocular and cochlear disorders, myocardial infarction and neurodegenerative disorders which can be all grouped under the umbrella of oxidative stress-related pathologies (Fig. 1). The phenotype of ko mice and the clinical features of the few human diseases with full inactivation of these DNA glycosylases support this conclusion (Table 1). Moreover, model mice defective in three DNA glycosylases (i.e. OGG1, NEIL1 and NEIL3) indicate that obesity and associated metabolic derangement may arise when these activities are defective. Further research should address the underlying mechanisms and the possible impact on human health. It is of interest that a large number of polymorphisms in DNA glycosylases are located in introns, often in exon-intron splice junctions (Table 2). This is also true for approximately 10% of all inherited pathological mutations [214]. This might be explained by the fact that introns represent a large mutational target and contain a multiplicity of functional elements that affect transcriptional activity or the splicing efficiency of their host genes and even of genes at distance. However, in most studies these polymorphisms are merely associated with impaired enzymatic activity and whether they are truly functional SNPs or a linkage disequilibrium effect remains to be addressed.