Detection of genetic variation using dual-labeled peptide nucleic acid (PNA) probe-based melting point analysis.

BACKGROUND: Thermal denaturation of probe-target hybrid is highly reproducible, and which makes probe melting point analysis reliable in the detection of mutations, polymorphisms and epigenetic differences in DNA. To improve resolution of these detections, we used dual-labeled (quencher and fluorescence), full base of peptide nucleic acid (PNA) probe for fluorescence probe based melting point analysis. Because of their uncharged nature and peptide bond-linked backbone, PNA probes have more favorable hybridization properties, which make a large difference in the melting temperature between specific hybridization and partial hybridization. RESULTS: Here, we have shown that full base dual-labeled PNA is apt material for fluorescence probe-based melting point analysis with large difference in the melting temperature between full specific hybridization and that of partial hybridization, including insertion and deletion. In case of narrowly distributed mutations, PNA probe effectively detects three mutations in a single reaction tube with three probes. Moreover, we successfully diagnose virus analogues with amplification and melting temperature signal. Lastly, Melting temperature of PNA oligomer can be easily adjusted just by adding gamma-modified PNA probe. CONCLUSIONS: The PNA probes offer advantage of improved flexibility in probe design, which could be used in various applications in mutation detection among a wide range of spectrums.

Cloning and characterization of a fish specific gelsolin family gene, ScinL, in olive flounder (Paralichthys olivaceus).

Scinderin like (ScinL) gene is a unique gelsolin family gene found only in fish. In this study ScinL gene was cloned in olive flounder for the first time and characterized its expression and function. Flounder ScinL cDNA consists of 2911 nucleotides encoding a putative protein of 720 amino acids (79.4 kDa). In phylogenetic analysis, flounder ScinL is closely related to ScinL of zebra fish, anableps, and fugu with the similarity of 51-72%. Fish ScinLs are positioned between gelsolin and scinderin of other species. Flounder ScinL protein has the highly conserved actin and PIP2 binding sites, Ca(2+) coordination site, and a C-terminal latch helix preventing the activation of ScinL protein in the absence of Ca(2+). Putative binding sites for NFAT and AP-1 were found in 5' flanking region. Constitutive ScinL expression was found in most organs and the expression level was higher in gill, head kidney, trunk kidney, spleen and skin than muscle, stomach, intestine and brain. In Q-PCR analysis ScinL and CYP1A1 gene expression were significantly upregulated by BaP in head kidney in vivo and in vitro, and in macrophage cells. Upregulated ScinL expression by BaP was blocked by EGTA, indicating a calcium dependent regulation of ScinL expression.

Analysis of immune gene expression modulated by benzo[a]pyrene in head kidney of olive flounder (Paralichthys olivaceus).

Poly aromatic hydrocarbons (PAHs) are known to cause functional disorder of fish immune responses. Alteration of inflammatory cytokines and other immune gene expressions by PAHs in immune organs may play a pivotal role in immunotoxicity. Thus this study aimed to elucidate the immunotoxic mechanism of PAH using benzo[a]pyrene (BaP) by analyzing the gene expression of cytokines (IL-1ß, TNFα, IL-6, IL-8, IFNγ, Mx), apoptosis (FasL, SOD) and other immune related substances (Lysozyme, IgM) in head kidney and macrophage in olive flounder. In Q-PCR analysis, proinflammatory cytokine (IL-1ß, IL-6, IL-8, TNFα) gene expressions were significantly upregulated by BaP while Mx and IgM gene expressions were significantly downregulated in head kidney by a longer exposure to BaP in vivo and in vitro. Lysozyme gene expression was initially upregulated but later downregulated in head kidney in vivo and in vitro. Inhibition test revealed that TNFα gene expression was upregulated by BaP via the AHR pathway as blocked by ANF while IL-6 and IFNγ gene expressions were upregulated by a calcium dependent pathway (i.e. NFAT) as blocked by EGTA. In primary macrophage cells, only IL-8 gene expression was significantly upregulated among proinflammatory cytokines while IFNγ, lysozyme and IgM gene expressions were downregulated by BaP. FasL and SOD expressions were not altered in head kidney cells but significantly upregulated in macrophage cells, indicating apoptosis and oxidative stress. These results indicate that exposure to BaP causes the downregulation of immune response by triggering the death of macrophage cells, the reduction of effectors like IgM and lysozyme, and the decrease of macrophage cell activity.