Bead-probe complex capture a couple of SINE and LINE family from genomes of two closely related species of East Asian cyprinid directly using magnetic separation

Background: Short and long interspersed elements (SINEs and LINEs, respectively), two types of retroposons, are active in shaping the architecture of genomes and powerful tools for studies of phylogeny and population biology. Here we developed special protocol to apply biotin-streptavidin bead system into isolation of interspersed repeated sequences rapidly and efficiently, in which SINEs and LINEs were captured directly from digested genomic DNA by hybridization to bead-probe complex in solution instead of traditional strategy including genomic library construction and screening. Results: A new couple of SINEs and LINEs that shared an almost identical 3'tail was isolated and characterized in silver carp and bighead carp of two closely related species. These SINEs (34 members), designated HAmo SINE family, were little divergent in sequence and flanked by obvious TSD indicated that HAmo SINE was very young family. The copy numbers of this family was estimated to 2 x 10(5) and 1.7 x 10(5) per haploid genome by Real-Time qPCR, respectively. The LINEs, identified as the homologs of LINE2 in other fishes, had a conserved primary sequence and secondary structures of the 3'tail region that was almost identical to that of HAmo SINE. These evidences suggest that HAmo SINEs are active and amplified recently utilizing the enzymatic machinery for retroposition of HAmoL2 through the recognition of higher-order structures of the conserved 42-tail region. We analyzed the possible structures of HAmo SINE that lead to successful amplification in genome and then deduced that HAmo SINE, SmaI SINE and FokI SINE that were similar in sequence each other, were probably generated independently and created by LINE family within the same lineage of a LINE phylogeny in the genomes of different hosts. Conclusion: The presented results show the advantage of the novel method for retroposons isolation and a pair of young SINE family and its partner LINE family in two carp fishes, which strengthened the hypotheses containing the slippage model for initiation of reverse transcription, retropositional parasitism of SINEs on LINEs, the formation of the stem loop structure in 3'tail region of some SINEs and LINEs and the mechanism of template switching in generating new SINE family.

Real-time PCR microfluidic devices with concurrent electrochemical detection

Electrochemistry-based detection methods hold great potential towards development of hand-held nucleic-acid analyses instruments. In this work, we demonstrate the implementation of in situ electrochemical (EC) detection method in a microfluidic flow-through EC-qPCR (FTEC-qPCR) device, where both the amplification of the target nucleic-acid sequence and subsequent EC detection of the PCR amplicon are realized simultaneously at selected PCR cycles in the same device. The FTEC-qPCR device utilizes methylene blue (MB), an electroactive DNA intercalator, for electrochemical signal measurements in the presence of PCR reagent components. Our EC detection method is advantageous, when compared to other existing EC methods for PCR amplicon analysis, since FTEC-qPCR does not require probe-modified electrodes, or asymmetric PCR, or solid-phase PCR. Key technical issues related to surface passivation, electrochemical measurement, PCR inhibition by metal electrode, bubble-free PCR, were investigated. By controlling the concentration of MB and the exposure of PCR mixture to the bare metal electrode, we successfully demonstrated electrochemical measurement of MB in solution-phase, symmetric PCR by amplifying a fragment of lambda phage DNA.

CLONING AND QUANTITATIVE ANALYSIS OF THE CARBONIC ANHYDRASE GENE FROM PORPHYRA YEZOENSIS

Carbonic anhydrase (CA), an enzyme that catalyzes the interconversion of CO2 and HCO3-, has a critical role in inorganic carbon acquisition in many kingdoms, including animals, plants, and bacteria. In this study, the full-length cDNA of the CA gene from Porphyra yezoensis Ueda (denoted as PyCA) was cloned by using an expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE). The nucleotide sequence of PyCA consists of 1,153 bp, including a 5' untranslated region (UTR) of 177 bp, a 3' UTR of 151 bp, and an open reading frame (ORF) of 825 bp that can be translated into a 274-amino-acid putative peptide with a molecular mass (M) of 29.8 kDa and putative isoelectric point (pI) of 8.51. The predicted polypeptide has significant homology to the beta-CA from bacteria and unicellular algae, such as Porphyridium purpureum. The mRNA in filamentous thalli, leafy thalli, and conchospores was examined, respectively, by real-time fluorescent quantitative PCR (qPCR), and the levels of PyCA are different at different stages of the life cycle. The lowest level of mRNA was observed in leafy thalli, and the level in filamentous thalli and in the conchospores was 4-fold higher and 10-fold higher, respectively.

VEGF gene silencing by cytomegalovirus promoter driven ShRNA expression vector results in vascular development defects in zebrafish

Zebrafish has been generally considered as an excellent model in case of drug screening, disease model establishment, and vertebrate embryonic development study. In this work, the ability of human cytomegalovirus immediate early promoter (CMV promoter)-driven short hairpin RNA (shRNA) expression vector to induce shRNA against VEGF gene in zebrafish was tested, and its effect on vascular development was assed, too. Using RT-qPCR, blood vessel staining, and in situ hybridization, we confirmed certain transcriptional activity and down regulation of gene expression by the vector. In situ hybridization analysis indicated selective inhibition of NRP1 expression in the VEGF gene loss of function model, which might imply in turn that VEGF could not only activate endothelial cells directly but also could contribute to stimulating angiogenesis in vivo by a mechanism that involved up-regulation of its cognate receptor expression in zebrafish. This contributed to a better understanding of molecular mechanisms of cardiovascular development. The system improved the success rate in making inducible knockdown and widened the possibilities for better therapeutic targets in zebrafish.