Removal of PCR Error Products and Unincorporated Primers by Metal-Chelate Affinity Chromatography

Immobilized Metal Affinity Chromatography (IMAC) has been used for decades to purify proteins on the basis of amino acid content, especially surface-exposed histidines and “histidine tags” genetically added to recombinant proteins. We and others have extended the use of IMAC to purification of nucleic acids via interactions with the nucleotide bases, especially purines, of single-stranded RNA and DNA. We also have demonstrated the purification of plasmid DNA from contaminating genomic DNA by IMAC capture of selectively-denatured genomic DNA. Here we describe an efficient method of purifying PCR products by specifically removing error products, excess primers, and unincorporated dNTPs from PCR product mixtures using flow-through metal-chelate affinity adsorption. By flowing a PCR product mixture through a Cu2+-iminodiacetic acid (IDA) agarose spin column, 94–99% of the dNTPs and nearly all the primers can be removed. Many of the error products commonly formed by Taq polymerase also are removed. Sequencing of the IMAC-processed PCR product gave base-calling accuracy comparable to that obtained with a commercial PCR product purification method. The results show that IMAC matrices (specifically Cu2+-IDA agarose) can be used for the purification of PCR products. Due to the generality of the base-specific mechanism of adsorption, IMAC matrices may also be used in the purification of oligonucleotides, cDNA, mRNA and micro RNAs

Investigation of highly unsaturated fatty acid metabolism in the Asian sea bass, Lates calcarifer

Lates calcarifer, commonly known as the Asian sea bass or barramundi, is an interesting species that has great aquaculture potential in Asia including Malaysia and also Australia. We have investigated essential fatty acid metabolism in this species, focusing on the endogenous highly unsaturated fatty acid (HUFA) synthesis pathway using both biochemical and molecular biological approaches. Fatty acyl desaturase (Fad) and elongase (Elovl) cDNAs were cloned and functional characterization identified them as ∆6 Fad and Elovl5 elongase enzymes, respectively. The ∆6 Fad was equally active towards 18:3n-3 and 18:2n-6, and Elovl5 exhibited elongation activity for C18-20 and C20-22 elongation and a trace of C22-24 activity. The tissue profile of gene expression for ∆6 fad and elovl5 genes, showed brain to have the highest expression of both genes compared to all other tissues. The results of tissue fatty acid analysis showed that the brain contained more docosahexaenoic acid (DHA, 22:6n-3) than flesh, liver and intestine. The HUFA synthesis activity in isolated hepatocytes and enterocytes using [1-14C]18:3n-3 as substrate was very low with the only desaturated product detected being 18:4n-3. These findings indicate that L. calcarifer display an essential fatty acid pattern similar to other marine fish in that they appear unable to synthesize HUFA from C18 substrates. High expression of ∆6 fad and elovl5 genes in brain may indicate a role for these enzymes in maintaining high DHA levels in neural tissues through conversion of 20:5n-3

Genomic organization and evolution of the Atlantic salmon hemoglobin repertoire

<p>Abstract</p> <p>Background</p> <p>The genomes of salmonids are considered pseudo-tetraploid undergoing reversion to a stable diploid state. Given the genome duplication and extensive biological data available for salmonids, they are excellent model organisms for studying comparative genomics, evolutionary processes, fates of duplicated genes and the genetic and physiological processes associated with complex behavioral phenotypes. The evolution of the tetrapod hemoglobin genes is well studied; however, little is known about the genomic organization and evolution of teleost hemoglobin genes, particularly those of salmonids. The Atlantic salmon serves as a representative salmonid species for genomics studies. Given the well documented role of hemoglobin in adaptation to varied environmental conditions as well as its use as a model protein for evolutionary analyses, an understanding of the genomic structure and organization of the Atlantic salmon α and β hemoglobin genes is of great interest.</p> <p>Results</p> <p>We identified four bacterial artificial chromosomes (BACs) comprising two hemoglobin gene clusters spanning the entire α and β hemoglobin gene repertoire of the Atlantic salmon genome. Their chromosomal locations were established using fluorescence <it>in situ </it>hybridization (FISH) analysis and linkage mapping, demonstrating that the two clusters are located on separate chromosomes. The BACs were sequenced and assembled into scaffolds, which were annotated for putatively functional and pseudogenized hemoglobin-like genes. This revealed that the tail-to-tail organization and alternating pattern of the α and β hemoglobin genes are well conserved in both clusters, as well as that the Atlantic salmon genome houses substantially more hemoglobin genes, including non-Bohr β globin genes, than the genomes of other teleosts that have been sequenced.</p> <p>Conclusions</p> <p>We suggest that the most parsimonious evolutionary path leading to the present organization of the Atlantic salmon hemoglobin genes involves the loss of a single hemoglobin gene cluster after the whole genome duplication (WGD) at the base of the teleost radiation but prior to the salmonid-specific WGD, which then produced the duplicated copies seen today. We also propose that the relatively high number of hemoglobin genes as well as the presence of non-Bohr β hemoglobin genes may be due to the dynamic life history of salmon and the diverse environmental conditions that the species encounters.</p> <p>Data deposition: BACs S0155C07 and S0079J05 (fps135): GenBank <ext-link ext-link-id="GQ898924" ext-link-type="gen">GQ898924</ext-link>; BACs S0055H05 and S0014B03 (fps1046): GenBank <ext-link ext-link-id="GQ898925" ext-link-type="gen">GQ898925</ext-link></p

Analysis of BAC-end sequences in rainbow trout: Content characterization and assessment of synteny between trout and other fish genomes

<p>Abstract</p> <p>Background</p> <p>Rainbow trout (<it>Oncorhynchus mykiss</it>) are cultivated worldwide for aquaculture production and are widely used as a model species to gain knowledge of many aspects of fish biology. The common ancestor of the salmonids experienced a whole genome duplication event, making extant salmonids such as the rainbow trout an excellent model for studying the evolution of tetraploidization and re-diploidization in vertebrates. However, the lack of a reference genome sequence hampers research progress for both academic and applied purposes. In order to enrich the genomic tools already available in this species and provide further insight on the complexity of its genome, we sequenced a large number of rainbow trout BAC-end sequences (BES) and characterized their contents.</p> <p>Results</p> <p>A total of 176,485 high quality BES, were generated, representing approximately 4% of the trout genome. BES analyses identified 6,848 simple sequence repeats (SSRs), of which 3,854 had high quality flanking sequences for PCR primers design. The first rainbow trout repeat elements database (INRA RT rep1.0) containing 735 putative repeat elements was developed, and identified almost 59.5% of the BES database in base-pairs as repetitive sequence. Approximately 55% of the BES reads (97,846) had more than 100 base pairs of contiguous non-repetitive sequences. The fractions of the 97,846 non-repetitive trout BES reads that had significant BLASTN hits against the zebrafish, medaka and stickleback genome databases were 15%, 16.2% and 17.9%, respectively, while the fractions of the non-repetitive BES reads that had significant BLASTX hits against the zebrafish, medaka, and stickleback protein databases were 10.7%, 9.5% and 9.5%, respectively. Comparative genomics using paired BAC-ends revealed several regions of conserved synteny across all the fish species analyzed in this study.</p> <p>Conclusions</p> <p>The characterization of BES provided insights on the rainbow trout genome. The discovery of specific repeat elements will facilitate analyses of sequence content (e.g. for SNPs discovery and for transcriptome characterization) and future genome sequence assemblies. The numerous microsatellites will facilitate integration of the linkage and physical maps and serve as valuable resource for fine mapping QTL and positional cloning of genes affecting aquaculture production traits. Furthermore, comparative genomics through BES can be used for identifying positional candidate genes from QTL mapping studies, aid in future assembly of a reference genome sequence and elucidating sequence content and complexity in the rainbow trout genome.</p

Comparative mapping of expressed sequence tags containing microsatellites in rainbow trout (Oncorhynchus mykiss)

BACKGROUND: Comparative genomics, through the integration of genetic maps from species of interest with whole genome sequences of other species, will facilitate the identification of genes affecting phenotypes of interest. The development of microsatellite markers from expressed sequence tags will serve to increase marker densities on current salmonid genetic maps and initiate in silico comparative maps with species whose genomes have been fully sequenced. RESULTS: Eighty-nine polymorphic microsatellite markers were generated for rainbow trout of which at least 74 amplify in other salmonids. Fifty-five have been associated with functional annotation and 30 were mapped on existing genetic maps. Homologous sequences were identified for 20 of the EST containing microsatellites to identify comparative assignments within the tetraodon, mouse, and/or human genomes. CONCLUSION: The addition of microsatellite markers constructed from expressed sequence tag data will facilitate the development of high-density genetic maps for rainbow trout and comparative maps with other salmonids and better studied species

Predictors of problem drinking in adolescence and young adulthood. A longitudinal twin-family study

We examined drinking behavior of parents, siblings, and friends of twins as predictors of adolescent and young adult problem drinking over a period of 2 and a period of 7 years. Data of 12 to 30-year-old twins and their family members from the Netherlands Twin Register were analyzed. Problem drinking in twins was assessed in 1995 and 2000 and was defined based on the CAGE and amount of drinking. Data on alcohol use of parents, siblings and friends were collected in 1993. Multinomial logistic regression analyses were used to examine the short-term (1993-1995; n = 2,994) and the long-term longitudinal predictors (1993-2000; n = 1,796) of problem drinking. Age, sex and own alcohol use in 1993 explained 25% of the variance in adolescent and young adult problem drinking. Moreover, adolescents and young adults with fathers who drank frequently and with a large numbers of drinking friends, were at the highest risk for problem drinking 2 years later. Over a period of 7 years the number of drinking friends was no longer a risk factor, but few times a week or daily alcohol use of fathers remained a risk factor for later problem drinking. Drinking behavior of mother and siblings did not substantially predict problem drinking. Sex and age did not moderate these effect