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We attempted to identify phage display cDNA clones that exhibited proteins with carbohydrate affinity, using libraries constructed in mid copy display T7Select 10-3 vector. Although we hoped that higher affinity with multiple number of capsid fusions would facilitate the screening of those clones, the attempts have been unsuccessful.

We attempted to identify phage display cDNA clones that exhibited proteins with carbohydrate affinity, using libraries constructed in mid copy display T7Select 10-3 vector. Although we hoped that higher affinity with multiple number of capsid fusions would facilitate the screening of those clones, the attempts have been unsuccessful.

The serpulid polychaete, Hydroides elegans is a problem fouler in warm water ports throughout the world and is an excellent candidate for investigating molecular reception during larval settlement and metamorphosis. H. elegans is induced to settle and metamorphose when given the appropriate bacterial cue. This study utilized transcriptome sequencing of H. elegans and genome sequencing of two inductive bacterial strains (i.e., Pseudoalteromonas luteoviolacea and Cellulophaga lytica) to provide a means to examine mechanisms and/or genes that may be significant in understanding the interaction between bacteria and larvae that result in biofouling. Transcriptome analysis and in-situ hybridization techniques determined the genetic expression and location of genes in H. elegans as well as important biological pathways involved in metamorphic competence. Likewise, the genome sequencing of P. luteoviolacea and C. lytica determined significant coding sequences for structures that play a role in the settlement and metamorphosis of H. elegans.

This article is from PLoS ONE , volume 9 . Abstract Sugarcane is a major crop used for food and bioenergy production. Modern cultivars are hybrids derived from crosses between Saccharum officinarum and Saccharum spontaneum. Hybrid cultivars combine favorable characteristics from ancestral species and contain a genome that is highly polyploid and aneuploid, containing 100–130 chromosomes. These complex genomes represent a huge challenge for molecular studies and for the development of biotechnological tools that can facilitate sugarcane improvement. Here, we describe full-length enriched cDNA libraries for Saccharum officinarum, Saccharum spontaneum, and one hybrid genotype (SP803280) and analyze the set of open reading frames (ORFs) in their genomes (i.e., their ORFeomes). We found 38,195 (19%) sugarcane-specific transcripts that did not match transcripts from other databases. Less than 1.6% of all transcripts were ancestor-specific (i.e., not expressed in SP803280). We also found 78,008 putative new sugarcane transcripts that were absent in the largest sugarcane expressed sequence tag database (SUCEST). Functional annotation showed a high frequency of protein kinases and stress-related proteins. We also detected natural antisense transcript expression, which mapped to 94% of all plant KEGG pathways; however, each genotype showed different pathways enriched in antisense transcripts. Our data appeared to cover 53.2% (17,563 genes) and 46.8% (937 transcription factors) of all sugarcane full-length genes and transcription factors, respectively. This work represents a significant advancement in defining the sugarcane ORFeome and will be useful for protein characterization, single nucleotide polymorphism and splicing variant identification, evolutionary and comparative studies, and sugarcane genome assembly and annotation.

This article is from BMC Genomics , volume 15 . Abstract Background: A major hurdle to transcriptome profiling by deep-sequencing technologies is that abundant transcripts, such as rRNAs, can overwhelm the libraries, severely reducing transcriptome-wide coverage. Methods for depletion of such unwanted sequences typically require treatment of RNA samples prior to library preparation, are costly and not suited to unusual species and applications. Here we describe Probe-Directed Degradation (PDD), an approach that employs hybridisation to DNA oligonucleotides at the single-stranded cDNA library stage and digestion with Duplex-Specific Nuclease (DSN). Results: Targeting Saccharomyces cerevisiae rRNA sequences in Illumina HiSeq libraries generated by the split adapter method we show that PDD results in efficient removal of rRNA. The probes generate extended zones of depletion as a function of library insert size and the requirements for DSN cleavage. Using intact total RNA as starting material, probes can be spaced at the minimum anticipated library size minus 20 nucleotides to achieve continuous depletion. No off-target bias is detectable when comparing PDD-treated with untreated libraries. We further provide a bioinformatics tool to design suitable PDD probe sets. Conclusion: We find that PDD is a rapid procedure that results in effective and specific depletion of unwanted sequences from deep-sequencing libraries. Because PDD acts at the cDNA stage, handling of fragile RNA samples can be minimised and it should further be feasible to remediate existing libraries. Importantly, PDD preserves the original RNA fragment boundaries as is required for nucleotide-resolution footprinting or base-cleavage studies. Finally, as PDD utilises unmodified DNA oligonucleotides it can provide a low-cost option for large-scale projects, or be flexibly customised to suit different depletion targets, sample types and organisms. Electronic supplementary material: The online version of this article (doi: 10.1186/1471-2164-15-401) contains supplementary material, which is available to authorized users.

This article is from BMC Genomics , volume 15 . Abstract Background: A major hurdle to transcriptome profiling by deep-sequencing technologies is that abundant transcripts, such as rRNAs, can overwhelm the libraries, severely reducing transcriptome-wide coverage. Methods for depletion of such unwanted sequences typically require treatment of RNA samples prior to library preparation, are costly and not suited to unusual species and applications. Here we describe Probe-Directed Degradation (PDD), an approach that employs hybridisation to DNA oligonucleotides at the single-stranded cDNA library stage and digestion with Duplex-Specific Nuclease (DSN). Results: Targeting Saccharomyces cerevisiae rRNA sequences in Illumina HiSeq libraries generated by the split adapter method we show that PDD results in efficient removal of rRNA. The probes generate extended zones of depletion as a function of library insert size and the requirements for DSN cleavage. Using intact total RNA as starting material, probes can be spaced at the minimum anticipated library size minus 20 nucleotides to achieve continuous depletion. No off-target bias is detectable when comparing PDD-treated with untreated libraries. We further provide a bioinformatics tool to design suitable PDD probe sets. Conclusion: We find that PDD is a rapid procedure that results in effective and specific depletion of unwanted sequences from deep-sequencing libraries. Because PDD acts at the cDNA stage, handling of fragile RNA samples can be minimised and it should further be feasible to remediate existing libraries. Importantly, PDD preserves the original RNA fragment boundaries as is required for nucleotide-resolution footprinting or base-cleavage studies. Finally, as PDD utilises unmodified DNA oligonucleotides it can provide a low-cost option for large-scale projects, or be flexibly customised to suit different depletion targets, sample types and organisms. Electronic supplementary material: The online version of this article (doi: 10.1186/1471-2164-15-401) contains supplementary material, which is available to authorized users.

Delos provides cDNA libraries , histological tissue samples and PCR reagents to research laboratories.

Delos provides cDNA libraries , histological tissue samples and PCR reagents to research laboratories.

We proposed to identify, by using a novel phage display technique, carbohydrate-binding proteins (lectins), which may play an important role in progression%on and metastasis of breast cancer. Using blood-group H-expressing glycoprotein fraction as a bait, we observed selective amplification from a HeLa cell transformant phage display cDNA library of phage clones expressing sequences from galectin-3, a lectin with an affinity with the blood-group substance. We then constructed additional libraries including MCF-7 human breast carcinoma and EJG bovine capillary endothelial cell libraries. We have performed in vitro biopanning experiments using purified fractions of glycoproteins and glycolipids as baits and obtained several dozens of candidate phage% clones. However, except for galectin-3, none of these candidates contained cDNA sequences encoding the same proteins. In vivo biopanning was performed using cultured EJG cells as bait. PCR amplification of the inserts from the phage lysates produced a smear of fragments rather than discrete bands even after the fifth/sixth round of selections, indicating that the numbers of clones in the selected populations were still large. We will identify the clones of interest to pursue and characterize those clones in the second year.

This article is from BMC Biotechnology , volume 14 . Abstract Background: Construction of high quality cDNA libraries from the usually low amounts of eukaryotic mRNA extracted from environmental samples is essential in functional metatranscriptomics for the selection of functional, full-length genes encoding proteins of interest. Many of the inserts in libraries constructed by standard methods are represented by truncated cDNAs due to premature stoppage of reverse transcriptase activity and preferential cloning of short cDNAs. Results: We report here a simple and cost effective technique for preparation of sized eukaryotic cDNA libraries from as low as three microgram of total soil RNA dominated by ribosomal and bacterial RNA. cDNAs synthesized by a template switching approach were size-fractionated by two dimensional agarose gel electrophoresis prior to PCR amplification and cloning. Effective size selection was demonstrated by PCR amplification of conserved gene families specific of each size class. Libraries of more than one million independent inserts whose sizes ranged between one and four kb were thus produced. Up to 80% of the insert sequences were homologous to eukaryotic gene sequences present in public databases. Conclusions: A simple and cost effective technique has been developed to construct sized eukaryotic cDNA libraries from environmental samples. This technique will facilitate expression cloning of environmental eukaryotic genes and contribute to a better understanding of basic biological and/or ecological processes carried out by eukaryotic microbial communities.

This article is from Frontiers in Immunology , volume 4 . Abstract High-throughput sequencing has the power to reveal the nature of adaptive immunity as represented by the full complexity of T-cell receptor (TCR) and antibody (IG) repertoires, but is at present severely compromised by the quantitative bias, bottlenecks, and accumulated errors that inevitably occur in the course of library preparation and sequencing. Here we report an optimized protocol for the unbiased preparation of TCR and IG cDNA libraries for high-throughput sequencing, starting from thousands or millions of live cells in an investigated sample. Critical points to control are revealed, along with tips that allow researchers to minimize quantitative bias, accumulated errors, and cross-sample contamination at each stage, and to enhance the subsequent bioinformatic analysis. The protocol is simple, reliable, and can be performed in 1–2 days.

It is difficult to obtain good quality cDNA from RNA obtained from microdissected cells from pathological sections, particularly from paraffin sections. We are studying ways of isolating extremely small quantities of mRNA and/or 1st strand cDNA with magnetic beads. We are developing techniques for increasing the yield of 1st strand and 2nd strand synthesis reactions. In addition, we are studying 2 methods of amplification: the polymerase chain reaction and amplified RNA methods. Most of our work to date has been carried out with total RNA isolated by traditional methods. In the coming year, we will apply our findings to RNA or 1st strand cDNA obtained from pathological sections.

This article is from Breeding Science , volume 62 . Abstract Tea is one of the most popular beverages in the world and the tea plant, Camellia sinensis (L.) O. Kuntze, is an important crop in many countries. To increase the amount of genomic information available for C. sinensis, we constructed seven cDNA libraries from various organs and used these to generate expressed sequence tags (ESTs). A total of 17,458 ESTs were generated and assembled into 5,262 unigenes. About 50% of the unigenes were assigned annotations by Gene Ontology. Some were homologous to genes involved in important biological processes, such as nitrogen assimilation, aluminum response, and biosynthesis of caffeine and catechins. Digital northern analysis showed that 67 unigenes were expressed differentially among the seven organs. Simple sequence repeat (SSR) motif searches among the unigenes identified 1,835 unigenes (34.9%) harboring SSR motifs of more than six repeat units. A subset of 100 EST-SSR primer sets was tested for amplification and polymorphism in 16 tea accessions. Seventy-one primer sets successfully amplified EST-SSRs and 70 EST-SSR loci were polymorphic. Furthermore, these 70 EST-SSR markers were transferable to 14 other Camellia species. The ESTs and EST-SSR markers will enhance the study of important traits and the molecular genetics of tea plants and other Camellia species.

This article is from Breeding Science , volume 62 . Abstract Tea is one of the most popular beverages in the world and the tea plant, Camellia sinensis (L.) O. Kuntze, is an important crop in many countries. To increase the amount of genomic information available for C. sinensis, we constructed seven cDNA libraries from various organs and used these to generate expressed sequence tags (ESTs). A total of 17,458 ESTs were generated and assembled into 5,262 unigenes. About 50% of the unigenes were assigned annotations by Gene Ontology. Some were homologous to genes involved in important biological processes, such as nitrogen assimilation, aluminum response, and biosynthesis of caffeine and catechins. Digital northern analysis showed that 67 unigenes were expressed differentially among the seven organs. Simple sequence repeat (SSR) motif searches among the unigenes identified 1,835 unigenes (34.9%) harboring SSR motifs of more than six repeat units. A subset of 100 EST-SSR primer sets was tested for amplification and polymorphism in 16 tea accessions. Seventy-one primer sets successfully amplified EST-SSRs and 70 EST-SSR loci were polymorphic. Furthermore, these 70 EST-SSR markers were transferable to 14 other Camellia species. The ESTs and EST-SSR markers will enhance the study of important traits and the molecular genetics of tea plants and other Camellia species.

Approved tor public release; distribution unlimited 13. ABSTRACT (Maximum 200 Words) We proposed to identify%, by using a novel phage display technique, carbohydrate-binding proteins (lectins), which may play an important role in progression and metastasis of breast cancer. Using blood- group H-expressing glycoprotein fraction as bait, we observed enrichment of phage clones expressing sequences from galectin-3, a lectin with an affinity with the blood-group substance. Since we obtained a multiple number of different clones encoding this protein, the binding was selective and real. We then constructed additional libraries including MCF-7 human breast carcinoma and EJG bovine capillary endothelial cell libraries. We performed in vitro biopanning experiments using as bait purified fractions of glycoproteins and glycolipids, as well as antibodies against plant lectins, and obtained several dozens of candidate phage clones.