Direct molecular haplotyping by melting curve analysis of hybridization probes: beta 2-adrenergic receptor haplotypes as an example

Direct determination of the association of multiple genetic polymorphisms, or haplotyping, in individual samples is challenging because of chromosome diploidy. Here, we describe the ability of hybridization probes, commonly used as genotyping tools, to establish single nucleotide polymorphism (SNP) haplotypes in a single step. Three haplotypes found in the beta 2-adrenergic receptor (β2AR) gene and characterized by three different SNPs combinations are presented as examples. Each combination of SNPs has a unique stability, recorded by its melting temperature, even when intervening sequences from the template must loop out during probe hybridization. In the course of this study, two haplotypes in β2AR not described previously were discovered. This approach provides a tool for molecular haplotyping that should prove useful in clinical molecular genetics diagnostics and pharmacogenetic research where methods for direct haplotyping are needed

An efficient method for multi-locus molecular haplotyping

Many methods exist for genotyping—revealing which alleles an individual carries at different genetic loci. A harder problem is haplotyping—determining which alleles lie on each of the two homologous chromosomes in a diploid individual. Conventional approaches to haplotyping require the use of several generations to reconstruct haplotypes within a pedigree, or use statistical methods to estimate the prevalence of different haplotypes in a population. Several molecular haplotyping methods have been proposed, but have been limited to small numbers of loci, usually over short distances. Here we demonstrate a method which allows rapid molecular haplotyping of many loci over long distances. The method requires no more genotypings than pedigree methods, but requires no family material. It relies on a procedure to identify and genotype single DNA molecules, and reconstruction of long haplotypes by a ‘tiling’ approach. We demonstrate this by resolving haplotypes in two regions of the human genome, harbouring 20 and 105 single-nucleotide polymorphisms, respectively. The method can be extended to reconstruct haplotypes of arbitrary complexity and length, and can make use of a variety of genotyping platforms. We also argue that this method is applicable in situations which are intractable to conventional approaches

PHUSER (Primer Help for USER): a novel tool for USER fusion primer design

Uracil-Specific Exision Reagent (USER) fusion is a recently developed technique that allows for assembly of multiple DNA fragments in a few simple steps. However, designing primers for USER fusion is both tedious and time consuming. Here, we present the Primer Help for USER (PHUSER) software, a novel tool for designing primers specifically for USER fusion and USER cloning applications. We also present proof-of-concept experimental validation of its functionality. PHUSER offers quick and easy design of PCR optimized primers ensuring directionally correct fusion of fragments into a plasmid containing a customizable USER cassette. Designing primers using PHUSER ensures that the primers have similar annealing temperature (Tm), which is essential for efficient PCR. PHUSER also avoids identical overhangs, thereby ensuring correct order of assembly of DNA fragments. All possible primers are individually analysed in terms of GC content, presence of GC clamp at 3′-end, the risk of primer dimer formation, the risk of intra-primer complementarity (secondary structures) and the presence of polyN stretches. Furthermore, PHUSER offers the option to insert linkers between DNA fragments, as well as highly flexible cassette options. PHUSER is publicly available at http://www.cbs.dtu.dk/services/phuser/

Thermodynamic contributions of single internal rA·dA, rC·dC, rG·dG and rU·dT mismatches in RNA/DNA duplexes

The thermodynamic contributions of rA·dA, rC·dC, rG·dG and rU·dT single internal mismatches were measured for 54 RNA/DNA duplexes in a 1 M NaCl buffer using UV absorbance thermal denaturation. Thermodynamic parameters were obtained by fitting absorbance versus temperature profiles using the curve-fitting program Meltwin. The weighted average thermodynamic data were fit using singular value decomposition to determine the eight non-unique nearest-neighbor parameters for each internal mismatch. The new parameters predict the ΔG°37, ΔH° and melting temperature (Tm) of duplexes containing these single mismatches within an average of 0.33 kcal/mol, 4.5 kcal/mol and 1.4°C, respectively. The general trend in decreasing stability for the single internal mismatches is rG·dG > rU·dT > rA·dA > rC·dC. The stability trend for the base pairs 5′ of the single internal mismatch is rG·dC > rC·dG > rA·dT > rU·dA. The stability trend for the base pairs 3′ of the single internal mismatch is rC·dG > rG·dC >> rA·dT > rU·dA. These nearest-neighbor values are now a part of a complete set of single internal mismatch thermodynamic parameters for RNA/DNA duplexes that are incorporated into the nucleic acid assay development software programs Visual oligonucleotide modeling platform (OMP) and ThermoBLAST

Evidence for a Fourteenth mtDNA-Encoded Protein in the Female-Transmitted mtDNA of Marine Mussels (Bivalvia: Mytilidae)

BACKGROUND: A novel feature for animal mitochondrial genomes has been recently established: i.e., the presence of additional, lineage-specific, mtDNA-encoded proteins with functional significance. This feature has been observed in freshwater mussels with doubly uniparental inheritance of mtDNA (DUI). The latter unique system of mtDNA transmission, which also exists in some marine mussels and marine clams, is characterized by one mt genome inherited from the female parent (F mtDNA) and one mt genome inherited from the male parent (M mtDNA). In freshwater mussels, the novel mtDNA-encoded proteins have been shown to be mt genome-specific (i.e., one novel protein for F genomes and one novel protein for M genomes). It has been hypothesized that these novel, F- and M-specific, mtDNA-encoded proteins (and/or other F- and/or M-specific mtDNA sequences) could be responsible for the different modes of mtDNA transmission in bivalves but this remains to be demonstrated. METHODOLOGY/PRINCIPAL FINDINGS: We investigated all complete (or nearly complete) female- and male-transmitted marine mussel mtDNAs previously sequenced for the presence of ORFs that could have functional importance in these bivalves. Our results confirm the presence of a novel F genome-specific mt ORF, of significant length (>100aa) and located in the control region, that most likely has functional significance in marine mussels. The identification of this ORF in five Mytilus species suggests that it has been maintained in the mytilid lineage (subfamily Mytilinae) for ∼13 million years. Furthermore, this ORF likely has a homologue in the F mt genome of Musculista senhousia, a DUI-containing mytilid species in the subfamily Crenellinae. We present evidence supporting the functionality of this F-specific ORF at the transcriptional, amino acid and nucleotide levels. CONCLUSIONS/SIGNIFICANCE: Our results offer support for the hypothesis that "novel F genome-specific mitochondrial genes" are involved in key biological functions in bivalve species with DUI

Deletion of the N-terminus of SF2/ASF Permits RS-Domain-Independent Pre-mRNA Splicing

Serine/arginine-rich (SR) proteins are essential splicing factors with one or two RNA-recognition motifs (RRMs) and a C-terminal arginine- and serine-rich (RS) domain. SR proteins bind to exonic splicing enhancers via their RRM(s), and from this position are thought to promote splicing by antagonizing splicing silencers, recruiting other components of the splicing machinery through RS-RS domain interactions, and/or promoting RNA base-pairing through their RS domains. An RS domain tethered at an exonic splicing enhancer can function as a splicing activator, and RS domains play prominent roles in current models of SR protein functions. However, we previously reported that the RS domain of the SR protein SF2/ASF is dispensable for in vitro splicing of some pre-mRNAs. We have now extended these findings via the identification of a short inhibitory domain at the SF2/ASF N-terminus; deletion of this segment permits splicing in the absence of this SR protein's RS domain of an IgM pre-mRNA substrate previously classified as RS-domain-dependent. Deletion of the N-terminal inhibitory domain increases the splicing activity of SF2/ASF lacking its RS domain, and enhances its ability to bind pre-mRNA. Splicing of the IgM pre-mRNA in S100 complementation with SF2/ASF lacking its RS domain still requires an exonic splicing enhancer, suggesting that an SR protein RS domain is not always required for ESE-dependent splicing activation. Our data provide additional evidence that the SF2/ASF RS domain is not strictly required for constitutive splicing in vitro, contrary to prevailing models for how the domains of SR proteins function to promote splicing

Gene Discovery in the Threatened Elkhorn Coral: 454 Sequencing of the Acropora palmata Transcriptome

BACKGROUND: Cnidarians, including corals and anemones, offer unique insights into metazoan evolution because they harbor genetic similarities with vertebrates beyond that found in model invertebrates and retain genes known only from non-metazoans. Cataloging genes expressed in Acropora palmata, a foundation-species of reefs in the Caribbean and western Atlantic, will advance our understanding of the genetic basis of ecologically important traits in corals and comes at a time when sequencing efforts in other cnidarians allow for multi-species comparisons. RESULTS: A cDNA library from a sample enriched for symbiont free larval tissue was sequenced on the 454 GS-FLX platform. Over 960,000 reads were obtained and assembled into 42,630 contigs. Annotation data was acquired for 57% of the assembled sequences. Analysis of the assembled sequences indicated that 83-100% of all A. palmata transcripts were tagged, and provided a rough estimate of the total number genes expressed in our samples (~18,000-20,000). The coral annotation data contained many of the same molecular components as in the Bilateria, particularly in pathways associated with oxidative stress and DNA damage repair, and provided evidence that homologs of p53, a key player in DNA repair pathways, has experienced selection along the branch separating Cnidaria and Bilateria. Transcriptome wide screens of paralog groups and transition/transversion ratios highlighted genes including: green fluorescent proteins, carbonic anhydrase, and oxidative stress proteins; and functional groups involved in protein and nucleic acid metabolism, and the formation of structural molecules. These results provide a starting point for study of adaptive evolution in corals. CONCLUSIONS: Currently available transcriptome data now make comparative studies of the mechanisms underlying coral's evolutionary success possible. Here we identified candidate genes that enable corals to maintain genomic integrity despite considerable exposure to genotoxic stress over long life spans, and showed conservation of important physiological pathways between corals and bilaterians

Characteristics of the nuclear (18S, 5.8S, 28S and 5S) and mitochondrial (12S and 16S) rRNA genes of Apis mellifera (Insecta: Hymenoptera): structure, organization, and retrotransposable elements

As an accompanying manuscript to the release of the honey bee genome, we report the entire sequence of the nuclear (18S, 5.8S, 28S and 5S) and mitochondrial (12S and 16S) ribosomal RNA (rRNA)-encoding gene sequences (rDNA) and related internally and externally transcribed spacer regions of Apis mellifera (Insecta: Hymenoptera: Apocrita). Additionally, we predict secondary structures for the mature rRNA molecules based on comparative sequence analyses with other arthropod taxa and reference to recently published crystal structures of the ribosome. In general, the structures of honey bee rRNAs are in agreement with previously predicted rRNA models from other arthropods in core regions of the rRNA, with little additional expansion in non-conserved regions. Our multiple sequence alignments are made available on several public databases and provide a preliminary establishment of a global structural model of all rRNAs from the insects. Additionally, we provide conserved stretches of sequences flanking the rDNA cistrons that comprise the externally transcribed spacer regions (ETS) and part of the intergenic spacer region (IGS), including several repetitive motifs. Finally, we report the occurrence of retrotransposition in the nuclear large subunit rDNA, as R2 elements are present in the usual insertion points found in other arthropods. Interestingly, functional R1 elements usually present in the genomes of insects were not detected in the honey bee rRNA genes. The reverse transcriptase products of the R2 elements are deduced from their putative open reading frames and structurally aligned with those from another hymenopteran insect, the jewel wasp Nasonia (Pteromalidae). Stretches of conserved amino acids shared between Apis and Nasonia are illustrated and serve as potential sites for primer design, as target amplicons within these R2 elements may serve as novel phylogenetic markers for Hymenoptera. Given the impending completion of the sequencing of the Nasonia genome, we expect our report eventually to shed light on the evolution of the hymenopteran genome within higher insects, particularly regarding the relative maintenance of conserved rDNA genes, related variable spacer regions and retrotransposable elements