Real-time PCR/MCA assay using fluorescence resonance energy transfer for the genotyping of resistance related DHPS-540 mutations in Plasmodium falciparum

BACKGROUND: Sulphadoxine-pyrimethamine has been abandoned as first- or second-line treatment by most African malaria endemic countries in favour of artemisinin-based combination treatments, but the drug is still used as intermittent preventive treatment during pregnancy. However, resistance to sulphadoxine-pyrimethamine has been increasing in the past few years and, although the link between molecular markers and treatment failure has not been firmly established, at least for pregnant women, it is important to monitor such markers. METHODS: This paper reports a novel sensitive, semi-quantitative and specific real-time PCR and melting curve analysis (MCA) assay using fluorescence resonance energy transfer (FRET) for the detection of DHPS-540, an important predictor for SP resistance. FRET/MCA was evaluated using 78 clinical samples from malaria patients and compared to PCR-RFLP. RESULTS: Sixty-two samples were in perfect agreement between both assays. One sample showed a small wild type signal with FRET/MCA that indicates a polyclonal infection. Four samples were not able to generate enough material in both assays to distinguish mutant from wild-type infection, six samples gave no signal in PCR-RFLP and five samples gave no amplification in FRET/MCA. CONCLUSION: FRET/MCA is an effective tool for the identification of SNPs in drug studies and epidemiological surveys on resistance markers in general and DHPS-540 mutation in particular

Sperm design and variation in the New World blackbirds (Icteridae)

Post-copulatory sexual selection (PCSS) is thought to be one of the evolutionary forces responsible for the rapid and divergent evolution of sperm design. However, whereas in some taxa particular sperm traits are positively associated with PCSS, in other taxa, these relationships are negative, and the causes of these different patterns across taxa are poorly understood. In a comparative study using New World blackbirds (Icteridae), we tested whether sperm design was influenced by the level of PCSS and found significant positive associations with the level of PCSS for all sperm components but head length. Additionally, whereas the absolute length of sperm components increased, their variation declined with the intensity of PCSS, indicating stabilizing selection around an optimal sperm design. Given the diversity of, and strong selection on, sperm design, it seems likely that sperm phenotype may influence sperm velocity within species. However, in contrast to other recent studies of passerine birds, but consistent with several other studies, we found no significant link between sperm design and velocity, using four different species that vary both in sperm design and PCSS. Potential reasons for this discrepancy between studies are discussed

Postcopulatory Sexual Selection Is Associated with Reduced Variation in Sperm Morphology

The evolutionary role of postcopulatory sexual selection in shaping male reproductive traits, including sperm morphology, is well documented in several taxa. However, previous studies have focused almost exclusively on the influence of sperm competition on variation among species. In this study we tested the hypothesis that intraspecific variation in sperm morphology is driven by the level of postcopulatory sexual selection in passerine birds.Using two proxy measures of sperm competition level, (i) relative testes size and (ii) extrapair paternity level, we found strong evidence that intermale variation in sperm morphology is negatively associated with the degree of postcopulatory sexual selection, independently of phylogeny.Our results show that the role of postcopulatory sexual selection in the evolution of sperm morphology extends to an intraspecific level, reducing the variation towards what might be a species-specific 'optimum' sperm phenotype. This finding suggests that while postcopulatory selection is generally directional (e.g., favouring longer sperm) across avian species, it also acts as a stabilising evolutionary force within species under intense selection, resulting in reduced variation in sperm morphology traits. We discuss some potential evolutionary mechanisms for this pattern

Nucleic acid-based fluorescent probes and their analytical potential

It is well known that nucleic acids play an essential role in living organisms because they store and transmit genetic information and use that information to direct the synthesis of proteins. However, less is known about the ability of nucleic acids to bind specific ligands and the application of oligonucleotides as molecular probes or biosensors. Oligonucleotide probes are single-stranded nucleic acid fragments that can be tailored to have high specificity and affinity for different targets including nucleic acids, proteins, small molecules, and ions. One can divide oligonucleotide-based probes into two main categories: hybridization probes that are based on the formation of complementary base-pairs, and aptamer probes that exploit selective recognition of nonnucleic acid analytes and may be compared with immunosensors. Design and construction of hybridization and aptamer probes are similar. Typically, oligonucleotide (DNA, RNA) with predefined base sequence and length is modified by covalent attachment of reporter groups (one or more fluorophores in fluorescence-based probes). The fluorescent labels act as transducers that transform biorecognition (hybridization, ligand binding) into a fluorescence signal. Fluorescent labels have several advantages, for example high sensitivity and multiple transduction approaches (fluorescence quenching or enhancement, fluorescence anisotropy, fluorescence lifetime, fluorescence resonance energy transfer (FRET), and excimer-monomer light switching). These multiple signaling options combined with the design flexibility of the recognition element (DNA, RNA, PNA, LNA) and various labeling strategies contribute to development of numerous selective and sensitive bioassays. This review covers fundamentals of the design and engineering of oligonucleotide probes, describes typical construction approaches, and discusses examples of probes used both in hybridization studies and in aptamer-based assays

Triple-FRET Technique for Energy Transfer Between Conjugated Polymer and TAMRA Dye with Possible Applications in Medical Diagnostics

Three-component Förster resonance energy transfer (FRET) has been used to obtain efficient FRET between the cationic conjugated polymer (CCP) as donor and 5-carboxy tetramethylrhodamine (TAMRA) dye as acceptor, by using an intermediate donor, fluorescein. In spite of the fact that there is enough overlap between the emission spectra of CCP and absorption spectra of TAMRA, the efficiency of FRET between CCP and TAMRA is poor. The reason for this is that while the Förster critical distance is not very sensitive to the overlap, the FRET efficiency is extremely sensitive to it. However, it is observed that the FRET efficiency between CCP and TAMRA improves considerably when fluorescein is introduced in the solution. The triple FRET so obtained can be used for deoxyribonucleic acid sequence detection in medical diagnostics because the fluorescence emission from TAMRA is pH-insensitive