Sequence Organization in Animal DNA’s

Recognition of poly(C) DNA and RNA sequences in mammalian cells is achieved by a subfamily of the KH (hnRNP K homology) domain-containing proteins known as poly(C)-binding proteins (PCBPs). To reveal the molecular basis of poly(C) sequence recognition, we have determined the crystal structure, at 1.7-A resolution, of PCBP2 KH1 in complex with a 7-nucleotide DNA sequence (5'-AACCCTA-3') corresponding to one repeat of the human C-rich strand telomeric DNA. The protein-DNA interaction is mediated by the combination of several stabilizing forces including hydrogen bonding, electrostatic interactions, van der Waals contacts, and shape complementarities. Specific recognition of the three cytosine residues is realized by a dense network of hydrogen bonds involving the side chains of two conserved lysines and one glutamic acid. The co-crystal structure also reveals a protein-protein dimerization interface of PCBP2 KH1 located on the opposite side of the protein from the DNA binding groove. Numerous stabilizing protein-protein interactions, including hydrophobic contacts, stacking of aromatic side chains, and a large number of hydrogen bonds, indicate that the protein-protein interaction interface is most likely genuine. Interaction of PCBP2 KH1 with the C-rich strand of human telomeric DNA suggests that PCBPs may participate in mechanisms involved in the regulation of telomere/telomerase functions.

Repetitive and non-repetitive sequence in sea urchin heterogeneous nuclear RNA

The Evolution of Nonrepetitive DNA in Sea Urchins

Morphogenesis, the process by which the tissues and organs of the embryo are properly shaped, is a fundamental feature of development. In the sea urchin, the formation of the calcified enodoskeleton is a major morphogenetic event. Differentiation of the skeletogenic primary mesenchyme cells (PMCs) has been considered to occur in two phases: the autonomous specification of PMCs followed by signal-dependent patterning of PMCs and the embryonic skeleton they produce. Autonomous specification creates a homogenous population of PMCs, but the later differentiation of these cells is influenced by extrinsic signals that provide essential positional information. Recent studies showed that ectodermal growth factors are critically involved in the guidance of PMC migration and skeletal differentiation. However, a better understanding of the various signaling pathways that regulate skeletogenesis and their role in PMC gene expression remains to be established. This study examines the regulation of morphogenesis by signaling pathways, using skeletogenesis in the sea urchin embryo as a model. The aim of this study was to identify and study the roles of extrinsic signals in regulating PMC gene expression, focusing on the later, signal-dependent phase of PMC differentiation. By analyzing and classifying spatial expression patterns of 39 genes preferentially expressed in PMCs, I find that: 1) these genes are expressed non-uniformly within the PMC syncytium, reflecting a widespread influence of locally activated signals; 2) regions with elevated gene expression correlate with sites of rapid biomineral deposition at each stage; 3) non-uniform expression of genes within the PMC syncytium is controlled by multiple signal in a precise temporal sequence. I also provide evidence that ectoderm-derived VEGF signaling regulates gene expression in PMCs via the MAPK pathway on the ventral side of the embryo. Additionally, my work has identified an essential role for TGF-β signaling in skeletogenesis. Previous studies indicate that a complete repertoire of TGF-β signaling components is present in the sea urchin genome and TgfbrII mRNA is preferentially expressed in PMCs at the early gastrula stage. In this study, I show that TgfbrII mRNA is specifically expressed in the PMC lineage from the hatched blastula to the mid-gastrula stage. Perturbation experiments indicate that TgfbrII is activated by the single, sensu stricto TGF-β ligand in sea urchins and is required for skeletogenesis in the sea urchin embryo. I also show that the late activity of Alk4/5/7, the putative Type I receptor, regulates skeletogenesis in a dose-dependent manner. Isolation and in vitro culture of PMCs demonstrates that both Alk4/5/7 and TgfbrII function cell autonomously in these cells. I provide evidence that TGF-β-TgfbrII signaling is not involved in dorsal-ventral axis patterning or PMC specification; instead, this pathway plays a selective role in later skeletal patterning. Taken as a whole, my research demonstrates that skeletogenesis is regulated by a much more diverse suite of signaling pathways than was previously appreciated. These findings significantly expand our understanding of the complex regulation of skeletal morphogenesis by extrinsic signals during embryonic development.

Due to high cost, availability of human immunodeficiency virus type 1 (HIV-1) drug resistance testing in resource-poor settings is still limited. We therefore evaluated the usefulness of viral DNA extracted from either whole blood or dried blood spots (DBS). Samples were collected from 50 patients receiving therapy and 10 therapy-naïve patients. Amplification and sequencing of RNA and DNA was performed using an in-house assay. Protease (PR) and reverse transcriptase (RT) sequences of plasma viral RNA were obtained for 96.6% and 89.7%, respectively, of the 29 patients with a detectable viral load. For cellular viral DNA, useful PR and RT sequences were obtained for 96.6% and 93.1% of the whole-blood-cell samples and for 93.1% and 93.1% of the DBS samples, respectively. For the 31 patients with an undetectable viral load, PR and RT sequences were obtained for 67.7% and 61.3% of the whole-blood-cell DNA preparations and for 54.8% and 58.1% of the DBS DNA preparations, respectively. A good correlation between RNA and DNA sequences was found; most discordances were caused by the detection of mixed amino acids. Of the RT drug-resistant mutations, 13 (38.2%) were seen in RNA only, 6 (17.6%) in DNA only, and 15 (44.1%) in both. Repeated amplification and sequencing of DNA extracts revealed a lack of reproducibility for the detection of drug resistance mutations in a number of samples, indicating a possible founder effect. In conclusion, this study shows the feasibility of genotypic drug resistance testing on whole blood cells or DBS and its possible usefulness for HIV-1 subtyping or examining the overall distribution of drug resistance in a population. For individual patients, RNA sequencing was shown to be superior to DNA sequencing, especially for patients who experienced early treatment failure. The use of DNA extracted from whole blood or DBS for the detection of archived drug resistance mutations deserves further study.

Fil: Rubilar Panasiuk, Cynthia Tamara. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Centro Nacional Patagonico. Centro para el Estudio de Sistemas Marinos; Argentina

Non-repetitive DNA of anciently tetraploid teleostean species was analysed for the presence of duplicated sequences. Closely related diploid species were investigated in comparison. From the reassociation kinetics of total nuclear DNA, rate constants and fraction sizes of classes of repetitive and non-repetitive sequences were determined. DNA fractions enriched in the slowest renaturing sequence class were determined. DNA fractions enriched in the slowest renaturing sequence class were prepared and subjected to reassociation. The rate constants of these reactions were compared with the values expected for single-copy DNA from analytical genome size determinations. From reassociated DNA enriched in non-repetitive sequences also the melting temperatures were determined as a measure of internal base sequence heterogeneity. It has been shown that the two ancient tetraploids Cyprinus carpio and Thymallus thymallus are, with regard to the thermal stability of reassociated non-repetitive DNA, and with regard to the correspondence of reaction rates with the values expected for single copy DNA, indistinguishable from diploid controls (Rutilus rutilus, Clupea harengus and Sprattus sprattus). The tetraploid species Salmo irideus, Salvelinus fontinalis and Coregonus lavaretus appear as very recent tetraploids with regard to these criteria. The significance of the results for estimating the time of occurrence of polyploidisation events in these taxa is discussed.

### Widespread RNA and DNA Sequence Differences in the Human Transcriptome Li et al Science . 2011;333:53–58. New genomic studies show that the transfer of genetic information from DNA to RNA within humans is not very faithful. If true, frequent, or purposeful, then the implications for human health and disease are significant, but the burden of proof required is significant. Commentary: Molecular biology and genetics has a “central dogma.” First postulated by Francis Crick1,2 in 1958, shortly after the discovery of the double helix, it states that the molecular transfer or flow of genetic information occurs only as DNA→RNA→ protein. This argument is intrinsic to our understanding of genotypes, phenotypes, and human disease. The central “dogma,” a word whose precise meaning Crick acknowledged he did not precisely know3 and that could have been given the less glamorous moniker “hypothesis,” is frequently misunderstood as also implying high accuracy or even inflexibility.2 We now know that genetic information flow can be sometimes corrupted and can also occur in other directions in particular biological systems. A recent study by Li and colleagues,4 from the laboratory of Vivian Cheung at the University of Pennsylvania, now demonstrates apparent widespread DNA→RNA sequence differences in humans. The experiments they conducted involved sequencing reverse-transcribed RNA from cultured B cells isolated from 27 different humans and comparing them with their host genome sequences. Surprisingly, after appropriate quality controls, they discovered numerous base positions at which the RNA sequence was different from the DNA sequence in multiple molecules, a phenomenon they refer to as RDD (RNA-DNA difference). Specifically, they identified 28766 RDDs in the 27 individuals at 10210 exonic sites within 4741 genes. Each individual had high-quality RNA-based sequence data at 9.8 million sites: so, RDD was detected at a rate of 1.1×10−4 per exonic site (base) across human genes. Interestingly, they observed all 12 types …

The arrangement of repetitive and nonrepetitive DNA sequences in the soybean genome was ascertained by a comparison of the reassociation kinetics of short (250 nucleotides) and long (2700 nucleotides) DNA fragments, the size distribution of S-1 nuclease resistant repetitive duplexes, and a direct assay of the spectrum of DNA sequences present on long DNA fragments enriched in repetitive DNA. These measurements reveal the following: (1) The 1N genome size of the soybean plant is 1.97 pg. (2) Approximately 40% of the soybean genome consists of nonrepetitive or single-copy DNA sequences, while 60% is repetitive DNA. (3) The repetitive DNA is partitioned into three discrete classes termed "very fast," "fast," and "slow," containing DNA sequences repeated an average of 290,000, 2800, and 19 times each. (4) Approximately 35--50% of the soybean genome is arranged in a short-period interspersion pattern of 250 nucleotide slow sequences and single-copy DNA averaging up to 2700 nucleotides in length. (5) From 30% to 45% of the soybean genome is organized into long stretches of repetitive DNA at least 1500 nucleotides in length. (6) Minimal interspersion of repetitive sequence classes occurs in soybean DNA.

The detection of regulatory sequences in DNA is a challenging problem, especially when considered in the context of whole genomes. The degree of sequence conservation of regulatory protein binding sites is often weak, and the sites are obscured by surrounding intergenic sequence. Since structural interactions are vital for protein-DNA interactions, structural representations of regulatory sites can yield a more accurate model and a better understanding of within-site variability. However, the use of multiple alternative representations of DNA introduces a requirement for novel algorithms that can create and test different combinations of DNA features. The Genetic Algorithm Neural Network (GANN) was designed to identify combinations of patterns that can be used to distinguish between different classes of training sequence. GANN trains a set of artificial neural networks to classify sets of sequence using either backpropagation or a genetic algorithm, and uses an ‘outer genetic algorithm’ to choose the best inputs from a pool of DNA features that can include sequence, structure, and weight matrix representations. When trained with a subset of upstream sequences from a whole genome, GANN was able to detect patterns such as the Shine-Dalgarno sequence in Escherichia coli K12, and sequences consistent with archaeal promoters in the archaeon Sulfolobus solfataricus P2. The Motif Genetic Algorithm (MGA) constructs motif representations by concatenating minimal units of DNA sequence and structure. This algorithm was used to model conserved patterns in DNA, including the binding sites for E. coli cyclic AMP activated protein (CAP), integration host factor (IHF), and two different promoter types recognized by alternative bacterial sigma factors. The CAP models were used to detect other putative binding sites in upstream regions of the E. coli K12 genome, while attempts to train an accurate model of IHF binding sites revealed an important role for structural representations in motif modeling.

A quantitative and highly sensitive, yet simple and rapid, biosensor system was developed for the detection of nucleic acid sequences that can also be adapted to the detection of antigens. A dipstick-type biosensor with liposome amplification, based on a sandwich assay format with optical detection, was combined with a simple coupling reaction that allows the transformation of the generic biosensor components to target specific ones by a mere incubation step. This biosensor platform system was developed and optimized, and its principle was proven using DNA oligonucleotides that provided a nucleic acid biosensor for the specific detection of RNA and DNA sequences. However, the coupling reaction principle chosen can also be used for the immobilization of antibodies or receptor molecules, and therefore for the development of immunosensors and receptor-based biosensors. The generic biosensor consists of liposomes entrapping sulforhodamine B that are coated with streptavidin on the outside, and polyethersulfone membranes with anti-fluorescein antibodies immobilized in the detection zone. In order to transform the generic biosensor into a specific DNA/RNA biosensor, two oligonucleotides that are able to hybridize to the target sequence were labeled with a biotin and a fluorescein molecule, respectively. By simultaneously incubating the liposomes, both oligonucleotides, and the target sequence in a hybridization buffer for 20-30 min at 42 degrees C, a sandwich complex was formed. The mixture was applied to the polyethersulfone membrane. The complex was captured in the detection zone and quantified using a hand-held reflectometer. The system was tested using RNA sequences from B. anthracis, C. parvum and E. coli. Quantitation of concentrations between 10 fmol and 1000 fmol (10-1000 nM) was possible without altering any biosensor assay conditions. In addition, no changes to hybridization conditions were required when using authentic nucleic acid sequence-based amplified RNA sequences, and the generic biosensor compared favorably with those previously developed specifically for the RNA sequences. Therefore, the universal biosensor described is an excellent tool, for use in laboratories or at test sites, for rapidly investigating and quantifying any nucleic acid sequence of interest, as well as potentially any antigen of interest that can be bound by two antibodies simultaneously.

Freshwater ecosystems represent hotspots for the world’s total diversity and human well-being. However, they are also subjected to threats across the globe as a result of localised human activities, broad scale catchment clearance, climate change and invasive species. The increased degradation of freshwater habitats and their ecological functions as a consequence of these threats, at local and global scales, has led to significant freshwater problems for human existence and the world’s biodiversity. There is growing evidence that the loss of biodiversity is one of the most complex environmental issues facing the world; however, the importance of understanding species distribution patterns and the ecological differentiation among species that are reflected as species-specific responses or tolerances to environmental drivers is less well understood. In particular, when a morphological approach is used as a taxonomic tool for investigating species diversity and species level responses to environmental drivers, the diversity of responses hidden within species complexes may not be realized, and the conclusion of generality may mask specific cryptic species responses. In South-East Queensland, Australia, European occupation since the mid 1800’s has seen large scale clearing of native vegetation along streams and rivers in nearly all catchments. As a consequence of this land-use change catchment hydrology has been substantially altered, which, combined with the presence of dams and weirs, has resulted in a decline in water quality of streams in some catchments, which is of growing concern for conservation of species biodiversity. This study aimed to explore cryptic diversity in two species complexes of freshwater aytid shrimps common in South-East Queensland and elucidate species level responses to environmental variation that could explain their spatial distribution. This broad aim was met through three specific studies. First, using regional scale data of cryptic species diversity and water quality, the importance of species-specific responses to environmental conditions in determining spatial distribution patterns and environmental relationships of cryptic species in the Caridina indistincta and Paratya australiensis species complexes was examined. To accomplish this aim, DNA sequences were used to identify shrimp specimens from 89 sites in 17 catchments spanning the study area. In addition, an assessment of eight morphological traits was used to test whether these cryptic species could be morphologically identified. Use of these eight traits did allow species level identification, at least in South-East Queensland. However, caution is suggested in the use of these morphological traits for recognising species, due to the probability of morphological plasticity within a species across broad spatial scales. Ordination analysis of presence-absence data showed that the five cryptic species within the two species complexes showed spatially distinct distributions across streams in SEQ, with each cryptic species displaying different relationships with individual environmental variables. For species in the Caridina indistincta complex, C. indistincta sp. B was significantly associated with elevation, C. indistincta sp. D was significantly correlated with dissolved oxygen range, whilst, individuals of C. indistincta sp. A were negatively associated with elevation and dissolved oxygen range. This may indicate that C. indistincta sp. A tended to inhabit sites with low elevation and perhaps having a higher tolerance to a low range of dissolved oxygen. For the Paratya australiensis species complex, P. australiensis lineage 4 and 6 showed significant correlations with elevation and conductivity, respectively. The second broad aim of the study was to explore these spatial patterns at smaller geographical scales and with greater detail about water quality to understand and quantify the fundamental environmental factors (e.g., physical chemical water parameters and concentrations of heavy metals) that are potentially shaping the current distribution patterns and abundance of cryptic species within the two species complexes. To explore this aim, sediment samples from 22 sites in 13 catchments in SEQ were analysed to determine concentrations (mg/kg dry weight) of 11 heavy metals. Additionally, a number of water quality variables were measured in situ, including: elevation, stream width, stream temperature, dissolved oxygen, conductivity, pH, total dissolved solids, and turbidity. Also, a water sample was taken from each site for laboratory analysis of: Ammonium nitrogen (NH4-N), Dissolved oxidized nitrogen (Nitrate+Nitrite) (NOX-N), Total nitrogen (TN), Total kjeldahl nitrogen (TKN), Total kjeldahl phosphorus (TKP), Orthophosphate-P (PO4-P). Shrimps were collected from each site and identified to species using both morphology and DNA sequencing. The morphological identification of each adult individual (except juveniles which were genetically analysed) was used as a measure of absolute abundance and the genetic ‘checking’ of a set number of individuals in each sample was used to compute relative abundance. Redundancy analysis (RDA) showed that the spatial distribution and absolute and relative abundance of C. indistincta sp. D and sp. B were significantly positively influenced by elevation, while the relative abundance of P. australiensis Lin.6 was significantly positively affected by the concentration of manganese (Mn). Stream Total nitrogen (TN) was significantly positive driver of the spatial distribution and relative abundance of C. indistincta sp. A, while Orthophosphate-P (PO4-P) was significantly positive driver for the absolute and relative abundance of this species. Further analysis, this study confirms that P. australiensis Lin.6 was more tolerant of heavy metal concentrations compared with other cryptic species, as its distribution and absolute and relative abundance were significantly positively correlated with the concentrations of manganese, iron and cobalt. In contrast, C. indistincta sp. A was more sensitive to these metals than other study species. These results demonstrated that cryptic species of freshwater atyid shrimps of the C. indistincta and P. australiensis species complexes were different in their environmental requirements. As well, the cryptic species of both complexes were identified to have different associations with heavy metal concentrations, indicating that these species were different in their tolerance to toxicants. Finally, the third aim of the study was to further examine the differences in sensitivity to heavy metals (Copper and Zinc) among cryptic species of the two study complexes experimentally in the laboratory. Two cryptic species of each complex were used as study species, C. indistincta sp. A and sp. D and P. australiensis Lin.4 and Lin.6. The field studies showed differences among these species in their correlations with metal concentrations, and therefore they were seen as good candidate species for testing differences in the sensitivity to metal toxicants. Each cryptic species was exposed to six concentrations of each metal Cu or Zn using an acute (96-h) toxicity test. The results from this study were generally showed contrasting correlation between species and heavy metals; P. australiensis Lin.6 was the most tolerant species to both study metals, while C. indistincta sp. A was more sensitive to copper, and C. indistincta sp. D was more sensitive to Zn compared with the other tested species. Furthermore, the exposure of individuals of each species to the heavy metals caused changes in both their behaviour and their colour during exposure time. Overall, this study has shown cryptic species within broad species complexes can vary in their spatial distribution and their tolerance and response to water quality parameters. This highlights the advantage of using analyses of biotic and abiotic variables for ecological management and biodiversity conservation and the need to understand true species diversity when looking at species level responses to environmental degradation.

Chapter Twenty - Reverse Transcriptase Dideoxy Sequencing of RNA

Identification of two families of satellite-like repetitive DNA sequences from the zebrafish ( Brachydanio rerio)

The ability of HIV-1 to develop resistance to antiretroviral drugs has limited the overall efficacy of combination therapy to suppress viral replication. Consequently, drug resistance testing has become an integral tool for many HIV specialists, particularly when managing patients who experience failure of antiretroviral therapy. Two principal methodologies are in current use for assessing resistance to antiviral agents: Genotyping and Phenotyping. The relative utility of the two types of resistance tests is a topic of a heated debate. The phenotypic technique, derived directly from patient leukocytes, is more labour-intensive and time-consuming. Nevertheless, it raised much interest with recent developments such as replicative phenotyping, which involve the constitution of recombinant virus genomes containing sequences from patient-derived viral protease and/or reverse transcriptase genes. 
\n
\nOne aim of this study was to validate the PhenoTecT system, a “rPhenotyping”, as diagnostic tool applicable for detecting and assigning HIV resistance in clinical specimens. This methodology can also be performed where known limitations of standard Genotyping pose obstacles, e.g. its low sensitivity for detecting viral minority variants or its limits in dissecting mixed virus populations. 
\nThe questions to elucidate in this study were the following:
\n1.\tIs rPhenotyping as powerful as Genotyping to reveal HIV-1 resistance in routine use? (Chapter I)
\n2.\tCan rPhenotyping overcome the limitations of standard Genotyping? 
\n-\tHow do viral mixtures influence a resistance test? (Chapter II)
\n-\tAre clinically critical HIV minorities revealed by replicative Phenotyping? (Chapter III) 
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\nStatistical analysis of more than 1,000 patient data sets from the InPheno database (PhenoBase®) identified relevant discordances between genotyping (Stanford DB) and phenotyping (PhenoTecT) in the interpretation of resistance profiles. Discordance was defined as determination of reduced susceptibility by one but sensitivity by the other test (Geno-R/Pheno-S), or vice versa (Geno-S/Pheno-R). The set of data used in this study was all diagnostic analyses from 2002 to 2007The ability of HIV-1 to develop resistance to antiretroviral drugs has limited the overall efficacy of combination therapy to suppress viral replication. Consequently, drug resistance testing has become an integral tool for many HIV specialists, particularly when managing patients who experience failure of antiretroviral therapy. Two principal methodologies are in current use for assessing resistance to antiviral agents: Genotyping and Phenotyping. The relative utility of the two types of resistance tests is a topic of a heated debate. The phenotypic technique, derived directly from patient leukocytes, is more labour-intensive and time-consuming. Nevertheless, it raised much interest with recent developments such as replicative phenotyping, which involve the constitution of recombinant virus genomes containing sequences from patient-derived viral protease and/or reverse transcriptase genes. 
\n
\nOne aim of this study was to validate the PhenoTecT system, a “rPhenotyping”, as diagnostic tool applicable for detecting and assigning HIV resistance in clinical specimens. This methodology can also be performed where known limitations of standard Genotyping pose obstacles, e.g. its low sensitivity for detecting viral minority variants or its limits in dissecting mixed virus populations. 
\nThe questions to elucidate in this study were the following:
\n1.\tIs rPhenotyping as powerful as Genotyping to reveal HIV-1 resistance in routine use? (Chapter I)
\n2.\tCan rPhenotyping overcome the limitations of standard Genotyping? 
\n-\tHow do viral mixtures influence a resistance test? (Chapter II)
\n-\tAre clinically critical HIV minorities revealed by replicative Phenotyping? (Chapter III) 
\n
\nStatistical analysis of more than 1,000 patient data sets from the InPheno database (PhenoBase®) identified relevant discordances b. For a fair comparison each patient’s sample was re-interpreted with the same version of algorithm. As genotypic algorithms are self-optimising systems that improve their performance over time we could observe that some of the discordances previously found between genotyping and phenotyping did not hold up in later versions. Moreover, three independent genotypic prediction systems were used in parallel to assess potential differences for the same patient samples. Significant discordances were observed between interpretations inferred by these different genotyping algorithms. This suggests the urgent requirement to define common rules and possibly even new criteria to be considered and will increase coherence of results and therapy advices within national and international studies. 
\nNevertheless some intrinsic shortcoming remain that necessitate the use of alternative dissecting methods such as rPhenotyping: Genotyping principally fails to assign new mutations or novel resistance patterns associated with new drugs, and systems are limited in the ability to follow viral speciation during extended treatment periods as this leads to the formation of viral minorities. The evolution of a growing complexity of viral genomes in any given patient requires dissection and assignment of mutations to individual viral genomes. In our study we hypothesised that these limitations could result in discordances between genotypic and phenotypic resistance profiling. To particularly focus on Geno-R/Pheno-S discordances we generated by point mutagenesis infectious, otherwise isogenic HIV-1 clones carrying selected combinations of one to four mutations known to decrease susceptibility to the drug AZT, and we investigated their phenotypic resistance profiles. As expected: analyses of virus mixtures each expressing at least three of the selected mutations was categorised as resistant to AZT by both testing whereas those with a mixture of single mutated virus was interpreted as AZT resistant only by genotypic algorithm whereas PhenoTecT was able to dissect viral mixtures. For Geno-S/Pheno-R samples, we speculated that rPhenotyping is able to detect resistant minority species not scored by genotyping. We prepared defined mixes of wild-type and M184V mutant which were analysed by rPhenotyping or standard genotyping. 
\nAllele-specific and quantitative polymerase chain reaction (PCR) set detection and quantification limits for minor virus populations in vitro and in authentic clinical samples showing geno-/pheno discrepant lamivudine resistance. Data presented in this study demonstrate that the principle of a functional assessment by rPhenotyping can strongly detect: (i) minor HIV populations with a sensitivity level of less than 1%, (ii) viral mixtures by analysing each single virus separately and (iii) viruses presenting mutations with synergistic or antagonistic interactions. This renders it, for these applications, superior to Genotyping. Nevertheless further studies will have to show how this gain in sensitivity will translate into clinical utility and improved disease management (ongoing project with the Swiss HIV cohort and the group of Prof. M.Battegay,USB).
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\nIn the future patients should benefit from those analytical improvements by assisting in improved identification of new drug-options. In addition, these observations will form the basis for applications in other viral diseases, notably HBV and HCV, where similar issues are likely to arise with the currently ongoing introduction of potent antiviral drugs. 
\n