A novel study of copy number variations in Hirschsprung disease using the multiple ligation-dependent probe amplification (MLPA) technique.

BACKGROUND Hirschsprung disease (HSCR) is a congenital malformation of the hindgut produced by a disruption in neural crest cell migration during embryonic development. HSCR has a complex genetic etiology and mutations in several genes, mainly the RET proto-oncogene, have been related to the disease. There is a clear predominance of missense/nonsense mutations in these genes whereas copy number variations (CNVs) have been seldom described, probably due to the limitations of conventional techniques usually employed for mutational analysis. METHODS In this study we have aimed to analyze the presence of CNVs in some HSCR genes (RET, EDN3, GDNF and ZFHX1B) using the Multiple Ligation-dependent Probe Amplification (MLPA) approach. RESULTS Two alterations in the MLPA profiles of RET and EDN3 were detected, but a detailed inspection showed that the decrease in the corresponding dosages were due to point mutations affecting the hybridization probes regions. CONCLUSION Our results indicate that CNVs of the gene coding regions analyzed here are not a common molecular cause of Hirschsprung disease. However, further studies are required to determine the presence of CNVs affecting non-coding regulatory regions, as well as other candidate genes.

Laboratory detection of respiratory viruses by automated techniques.

Advances in clinical virology for detecting respiratory viruses have been focused on nucleic acids amplification techniques, which have converted in the reference method for the diagnosis of acute respiratory infections of viral aetiology. Improvements of current commercial molecular assays to reduce hands-on-time rely on two strategies, a stepwise automation (semi-automation) and the complete automation of the whole procedure. Contributions to the former strategy have been the use of automated nucleic acids extractors, multiplex PCR, real-time PCR and/or DNA arrays for detection of amplicons. Commercial fully-automated molecular systems are now available for the detection of respiratory viruses. Some of them could convert in point-of-care methods substituting antigen tests for detection of respiratory syncytial virus and influenza A and B viruses. This article describes laboratory methods for detection of respiratory viruses. A cost-effective and rational diagnostic algorithm is proposed, considering technical aspects of the available assays, infrastructure possibilities of each laboratory and clinic-epidemiologic factors of the infection.

Application of Molecular Diagnostic Techniques for Viral Testing.

Nucleic acid amplification techniques are commonly used currently to diagnose viral diseases and manage patients with this kind of illnesses. These techniques have had a rapid but unconventional route of development during the last 30 years, with the discovery and introduction of several assays in clinical diagnosis. The increase in the number of commercially available methods has facilitated the use of this technology in the majority of laboratories worldwide. This technology has reduced the use of some other techniques such as viral culture based methods and serological assays in the clinical virology laboratory. Moreover, nucleic acid amplification techniques are now the methods of reference and also the most useful assays for the diagnosis in several diseases. The introduction of these techniques and their automation provides new opportunities for the clinical laboratory to affect patient care. The main objectives in performing nucleic acid tests in this field are to provide timely results useful for high-quality patient care at a reasonable cost, because rapid results are associated with improvements in patients care. The use of amplification techniques such as polymerase chain reaction, real-time polymerase chain reaction or nucleic acid sequence-based amplification for virus detection, genotyping and quantification have some advantages like high sensitivity and reproducibility, as well as a broad dynamic range. This review is an up-to-date of the main nucleic acid techniques and their clinical applications, and special challenges and opportunities that these techniques currently provide for the clinical virology laboratory.

Strategy for optimizing DNA amplification in a peripheral blood PCR assay used for diagnosis of human brucellosis.

We studied two of the possible factors which can interfere with specific DNA amplification in a peripheral-blood PCR assay used for the diagnosis of human brucellosis. We found that high concentrations of leukocyte DNA and heme compounds inhibit PCR. These inhibitors can be efficiently suppressed by increasing the number of washings to four or five and decreasing the amount of total DNA to 2 to 4 microg, thereby avoiding false-negative results.

Identification of differentially expressed mRNA in prokaryotic organisms by customized amplification libraries (DECAL): The effect of isoniazid on gene expression in Mycobacterium tuberculosis

Understanding the effects of the external environment on bacterial gene expression can provide valuable insights into an array of cellular mechanisms including pathogenesis, drug resistance, and, in the case of Mycobacterium tuberculosis, latency. Because of the absence of poly(A)+ mRNA in prokaryotic organisms, studies of differential gene expression currently must be performed either with large amounts of total RNA or rely on amplification techniques that can alter the proportional representation of individual mRNA sequences. We have developed an approach to study differences in bacterial mRNA expression that enables amplification by the PCR of a complex mixture of cDNA sequences in a reproducible manner that obviates the confounding effects of selected highly expressed sequences, e.g., ribosomal RNA. Differential expression using customized amplification libraries (DECAL) uses a library of amplifiable genomic sequences to convert total cellular RNA into an amplified probe for gene expression screens. DECAL can detect 4-fold differences in the mRNA levels of rare sequences and can be performed on as little as 10 ng of total RNA. DECAL was used to investigate the in vitro effect of the antibiotic isoniazid on M. tuberculosis, and three previously uncharacterized isoniazid-induced genes, iniA, iniB, and iniC, were identified. The iniB gene has homology to cell wall proteins, and iniA contains a phosphopantetheine attachment site motif suggestive of an acyl carrier protein. The iniA gene is also induced by the antibiotic ethambutol, an agent that inhibits cell wall biosynthesis by a mechanism that is distinct from isoniazid. The DECAL method offers a powerful new tool for the study of differential gene expression.

Quasi-lossless data transmission with ultra-long Raman fibre laser based amplification

The project consists of an experimental and numerical modelling study of the applications of ultra-long Raman fibre laser (URFL) based amplification techniques for high-speed multi-wavelength optical communications systems. The research is focused in telecommunications C-band 40 Gb/s transmission data rates with direct and coherent detection. The optical transmission performance of URFL based systems in terms of optical noise, gain bandwidth and gain flatness for different system configurations is evaluated. Systems with different overall span lengths, transmission fibre types and data modulation formats are investigated. Performance is compared with conventional Erbium doped fibre amplifier based system to evaluate system configurations where URFL based amplification provide performance or commercial advantages.

Advanced techniques for the improvement of optical transmission systems

This thesis presents the experimental investigation into two novel techniques which can be incorporated into current optical systems. These techniques have the capability to improve the performance of transmission and the recovery of the transmitted signal at the receiver. The experimental objectives are described and the results for each technique are presented in two sections: The first experimental section is on work related to Ultra-long Raman Fibre lasers (ULRFLs). The fibre lasers have become an important research topic in recent years due to the significant improvement they give over lumped Raman amplification and their potential use in the development of system with large bandwidths and very low losses. The experiments involved the use of ASK and DPSK modulation types over a distance of 240km and DPSK over a distance of 320km. These results are compared to the current state of-the-art and against other types of ultra-long transmission amplification techniques. The second technique investigated involves asymmetrical, or offset, filtering. This technique is important because it deals with the strong filtering regimes that are a part of optical systems and networks in modern high-speed communications. It allows the improvement of the received signal by offsetting the central frequency of a filter after the output of a Delay Line Interferometer (DLI), which induces significant improvement in BER and/or Qvalues at the receiver and therefore an increase in signal quality. The experimental results are then concluded against the objectives of the experimental work and potential future work discussed.

Transmission comparison of ultra-long Raman fibre laser based amplification with first and dual order Raman amplification using 10×118 Gbit/s DP-QPSK

Experimental investigations of 10×118 Gbit/s DP-QPSK WDM transmission using three types of distributed Raman amplification techniques are presented. Novel ultra-long Raman fibre laser based amplification with second order counter-propagated pumping is compared with conventional first order and dual order counter-pumped Raman amplification. We demonstrate that URFL based amplification can extend the transmission reach up to a distance of 7520 km in comparison with 5010 km and 6180 km using first order and dual order Raman amplification respectively. © 2014 IEEE.

Extended reach of 116 Gb/s DP-QPSK transmission using random DFB fiber laser based Raman amplification and bidirectional second-order pumping

We propose a novel random DFB fiber laser based Raman amplification using bidirectional second-order pumping. This extends the reach of 116 Gb/s DP-QPSK WDM transmission up to 7915 km, compared with other Raman amplification techniques.

RIN mitigation in second-order pumped Raman fibre laser based amplification

We experimentally investigate three Raman fibre laser based amplification techniques with second-order bidirectional pumping. Relatively intensity noise (RIN) being transferred to the signal can be significantly suppressed by reducing first-order reflection near the input end. © 2015 OSA.

Raman fibre laser based amplification in coherent transmission systems

The thesis presents a detailed study of different Raman fibre laser (RFL) based amplification techniques and their applications in long-haul/unrepeatered coherent transmission systems. RFL based amplifications techniques were characterised from different aspects, including signal/noise power distributions, relative intensity noise (RIN), mode structures of induced Raman fibre lasers, and so on. It was found for the first time that RFL based amplification techniques could be divided into three categories in terms of the fibre laser regime, which were Fabry-Perot fibre laser with two FBGs, weak Fabry-Perot fibre laser with one FBG and very low reflection near the input, and random distributed feedback (DFB) fibre laser with one FBG. It was also found that lowering the reflection near the input could mitigate the RIN of the signal significantly, thanks to the reduced efficiency of the Stokes shift from the FW-propagated pump. In order to evaluate the transmission performance, different RFL based amplifiers were evaluated and optimised in long-haul coherent transmission systems. The results showed that Fabry-Perot fibre laser based amplifier with two FBGs gave >4.15 dB Q factor penalty using symmetrical bidirectional pumping, as the RIN of the signal was increased significantly. However, random distributed feedback fibre laser based amplifier with one FBG could mitigate the RIN of the signal, which enabled the use of bidirectional second order pumping and consequently give the best transmission performance up to 7915 km. Furthermore, using random DFB fibre laser based amplifier was proved to be effective to combat the nonlinear impairment, and the maximum reach was enhanced by >28% in mid-link single/dual band optical phase conjugator (OPC) transmission systems. In addition, unrepeatered transmission over >350 km fibre length using RFL based amplification technique were presented experimentally using DP-QPSK and DP-16QAM transmitter.

Nucleic Acid Diagnostics using Isotachophoresis and Isothermal Amplification

Thesis (Ph.D.)--University of Washington, 2016-08

PCR-based identification of Burkholderia pseudomallei

DNA amplification techniques are being used increasingly in clinical laboratories to confirm the identity of medically important bacteria. A PCR-based identification method has been in use in our centre for 10 years for Burkholderia pseudomallei and was used to confirm the identity of bacteria isolated from cases of melioidosis in Ceará since 2003. This particular method has been used as a reference standard for less discriminatory methods. In this study we evaluated three PCR-based methods of B. pseudomallei identification and used DNA sequencing to resolve discrepancies between PCR-based results and phenotypic identification methods. The established semi-nested PCR protocol for B. pseudomallei 16-23s spacer region produced a consistent negative result for one of our 100 test isolates (BCC #99), but correctly identified all 71 other B. pseudomallei isolates tested. Anomalous sequence variation was detected at the inner, reverse primer binding site for this method. PCR methods were developed for detection of two other B. pseudomallei bacterial metabolic genes. The conventional lpxO PCR protocol had a sensitivity of 0.89 and a specificity of 1.00, while a real-time lpxO protocol performed even better with sensitivity and specificity of 1.00, and 1.00. This method identified all B. pseudomallei isolates including the PCR-negative discrepant isolate. The phaC PCR protocol detected the gene in all B. pseudomallei and all but three B. cepacia isolates, making this method unsuitable for PCR-based identification of B. pseudomallei. This experience with PCR-based B. pseudomallei identification methods indicates that single PCR targets should be used with caution for identification of these bacteria, and need to be interpreted alongside phenotypic and alternative molecular methods such as gene sequencing.

Evidence for transcript networks composed of chimeric RNAs in human cells.

The classic organization of a gene structure has followed the Jacob and Monod bacterial gene model proposed more than 50 years ago. Since then, empirical determinations of the complexity of the transcriptomes found in yeast to human has blurred the definition and physical boundaries of genes. Using multiple analysis approaches we have characterized individual gene boundaries mapping on human chromosomes 21 and 22. Analyses of the locations of the 5' and 3' transcriptional termini of 492 protein coding genes revealed that for 85% of these genes the boundaries extend beyond the current annotated termini, most often connecting with exons of transcripts from other well annotated genes. The biological and evolutionary importance of these chimeric transcripts is underscored by (1) the non-random interconnections of genes involved, (2) the greater phylogenetic depth of the genes involved in many chimeric interactions, (3) the coordination of the expression of connected genes and (4) the close in vivo and three dimensional proximity of the genomic regions being transcribed and contributing to parts of the chimeric RNAs. The non-random nature of the connection of the genes involved suggest that chimeric transcripts should not be studied in isolation, but together, as an RNA network.