eine neue SNP-Genotypisierungsmethode

1\. TITLE PAGE, TABLE OF CONTENTS, ABBREVIATIONS 6 2\. INTRODUCTION 7 2.1. SINGLE NUCLEOTIDE POLYMORPHISM - DEFINITION AND APPLICATIONS 7 2.1.1. What are SNPs? 7 2.1.2. SNP maps 8 2.1.3. Technology development for SNP detection 9 2.2. CURRENT STATE IN THE FIELD OF SNP DETECTION 9 2.2.1. Hybridization-based methods 10 2.2.1.1. Microarrays 10 2.2.1.2. DASH 12 2.2.1.3. Homogenous assays 14 2.2.2. Enzymatic methods 17 2.2.2.1. SNP discrimination by polymerase 17 2.2.2.1.1. Allele-specific PCR 17 2.2.2.1.2. Primer extension 19 2.2.2.1.2.1. Primer extension on microarrays 19 2.2.2.1.2.2. Homogenous primer extension assays 21 2.2.2.1.2.3. Primer extension with MALDI-TOF detection 22 2.2.2.1.2.4. Pyrosequencing 23 2.2.2.2. SNP discrimination by ligase 25 2.2.2.3. Invasive cleavage 29 2.2.2.4. Site-specific cleavage 31 2.2.3. Chemical ligation 31 3\. MATERIALS AND METHODS 34 3.1. MATERIALS 34 3.1.1. Oligonucleotides 35 3.1.2. DNA samples 35 3.1.3. SNP loci 35 3.2. METHODS 35 3.2.1. Universal oligonucleotides 35 3.2.2. Design of locus-specific oligonucleotides 36 3.2.3. Preparation of detector oligonucleotides 37 3.2.4. Ligation detection reaction (LDR) - TaqMan SNP detection 38 3.2.4.1. One tube - one locus procedure 38 3.2.4.2. One tube - many loci procedure 39 3.2.4.3. Determination of minimal concentrations of DOs 39 3.2.4.4. Influence of PEG on the hybridization of DOs 39 3.2.5. Hot start Taq polymerase preparation 40 3.2.5.1. Purification of Taq polymerase. 40 3.2.5.2. Determination of Taq polymerase activity 41 3.2.5.3. Preparation of hot start Taq polymerase 42 3.2.5.4. Estimation of the efficiency of formaldehyde inactivation 42 3.2.6. Pfu DNA Ligase preparation 43 3.2.6.1. Purification of Pfu DNA ligase. 43 3.2.6.2. Estimation of Pfu DNA Ligase activity 44 3.2.7. T4 DNA Ligase preparation 44 3.2.8. Genomic DNA purification 44 3.2.8.1. Plant Genomic DNA purification using CTAB buffer 44 3.2.8.21. Plant Genomic DNA purification using homemade silica spin-columns. 45 3.2.8.3. Genomic DNA purification from saliva. 46 4\. RESULTS AND DISCUSSION 48 4.1. BACKGROUND 48 4.2. LDR-TAQMAN SNP DETECTION METHOD 50 4.2.1. The structure of detector oligonucleotides 51 4.2.2. The "one tube - one locus" procedure (Figure 13) 53 4.2.3. The "one tube - many loci" procedure (Figure 14) 55 4.2.4. Discussion of the LDR-TaqMan protocol 58 4.3. LDR-TAQMAN GENOTYPING KITS 64 4.3.1. Preparation of enzymes and Detector oligonucleotides 64 4.3.1.1. Homemade Pfu DNA ligase 65 4.3.1.2. Homemade hot start Taq DNA polymerase 65 4.3.1.3. Plastic consumables 66 4.3.1.4. Plant and human genomic DNA isolation using homemade spin columns 66 4.3.1.5. Ligation-based synthesis of Detector Oligonucleotides (DOs) 66 4.3.2. Arabidopsis thaliana SNP kit 69 4.3.3. Human SNP kit 75 5\. SUMMARY 79 5.1. ZUSAMMENFASSUNG 80 6\. REFERENCES 81 7\. SUPPLEMENTS 98The LDR-TaqMan SNP genotyping method was elaborated and patented [Borodina et al., 2004; EP03019521]. The method is based on the ligation detection reaction (LDR), which is performed directly on genomic DNA. During the LDR, the biallelic state of an SNP locus is converted into the bimarker state of ligated detector oligonucleotides. The state of the markers is then determined by the 5'-nuclease assay (TaqMan) with universal fluorescent probes. The technology is sensitive, has high discrimination power, needs no locus- specific optimization and is applicable both for individual and multiplex SNP analyses. To transfer the technology to scientific partners, the following additional tasks were solved: * a method for cost-effective synthesis of long (60-120nt) oligonucleotides with block structure was developed and patented [Borodina et al., 2003; PCT/EP 2004/003921]; * a procedure for DNA isolation from plant material and saliva on home-made silica spin-columns was established [Borodina et al., 2003a]; * enzymes required for the method (thermostable Pfu DNA ligase and hot start Taq DNA polymerase) were cloned and expressed. The LDR-TaqMan method was applied for SNP genotyping of human and Arabidopsis thaliana DNA samples: * a kit for genotyping of 9 clinically important human SNPs was prepared and used for determination of allele frequencies of these SNPs in two East European populations (Ukrainians and Belorussians, 216 DNA samples) [Kozhekbaeva et al., 2004]; * a 138-loci genotyping kit for Arabidopsis thaliana accessions Columbia (Col-0) and C24 was prepared and tested in the blind genotyping of 10 plants. A comparison of the LDR-TaqMan genotyping results with those obtained by conventional methods (sequencing, RFLP, SNaPshot, Amplifluor [Rickert et al., 2004]) demonstrated the efficiency and reliability of the LDR-TaqMan procedure. Ready-to-use genotyping kits (including enzymes, buffers and sets of detector oligonucleotides) were transferred to collaborators in Golm (Germany) and Moscow (Russia).Im Rahmen der Arbeit wurde die LDR-TaqMan SNP-Genotypisierungsmethode entwickelt und patentiert [Borodina et al., 2004; EP03019521]. Die Methode basiert auf der direkt mit genomischer DNA durchgefuhrten Ligationsdetektionsreaktion (LDR). Abhangig vom allelischen Zustand des SNP Locus wird eine korrespondierende Markersequenz in das Ligationsprodukt integriert. Die Markersequenz wird im folgenden 5'-Exonukleaseassay (TaqMan) durch universelle Sonden identifiziert. Die Methode ist sensitiv, hat ein hohes Diskriminierungsvermogen, bedarf keiner lokusspezifischen Optimierung und ist in Single- als auch Multiplexanalysen anwendbar. Um die Technologie unseren wissenschaftlichen Partnern zur Verfugung zu stellen, wurden folgende Aufgaben gelost: * die kosteneffiziente Synthese langer (60 - 120nt) Oligonukleotide mit Blockstruktur [Borodina et al., 2003; PCT/EP 2004/003921]; * Entwicklung einer effektiven Methode zur DNA Isolation aus pflanzlichem Material und Speichel in Silica basierten Saulen [Borodina et al., 2003a]; * Klonierung und Expression von fur die Methode benotigten Enzymen (thermostabile Pfu DNA Ligase und HotStart-DNA Polymerase). Die LDR-TaqMan Methode wurde zur SNP-Genotypisierung von humanen und Arabidopsis thaliana Proben verwendet: * ein Genotypisierungskit fur 9 klinisch bedeutenden humanen SNPs wurde angefertigt und zur Bestimmung der Allelfhaufigkeit dieser SNPs in zwei Osteuropaischen Populationen verwendet (Ukrainer und Wei?russen - 216 DNA Proben) [Kozhekbaeva et al., 2004]; * ein 138-Loci Genotypisierungskit fur Arabidopsis thaliana der Okotypen Columbia (Col-0) und C24 wurde angefertigt und in einem Genotypisierungsversuch mit 10 Pflanzen getestet. Der Vergleich der Genotypisierungsergebnisse mit den Ergebnissen von komerziellen Produkten (Sequenzierung, PDRF, SnaPshot, Amplifluor [Rickert et al., 2004]) bestatigte die Effizienz und Zuverlassigkeit der LDR-TaqMan Methode. Daraufhin wurden gebrauchsfertige Genotypisierungskits (Enzyme, Puffer und lokusspezifische Oligonukleotide) Partnern in Golm (Deutschland) und Moskau (Russland) zur Verfugung gestellt

Estimation and Computer Simulation of the Effective Doses in the Dose-Effect Dependence Over Random Experiment Plans

In this article for the model of dose-effect dependence we propose estimators of the effective dose level based on a random experiment plans which are consistency and asymptotic normality. We also compare the proposed estimators with an DNP-estimate by means of computer simulation

Matrin 3 Binds and Stabilizes mRNA

Matrin 3 (MATR3) is a highly conserved, inner nuclear matrix protein with two zinc finger domains and two RNA recognition motifs (RRM), whose function is largely unknown. Recently we found MATR3 to be phosphorylated by the protein kinase ATM, which activates the cellular response to double strand breaks in the DNA. Here, we show that MATR3 interacts in an RNA-dependent manner with several proteins with established roles in RNA processing, and maintains its interaction with RNA via its RRM2 domain. Deep sequencing of the bound RNA (RIP-seq) identified several small noncoding RNA species. Using microarray analysis to explore MATR3′s role in transcription, we identified 77 transcripts whose amounts depended on the presence of MATR3. We validated this finding with nine transcripts which were also bound to the MATR3 complex. Finally, we demonstrated the importance of MATR3 for maintaining the stability of several of these mRNA species and conclude that it has a role in mRNA stabilization. The data suggest that the cellular level of MATR3, known to be highly regulated, modulates the stability of a group of gene transcripts

Comparing Three Approaches

Hybridization-based target enrichment protocols require relatively large starting amounts of genomic DNA, which is not always available. Here, we tested three approaches to pre-capture library preparation starting from 10 ng of genomic DNA: (i and ii) whole-genome amplification of DNA samples with REPLI-g (Qiagen) and GenomePlex (Sigma) kits followed by standard library preparation, and (iii) library construction with a low input oriented ThruPLEX kit (Rubicon Genomics). Exome capture with Agilent SureSelectXT2 Human AllExon v4+UTRs capture probes, and HiSeq2000 sequencing were performed for test libraries along with the control library prepared from 1 µg of starting DNA. Tested protocols were characterized in terms of mapping efficiency, enrichment ratio, coverage of the target region, and reliability of SNP genotyping. REPLI-g- and ThruPLEX-FD-based protocols seem to be adequate solutions for exome sequencing of low input sample

Diverse but unique astrocytic phenotypes during embryonic stem cell differentiation, culturing and development

Astrocytes are resident glia cells of the central nervous system (CNS) that play complex and heterogeneous roles in brain development, homeostasis and disease. Since their vast involvement in health and disease is becoming increasingly recognized, suitable and reliable tools for studying these cells in vivo and in vitro are of utmost importance. One of the key challenges hereby is to adequately mimic their context-dependent in vivo phenotypes and functions in vitro. To better understand the spectrum of astrocytic variations in defined settings we performed a side-by-side-comparison of embryonic stem cell (ESC)-derived astrocytes as well as primary neonatal and adult astrocytes, revealing major differences on a functional and transcriptomic level, specifically on proliferation, migration, calcium signaling and cilium activity. Our results highlight the need to carefully consider the choice of astrocyte origin and phenotype with respect to age, isolation and culture protocols based on the respective biological question

Transcriptome analysis by strand-specific sequencing of complementary DNA

High-throughput complementary DNA sequencing (RNA-Seq) is a powerful tool for whole-transcriptome analysis, supplying information about a transcript's expression level and structure. However, it is difficult to determine the polarity of transcripts, and therefore identify which strand is transcribed. Here, we present a simple cDNA sequencing protocol that preserves information about a transcript's direction. Using Saccharomyces cerevisiae and mouse brain transcriptomes as models, we demonstrate that knowing the transcript's orientation allows more accurate determination of the structure and expression of genes. It also helps to identify new genes and enables studying promoter-associated and antisense transcription. The transcriptional landscapes we obtained are available online

Long-read sequencing identifies a common transposition haplotype predisposing for CLCNKB deletions

BACKGROUND: Long-read sequencing is increasingly used to uncover structural variants in the human genome, both functionally neutral and deleterious. Structural variants occur more frequently in regions with a high homology or repetitive segments, and one rearrangement may predispose to additional events. Bartter syndrome type 3 (BS 3) is a monogenic tubulopathy caused by deleterious variants in the chloride channel gene CLCNKB, a high proportion of these being large gene deletions. Multiplex ligation-dependent probe amplification, the current diagnostic gold standard for this type of mutation, will indicate a simple homozygous gene deletion in biallelic deletion carriers. However, since the phenotypic spectrum of BS 3 is broad even among biallelic deletion carriers, we undertook a more detailed analysis of precise breakpoint regions and genomic structure. METHODS: Structural variants in 32 BS 3 patients from 29 families and one BS4b patient with CLCNKB deletions were investigated using long-read and synthetic long-read sequencing, as well as targeted long-read sequencing approaches. RESULTS: We report a ~3 kb duplication of 3'-UTR CLCNKB material transposed to the corresponding locus of the neighbouring CLCNKA gene, also found on ~50 % of alleles in healthy control individuals. This previously unknown common haplotype is significantly enriched in our cohort of patients with CLCNKB deletions (45 of 51 alleles with haplotype information, 2.2 kb and 3.0 kb transposition taken together, p=9.16×10-9). Breakpoint coordinates for the CLCNKB deletion were identifiable in 28 patients, with three being compound heterozygous. In total, eight different alleles were found, one of them a complex rearrangement with three breakpoint regions. Two patients had different CLCNKA/CLCNKB hybrid genes encoding a predicted CLCNKA/CLCNKB hybrid protein with likely residual function. CONCLUSIONS: The presence of multiple different deletion alleles in our cohort suggests that large CLCNKB gene deletions originated from many independently recurring genomic events clustered in a few hot spots. The uncovered associated sequence transposition haplotype apparently predisposes to these additional events. The spectrum of CLCNKB deletion alleles is broader than expected and likely still incomplete, but represents an obvious candidate for future genotype/phenotype association studies. We suggest a sensitive and cost-efficient approach, consisting of indirect sequence capture and long-read sequencing, to analyse disease-relevant structural variant hotspots in general

Increased risk of severe clinical course of COVID-19 in carriers of HLA-C*04:01

Background: Since the beginning of the coronavirus disease 2019 (COVID-19) pandemic, there has been increasing urgency to identify pathophysiological characteristics leading to severe clinical course in patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Human leukocyte antigen alleles (HLA) have been suggested as potential genetic host factors that affect individual immune response to SARS-CoV-2. We sought to evaluate this hypothesis by conducting a multicenter study using HLA sequencing. Methods: We analyzed the association between COVID-19 severity and HLAs in 435 individuals from Germany (n = 135), Spain (n = 133), Switzerland (n = 20) and the United States (n = 147), who had been enrolled from March 2020 to August 2020. This study included patients older than 18 years, diagnosed with COVID19 and representing the full spectrum of the disease. Finally, we tested our results by meta-analysing data from prior genome-wide association studies (GWAS). Findings: We describe a potential association of HLA-C*04:01 with severe clinical course of COVID-19. Carriers of HLA-C*04:01 had twice the risk of intubation when infected with SARS-CoV-2 (risk ratio 1.5 [95% CI 1.1-2.1], odds ratio 3.5 [95% CI 1.9-6.6], adjusted p-value = 0.0074). These findings are based on data from four countries and corroborated by independent results from GWAS. Our findings are biologically plausible, as HLA-C*04:01 has fewer predicted bindings sites for relevant SARS-CoV-2 peptides compared to other HLA alleles. Interpretation: HLA-C*04:01 carrier state is associated with severe clinical course in SARS-CoV-2. Our findings suggest that HLA class I alleles have a relevant role in immune defense against SARS-CoV-2. Funding: Funded by Roche Sequencing Solutions, Inc

Nanomedicine for Treating Diabetic Retinopathy Vascular Degeneration

The incidence of diabetes and the pathological conditions associated with chronic hyperglycemia is increasing worldwide. Among them, diabetic retinopathy represents a leading cause of vision loss, causing a significant structural and functional impairment of the retinal and choroidal capillary network. Current therapies include anti-angiogenic and anti-inflammatory drugs administered through repetitive and invasive intraocular injections, and associated with significant adverse effects. The presence of ocular barriers affects the efficiency of topically administered therapeutics for treating the posterior segment of the eye. In this scenario, nanomedicine could improve current therapies for diabetic retinopathy by providing tools that can decrease the number of injections thanks to their controlled release properties, while some materials showed a natural ability to mitigate pathological neo-angiogenesis. Moreover, specific surface modifications could open new scenarios for the development of topical treatments. This review describes current advances in generating nanomedicine for diabetic retinopathy, focusing on the properties of the different materials tested explicitly for this purpose