1. Probenentnahme <ol><li> Sammeln von Blutproben von Personen mit entsprechenden Einwilligung. Für jeden einzelnen, ziehen 4 bis 5 ml Blut in ein Röhrchen P100 (BD Diagnostics, Franklin Lake, NY, USA). Zu Vergleichszwecken wurden gleichzeitig sammeln Blut in einem EDTA-Röhrchen. </li><li> Die BD P100 Röhrchen enthalten 15,8 ml sprühgetrockneten K2EDTA und eine lyophilisierte proprietären breites Spektrum Cocktail von Proteaseinhibitoren zur Gerinnung zu verhindern und zur Stabilisierung der Plasmaproteine. Sie enthalten auch einen mechanischen Abscheider. Nach dem Sammeln der Vollblut, halten beide Röhrchen bei Raumtemperatur für 60 min über Nacht bis zur Verarbeitung auftritt. </li><li> Zentrifugieren Sie die Röhrchen BD P100 bei 2.500 g für 15 Minuten bei Raumtemperatur. Übertragen Sie das Plasma über die mechanische Abscheider in Mikrozentrifugengefäße gesammelt. </li><li> Bewahren Sie die P100 Rohre, die das Blut unter dem Separator verdichtet, bei -80 ° C, bis Sie bereit sind, die DNA ext beginnenraction. </li></ol> 2.1 Von Blut P100 Rohr (P100_DNA) <ol><li> Die P100 Röhrchen werden bei -80 ° C nach dem Plasma-Extraktion. Innerhalb von 3 bis 4 Monate, Abrufen der P100 Röhrchen mit dem verdichteten Blut aus dem Gefrierfach und Auftauen bei 37 ° C in einem Wasserbad für 5 min (1A). Entfernen Sie alle Restplasma, die über dem Abscheider sitzt. 2 ml 17% Saccharose-Lösung (Gradient Lösung im Haus getestet - unveröffentlichte Daten) in den P100 Rohre über dem Separator (Abbildung 1B). Die Röhrchen bei Raumtemperatur für 20 min bei 2.500 g; dieser Vorgang drückt die mechanische Abscheider zum oberen Ende des Rohres und liefert drei Schichten aus Lösung, die das buffy coat (Abbildung 1C). Extrahieren Sie manuell den Separator mit einer steriled Haken Metall Büroklammer (1D). </li><li> Nach dem Abrufen der mechanischen Abscheider aus jedem Röhrchen (1D), zu entfernen und discard die obere Schicht Saccharose. Übertragen Sie die mittlere Schicht, die die Buffy-Coat (BC)-Schicht, in ein steriles 15 ml konischen Röhrchen. In Phosphate Buffer Saline (PBS), die Buffy-Coat-Volumen bis 3 ml zu erhöhen. Extrahieren Sie die DNA aus dem Buffy-Coat mit Reagenzien aus dem Qiagen Puregene Blood Kit nach der Empfehlung des Herstellers, ausgenommen einer Zentrifugationsgeschwindigkeit von 2.500 g (anstelle von 2.000 g). </li><li> Um lysieren und entfernen Sie die roten Blutkörperchen (RBC), fügen 9 ml RBC Lyse bis 3 ml Buffy-Coat. Umkehren jedes Rohr mehrmals und Inkubation bei Raumtemperatur für 10 min mit gelegentlichen invertierenden während der Inkubation. Drehe jede Mischung unten bei 2.500 g für 5 min und den Überstand verwerfen. Behandeln Sie die verbleibende Pellet in jedem Röhrchen mit 3 ml Zell-Lyse-Lösung und Vortex für 15 Sekunden. Behandeln Sie das Lysat mit 1 ml Protein Fällungslösung und Vortex für 15 Sekunden. </li><li> Zentrifuge bei 2500 g für 5 min und den Überstand in ein frisches 15 ml konischen Röhrchen mit3 ml 100% Isopropanol. Kehren Sie die neuen Rohre 10-mal und Zentrifuge bei 2.500 g für 3 min. Überstand verwerfen und 1 ml 70% Ethanol. Kehren Sie die konische Röhrchen ein paar Mal zu lösen und waschen das DNA-Pellet. Zentrifuge bei 2.500 g für 1 min. Die Tablette für 3 min (das Reagenzglas auf den Kopf) bei Raumtemperatur trocknen und dann setzt die DNA mit 250 ml Rehydrationslösung bei 37 ° C für 10 min und Transfer zu einem pre-markierten 1,7 ml Mikroröhre. Bewahren Sie die Röhrchen bei -80 ° C bis zur Verwendung. </li></ol> 2.2 aus dem Blut von EDTA Ttube (EDTA_DNA) <ol><li> Das Vollblut für die routinemäßige Prüfung Immunhämatologie in Kunststoff vaccutainer Röhrchen gesammelt mit 10,8 mg K 2 EDTA-Spray aufgetragen und bei -80 ° C. Da die Rohre nicht enthalten einen mechanischen Abscheider, wird die DNA-Extraktion nicht die Schritte, die die mechanischen Abscheider (2.1.1 und 2.1.2). </li><li> Innerhalb von 3 bis 4 Monaten von Blut Lagerung, die DNA extrahiert mit der KingFisher Flex (Thermo Fisher Scientific, Waltham, MA, USA). Es ist eine Automatisierung DNA Absaugsystem Magnetpartikel-Technologie und besteht aus Zelllyse / DNA-Bindung, mehreren Waschschritten und DNA-Elution. </li><li> Das Kit in der DNA-Extraktion verwendet wird, die KingFisher Flex 24 DNA Blood Kit (Qiagen, Deutschland). </li></ol> 3. DNA Menge and Quality Assessment Die Menge, Qualität und Integrität der genomischen DNA durch eine Kombination von Verfahren, die PicoGreen, Spektroskopie und Elektrophorese umfasst beurteilt. <ol><li> Die Konzentration der genomischen DNA durch Nanodrop und PicoGreen Quantifizierung bestimmt. </li><li> Die Reinheit wird aus dem 260/280 Verhältnis Messung am Nanodrop Spektralphotometer bestimmt. </li><li> Die Probe (500 ng) wird ebenfalls auf 1% Agarose-Gel geladen, um die Integrität (Degradation) der DNA zu beurteilen. </li></ol> 4. Genotyping Assays und Qualitätskontrolle <ol><li> Fünf P100_DNA und ihre entsprechenden EDTA_DNA Proben werden nach dem Zufallsprinzip für die weitere Analyse mithilfe des benutzerdefinierten Immunochip (Immuno DNA Analysis BeadChip) ausgewählt. Immunochip ist ein Infinium Genotypisierung Chip mit 196.524 Polymorphismen und ist für 5 immungenetischen Studien konzipiert. </li><li> Für jede Probe werden 200 ng DNA amplifiziert, fragmentiert, gefällt und in die entsprechende Hybridisierungspuffer. </li><li> Die denaturierten Proben werden auf Immunochips bei 48 ° C für mindestens 16 Stunden hybridisiert. </li><li> Nach der Hybridisierung werden die Immunochips für Ein-Basen-Extension Reaktionen und gefärbten verarbeitet. </li><li> Die immunochips werden dann aufgenommen mit Illumina Bead Array HiScan Leser und die normalisierte Intensität Wulst Daten in den Illumina GenomeStudio Software geladen und in Genotypen. Genotypen werden als mit dem Algorithmus von autocalling GenomeStudio. Die Qualitätskontrolle ines spricht Ausschluss von Einzel-Nukleotid-Polymorphismen (SNPs) mit einem Aufruf Rate &lt;95%. </li><li> Die SNP anrufen wird als Maß für die Effizienz immunochip Assay verwendet. Es wird als Prozentsatz der Gesamtzahl von Assays, die auf dem Chip (196.524 SNPs auf jedem Chip codiert), die eine ausreichende Signalstärke und Qualität zu erzielen, eine automatische Zuordnung Genotyp erhalten berechnet. Hochwertige DNA (260/280 von 1,8 oder höher und Abwesenheit von Abbau) wird erwartet, dass mindestens die gleiche anrufen als HapMap Kontroll-DNA in dem Test eingeschlossen immunochip erreichen. Qualitätskontrolle beinhaltet Ausschluss von SNPs mit einem Aufruf Rate &lt;95% in jedem Datensatz. </li></ol>

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Keine Interessenskonflikte erklärt.

Viele menschliche Erkrankungen genetische Ursachen haben und die Erforschung der genetischen Grundlagen von Erkrankungen des Menschen verlangt eine zunehmende hochwertige DNA. Die häufigste Quelle von DNA in genetischen Studien verwendet wird Vollblut Antikoagulation. Da die meisten klinischen Labors biochemische, serologische und viraler Tests durchzuführen, wie in einem ersten Schritt phänotypische Untersuchung Ergebnis wird das Blut oft in einem Plasma-Röhrchen gesammelt. Nach der Entnahme des Plasmas wird der ve...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent/equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>BD P100 tubes</td>
 <td>BD Diagnostics</td>
 <td>366448</td>
 </tr>
 <tr>
 <td>EDTA tubes</td>
 <td>BD Diagnostics</td>
 <td>367863</td>
 </tr>
 <tr>
 <td>Sucrose</td>
 <td>Sigma</td>
 <td>S9378</td>
 </tr>
 <tr>
 <td>Paper clip</td>
 <td>Office product</td>
 <td></td>
 </tr>
 <tr>
 <td>15 ml tube</td>
 <td>Corning</td>
 <td>430052</td>
 </tr>
 <tr>
 <td>Red blood cells (RBC)</td>
 <td>Qiagen</td>
 <td>158389 (kit)</td>
 </tr>
 <tr>
 <td>Phosphate Buffer Saline (PBS)</td>
 <td>Thermo Scientific</td>
 <td>SH30256.01</td>
 </tr>
 <tr>
 <td>BioSprint 96 DNA Blood Kit (48) </td>
 <td>Qiagen</td>
 <td>940054</td>
 </tr>
 <tr>
 <td>1.7 ml microtubes</td>
 <td>Axygen</td>
 <td>MCT-175-C</td>
 </tr>
 <tr>
 <td>Human Immuno DNA Analysis BeadChip Kit</td>
 <td>Illumina</td>
 <td>WG-352-1001</td>
 </tr>
 <tr>
 <td>Bead array reader</td>
 <td>Illumina</td>
 <td>NA</td>
 </tr>
 <tr>
 <td>GenomeStudio Software</td>
 <td>Illumina</td>
 <td>N/A</td>
 </tr>
 </tbody>
</table>

a practical and novel method to extract genomic dna from blood collection kits for plasma protein preservation

Labortests können auf zellulärer oder Fluidanteile des Blutes erfolgen. Die Verwendung unterschiedlicher Blutsammelröhrchen bestimmt den Anteil des Blutes, das analysiert werden kann (Vollblut, Plasma oder Serum). Laboratories in das Studium der genetischen Grundlagen menschlicher Erkrankungen beteiligt vertrauen auf antikoaguliertes Vollblut in EDTA-haltige vacutainer als Quelle der DNA für genetische / genomische Analyse gesammelt. Da die meisten klinischen Laboratorien, biochemische und serologische viraler Tests in einem ersten Schritt in phänotypischen Ergebnis Untersuchung durchzuführen, wird auch in Antikoagulans Heparin-haltigen Rohr (Plasmarohr) gesammelt. Daher, wenn die DNA-und Plasma für simultane und parallele Analysen sowohl Genomik und Proteomik-Daten benötigt werden, ist es üblich, Blut sowohl EDTA und Heparin Röhrchen zu sammeln. Wenn Blut in einem Röhrchen gesammelt werden konnte und als Quelle für Plasma und DNA, würde das Verfahren als eine Weiterentwicklung bestehender meth werdenODS. Die Verwendung des verdichteten Blut nach Plasmaextraktion stellt eine alternative Quelle für genomische DNA und damit Minimierung der Menge von Blutproben verarbeitet und die Verringerung der Anzahl von Proben von jedem Patienten erforderlich. Dies würde letztendlich Zeit und Ressourcen sparen. Der BD P100 Blutentnahme für Plasmaprotein Erhaltung wurden als ein verbessertes Verfahren gegenüber früheren Plasma oder Serum-Röhrchen 1 erstellt haben, um den Proteingehalt des Blutes zu stabilisieren, was eine bessere Protein Biomarkern und Proteomik Experimente aus menschlichem Blut. Die BD P100 Röhrchen enthalten 15,8 ml sprühgetrockneten K2EDTA und eine lyophilisierte proprietären breites Spektrum Cocktail von Protease-Inhibitoren zur Verhinderung der Koagulation und Stabilisierung der Plasmaproteine. Sie umfassen auch eine mechanische Trennvorrichtung, die eine physische Barriere zwischen Plasma und Zellpellets nach Zentrifugation stellt. Nur wenige Methoden wurden entwickelt, um DNA aus geronnenes Blut sa extrahierenmples in alten Plasmaröhrchen 2-4 gesammelt. Herausforderungen von diesen Methoden wurden hauptsächlich mit der Art der Separator im Inneren der Rohre (Gel-Separator) zugeordnet ist und inklusive Schwierigkeiten bei der Gewinnung des geronnenen Blut, die Unannehmlichkeiten der Fragmentierung oder Dispergieren des Gerinnsels und Behinderung des Gerinnsels durch Extraktion des Trenngels. Wir präsentieren die erste Methode, die extrahiert und reinigt genomischer DNA aus Blut in den neuen BD P100 Rohre gezogen. Wir vergleichen die Qualität der DNA-Probe von P100 Röhren dass von EDTA. Unser Ansatz ist einfach und effizient. Es handelt sich um vier große Schritte wie folgt: 1) die Verwendung eines Plasmas BD P100 (BD Diagnostics, Sparks, MD, USA) Rohr mit mechanischen Abscheider für die Blutentnahme, 2) die Entfernung des mechanischen Abscheider mit einer Kombination aus Saccharose und eine sterile Büroklammer Metallhakgen, 3) die Abtrennung des buffy coat Schicht, die die weißen Zellen und 4) die Isolierung der genomischen DNA aus derBuffy-Coat mit einem normalen kommerziellen DNA-Extraktions-Kits oder eine ähnliche Standard-Protokoll.

DNA Qualitätsbewertung und Konzentrationsmessung Die Ausbeuten an P100_DNAs waren signifikant niedriger als die der EDTA_DNAs (Tabellen 1 und 2). Die DNA-Reinheit durch seine 260/280 Verhältnis Wert dargestellt ist ähnlich für beide P100_DNA und EDTA_DNA Probe-Set (Tabellen 1 und 2). Die Qualität der DNA ist ziemlich einheitlich in jedem Satz der Probe, obwohl einige Verschlechterung der EDTA_DNA Proben gesehen, wie durch ...

Watch this Scientific Journal Video about A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation at JoVE.com

A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation

Wir beschreiben eine neue Methode zur Isolierung von genomischer DNA aus Vollblut für Plasma / Serologie gesammelt. Nach Plasma-Sammlung wird die verdichtete Blut in der Regel verworfen. Unsere neue Methode stellt eine signifikante Verbesserung gegenüber bestehenden Verfahren und macht DNA und Plasma von einem einzigen Sammlung, ohne Anforderung zusätzlicher Blut.

Eine praktische und neuartige Methode, um genomische DNA aus Blutentnahme Kits Auszug für Plasma Protein Preservation

Laboratory tests can be done on the cellular or  fluid portions of the blood. The use of different blood collection tubes  determines the portion of the ...

a-practical-novel-method-to-extract-genomic-dna-from-blood-collection

Research

JoVE Journal

Biology

41.9K Aufrufe.  Emory University School of Medicine and Children's Health Care of Atlanta. Wir beschreiben eine neue Methode zur Isolierung von genomischer DNA aus Vollblut für Plasma / Serologie gesammelt. Nach Plasma-Sammlung wird die verdichtete Blut in der Regel verworfen. Unsere neue Methode stellt eine signifikante Verbesserung gegenüber bestehenden Verfahren und macht DNA und Plasma von einem einzigen Sammlung, ohne Anforderung zusätzlicher Blut.

Serie: A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation

Isolation of Genomic DNA from Mouse Tails

Isolierung genomischer DNA aus Maus-Tails

Forschung

Biologie

Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate. The study of immunity in horseshoe crabs has been facilitated by the ease in collecting large volumes of blood and from the simplicity of the blood. Horseshoe crabs show only a single cell type in the general circulation, the granular amebocyte. The plasma has the salt content of sea water and only three abundant proteins, hemocyanin, the respiratory protein, the C-reactive proteins, which function in the cytolytic destruction of foreign cells, including bacterial cells, and &alpha;2-macroglobulin, which inhibits the proteases of invading pathogens. Blood is collected by direct cardiac puncture under conditions that minimize contamination by lipopolysaccharide (a.k.a., endotoxin, LPS), a product of the Gram-negative bacteria. A large animal can yield 200 - 400 mL of blood. For the study of the plasma, blood cells are immediately removed from the plasma by centrifugation and the plasma can then be fractionated into its constituent proteins. The blood cells are conveniently studied microscopically by collecting small volumes of blood into LPS-free isotonic saline (0.5 M NaCl) under conditions that permit direct microscopic examination by placing one of more LPS-free coverglasses on the culture dish surface, then mounting those coverglasses in simple observation chambers following cell attachment. A second preparation for direct observation is to collect 3 - 5 mL of blood in a LPS-free embryo dish and then explanting fragments of aggregated amebocytes to a chamber that sandwiches the tissue between a slide and a coverglass. In this preparation, the motile amebocytes migrate onto the coverglass surface, where they can readily be observed. The blood clotting system involves aggregation of amebocytes and the formation of an extracellular clot of a protein, coagulin, which is released from the secretory granules of the blood cells. Biochemical analysis of washed blood cells requires that aggregation and degranulation does not occur, which can be accomplished by collecting blood into 0.1 volumes of 2% Tween-20, 0.5 M LPS-free NaCl, followed by centrifugation of the cells and washing with 0.5 M NaCl.

The American horseshoe crab, Limulus polyphemus, is arguably the most convenient source for large quantities of blood of any invertebrate. The blood is simple in composition, with only one cell-type in the general circulation, the granular amebocyte, and only three abundant proteins in the plasma, hemocyanin, the C-reactive proteins, and &alpha;2-macroglobulin. Blood is collected from the heart and the blood cells and plasma are separated by centrifugation.

Blutentnahme aus der amerikanischen Horseshoe Crab, Limulus Polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate.  The study of immunity in horseshoe crabs has been facilitated ...

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. 
The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (&beta;-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.

Extraktion von High Molecular Weight genomischer DNA aus Böden und Sedimenten

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

Lipidvesikel-vermittelte Affinitätschromatographie unter Verwendung von Magnetic Activated Cell Sorting (LIMACS): Eine neue Methode zur Protein-Lipid-Wechselwirkungen Analyze

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in their nucleotide composition and consist of a complex mosaic of sequence patterns. These patterns include so-called Mid-Range Inhomogeneity (MRI) regions -- sequences 30-10000 nucleotides in length that are enriched by a particular base or combination of bases (e.g. (G+T)-rich, purine-rich, etc.). MRI regions are associated with unusual (non-B-form) DNA structures that are often involved in regulation of gene expression, recombination, and other genetic processes (Fedorova &amp; Fedorov 2010). The existence of a strong fixation bias within MRI regions against mutations that tend to reduce their sequence inhomogeneity additionally supports the functionality and importance of these genomic sequences (Prakash et al. 2009).
Here we demonstrate a freely available Internet resource -- the Genomic MRI program package -- designed for computational analysis of genomic sequences in order to find and characterize various MRI patterns within them (Bechtel et al. 2008). This package also allows generation of randomized sequences with various properties and level of correspondence to the natural input DNA sequences. The main goal of this resource is to facilitate examination of vast regions of non-coding DNA that are still scarcely investigated and await thorough exploration and recognition.

We present a public computational web site for the analysis of genomic sequences. It detects DNA sequence patterns with various non-random nucleotide compositions. This resource also generates randomized sequences with diverse levels of complexity.

Genomic MRI - eine öffentliche Ressource für ein Studium Sequence Patterns in genomischer DNA

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

Selektive Aufnahme von 5-Hydroxymethylcytosin aus genomischer DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

A Practical Guide to Phylogenetics for Nonexperts

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this topic and so it presents inherent problems. Here we compile a practical introduction to phylogenetics for nonexperts.&#160;We outline in a step-by-step manner, a pipeline for generating reliable phylogenies from gene sequence datasets. We begin with a user-guide for similarity search tools via online interfaces as well as local executables. Next, we explore programs for generating multiple sequence alignments followed by protocols for using software to determine best-fit models of evolution. We then outline protocols for reconstructing phylogenetic relationships via maximum likelihood and Bayesian criteria&#160;and finally describe tools for visualizing phylogenetic trees. While this is not by any means an exhaustive description of phylogenetic approaches, it does provide the reader with practical starting information on key software applications commonly utilized by phylogeneticists. The vision for this article would be that it could serve as a practical training tool for researchers embarking on phylogenetic studies&#160;and also serve as an educational resource that could be incorporated into a classroom or teaching-lab.

Here we describe a step-by-step pipeline for generating reliable phylogenies from nucleotide or amino acid sequence datasets. This guide aims to serve researchers or students new to phylogenetic analysis.&#160;

A Practical Guide to Phylogenetics für Nichtexperten

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this ...

A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the potential of targeting the kinase in cancers by effectively treating the CML patients. This observation revolutionized drug development to target the oncogenic kinases implicated in various other malignancies, such as, EGFR, B-RAF, KIT and PDGFRs. However, one major drawback of anti-kinase therapies is the emergence of drug resistance mutations rendering the target to have reduced or lost affinity for the drug. Understanding the mechanisms employed by resistant variants not only helps in developing the next generation inhibitors but also gives impetus to clinical management using personalized medicine. We reported a retroviral vector based screening strategy to identify the spectrum of resistance conferring mutations in BCR/ABL, which has helped in developing the next generation BCR/ABL inhibitors. Using Ruxolitinib and JAK2 as a drug target pair, here we describe in vitro screening methods that utilizes the mouse BAF3 cells expressing the random mutation library of JAK2 kinase.

Emergence of genetic resistance against kinase inhibitor therapy poses significant challenge for effective cancer therapy. Identification and characterization of resistant mutations against a newly developed drug helps in better clinical management and next generation drug design. Here, we describe our protocol for in vitro screening and validation of resistant mutations.

Verfahren zum Screening und Validierung von Beständig Mutationen Vor-Inhibitoren

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the ...

Repeated Blood Collection for Blood Tests in Adult Zebrafish

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from zebrafish has not been established. The previous methods for blood collection from zebrafish are lethal, such as lateral incision, decapitation and tail ablation. Thus we have developed a novel &#8220;repeated&#8221; blood collection method, and present here a detailed protocol outlining this procedure. This method is minimally invasive and results in a very low mortality rate (2.3%) for zebrafish, thus enabling repeated blood sampling from the same individual. The maximum volume of blood sampling is dependent on body weight of the fish. The volume for repeated blood sampling at intervals should be&#160;&#8804;0.4% of body weight every week or &#8804;1% every 2 weeks, which were evaluated by measurements of blood hemoglobin. Additionally, hemoglobin, fasting blood glucose, plasma triacylglycerol (TG) and total cholesterol levels in male and female adult zebrafish were measured. We also applied this method to investigate the dysregulation of glucose metabolism in diet-induced obesity. This blood collection method will allow many applications, including glucose and lipid metabolism and hematological studies, which will increase the use of zebrafish as a human disease model organism.

Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

Wiederholte Blutentnahme für Bluttests in Adult Zebrafisch

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Amplification, Next-Generation-Sequenzierung und genomischer DNA Mapping von Retrovirale Integration Seiten

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...