Wir wünschen den Mitgliedern des Wan Lam Lab BAC-Array-Team vor allem Miwa Suzuki und Bryan Chi für die Vorbereitung dieses Artikels danken. Diese Arbeit wurde durch Mittel aus Canadian Institutes for Health Research, Genome Canada / Genome British Columbia, und NIH / NIDCR gewähren RO1 DE15965-01 unterstützt.

Sondenmarkierung Hinweis: Begrenzung der Exposition Cye Farbstoffe, um Licht zu jeder Zeit (dies kann durch die Arbeit in einem abgedunkelten Raum oder durch Abschirmung der Rohre mit einer Abdeckung wie Aluminiumfolie erreicht werden) <ol><li> Kombinieren Sie: (Setup 1 Reaktionsgefäß als Referenz und 1 Reaktionsrohr Probe) <ul><li> DNA (25-400 ng) </li><li> 5 ul 5X random Primer (Endkonzentration: 5X Promega Klenow-Puffer und 7 ug / ul random Octamere) </li><li> Verdünnen auf 17,0 ul Gesamtvolumen mit destilliertem H 2 O </li></ul></li><li> Kochen für 10 Minuten bei 100 ° C. Sofort auf Eis für 1 Minute. </li><li> Add 4 ul 10x dNTP-Mix (2 mM dATP, dGTP, dTTP, dCTP 1,2 mm). </li><li> Add CyeDyes: <ul><li> Add 2 ul (2 nmol) von Cy-3 markiertem dCTP zu Proben-DNA </li><li> Add 2 ul (2 nmol) von Cy-5 markierten dCTP zu Referenz-DNA (zB Novagen humanen genomischen DNA) </li></ul></li><li> Add 2,5 ul Klenow (9 U / ul, Promega) und mischen. </li><li> Bei 37 ° C über Nacht * (~ 18 Stunden). * Die Hybridisierung über Nacht kann zwischen 14 bis 24 Stunden ohne Beeinträchtigung der Labelling-Verfahren in ein Labor Zeitplan passen eingestellt werden. </li></ol> Probenaufbereitung (Combined Sonde clean-up und Vorbereitung für die Hybridisierung) <ol><li> Mit einem Microcon YM-30 Spalte: <ul><li> 100 l Cot-1 DNA (1μg/μL) in die Spalte. Berühren Sie nicht Membran mit Pipettenspitze. </li><li> Pool der Reaktionen (Referenz und Probe) und fügen Sie in die Spalte. </li><li> Legen Spalte in sofern Rohr und Spin bei 13000 g für 10 Minuten. </li><li> Geben Sie 200 ul destilliertem H 2 0 auf die Membran und wiederholen Spin zu waschen. </li><li> Discard Tube und füge 45 ul Hybridisierungslösung: (Roche DIG Easy) </li></ul> Optional: add 5μL geschert Heringssperma DNA (10μg/μL Einheit, Promega) <ul><li> Invert Microcon, Platz in einer neuen Bezeichnung Rohr und drehen sich mit 3000 g für 3 Minuten. </li></ul></li></ol> BERECHNUNG Eingemeindungen <ol><li> Entfernen 1,5 ul und messen Fluorophor Einbau mit NanoDrop Spektralphotometer. Mit Ihrem Hybridisierungspuffer wie eine leere (DIG Easy) folgen Sie den Anweisungen auf dem Bildschirm. (Sie können die Probe aus dem Sockel zu erholen, falls erforderlich, nach der Messung.) (Hinweis:. Incorporations unter 3,0 pmol / ul in einem der Kanäle zeigten unterschiedliche Ergebnisse) </li><li> Denaturieren bei 85 ° C für 10 Minuten. </li><li> Legen Sonde bei 45 ° C für 30 Minuten bis 1 Stunde (erlaubt Cot-1 DNA Glühen.) </li></ol> ARRAY HYBRIDIZATION <ol><li> Platz 44 ul Probe-Lösung auf das Deckglas (22 mm x 60 mm) (Fisher Scientific). Falls vorhanden, legen Sie das Deckglas auf einem Objektträger wärmer oder Heizblock auf 45 vorgewärmten ° C auf Temperatur zu halten. </li><li> Richten Sie gleiten über Deckglas und Probe-Lösung, untere Kante der Folie ermöglicht den Kontakt mit der Hybridisierungslösung. Weiter zu senken, bis das Deckglas auf dem Objektträger mit der Oberflächenspannung verbunden ist. Invert gleiten. </li><li> Legen Sie die Folie in Hybridisierung Kassette (Telechem), vorgewärmt auf 45 ° C, und fügen Sie 10 l Wasser in die untere Nut (gegen Feuchtigkeit während der Hybridisierung Steuerung). </li><li> Seal Kassette und inkubieren 36 bis 40 Stunden bei 45 ° C. </li></ol> ARRAY WASCHEN Hinweis: Alle Waschlösungen werden bei pH 7,0 Das Entfernen der Folie aus der Hybridisierung Kassette ist kritisch. DIG Easy schnell kristallisiert bei Raumtemperatur. Der Schlitten sollte sofort eingetaucht werden und das Deckglas in der Waschlösung entfernt. <ol><li> Fügen Sie ca. 60 ml 0.1XSSC, 0,1% SDS (pH 7,0, 45 ° C) waschen Lösung für ein Coplin Glas vor dem Öffnen der Hybridisierung Kammer. </li><li> Offene Kammer, fügen Folie Lösung zu waschen und zu entfernen Deckglas (es sollte abrutschen). </li><li> Waschen Sie die Folie 3 mal in 0.1XSSC + 0,1% SDS bei 45 ° C für 1 Minute unter Rühren. </li><li> Spülen Sie die Folie 3 mal in frischem 0.1XSSC *. Es sollte kein Rest-Blasen aus dem SDS waschen sichtbar werden. * Die Folien können in der letzten Waschlösung bis zu seiner Zentrifuge und Scannen (~ 15 Minuten) bleiben. </li><li> Zentrifugieren Sie die Folie in einer 50 ml Falcon-Röhrchen bei 700 g für 3 Minuten. </li><li> Unmittelbar scannen die Dias (Signalintensitäten im Laufe der Zeit abnehmen.) </li></ol>

<ol>
	<li>Lockwood, W. W., Chari, R., Chi, B., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+array+comparative+genomic+hybridization+technologies+and+their+applications+in+human+genetics.">Recent advances in array comparative genomic hybridization technologies and their applications in human genetics.</a> European Journal of Human Genetics. 14, 139-1x`48 (2006).</li><li>Ishkanian, A. S., Malloff, C. A., Watson, S. K., deLeeuw, R. J., Chi, B., Coe, B. P., Snijders, A., Albertson, D. G., Pinkel, D., Marra, M. A., Ling, V., MacAulay, C., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tiling+resolution+DNA+microarray+with+complete+coverage+of+the+human+genome.">A tiling resolution DNA microarray with complete coverage of the human genome.</a> Nature Genetics. 36, 299-303 (2004).</li><li>Watson, S. K., DeLeeuw, R. J., Ishkanian, A. S., Malloff, C. A., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+high+throughput+validation+of+amplified+fragment+pools+of+BAC+DNA+for+constructing+high+resolution+CGH+arrays.">Methods for high throughput validation of amplified fragment pools of BAC DNA for constructing high resolution CGH arrays.</a> BMC Genomics. 5, 6 (2004).</li><li>Watson, S. K., DeLeeuw, R. J., Horsman, D. E., Squire, J. A., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytogenetically+balanced+translocations+are+associated+with+focal+copy+number+alterations.">Cytogenetically balanced translocations are associated with focal copy number alterations.</a> Human Genetics. 120, 795-805 (2007).</li><li>Chi, B., DeLeeuw, R. J., Coe, B. P., MacAulay, C., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SeeGH+--+a+software+tool+for+visualization+of+whole+genome+array+comparative+genomic+hybridization+data.">SeeGH -- a software tool for visualization of whole genome array comparative genomic hybridization data.</a> BMC Bioinformatics. 5, 13 (2004).</li><li>Chi, B., DeLeeuw, R. J., Coe, B. P., Ng, R. T., MacAulay, C., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MD-SeeGH:+a+platform+for+integrative+analysis+of+multi-dimensional+genomic+data.">MD-SeeGH: a platform for integrative analysis of multi-dimensional genomic data.</a> BMC Bioinformatics. 9, 243 (2008).</li><li>Coe, B. P., Ylstra, B., Carvalho, B., Meijer, G. A., Macaulay, C., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolving+the+resolution+of+array+CGH.">Resolving the resolution of array CGH.</a> Genomics. 89, 647-653 (2007).</li><li>Garnis, C., Coe, B. P., Lam, S. L., MacAulay, C., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+array+CGH+increases+heterogeneity+tolerance+in+the+analysis+of+clinical+samples.">High-resolution array CGH increases heterogeneity tolerance in the analysis of clinical samples.</a> Genomics. 85, 790-793 (2005).</li></ol>

Schlechte Qualität DNA wird nicht eine gute Hybridisierung Profil. Es ist wichtig, dass die Probe zu gewährleisten und Referenz-DNA sind frei von Verunreinigungen wie Phenol, RNA, Salz, etc, die mit dem random prime Kennzeichnung Schritt vor Beginn einer Hybridisierung Experiment stören können. Zum Beispiel die Resuspension der DNA in Tris - EDTA (TE) anstelle von Wasser ist nicht so hohe Salzkonzentration empfohlen hemmen können die Kennzeichnung Reaktion. Wir empfehlen Testen DNA-Qualität mit dem Nanodrop Spektralphotometer, um sowohl die DNA-Menge sowie allgemeine Form der Absorptionskurve und 260/280, 260/230 Verhältnis zu messen. Waschen: Das Entfernen der Folie aus der Hybridisierung Kassette und die Übertragung auf die beheizte Waschlösung ist ein kritischer Schritt bei der Hybridisierung Prozess wie der DIG Easy Hybridisierungslösung kristallisiert sehr schnell. Es ist wichtig, dass die Waschlösungen bei einem neutralen pH und die Temperatur der Waschlösung für Stringenz wichtig beim Entfernen der ungebundenen Sonde. Nach dem Trocknen gleitet, ist es wichtig, sie im Dunkeln zu halten, wenn nicht das Scannen sofort, oder nach dem Scannen, wenn Sie sie zu einem späteren Zeitpunkt erneut prüfen wollen. Ozon: verfügt über ein weltweites Anliegen bei der Durchführung von Array-CGH. Ozonwerte über 5 ppm haben gezeigt, dass sich negativ auf die Cye Farbstoffe. Cye 5 ist besonders empfindlich auf Ozon-Abbau. Minimierung der Ozonbelastung ist der Schlüssel zur Verhinderung Cye Farbstoff Abbau und Verlust des Signals vor und während des Scanvorgangs. Scanning in der Nacht zum Beispiel, wenn die Umwelt Ozon in der Regel niedriger ist, ist eine mögliche Option. Ozonfreie Gehäuse für automatisierte Dia Waschen und Scannen und verschiedene chemische Dia-Behandlungen sind im Handel erhältlich. Ozonbeständig Cye Farbstoffe haben auch vor kurzem entwickelt worden.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>5X Klenow Buffer</td>
<td>Reagent</td>
<td>Promega</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Random Octamers </td>
<td>Reagent</td>
<td>Alpha DNA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10X dNTP mix</td>
<td>Reagent</td>
<td>Promega</td>
<td>&nbsp;</td>
<td>2mM dATP, dGTP, dTTP, 1.2mM dCTP</td>
</tr>
<tr>
<td>Cy-3 labeled dCTP</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cy-5 labeled dCTP</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Human Genomic DNA Reference</td>
<td>Reagent</td>
<td>Novagen</td>
<td>&nbsp;</td>
<td>Example of a possible reference</td>
</tr>
<tr>
<td>Klenow</td>
<td>Reagent</td>
<td>Promega</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>YM-30 Column</td>
<td>Reagent</td>
<td>Millipore</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cot-1 DNA</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>DIG Easy</td>
<td>Reagent</td>
<td>Roche</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Sheared Herring Sperm DNA</td>
<td>Reagent</td>
<td>Promega</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Coverslip</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>&nbsp;</td>
<td>22mm x 60mm</td>
</tr>
<tr>
<td>Hybridization Cassette</td>
<td>Tool</td>
<td>Telechem</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

technical demonstration of whole genome array comparative genomic hybridization

Array komparative genomische Hybridisierung (Array-CGH) ist eine Methode zum Nachweis von Gewinnen und Verlusten von DNA-Segmenten oder Gen-Dosierung in das Genom 1. Jüngste Fortschritte in dieser Technologie sind mit hochauflösenden Vergleich ganzer Genome für die Identifizierung genetischer Veränderungen bei Krebs und anderen genetischen Erkrankungen 2 aktiviert. Die Sub-Megabase Resolution Tiling-set array (oder SMRT)-Array besteht aus einer Reihe von rund 30.000 überlappenden bakterielle künstliche Chromosom (BAC) Klone, die das menschliche Genom in ~ 100 Kilobasenpaar (kb) Segmente 2 span zusammen. Diese BAC-Ziele werden individuell hergestellt und entdeckte in zweifacher Ausfertigung auf einem einzigen Objektträger 2-4. Array-CGH ist auf dem Prinzip der kompetitiven Hybridisierung. Proben-und Referenz-DNA werden differentiell mit Cyanin-3 bezeichnet und Cyanine-5 Fluoreszenzfarbstoffe, und das Array co-hybridisiert. Nach einer Inkubationszeit die ungebundenen Proben werden von der Folie gewaschen und das Array abgebildet. Eine frei verfügbare kundenspezifische Software-Paket namens SeeGH (www.flintbox.ca) wird verwendet, um die große Menge an Daten gesammelt Prozess - einem einzigen Experiment erzeugt 53.892 Datenpunkte. SeeGH visualisiert die log2 Signalintensität Verhältnis zwischen den 2 Proben an jedem BAC Ziel, die vertikal mit chromosomalen Position 5,6 ausgerichtet ist. Der SMRT Array erkennt Änderungen so klein wie 50 kb in Größe 7. Der SMRT Array erkennt eine Vielzahl von DNA Umlagerung Veranstaltungen, darunter DNA Gewinne, Verluste, Amplifikationen und homozygote Deletionen. Ein einzigartiger Vorteil der SMRT Array ist, dass man DNA aus Formalin-fixierten Paraffin eingebetteten Proben isoliert zu verwenden. In Verbindung mit dem niedrigen Eingang Anforderungen der unverstärkten DNA (25-100 ng) kombiniert dies ermöglicht Profilierung der wertvollen Proben, wie sie durch Mikrodissektion 7,8 produziert. Dies ist auf die Größe der einzelnen BAC-Hybridisierung Ziel, dass die Bindung von ausreichend markierten Proben, um Signale für den Nachweis produzieren können zugeschrieben. Ein weiterer Vorteil dieser Plattform ist die Toleranz des Gewebes Heterogenität, der Bedarf an langwierige Gewebe Mikrodissektion 8. Dieses Video-Protokoll ist ein Schritt-für-Schritt-Anleitung von der Kennzeichnung der eingesetzten DNA bis hin zur Übernahme des gesamten Genoms Fliesen Weg SMRT Array Signal.

Watch this Scientific Journal Video about Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization at JoVE.com

Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization

Dieses Video ist eine technische Demonstration der Hybridisierung Protokoll für gesamte Genom Fliesen Weg Array-CGH, ​​die das gesamte menschliche Genom mit nur 25-100 ng DNA, die aus einer Vielzahl von Quellen, einschließlich Archivierung Formalin fixiert Material isoliert werden können Scans.

Technische Demonstration von Whole Genome Array Comparative Genomic Hybridization

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome 1.  Recent ...

technical-demonstration-whole-genome-array-comparative-genomic

Nanyang Technological University

Research

JoVE Journal

Biology

12.7K Aufrufe.  BC Cancer Research Centre. *The slides can remain in the 

Serie: Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

Doppel Whole Mount in situ Hybridisierung von Anfang Hühnerembryonen

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Forschung

Biologie

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

Zebrafisch Whole Mount High-Resolution Doppel Fluorescent In-situ Hybridisierung

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of specific mRNA transcripts within whole mount specimen. This technique (adapted from Albertson and Yelick, 2005) was used in an upper level undergraduate Comparative Vertebrate Biology laboratory classroom at Syracuse University. The first two thirds of the Comparative Vertebrate Biology lab course gave students the opportunity to study the embryology and gross anatomy of several organisms representing various chordate taxa primarily via traditional dissections and the use of models. The final portion of the course involved an innovative approach to teaching anatomy through observation of vertebrate development employing molecular techniques in which WISH was performed on zebrafish embryos. A heterozygous fibroblast growth factor 8 a (fgf8a) mutant line, ace, was used. Due to Mendelian inheritance, ace intercrosses produced wild type, heterozygous, and homozygous ace/fgf8a mutants in a 1:2:1 ratio. RNA probes with known expression patterns in the midline and in developing anatomical structures such as the heart, somites, tailbud, myotome, and brain were used. WISH was performed using zebrafish at the 13 somite and prim-6 stages, with students performing the staining reaction in class. The study of zebrafish embryos at different stages of development gave students the ability to observe how these anatomical structures changed over ontogeny. In addition, some ace/fgf8a mutants displayed improper heart looping, and defects in somite and brain development. The students in this lab observed the normal development of various organ systems using both external anatomy as well as gene expression patterns. They also identified and described embryos displaying improper anatomical development and gene expression (i.e., putative mutants).
For instructors at institutions that do not already own the necessary equipment or where funds for lab and curricular innovation are limited, the financial cost of the reagents and apparatus may be a factor to consider, as will the time and effort required on the part of the instructor regardless of the setting. Nevertheless, we contend that the use of WISH in this type of classroom laboratory setting can provide an important link between developmental genetics and anatomy. As technology advances and the ability to study organismal development at the molecular level becomes easier, cheaper, and increasingly popular, many evolutionary biologists, ecologists, and physiologists are turning to research strategies in the field of molecular biology. Using WISH in a Comparative Vertebrate Biology laboratory classroom is one example of how molecules and anatomy can converge within a single course. This gives upper level college students the opportunity to practice modern biological research techniques, leading to a more diversified education and the promotion of future interdisciplinary scientific research.

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) was used in an upper level undergraduate Comparative Vertebrate Biology course in addition to vertebrate dissections. This gave students the opportunity to study gene expression patterns as well as gross anatomy, linking the study of molecular and organismal biology within one course.

Mit Whole Berg In situ Hybridisierung zu Molecular Biology und Organismal Link-

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of ...

DNA Microarrays: Sample Quality Control, Array Hybridization and Scanning

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, different physiological or pathological states, response to therapy, and, in general, any condition that is being experimentally tested1. Expression profiling of neural tissues requires isolation of high quality RNA, amplification of the isolated RNA and hybridization to DNA microarrays. In this article we describe protocols for reproducible microarray experiments from brain tumor tissue2. We will start by performing a quality control analysis of isolated RNA samples with Agilent's 2100 Bioanalyzer "lab-on-a-chip" technology. High quality RNA samples are critical for the success of any microarray experiment, and the 2100 Bioanalyzer provides a quick, quantitative measurement of the sample quality. RNA samples are then amplified and labeled by performing reverse transcription to obtain cDNA, followed by in vitro transcription in the presence of labeled nucleotides to produce labeled cRNA. By using a dual-color labeling kit, we will label our experimental sample with Cy3 and a reference sample with Cy5. Both samples will then be combined and hybridized to Agilent's 4x44 K arrays. Dual-color arrays offer the advantage of a direct comparison between two RNA samples, thereby increasing the accuracy of the measurements, in particular for small changes in expression levels, because the two RNA samples are hybridized competitively to a single microarray. The arrays will be scanned at the two corresponding wavelengths, and the ratio of Cy3 to Cy5 signal for each feature will be used as a direct measurement of the relative abundance of the corresponding mRNA. This analysis identifies genes that are differentially expressed in response to the experimental conditions being tested.

We demonstrate the use of DNA microarrays for expression profiling of the nervous system. We describe RNA quality control, sample labeling, and array hybridization and scanning.

DNA Microarrays: Sample Quality Control, Array Hybridisierung und Scannen

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, ...

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

Ganze Berge In-situ Hybridisierung von E8.5 bis E11.5 Maus Embryonen

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

Competitive Genomic Screens von Barcoded Hefe Bibliotheken

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any two individuals is greater than previously thought3. In contrast, array-based genotyping has failed to identify a significant contribution of common sequence variants to the phenotypic variability of common disease4,5. Taken together, these observations have led to the evolution of the Common Disease / Rare Variant hypothesis suggesting that the majority of the "missing heritability" in common and complex phenotypes is instead due to an individual's personal profile of rare or private DNA variants6-8. However, characterizing how rare variation impacts complex phenotypes requires the analysis of many affected individuals at many genomic loci, and is ideally compared to a similar survey in an unaffected cohort. Despite the sequencing power offered by today's platforms, a population-based survey of many genomic loci and the subsequent computational analysis required remains prohibitive for many investigators.
To address this need, we have developed a pooled sequencing approach1,9 and a novel software package1 for highly accurate rare variant detection from the resulting data. The ability to pool genomes from entire populations of affected individuals and survey the degree of genetic variation at multiple targeted regions in a single sequencing library provides excellent cost and time savings to traditional single-sample sequencing methodology. With a mean sequencing coverage per allele of 25-fold, our custom algorithm, SPLINTER, uses an internal variant calling control strategy to call insertions, deletions and substitutions up to four base pairs in length with high sensitivity and specificity from pools of up to 1 mutant allele in 500 individuals. Here we describe the method for preparing the pooled sequencing library followed by step-by-step instructions on how to use the SPLINTER package for pooled sequencing analysis (<a href="http://www.ibridgenetwork.org/wustl/splinter" target="_blank">http://www.ibridgenetwork.org/wustl/splinter</a>). We show a comparison between pooled sequencing of 947 individuals, all of whom also underwent genome-wide array, at over 20kb of sequencing per person. Concordance between genotyping of tagged and novel variants called in the pooled sample were excellent. This method can be easily scaled up to any number of genomic loci and any number of individuals. By incorporating the internal positive and negative amplicon controls at ratios that mimic the population under study, the algorithm can be calibrated for optimal performance. This strategy can also be modified for use with hybridization capture or individual-specific barcodes and can be applied to the sequencing of naturally heterogeneous samples, such as tumor DNA.

Pooled DNA sequencing is a fast and cost-effective strategy to detect rare variants associated with complex phenotypes in large cohorts. Here we describe the computational analysis of pooled, next-generation sequencing of 32 cancer-related genes using the SPLINTER software package. This method is scalable, and applicable to any phenotype of interest.

Nachweis seltener Varianten von Genomic Pooled-Sequenzierung mit SPLINTER

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

Whole Berg RNA Fluorescent<em&gt; In situ</em&gt; Hybridisierung<em&gt; Drosophila</em&gt; Embryonen

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

Array Comparative Genomic Hybridization (Array CGH) for Detection of Genomic Copy Number Variants

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a clinical diagnostic service laboratory. DNA extracted from a patient&#8217;s sample (blood, saliva or other tissue types) is labeled with a fluorochrome (either cyanine 5 or cyanine 3). A reference DNA sample is labeled with the opposite fluorochrome. There follows a cleanup step to remove unincorporated nucleotides before the labeled DNAs are mixed and resuspended in a hybridization buffer and applied to an array comprising ~60,000 oligonucleotide probes from loci across the genome, with high probe density in clinically important areas. Following hybridization, the arrays are washed, then scanned and the resulting images are analyzed to measure the red and green fluorescence for each probe. Software is used to assess the quality of each probe measurement, calculate the ratio of red to green fluorescence and detect potential copy number variants.

Array Comparative Genomic Hybridization Array CGH for Detection of Genomic Copy Number Variants

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a diagnostic service laboratory.

Array Comparative Genomic Hybridization (Array-CGH) zum Nachweis von Genomic Copy Number Varianten

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its ...

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors&#160;induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, deep sequencing, and mapping. Extensive exonuclease treatment&#160;was&#160;required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by &#966;29 polymerase enriched for circular DNA over linear DNA. Validation of the Circle-Seq method on three S. cerevisiae CEN.PK populations of 1010 cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30,&#160;HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types.

This paper presents a sensitive method called Circle-Seq for purifying extrachromosomal circular DNA (eccDNA). The method encompasses column purification, removal of remaining linear chromosomal DNA, rolling-circle amplification and high-throughput sequencing. Circle-Seq is applicable to genome-scale screening of eukaryotic eccDNA and studying genome instability and copy-number variation.

Genomweite Reinigung von extrachromosomale zirkuläre DNA aus eukaryotischen Zellen

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs ...