עבודה זו נתמכה על ידי מענקים ED מן ס ג&#39;יימס מקדונל קרן 21 תוכנית פרס רח&#39; המאה, ואת פרס ממציא ניו מנהל של NIH. במודלים של GAB נתמך בחלקו על ידי מענק דוקטורט ממכון קליפורניה של רפואה רגנרטיבית.

<ol>
	<li>Diaz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One+Decade+Later:+What+has+Gene+Expression+Profiling+Told+us+About+Neuronal+Cell+Types,+Brain+Function+and+Disease.">One Decade Later: What has Gene Expression Profiling Told us About Neuronal Cell Types, Brain Function and Disease.</a> Curr Genomics. 10, 318-318 (2009).</li><li>Barisone, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Expression profiling in Neuroscience, edited by Ioannis Karamanos. , (2010).</li><li>Gelder, R. N. V. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amplified+RNA+synthesized+from+limited+quantities+of+heterogeneous+cDNA.">Amplified RNA synthesized from limited quantities of heterogeneous cDNA.</a> Proc Natl Acad Sci U S A. 87, 1663-1663 (1990).</li></ol>

אין ניגודי אינטרסים הכריז.

פרופיל הביטוי עם microarrays DNA מספק גישה פשוטה לזיהוי גנים הביע באופן דיפרנציאלי בין שתי דגימות ביולוגיות. פרופיל מוצלח ניסויים ביטוי דורשים איכות גבוהה דגימות רנ&quot;א, תיוג חזקים הכלאה. מניסיוננו, מערכי מסחריים ערכות תיוג של Agilent לספק נתונים באיכות גבוהה במחיר סביר. בעוד Agilent מציעה גם ההכלאה שלהם במכונות סריקה, אנחנו בעד מערכת הכלאה בין רוש-NimbleGen ואת 4000B GenePix סורק microarray מן התקנים מולקולריים. ראוי לציין, כי יחידת Roche-NimbleGen הכלאה היה ידוע בעבר בשם מערכת הכלאה מאווי. לאחר הרכישה האחרונה של החברה על ידי חברת רוש, מערבלי A4 שהופסקו. נכון לרגע כתיבת שורות אלה, הם עדיין יכולים למצוא דרך מוכרים אחרים כגון Kreatech אבחון ( <a href="http://www.kreatech.com/">www.kreatech.com</a> ; אתר זה מציע גם כלי נוח תאימות במערך), אבל לטווח ארוך זמינות מלאי אינה ודאית. מערכות הכלאה אחרים זמינים (לדוגמה, מ - Agilent), אולם אם מערבלי תואם ניתן למצוא את מערך בשימוש, אנו ממליצים על המערכת של רוש, NimbleGen עבור איכותו reproducibility.

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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>2100 Bioanalyzer</td>
<td>&nbsp;</td>
<td>Agilent Technologies</td>
<td>G2943CA</td>
<td>Includes chip priming station, vortexer, software and laptop computer</td>
</tr>
<tr>
<td>2100 Bioanalyzer electrophoresis set</td>
<td>&nbsp;</td>
<td>Agilent Technologies</td>
<td>G2947CA</td>
<td>&nbsp;</td>
<tr>
<td>RNA 6000 Nano kit</td>
<td>&nbsp;</td>
<td>Agilent Technologies</td>
<td>5067-1511</td>
<td>includes size ladder, marker, gel matrix, dye, electrode cleaners, spin filters and nanochips</td>
</tr>
<tr>
<td>RNase Zap</td>
<td>&nbsp;</td>
<td>Applied Biosystems &#x2013; Ambion</td>
<td>AM9780M</td>
<td>&nbsp;</td>
<tr>
<td>Quick Amp labeling kit, two-color</td>
<td>&nbsp;</td>
<td>Agilent Technologies</td>
<td>5190-0444</td>
<td>&nbsp;</td>
<tr>
<td>RNeasy mini kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>74104</td>
<td>&nbsp;</td>
<tr>
<td>Gene Expression Hybridization Kit</td>
<td>&nbsp;</td>
<td>Agilent Technologies</td>
<td>5188-542</td>
<td>Includes Blocking agent, Fragmentation buffer and Hybridization buffer</td>
</tr>
<tr>
<td>Gene Expression Wash Buffer Kit</td>
<td>&nbsp;</td>
<td>Agilent Technologies</td>
<td>5188-5327</td>
<td>Includes Wash Buffers 1 and 2 and Triton X-102</td>
</tr>
<tr>
<td>Hybridization Station</td>
<td>&nbsp;</td>
<td>Roche Nimblegen</td>
<td>05223652001</td>
<td>4-slide model</td>
</tr>
<tr>
<td>Hybridization System Accesory Kit</td>
<td>&nbsp;</td>
<td>Roche Nimblegen</td>
<td>05327695001</td>
<td>Contains verification assembly for testing mixing, replacement O-rings, disassembly tool, forceps and brayer</td>
</tr>
<tr>
<td>A4 mixer hybridization chambers</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Whole Human Genome (4x44) Oligo Microarray</td>
<td>&nbsp;</td>
<td>Agilent Technologies</td>
<td>G4112F</td>
<td>&nbsp;</td>
<tr>
<td>Positive displacement pipette</td>
<td>&nbsp;</td>
<td>Gilson</td>
<td>F148504</td>
<td>&nbsp;</td>
<tr>
<td>Capillary pistons for positive displacement pipette</td>
<td>&nbsp;</td>
<td>Gilson</td>
<td>F148415</td>
<td>&nbsp;</td>
<tr>
<td>Microarray dryer</td>
<td>&nbsp;</td>
<td>Nimblegen</td>
<td>05223636001</td>
<td>&nbsp;</td>
<tr>
<td>Microarray fluorescent scanner, Axon GenePix 4000B</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>GENEPIX4000B</td>
<td>&nbsp;</td>
<tr>
<td>GenePix Pro 6.0 software</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

dna microarrays: sample quality control, array hybridization and scanning

פרופיל microarray ביטוי של מערכת העצבים מספק גישה רב עוצמה לזיהוי פעילויות גן בשלבים שונים של פיתוח, או מצבים פיזיולוגיים פתולוגיים שונים, התגובה לטיפול, ובאופן כללי, כל מצב זה נבדק בניסוי 1. פרופיל הביטוי של רקמות עצביות דורש בידוד של איכות גבוהה RNA, הגברה של רנ&quot;א מבודד הכלאה כדי microarrays DNA. במאמר זה נתאר פרוטוקולים לניסויים microarray לשחזור מרקמת המוח הגידול 2. נתחיל ידי ביצוע בקרת איכות ניתוח של דגימות RNA בודד עם 2100 של Agilent Bioanalyzer טכנולוגיית &quot;מעבדה על שבב&quot;. איכות גבוהה דגימות RNA הם קריטיים להצלחת הניסוי כל microarray, ואת 2100 Bioanalyzer מספק מדידה מהירה, כמותית של איכות המדגם. דגימות RNA הם מוגבר אז שכותרתו ידי ביצוע שעתוק לאחור כדי לקבל cDNA, ואחריו ב שעתוק במבחנה בנוכחות של נוקלאוטידים שכותרתו לייצר Crna שכותרתו. באמצעות ערכת כפול צבע תיוג, אנו תווית מדגם הניסוי שלנו עם Cy3 ו מדגם התייחסות עם Cy5. דגימות הן מכן ניתן יהיה משולב הכלאה למערכי 4x44 של Agilent K. Dual-צבע מערכים להציע את היתרון של השוואה ישירה בין שתי דגימות רנ&quot;א, ובכך להגדיל את רמת הדיוק של המדידות, בפרט עבור שינויים קטנים ברמות הביטוי, כי שתי דגימות RNA הם הכלאה תחרותי כדי microarray יחיד. מערכים ייסרקו על שני אורכי הגל המתאים, יחס אות Cy3 כדי Cy5 עבור כל תכונה תשמש מדידה ישירה של השפע היחסי של mRNA המתאים. ניתוח זה מזהה את הגנים באים לידי ביטוי באופן דיפרנציאלי בתגובה תנאי הניסוי נבדק.

Watch this Scientific Journal Video about DNA Microarrays: Sample Quality Control, Array Hybridization and Scanning at JoVE.com

DNA Microarrays: Sample Quality Control, Array Hybridization and Scanning

אנו מדגימים את השימוש microarrays DNA עבור פרופיל הביטוי של מערכת העצבים. אנו מתארים לשלוט RNA איכות, תיוג המדגם, הכלאה מערך סריקה.

Microarrays DNA: בקרת איכות המדגם, הכלאת מערך סריקה ו

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

dna-microarrays-sample-quality-control-array-hybridization

Nanyang Technological University

Research

JoVE Journal

Biology

37.9K Views.  University of California, Davis. אנו מדגימים את השימוש microarrays DNA עבור פרופיל הביטוי של מערכת העצבים. אנו מתארים לשלוט RNA איכות, תיוג המדגם, הכלאה מערך סריקה.

Video: DNA Microarrays: Sample Quality Control, Array Hybridization and Scanning

Bacterial Gene Expression Analysis Using Microarrays

Gene Expression חיידקית ניתוח שימוש microarrays

Technical Demonstration of 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 advances in this technology have enabled high resolution comparison of whole genomes for the identification of genetic alterations in cancer and other genetic diseases 2. The Sub-Megabase Resolution Tiling-set array (or SMRT) array is comprised of a set of approximately thirty thousand overlapping bacterial artificial chromosome (BAC) clones that span the human genome in ~100 kilobase pair (kb) segments 2. These BAC targets are individually synthesized and spotted in duplicate on a single glass slide 2-4. Array CGH is based on the principle of competitive hybridization. Sample and reference DNA are differentially labeled with Cyanine-3 and Cyanine-5 fluorescent dyes, and co-hybridized to the array. After an incubation period the unbound samples are washed from the slide and the array is imaged. A freely available custom software package called SeeGH (www.flintbox.ca) is used to process the large volume of data collected - a single experiment generates 53,892 data points. SeeGH visualizes the log2 signal intensity ratio between the 2 samples at each BAC target which is vertically aligned with chromosomal position 5,6. The SMRT array can detect alterations as small as 50 kb in size 7. The SMRT array can detect a variety of DNA rearrangement events including DNA gains, losses, amplifications and homozygous deletions. A unique advantage of the SMRT array is that one can use DNA isolated from formalin fixed paraffin embedded samples. When combined with the low input requirements of unamplified DNA (25-100ng) this allows profiling of precious samples such as those produced by microdissection 7,8. This is attributed to the large size of each BAC hybridization target that allows the binding of sufficient labeled samples to produce signals for detection. Another advantage of this platform is the tolerance of tissue heterogeneity, decreasing the need for tedious tissue microdissection 8. This video protocol is a step-by-step tutorial from labeling the input DNA through to signal acquisition for the whole genome tiling path SMRT array.

This video is a technical demonstration of the hybridization protocol for whole genome tiling path array CGH, which scans the entire human genome using only 25-100 ng of DNA that can be isolated from a variety of sources, including archival formalin fixed material.

הפגנה טכנית של הכלאה שלמות מערך הגנום הגנום השוואתי

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 ...

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using noninvasive sampling techniques to investigate population genetics and demography of wild populations1. This approach has proven to be especially useful when dealing with rare or elusive species2. While a number of these methods have been developed to sample hair, feces and other biological material from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. In this video, we present a novel, inexpensive and noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We describe the general set-up of the hair snare, which consists of strips of packing tape arranged in a web-like fashion and placed along travelling routes in the pikas&#x2019; habitat. We illustrate the efficiency of the snare at collecting a large quantity of hair that can then be collected and brought back to the lab. We then demonstrate the use of the DNA IQ system (Promega) to isolate DNA and showcase the utility of this method to amplify commonly used molecular markers including nuclear microsatellites, amplified fragment length polymorphisms (AFLPs), mitochondrial sequences (800bp) as well as a molecular sexing marker. Overall, we demonstrate the utility of this novel noninvasive hair snare as a sampling technique for wildlife population biologists. We anticipate that this approach will be applicable to a variety of small mammals, opening up areas of investigation within natural populations, while minimizing impact to study organisms.

We present a noninvasive sampling approach to efficiently collect hair samples from elusive small mammals, as shown for the American pika. We demonstrate the utility of this method by extracting DNA from sampled hair and amplifying several types of molecular markers commonly used in studies of wildlife ecology and conservation.

שיער פולשנית דגימה טכניקה לקבל DNA באיכות גבוהה מ יונקים קטנים החמקמק

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

Fabrication and Use of MicroEnvironment microArrays (MEArrays)

The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, distinct microenvironments can impose differentiation, migration, and proliferation phenotypes, and on the tissue level the microenvironment processes as complex as morphogenesis and tumorigenesis1. Not only do the cell and molecular contents of microenvironments impact the cells within, but so do the elasticity2 and geometry3 of the tissue. Defined as the sum total of cell-cell, -ECM, and -soluble factor interactions, in addition to physical characteristics, the microenvironment is complex. The phenotypes of cells within a tissue are partially due to their genomic content and partially due to the combinatorial interactions with the microenviroment. A major challenge is to link specific combinations of microenvironmental components with distinctive behaviours.
Here, we present the microenvironment microarray (MEArray) platform for cell-based functional screening of interactions with combinatorial microenvironments4. The method allows for simultaneous control of the molecular composition and the elastic modulus, and combines the use of widely available microarray and micropatterning technologies. MEArray screens require as few as 10,000 cells per array, which facilitates functional studies of rare cell types such as adult progenitor cells. A limitation of the technology is that entire tissue microenvironments cannot be completely recapitulated on MEArrays. However, comparison of responses in the same cell type to numerous related microenvironments, for instance pairwise combinations of ECM proteins that characterize a given tissue, will provide insights into how microenvironmental components elicit tissue-specific functional phenotypes.
MEArrays can be printed using a wide variety of recombinant growth factors, cytokines, and purified ECM proteins, and combinations thereof. The platform is limited only by the availability of specific reagents. MEArrays are amenable to time-lapsed analysis, but most often are used for end point analyses of cellular functions that are measureable with fluorescent probes. For instance, DNA synthesis, apoptosis, acquisition of differentiated states, or production of specific gene products are commonly measured. Briefly, the basic flow of an MEArray experiment is to prepare slides coated with printing substrata and to prepare the master plate of proteins that are to be printed. Then the arrays are printed with a microarray robot, cells are allowed to attach, grow in culture, and then are chemically fixed upon reaching the experimental endpoint. Fluorescent or colorimetric assays, imaged with traditional microscopes or microarray scanners, are used to reveal relevant molecular and cellular phenotypes (Figure 1).

Fabrication and Use of MicroEnvironment microArrays MEArrays

A combinatorial functional screening method for gaining insights into the impacts of the molecular composition of microenvironments on cellular functions is described. The method takes advantage of existing microarray-based technologies to generate arrays of defined combinatorial microenvironments that support cell adhesion and functional analysis.

ייצור ושימוש של microarrays Microenvironment (MEArrays)

The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

דור של תבנית הכרומטין immunoprecipitation איכות גבוהה עבור ה-DNA רצף תפוקה גבוהה (שבב ואילך)

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

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.

מערך השוואתי הגנום הכלאה (Array CGH) לאיתור של הגנום גרסאות מספר עותק

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

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the &#8220;gold standard&#8221; enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

הפקת DNA שיטה היברידית להערכה איכותית וכמותית של הקהילות בקטריאלי מדוגמאות ייצור עופות

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses ...

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH.

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals.

שיטה פשוטה DNA הקרינה<em&gt; באתר</em&gt; הכלאה לכרומוזומים &quot;סתמת

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly ...

Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

Cryo-electron microscopy (cryoEM) entails flash-freezing a thin layer of sample on a support, and then visualizing the sample in its frozen hydrated state by transmission electron microscopy (TEM). This can be achieved with very low quantity of protein and in the buffer of choice, without the use of any stain, which is very useful to determine structure-function correlations of macromolecules. When combined with single-particle image processing, the technique has found widespread usefulness for 3D structural determination of purified macromolecules. 
The protocol presented here explains how to perform cryoEM and examines the causes of most commonly encountered problems for rational troubleshooting; following all these steps should lead to acquisition of high quality cryoEM images. The technique requires access to the electron microscope instrument and to a vitrification device. Knowledge of the 3D reconstruction concepts and software is also needed for computerized image processing. Importantly, high quality results depend on finding the right purification conditions leading to a uniform population of structurally intact macromolecules. 
The ability of cryoEM to visualize macromolecules combined with the versatility of single particle image processing has proven very successful for structural determination of large proteins and macromolecular machines in their near-native state, identification of their multiple components by 3D difference mapping, and creation of pseudo-atomic structures by docking of x-ray structures. The relentless development of cryoEM instrumentation and image processing techniques for the last 30 years has resulted in the possibility to generate de novo 3D reconstructions at atomic resolution level.

This paper describes the cryo-electron microscopy methodology, which is used to obtain high quality microscopic images of macromolecules in their near-native state. The method yields images suitable for further computerized processing using the single particle approach, devised to generate the 3D reconstruction of a macromolecule.

עשה ואל תעשה של מיקרוסקופ אלקטרוני cryo-: פריימר על הכנת דוגמאות וגבוהה איסוף נתונים איכות לשיקום Macromolecular 3D

Cryo-electron microscopy (cryoEM) entails flash-freezing a thin layer of sample on a support, and then visualizing the sample in its frozen hydrated state ...

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.

הגברה, רצף הדור הבא, ואת הגנום מיפוי ה- DNA של אתרי אינטגרצית retroviral

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