Эта работа была поддержана коалицией Грузии рака грант-R9028.

<ol>
	<li>Storici, F., Bebenek, K., Kunkel, T. A., Gordenin, D. A., Resnick, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-templated+DNA+repair.">RNA-templated DNA repair.</a> Nature. 447, 338-3341 (2007).</li><li>Storici, F., Resnick, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+delitto+perfetto+approach+to+in+vivo+site-directed+mutagenesis+and+chromosome+rearrangements+with+synthetic+oligonucleotides+in+yeast.">The delitto perfetto approach to in vivo site-directed mutagenesis and chromosome rearrangements with synthetic oligonucleotides in yeast.</a> Methods Enzymol. 409, 329-345 (2006).</li><li>Storici, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-mediated+DNA+modifications+and+RNA-templated+DNA+repair.">RNA-mediated DNA modifications and RNA-templated DNA repair.</a> Curr Opin Mol Ther. 10, 224-230 (2008).</li><li>Cerritelli, S. M., Crouch, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribonuclease+H:+the+enzymes+in+eukaryotes.">Ribonuclease H: the enzymes in eukaryotes.</a> FEBS J. 276, 1494-1505 (2009).</li></ol>

Видео-продукции для этой статьи была организована Thermo Fisher Scientific, которая производит реактивы и инструмент, используемые в настоящей публикации.

Тот факт, что РНК может передавать генетическую информацию непосредственно геномной ДНК в клетках было обнаружено использование использование синтетических РНК-содержащих олигонуклеотидов (Dharmacon синтезированных олигонуклеотидов) 1. Это было доказательством принципа, что клетки могут использовать РНК-содержащих молекул или РНК-последовательностей, только в качестве шаблонов для синтеза ДНК. Использовать РНК-содержащих олигонуклеотидов не только привело к демонстрации того, что генетическая информация может быть передана непосредственно от РНК к геномной ДНК без необходимости обратной транскрипции ДНК-копию промежуточного, но и доказательство того, что РНК может быть использован в качестве шаблона в гомологичных ремонт повреждений ДНК 1,3. Олигонуклеотидов могут быть изготовлены из РНК-содержащие или содержащие только одно рибонуклеотид встроенные в ДНК последовательности, как в примере, мы представили здесь. РНК-содержащих олиго имеет один встроенный рибонуклеотид направленных на корректировку нонсенс-мутации в геномной дефектных trp5 аллеля. Передачи генетической информации от рибонуклеотид в геномной ДНК проявляется изменением фенотипа (клетка, растущих на среде отсутствует триптофан) из клеток-мишеней. Частота гена коррекции, полученные с РНК-содержащих олиго измеряется и по сравнению с контролем ДНК-только олиго. Выполняя этот анализ генов коррекции в разных слоев клетки, где мы мутировать различных генов дрожжей, мы можем обнаружить, какой фактор / с затрагивающих таргетинг по РНК-содержащих олигонуклеотидов. Таким образом, мы можем выявить механизмы, как клетки регулируют стабильность РНК / ДНК гибридов. Просто проектирования различных вариантов РНК-содержащих олигонуклеотидов можно определить вероятность для конкретного тракта РНК в качестве шаблона в ДНК, модификации и мы можем определить устойчивость специфические последовательности РНК, встроенные в ДНК. Более того, мы можем определить, что является предпочтительным в естественных субстратов для факторов, взаимодействующих с РНК / ДНК гибридов. Хотя некоторые лабораторные исследования, в основном используя короткие РНК-содержащих олигонуклеотидов, были проведены, чтобы охарактеризовать функции факторов, которые могут распознать РНК гибрид с ДНК, например, ферментов РНКазы H 4, в естественных функций РНКазы H, а как личность других белков, которые могут повлиять РНК / ДНК гибридных стабильности остаются по большей части неизвестна. Возможность использовать РНК-содержащих олигонуклеотидов значительной длины (от 50 до 80-меров) и оптимального качества (например, Thermo Scientific Dharmacon РНК-содержащих олигонуклеотидов) имеет открыть путь к изучению широкого спектра молекулярных процессов непосредственно в естественных условиях в Клетки интерес. Как показано в этом видео, преобразование с помощью РНК-содержащих олигонуклеотидов требует по существу, лишь несколько дополнительных шагов по сравнению с преобразованием при помощи ДНК-молекул, в целях предотвращения деградации РНКазы. Таким образом, преобразование с помощью РНК-содержащих олигонуклеотидов не ограничивается дрожжей системы, но может быть применен к любой организм или тип клеток, где трансформация ДНК-олигонуклеотиды владеет. В заключение ориентации клеток РНК-содержащих олигонуклеотидов дает возможность получать РНК / ДНК гибридов и рибонуклеотидов встроенные в ДНК в естественных условиях в клетках. Стабильность, функции и последствия этих порожденных в естественных условиях РНК / ДНК гибриды могут быть проанализированы и характеризуется, потенциально раскрытие неизвестных механизмов репарации ДНК и выявление новых стратегий для генного таргетинга.

А. Преобразование реагентов и средств массовой информации (с изменениями от Storici и Резник, 2006 <ol><li> YPD (дрожжевой пептон декстроза): на 1 л, 10 г дрожжевого экстракта, 20 г соевого пептона, 20 г декстрозы. Добавьте 15 г агара, чтобы сделать YPD твердых средах. Автоклав перед использованием. Хранить при комнатной температуре. </li><li> Решение 1: 0,1 М ацетата лития. Подготовка непосредственно перед преобразованием. Раствор 1 рабочего раствора, таким образом, она готова прямо из порошка. Нет маточного раствора производится. Хранить при комнатной температуре. LiAc увеличивает стене дрожжевых клеток проницаемости для ДНК. </li><li> Решение 2: 0,1 М ацетата лития и 50% полиэтиленгликоля 4000. Кроме того, решение 2 является рабочим раствором и это сделано непосредственно из порошка. Нет маточный раствор готов. Хранить при комнатной температуре. PEG месторождений олигонуклеотидов на дрожжевых клеток. </li><li> РНК-содержащих олигонуклеотидов (Thermo Scientific Dharmacon), 50-80-меров, обессоленной, снимают защиту и не очищается. Ресуспендируют до 250 пмоль / мкл. Хранить при температуре -80 ° C. </li><li> ДНК-только олигонуклеотидов, 50-80-меров, обессоленной и неочищенные. Ресуспендируют до 50 пмоль / мкл. Хранить при -20 ° C. </li><li> SC-Trp (Синтетический полный средах без триптофана) твердых средах. </li><li> 0,5 см в диаметре стеклянные бусы, стерилизовать в автоклаве. </li><li> РНКазы матча РНКазы обеззараживание раствором. </li><li> ДНКазы / РНКазы бесплатно, стерильные пробирки центрифуги. </li><li> ДНКазы / РНКазы свободной, стерильные конические пробирки. </li><li> ДНКазы / РНКазы свободной, стерильные наконечники аэрозоля с ZAP: 1-200 мл, 100-1000 мл. </li></ol> Б. колонии PCR материалов. <ol><li> ДНК-праймеров, обессоленной и неочищенные. Растворяют в стерильной воде до 50 пмоль / мкл. Хранить при -20 ° C. </li><li> Taq-ДНК-полимераза, буфер, дНТФ. </li><li> ПЦР пробирки. </li></ol> С. ПЦР очистки. <ol><li> ПЦР очистки комплект. </li></ol> Д. гель-электрофореза. <ol><li> Агарозы. </li><li> 1 х КЭ работает буфера (45 мМ Трис-борат и 1 мМ этилендиамина тетраацетат) разводят с 10-кратным TBE. </li><li> Prestained маркер молекулярного веса. </li><li> ДНК загрузки красителя. </li></ol> E. Ограничение пищеварения. <ol><li> Ограничение ферменты, 10x буферов, BSA. </li></ol> Ф. Щелочные лечения РНК-содержащих олиго. <ol><li> 1 М раствора NaOH. </li><li> 1,2 М раствора соляной кислоты. </li><li> 1 М Трис-HCl, рН 7,4 раствором. </li></ol>

generation of rna/dna hybrids in genomic dna by transformation using rna-containing oligonucleotides

Синтетические короткие нуклеиновых кислот полимеры, олигонуклеотиды (олигонуклеотиды), являются наиболее функциональных и распространенных инструментов молекулярной биологии. Олигонуклеотидов могут быть произведены содержать любую ДНК или РНК, последовательность и могут быть готовы включить широкий спектр базовых и сахар модификаций. Более того, олигонуклеотиды могут быть разработаны для имитации конкретных нуклеиновые кислоты изменений и, таким образом, может служить важным инструментом для исследования последствий повреждения ДНК и механизмов ремонта. Мы обнаружили, что Thermo Scientific Dharmacon РНК-содержащих олигонуклеотидов с длиной от 50 до 80 нуклеотидов может быть особенно подходящим для изучения, в естественных условиях, функции и последствия хромосомных РНК / ДНК гибридов и рибонуклеотидов встроенные в ДНК. РНК / ДНК гибридов легко образуются во время репликации ДНК, ремонт и транскрипции, однако, очень мало известно о стабильности РНК / ДНК гибридов в клетках и в какой степени эти гибриды могут повлиять на генетической целостности клеток. РНК-содержащих олигонуклеотидов, следовательно, представляют собой идеальный вектор ввести рибонуклеотидов в хромосомной ДНК и создания РНК / ДНК гибридов выбранной длины и базового состава. Здесь мы приводим протокол для включения рибонуклеотидов в геном эукариотической дрожжей модели системы / Saccharomyces CEREVISIAE /. Тем не менее, наша лаборатория использовала Thermo Scientific Dharmacon РНК-содержащих олигонуклеотидов для создания РНК / ДНК гибридов на хромосомном уровне в различных клеточных систем, от бактерий до клеток человека.

Watch this Scientific Journal Video about Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides at JoVE.com

Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

Эта работа показывает, как форма РНК / ДНК гибридной на хромосомном уровне и выявить передачи генетической информации от РНК к геномной ДНК в клетках дрожжей.

Генерация РНК / ДНК гибридов в геномной ДНК с помощью преобразования с помощью РНК-содержащих олигонуклеотидов

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be ...

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Research

JoVE Journal

Biology

26.7K Views.  Georgia Institute of Technology. Эта работа показывает, как форма РНК / ДНК гибридной на хромосомном уровне и выявить передачи генетической информации от РНК к геномной ДНК в клетках дрожжей.

Video: Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

Isolation of Genomic DNA from Mouse Tails

Выделение геномной ДНК из хвостов мышей

Transformation of Plasmid DNA into E. coli Using the Heat Shock Method

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign plasmid or ligation product into bacteria. This video protocol describes the traditional method of transformation using commercially available chemically competent bacteria from Genlantis. After a short incubation in ice, a mixture of chemically competent bacteria and DNA is placed at 42&deg;C for 45 seconds (heat shock) and then placed back in ice. SOC media is added and the transformed cells are incubated at 37&deg;C for 30 min with agitation. To be assured of isolating colonies irrespective of transformation efficiency, two quantities of transformed bacteria are plated. This traditional protocol can be used successfully to transform most commercially available competent bacteria. The turbocells from Genlantis can also be used in a novel 3-minute transformation protocol, described in the instruction manual.

Преобразование плазмидной ДНК в E.coli, с использованием метода теплового шока

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign ...

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.

Добыча высокомолекулярных ДНК Геномная Вес от почвы и осадков

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

Generation of Transgenic C. elegans by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.

Generation of Transgenic C. elegans by Biolistic Transformation

Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.

Поколение трансгенных С. Элеганс По Biolistic преобразования

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

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.

Геномная МРТ - общественный ресурс для изучения последовательности Шаблоны в геномной ДНК

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

Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.

This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.

Выделение и характеристика РНК-содержащих экзосом

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of ...

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic redundancy affecting simple genetics in higher plants. The novel model organism Ostreococcus tauri is the smallest free-living eukaryote known to date, and possesses a greatly reduced genome size and cellular complexity1,2, manifested by the presence of just one of most organelles (mitochondrion, chloroplast, golgi stack) per cell, and a genome containing only ~8000 genes. Furthermore, the combination of unicellularity and easy culture provides a platform amenable to chemical biology approaches. Recently, Ostreococcus has been successfully employed to study basic mechanisms underlying circadian timekeeping3-6. Results from this model organism have impacted not only plant science, but also mammalian biology7. This example highlights how rapid experimentation in a simple eukaryote from the green lineage can accelerate research in more complex organisms by generating testable hypotheses using methods technically feasible only in this background of reduced complexity. Knowledge of a genome and the possibility to modify genes are essential tools in any model species. Genomic1, Transcriptomic8, and Proteomic9 information for this species is freely available, whereas the previously reported methods6,10 to genetically transform Ostreococcus are known to few laboratories worldwide.
In this article, the experimental methods to genetically transform this novel model organism with an overexpression construct by means of electroporation are outlined in detail, as well as the method of inclusion of transformed cells in low percentage agarose to allow selection of transformed lines originating from a single transformed cell. Following the successful application of Ostreococcus to circadian research, growing interest in Ostreococcus can be expected from diverse research areas within and outside plant sciences, including biotechnological areas. Researchers from a broad range of biological and medical sciences that work on conserved biochemical pathways may consider pursuing research in Ostreococcus, free from the genomic and organismal complexity of larger model species.

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

This article describes genetic transformation of the unicellular marine alga Ostreococcus tauri by electroporation. This eukaryotic organism is an effective model platform for higher plants, possesing greatly reduced genomic and cellular complexity and being readily amenable to both cell culture and chemical biology.

Геномная Трансформация Picoeukaryote<em&gt; Ostreococcus Тельца</em

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic ...

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.

Селективный захват 5-hydroxymethylcytosine из геномной ДНК

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 ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

В режиме реального времени изображений Единого Engineered РНК-транскриптов в живых клетках, используя Логометрический Бимолекулярные маяки

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific eukaryotic genome engineering. CRISPR/Cas9 is an inexpensive, facile, and efficient genome editing tool that allows genetic perturbation of genes and genetic elements. Here we present a simple methodology for CRISPR design, cloning, and delivery for the production of genomic deletions. In addition, we describe techniques for deletion, identification, and characterization. This strategy relies on cellular delivery of a pair of chimeric single guide RNAs (sgRNAs) to create two double strand breaks (DSBs) at a locus in order to delete the intervening DNA segment by non-homologous end joining (NHEJ) repair. Deletions have potential advantages as compared to single-site small indels given the efficiency of biallelic modification, ease of rapid identification by PCR, predictability of loss-of-function, and utility for the study of non-coding elements. This approach can be used for efficient loss-of-function studies of genes and genetic elements in mammalian cell lines.

CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9.

Генерация геномных делеций в линии клеток млекопитающих, через CRISPR / Cas9

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific ...