我们感谢M.迈尔的意见;克罗地亚科学和艺术学院和柏林 - 勃兰登堡科学院后勤和科学支持科学和马普学会主席创新基金的财政支持。 JMG是支持由美国国家科学基金会国际博士后奖学金（OISE - 0754461）。序列是存放在EBI的核苷酸数据库加入数字：1（Feldhofer 1）穴居人FM865407，尼安德特人2（Feldhofer 2）FM865408，Sidron 1253 FM865409，Vindija 33.25 FM865410，Mezmaiskaya 1 FM865411

使用这种方法是，在研究中报道<a href="http://www.sciencemag.org/cgi/content/full/325/5938/318">的百等。，325（5938）科学</a> 。 <a href="http://www.sciencemag.org/cgi/content/full/325/5938/318">318-321（2009） 。</a> 试剂要求： <ul><li> AmpliTaq黄金DNA聚合酶</li><li> 10X GeneAmp PCR缓冲液II </li><li> MgCl 2的 25MM </li><li> dNTP混合液，每25毫米</li><li> BSA，10毫克毫升水1 </li><li>分子生物学级水</li><li> EB缓冲液（用MinElute PCR纯化试剂盒提供），10毫米的Tris盐酸，pH值8.5 </li><li> MinElute PCR纯化试剂盒</li><li> M - 270链​​霉亲和素磁珠</li><li> 2个绑定和清洗（BW）缓冲液（2 M氯化钠，10毫米，1毫米EDTA，pH值8.0，0.2％吐温20的Tris - CL） </li><li> 1X绑定和清洗（BW）缓冲液（2X BindWash缓冲液稀释水2X） </li><li>热洗（硬件）缓冲液（1X的Taq黄金缓冲区，2.5mm的氯化镁，0.1％吐温20） </li></ul>关于非标准试剂和仪器的制造商信息，请参见后表。 1）图书馆放大<ol><li>这里的DNA为模板，是一个从低拷贝数的DNA中所述的源（Rohland和Hofreiter，2007年，2007年b）和（Maricic和帕博，2009年）编写的454库。要健全整个库，准备下面的PCR混合物： <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 试剂 </td><td> μL，每个反应 </td></tr><tr><td>水</td><td> 45 </td></tr><tr><td> 10X基因AMP PCR缓冲液II </td><td> 10 </td></tr><tr><td>氯化镁2（25MM） </td><td> 10 </td></tr><tr><td> BSA（10毫克/毫升） </td><td> 2 </td></tr><tr><td> dNTPs（各25毫米） </td><td> 1 </td></tr><tr><td> 454 emPCR FWD primer/10uM </td><td> 3 </td></tr><tr><td> 454 emPCR RVS primer/10uM </td><td> 3 </td></tr><tr><td> AmpliTaq黄金DNA聚合酶（5 U /μL） </td><td> 1 </td></tr><tr><td> 454 DNA库模板</td><td> 25 </td></tr><tr><td> 共有 </td><td> 100 </td></tr></tbody></table></li><li>使用以下的PCR 程序，14个循环扩增热循环仪<ol><li> 95 °彗星12分钟</li><li> 95 °彗星30秒</li><li> 60 ° C（或所需的退火温度）1分钟</li><li> 72℃1分钟</li><li> 2（× 13） </li><li> 72 °彗星5分钟</li><li> 10 °彗星∞ </li></ol></li><li>净化根据制造商的指示超过Qiagen公司MinElute离心柱的反应。洗脱50μL缓冲液EB。 </li><li>量化纯化扩增产物以及定量PCR（Meyer等人，2007年）的非放大库等分。如果扩增产物已超过1 × 10 12份，每UL，减少在下面的引物延伸的模板量，以确保不超过2 × 10 12副本添加到引物延伸反应。 </li></ol> 2）引物延伸<ol><li>准备一个反应所需数量的主结构。 <table border="0" cellspacing="0" cellpadding="0"><tbody><tr><td> 试剂 </td><td> μL，每个反应 </td></tr><tr><td>水</td><td> 45 </td></tr><tr><td> 10X基因AMP PCR缓冲液II </td><td> 10 </td></tr><tr><td>氯化镁2（25MM） </td><td> 10 </td></tr><tr><td> BSA（10毫克/毫升） </td><td> 2 </td></tr><tr><td> dNTPs（各25毫米） </td><td> 1 </td></tr><tr><td> 454 emPCR FWD primer/10uM </td><td> 3 </td></tr><tr><td> 454 emPCR RVS primer/10uM </td><td> 3 </td></tr><tr><td> AmpliTaq黄金DNA聚合酶（5 U /μL） </td><td> 1 </td></tr><tr><td> 454 DNA库模板</td><td> 25 </td></tr><tr><td> 共有 </td><td> 100 </td></tr></tbody></table></li><li> 单引物延伸反应的热循环运行以下程序： <ol><li> 95 °彗星12分钟</li><li> 60 ° C（或您的PEC的引物退火温度不同）1分钟</li><li> 72℃5分钟</li><li> 72 °彗星永远！ 关键的一步延伸反应后保持在72 ° C 。然后直接移液器150ul的PBI或PB缓冲液从加热块中取出之前QIAGEN公司MinElute管道进入净化。这一点很重要，以避免非特异性引物退火和捕获的混合物冷却。 </li></ol></li><li>净化超过Qiagen公司MinElute离心柱的反应，根据制造商的指示。在50μL的EB洗脱。 </li></ol> 3）延伸产品捕捉<ol><li>悬浮原液的M - 270珠震荡。取出25μL珠每个样品悬挂。珠两次洗500ul 2X BW缓冲和重悬在每个样品25μL2xBW缓冲区。 </li><li>从步骤2.3中添加25μL洗脱液25μL 步骤 3.1珠悬挂。然后旋转混合1在室温下5分钟。 建议 ：保持其余的步骤定量PCR定量2.3 5洗脱液UL。 </li><li>整个混合物转移到一个新的1.5毫升管（这有助于减少非目标库片段结转）。颗粒的上清液用磁性粒子收集器（MPC），弃上清。 </li><li>洗净用500μl1xBW缓冲区（许多清洗有助于消除尽可能多的背景）的5倍。在最后的洗涤步骤，降速简要上清液中删除的痕迹。 </li><li>加入500μL1X热洗（硬件）缓冲区。摇2分钟，在65 ° C（或以上PEC底漆TM 5C）在热块。快速去除上清，在此之后，，以尽量减少降温。 （这一步是删除仍然PEC的引物，但实际上并不在延伸扩展的背景片段）。除去上清的最后的痕迹。 </li><li>在30μL的EB缓冲液重悬磁珠颗粒。整个混合物转移到一个新的1.5毫升管（这有助于减少非目标库片段结转）。在95℃孵育3分钟，在热循环仪进行洗脱。放置在MPC和去除上清，照顾身后留下的珠。 </li></ol> 4）Capture产品放大<ol><li>准备一个样品所需数量的PCR扩增预混。 <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 试剂 </td><td> μL，每个反应 </td></tr><tr><td>水</td><td> 45 </td></tr><tr><td> 10X基因AMP PCR缓冲液II </td><td> 10 </td></tr><tr><td>氯化镁2（25MM） </td><td> 10 </td></tr><tr><td> BSA（10毫克/毫升） </td><td> 2 </td></tr><tr><td> dNTPs（各25毫米） </td><td> 1 </td></tr><tr><td> 454 emPCR FWD primer/10uM </td><td> 3 </td></tr><tr><td> 454 emPCR RVS primer/10uM </td><td> 3 </td></tr><tr><td> AmpliTaq黄金DNA聚合酶（5 U /μL） </td><td> 1 </td></tr><tr><td>洗脱捕捉产品</td><td> 25 </td></tr><tr><td> 共有 </td><td> 100 </td></tr></tbody></table></li><li>在14个循环扩增热循环仪使用以下的PCR程序： <ol><li> 95 °彗星12分钟</li><li> 95 °彗星30秒</li><li> 60 ° C（或所需的退火温度）1分钟</li><li> 72℃1分钟</li><li> 2（× 13） </li><li> 72 °彗星5分钟</li><li> 10 °彗星∞ </li></ol></li><li>超过Qiagen公司MinElute硅离心柱净化，根据制造商的指示反应。在50μL的EB缓冲液洗脱。该产品可现在使用的捕捉第二轮，开始在步骤2.1中，或直接进入454乳液PCR协议，进行测序。 </li></ol>代表性的成果： 强烈建议时与PEC协议开始执行第一个捕获反应与建议正常少量的阳性对照目标序列“（例如〜100bp的PCR产物 - 1皮克很容易不够）混合金额扩增454库模板（见第2.1步），并捕获与一个单一的PEC捕捉到的阳性对照产品设计引物进行。如果执行这种控制的反应是，qPCR的，既有积极的控制和454适配器特异性引物对可用于量化“控制目标”的量与纯化的引物延伸反应（1.3），引物延伸产品（ 2.3的背景）和洗脱磁珠捕获产品（3.6）。这样，无论是背景去除（“特殊性”），并在您的实验条件下的效率目标恢复（“敏感性”）的成效，可以直接测量。 一个成功的PEC协议通常具有以下属性 - 灵敏度（保留通过协议控制目标量测）： 的洗脱磁珠捕获产品的总量控制目标（3.6）=〜总额的 1-20％，在纯化的引物延伸反应（2.3）。 背景 （454 emPCR引物测）： （3.6）洗脱磁珠捕获产品总额的背景&lt;纯化的引物延伸反应（2.3）总额的 0.01 ％。 如果有小于1％的最终产品的剩余目标，引物延伸步骤可能已不成功，并且要追究。 如果有比0.01％的背景留在最终产品，洗涤步骤可能已被错误地执行，并应进行调查。

<ol>
	<li>Briggs, A. W., Good, J. M., Green, R. E., Krause, J., Maricic, T., Stenzel, U., Lalueza-Fox, C., Rudan, P., Brajkovic, D., Kucan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+retrieval+and+analysis+of+five+Neandertal+mtDNA+genomes.">Targeted retrieval and analysis of five Neandertal mtDNA genomes.</a> Science. 325 (5938), 318-321 (2009).</li><li>Rohland, N., Hofreiter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+and+optimization+of+ancient+DNA+extraction.">Comparison and optimization of ancient DNA extraction.</a> Biotechniques. 42 (3), 343-352 (2007).</li><li>Rohland, N., Hofreiter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ancient+DNA+extraction+from+bones+and+teeth.">Ancient DNA extraction from bones and teeth.</a> Nat Protoc. 2 (7), 1756-1762 (2007).</li><li>Maricic, T., P&#228;&#228;bo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+454+sequencing+library+preparation+from+small+amounts+of+DNA+permits+sequence+determination+of+both+DNA+strands.">Optimization of 454 sequencing library preparation from small amounts of DNA permits sequence determination of both DNA strands.</a> Biotechniques. 46 (1), 51-57 (2009).</li><li>Meyer, M., AW, B. r. i. g. g. s., Maricic, T., Hober, B., Hoffner, B., Krause, J., Weihmann, A., Paabo, S., Hofreiter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+micrograms+to+picograms:+quantitative+PCR+reduces+the+material+demands+of+high-throughput+sequencing.">From micrograms to picograms: quantitative PCR reduces the material demands of high-throughput sequencing.</a> Nucleic Acids Res. 36 (1), e5-e5 (2007).</li></ol>

The authors have nothing to disclose.

佩奇方法简单，快速，敏感性和特异性。因此，我们设想的作者的多个应用程序古代DNA外，如捕获的小分子RNA的RNA库的片段，审讯在样本或捕获16S（或其他位点）的多样性从宏基因组样品的结构变化。有一点要提的是，捕获灵敏度的PEC引物的多重捕获反应增加。因此，PEC是不是非常适合非常大（例如megabase或以上）的捕捉区域捕捉，但捕捉小目标的地区，许多人在快速时尚甚至SNP的立场是非常适合。

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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>
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<tr>
<td>AmpliTaq Gold polymerase with GeneAmp PCR buffer II and MgCl2</td>
<td>&nbsp;</td>
<td>Applied Biosystems</td>
<td>N8080241</td>
<td>&nbsp;</td>
<tr>
<td>QIAGEN MinElute PCR purification kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>28004</td>
<td>&nbsp;</td>
<tr>
<td>M-270 streptavidin beads</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>653.05</td>
<td>&nbsp;</td>
<tr>
<td>Tween-20</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P2287</td>
<td>&nbsp;</td>
<tr>
<td>DynaMag-2 magnetic particle separator</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>120.02D</td>
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primer extension capture: targeted sequence retrieval from heavily degraded dna sources

我们提出了有针对性的DNA DNA的来源，其中严重退化和污染的微生物DNA序列检索的方法，是典型的古代骨骼。该方法大大降低了样品的破坏和测序的要求相对于直接PCR或鸟枪测序方法。我们用这种方法来重建完整的线粒体DNA（mtDNA）的五个尼安德特人的基因组，来自全国各地其地理范围。后期尼安德特人线粒体DNA的遗传多样性比当代现代人类的大约三倍。连同线粒体蛋白质进化分析，这些数据表明，尼安德特人的长期有效的人口规模小于现代人类和现存的类人猿。

Watch this Scientific Journal Video about Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources at JoVE.com

Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources

我们提出了有针对性的古DNA序列检索，这是我们用来重建完整的线粒体基因组的5个尼安德特人的个人方法。现今人类的这些序列的比较表明，尼安德特人有一个长期的低有效的人口规模。

引物延伸捕获：从严重退化的DNA来源有针对性的顺序检索

We present a method of targeted DNA sequence retrieval from DNA sources which are heavily degraded and contaminated with microbial DNA, as is typical of ...

primer-extension-capture-targeted-sequence-retrieval-from-heavily

Research

JoVE Journal

Biology

20.0K 次观看.  Max-Planck Institute for Evolutionary Anthropology, Leipzig. 我们提出了有针对性的古DNA序列检索，这是我们用来重建完整的线粒体基因组的5个尼安德特人的个人方法。现今人类的这些序列的比较表明，尼安德特人有一个长期的低有效的人口规模。

视频: Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources

Retrieval of Mouse Oocytes

To date, only a few studies have reported successful manipulations of Peromyscus embryogenesis or reproductive biology.  Together with the Peromyscus Genetic Stock Center (http://stkctr.biol.sc.edu), we are characterizing the salient differences needed to develop this system.  A primary goal has been to optimize oocyte/early embryo retrieval.

This protocol illustrates the technique for extracting oocytes or early-stage fertilized embryos from the oviduct of mice. The ability to identify the infindibulum and insert a blunt end needle into it is essential to correctly performing the procedure.

检索小鼠卵母细胞

To date, only a few studies have reported successful manipulations of Peromyscus embryogenesis or reproductive biology.  Together with the Peromyscus ...

科研

生物学

Proboscis Extension Response (PER) Assay in Drosophila

Proboscis extension response (PER) is a taste behavior assay that has been used in flies as well as in honeybees.
On the surface of the fly's mouth (labellum), there are hair-like structures called sensilla which houses taste neurons. When an attractive substance makes contact to the labellum, the fly extends its proboscis to consume the material. Proboscis Extension Response (PER) assay measures this taste behavior response, and it is a useful method to learn about food preferences in a single fly. Solutions of various sugars, such as sucrose, glucose and fructose, are very attractive to the fly. The effect of aversive substances can also be tested as reduction of PER when mixed in a sweet solution.Despite the simplicity of the basic procedure, there are many things that can prevent it from working. One of the factors that requires attention is the fly's responsive state. The required starvation time to bring the fly to the proper responsive state varies drastically from 36 to 72 hours. We established a series of controls to evaluate the fly's state and which allows screening out of non-responsive or hyper-responsive individual animals. Another important factor is the impact level and the position of the contact to the labellum, which would be difficult to describe by words. This video presentation demonstrates all these together with several other improvements that would increase the reproducibility of this method.

Proboscis Extension Response PER Assay in Drosophila

Proboscis extension response or PER is a taste behavior assay that has been used in flies as well as in honeybees. When the proboscis makes contact with an attractive substance, the fly extends its proboscis to consume the substance. Solutions of various sugars are very attractive to the fly.

长鼻扩展响应（PER）的含量在果蝇

Proboscis extension response (PER) is a taste behavior assay that has been used in flies as well as in honeybees.
On the surface of the fly's mouth ...

Isolation of Genomic DNA from Mouse Tails

从鼠尾基因组DNA提取

Primer-Free Aptamer Selection Using A Random DNA Library

Aptamers are highly structured oligonucleotides (DNA or RNA) that can bind to targets with affinities comparable to antibodies 1. They are identified through an in vitro selection process called Systematic Evolution of Ligands by EXponential enrichment (SELEX) to recognize a wide variety of targets, from small molecules to proteins and other macromolecules 2-4. Aptamers have properties that are well suited for in vivo diagnostic and/or therapeutic applications: Besides good specificity and affinity, they are easily synthesized, survive more rigorous processing conditions, they are poorly immunogenic, and their relatively small size can result in facile penetration of tissues.
Aptamers that are identified through the standard SELEX process usually comprise ~80 nucleotides (nt), since they are typically selected from nucleic acid libraries with ~40 nt long randomized regions plus fixed primer sites of ~20 nt on each side. The fixed primer sequences thus can comprise nearly ~50% of the library sequences, and therefore may positively or negatively compromise identification of aptamers in the selection process 3, although bioinformatics approaches suggest that the fixed sequences do not contribute significantly to aptamer structure after selection 5. To address these potential problems, primer sequences have been blocked by complementary oligonucleotides or switched to different sequences midway during the rounds of SELEX 6, or they have been trimmed to 6-9 nt 7, 8. Wen and Gray 9 designed a primer-free genomic SELEX method, in which the primer sequences were completely removed from the library before selection and were then regenerated to allow amplification of the selected genomic fragments. However, to employ the technique, a unique genomic library has to be constructed, which possesses limited diversity, and regeneration after rounds of selection relies on a linear reamplification step. Alternatively, efforts to circumvent problems caused by fixed primer sequences using high efficiency partitioning are met with problems regarding PCR amplification 10.
We have developed a primer-free (PF) selection method that significantly simplifies SELEX procedures and effectively eliminates primer-interference problems 11, 12. The protocols work in a straightforward manner. The central random region of the library is purified without extraneous flanking sequences and is bound to a suitable target (for example to a purified protein or complex mixtures such as cell lines). Then the bound sequences are obtained, reunited with flanking sequences, and re-amplified to generate selected sub-libraries. As an example, here we selected aptamers to S100B, a protein marker for melanoma. Binding assays showed Kd s in the 10-7 - 10-8 M range after a few rounds of selection, and we demonstrate that the aptamers function effectively in a sandwich binding format.

SELEX protocols comprise multiple rounds of selection, each of which require regeneration of bound ligands, which in turn require fixed primer sequences flanking the random library regions. These fixed primer sequences can interfere with the selection process (false positives and negatives). Here we present a primer-free protocol.

引物的适体的选择，使用一个随机DNA文库

Aptamers are highly structured oligonucleotides (DNA or RNA) that can bind to targets with affinities comparable to antibodies 1. They are identified ...

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.

磁共振成像基因 - 公共资源为研究基因组DNA序列内模式

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

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

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

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

从基因组DNA选择性捕获的5  - 羟甲基

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.

单工程的RNA转录物的活细胞实时成像采用比例式双分子信标

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

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

Fluorescence based primer extension (FPE) is a molecular method to determine transcriptional starting points or processing sites of RNA molecules. This is achieved by reverse transcription of the RNA of interest using specific fluorescently labeled primers and subsequent analysis of the resulting cDNA fragments by denaturing polyacrylamide gel electrophoresis. Simultaneously, a traditional Sanger sequencing reaction is run on the gel to map the ends of the cDNA fragments to their exact corresponding bases. In contrast to 5&#39;-RACE (Rapid Amplification of cDNA Ends), where the product must be cloned and multiple candidates sequenced, the bulk of cDNA fragments generated by primer extension can be simultaneously detected in one gel run. In addition, the whole procedure (from reverse transcription to final analysis of the results) can be completed in one working day. By using fluorescently labeled primers, the use of hazardous radioactive isotope labeled reagents can be avoided and processing times are reduced as products can be detected during the electrophoresis procedure.
In the following protocol, we describe an in vivo fluorescent primer extension method to reliably and rapidly detect the 5&#39; ends of RNAs to deduce transcriptional starting points and RNA processing sites (e.g., by toxin-antitoxin system components) in S. aureus, E. coli and other bacteria.

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

We here describe a fluorescence based primer extension method to determine transcriptional starting points from bacterial transcripts and RNA processing in vivo using an automated gel sequencer.

基于荧光引物延伸法确定转录起点和核糖核酸酶的切割位点<em&gt;在体内</em

Fluorescence based primer extension (FPE) is a molecular method to determine transcriptional starting points or processing sites of RNA molecules. This is ...

Targeted DNA Methylation Analysis by Next-generation Sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

有针对性的DNA甲基化分析通过新一代测序

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

RNA Isolation from Cell Specific Subpopulations Using Laser-capture Microdissection Combined with Rapid Immunolabeling

Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires precise identification of cells subpopulations from a heterogeneous tissue. Identification of cells of interest for LCM is usually based on morphological criteria or on fluorescent protein reporters. The combination of LCM and rapid immunolabeling offers an alternative and efficient means to visualize specific cell types and to isolate them from surrounding tissue. High-quality RNA can then be extracted from a pure cell population and further processed for downstream applications, including RNA-sequencing, microarray or qRT-PCR. This approach has been previously performed and briefly described in few publications. The goal of this article is to illustrate how to perform rapid immunolabeling of a cell population while keeping RNA integrity, and how to isolate these specific cells using LCM. Herein, we illustrated this multi-step procedure by immunolabeling and capturing dopaminergic cells in brain tissue from one-day-old mice. We highlight key critical steps that deserve special consideration. This protocol can be adapted to a variety of tissues and cells of interest. Researchers from different fields will likely benefit from the demonstration of this approach.

Gene expression analysis of a subset of cells in a specific tissue represents a major challenge. This article describes how to isolate high-quality total RNA from a specific cell population by combining a rapid immunolabeling method with laser capture microdissection.

从细胞亚群特异性RNA提取使用激光捕获显微切割加上快速的免疫标记

Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires ...