Hırvat Bilimler ve Sanatlar Akademisi ve Berlin-Brandenburg lojistik ve bilimsel destek Bilimler Akademisi; ve mali destek için Max-Planck-Society Cumhurbaşkanlığı İnovasyon Fonu Biz M. Meyer tavsiye için teşekkür ederiz. JMG bir NSF uluslararası doktora sonrası burs (OISE 0.754.461) tarafından desteklenmiştir. Neandertal 1 (Feldhofer 1) FM865407, Neandertal 2 (Feldhofer 2) FM865408, Sidron 1253 FM865409 Vindija 33,25 FM865410 Mezmaiskaya 1 FM865411: Diziler üyelik numaraları ile EBI nükleotid veritabanı yatırılır

Bu yöntem bildirilen araştırmada kullanılan <a href="http://www.sciencemag.org/cgi/content/full/325/5938/318">Briggs ve ark, Bilim 325 (5938). 318-321 (2009)</a> . Reaktifler gereklidir: <ul><li> AmpliTaq Gold DNA Polimeraz </li><li> 10x GeneAmp PCR Buffer II </li><li> MgCl 2 25mm </li><li> dNTP karışımı, her biri 25 mM </li><li> BSA, 10 mg ml-1 su </li><li> Moleküler biyoloji sınıf su </li><li> EB tamponu (MinElute PCR Arıtma kiti ile birlikte); 10 mM Tris HCl, pH 8.5 </li><li> MinElute PCR Arıtma Kiti </li><li> M-270 streptavidin Dynabeads </li><li> 2x Ciltleme ve Yıkama (BW) buffer (2 M NaCl, 10 mM Tris-Cl, 1 mM EDTA, pH 8.0,% 0.2 Tween 20) </li><li> 1x Ciltleme ve Wash (BW) tampon (2x BindWash tampon su 2X seyreltilmiş) </li><li> Sıcak Wash (HW) tamponu (2.5mm MgCl, 1X Taq Gold tampon,% 0.1 Tween 20) </li></ul> Daha sonra standart olmayan reaktiflerin ve ekipmanlarla ilgili olarak üretici bilgi için tabloya bakınız. 1) Kütüphane amplifikasyon <ol><li> Burada DNA örneğinin, 454 kütüphane düşük bir kopya sayısı DNA olarak açıklanan kaynak (Rohland ve Hofreiter, 2007a, 2007b) ve (Maricic ve Pääbo, 2009) hazırlanan bir. Bütün kütüphane yükseltmek için, aşağıdaki PCR karışımı hazırlayın: <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Reaktif </td><td> her reaksiyon için ul </td></tr><tr><td> Su </td><td> 45 </td></tr><tr><td> 10X Gen Amp PCR tampon II. </td><td> 10 </td></tr><tr><td> MgCl 2 (25mm) </td><td> 10 </td></tr><tr><td> BSA (10 mg / ml) </td><td> 2 </td></tr><tr><td> dNTP (her biri 25 mM) </td><td> 1 </td></tr><tr><td> 454 emPCR ileri primer/10uM </td><td> 3 </td></tr><tr><td> 454 emPCR rvs primer/10uM </td><td> 3 </td></tr><tr><td> AmpliTaq Gold DNA polimeraz (5 U / ml) </td><td> 1 </td></tr><tr><td> 454 DNA kütüphane şablon </td><td> 25 </td></tr><tr><td> Toplam </td><td> 100 </td></tr></tbody></table></li><li> 14 döngüleri amplifikasyonu için bir Thermo cycler aşağıdaki PCR programı kullanın. <ol><li> 95 ° C 12min </li><li> 95 ° C 30s </li><li> 60 ° C (veya istenilen tavlama sıcaklığı) 1 dk. </li><li> 72 ° C 1 dakika </li><li> 2 (x 13) </li><li> 72 ° C 5min </li><li> 10 ° C ∞ </li></ol></li><li> Üreticinin talimatlarına göre bir Qiagen MinElute spin kolon üzerinde reaksiyon arındırın. 50 ul tampon EB Zehir. </li><li> Saflaştırılmış amplifiye ürünün yanı sıra, qPCR (Meyer ve ark, 2007) ile Yükseltilmemiş kütüphane bir kısım sayısal olmalıdır. Amplifiye ürün ul başına en fazla 1 X 10 12 kopya varsa, en fazla 2 X 10 12 kopya Primer Extension reaksiyon olduğunu sağlamak için aşağıdaki Primer Eklenti adım şablon miktarını azaltmak. </li></ol> 2) Primer Uzantısı <ol><li> Gerekli sayıda reaksiyon için bir ana karışımı hazırlayın. <table border="0" cellspacing="0" cellpadding="0"><tbody><tr><td> Reaktif </td><td> her reaksiyon için ul </td></tr><tr><td> Su </td><td> 45 </td></tr><tr><td> 10X Gen Amp PCR tampon II. </td><td> 10 </td></tr><tr><td> MgCl 2 (25mm) </td><td> 10 </td></tr><tr><td> BSA (10 mg / ml) </td><td> 2 </td></tr><tr><td> dNTP (her biri 25 mM) </td><td> 1 </td></tr><tr><td> 454 emPCR ileri primer/10uM </td><td> 3 </td></tr><tr><td> 454 emPCR rvs primer/10uM </td><td> 3 </td></tr><tr><td> AmpliTaq Gold DNA polimeraz (5 U / ml) </td><td> 1 </td></tr><tr><td> 454 DNA kütüphane şablon </td><td> 25 </td></tr><tr><td> Toplam </td><td> 100 </td></tr></tbody></table></li><li> Tek astar uzatma reaksiyonu için bir termalcycler aşağıdaki programı çalıştırın: <ol><li> 95 ° C 12min </li><li> 60 ° C (veya farklı ise PEC astar sıcaklığı tavlama) 1 dk </li><li> 72 ° C&#39;de 5 dakika </li><li> 72 ° C sonsuza kadar! KRİTİK ADIM 72 ° C uzantısı sonra reaksiyon edin Bundan sonra direkt olarak ısı blok çıkarmadan önce tüpler içine QIAGEN MinElute arıtma için pipet 150ul PBI veya PB tampon. Bu non-spesifik astar tavlama önlemek ve karışım soğutur olarak yakalamak için önemlidir. </li></ol></li><li> Üreticinin talimatlarına göre, bir Qiagen MinElute spin kolon üzerinde reaksiyon arındırın. 50 ul EB Zehir. </li></ol> 3) Uzatma Ürün Yakalama <ol><li> M-270 boncuk vorteks tarafından süspanse edin stok solüsyonu. Örnek başına 25 ul boncuk süspansiyonu çıkartın. Örnek başına 25 ul 2xBW tampon, boncuklar 500ul 2x BW tampon ve tekrar süspansiyon ile iki kez yıkayın. </li><li> 25 ul eluat 2.3 adım adım 3.1 &#39;den 25 ul boncuk süspansiyonu ekleyin. 1 döndürmek sonra karıştırın.Oda sıcaklığında 5 dakika. Önerilen: qPCR ölçümü için 2.3 adım 5 ul eluat kalan tutmak. </li><li> Taze bir 1.5ml tüp karışımın tamamı Transferi (hedef olmayan kütüphane parçalarının taşınmasını azaltmak için yardımcı olur). Pelet (PPK), Manyetik parçacık toplayıcı kullanarak süpernatant ve supernatant atın. </li><li> 500 ul 1xBW tampon (birçok yıkar, mümkün olduğu kadar çok arka plan olarak kaldırmak için yardımcı) ile 5 kez yıkayın. Son yıkama adımdan sonra, kısa bir süre aşağı spin ve süpernatant son izlerini kaldırmak. </li><li> 500μl 1x milyondan fazla Sıcak Yıkama (HW) tampon ekleyin. 2 dakika çalkalanır 65 ° C (veya yukarıda 5C PEC astar Tm) termal blok. Soğuma en aza indirmek için, bundan sonra hızlı bir şekilde süpernatantı. (Bu adım, hala PEC primerleri değil aslında uzantısı adım sırasında genişletilmiş ile ilişkili arka plan parçaları kaldırmak için). Süpernatant son izleri çıkarın. </li><li> 30 ul EB tampon boncuk pelletini tekrar. Taze bir 1.5ml tüp karışımın tamamı Transferi (hedef olmayan kütüphane parçalarının taşınmasını azaltmak için yardımcı olur). Yürütülmesi için bir termal cycler 3 dakika 95 ° C&#39;de inkübe edin. PPK yerleştirin ve boncuklar geride bırakmak özen süpernatant kaldırmak. </li></ol> 4) Ürün Amplifikasyon Yakalama <ol><li> Gerekli sayıda numune için PCR master miks hazırlayın. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Reaktif </td><td> her reaksiyon için ul </td></tr><tr><td> Su </td><td> 45 </td></tr><tr><td> 10X Gen Amp PCR tampon II. </td><td> 10 </td></tr><tr><td> MgCl 2 (25mm) </td><td> 10 </td></tr><tr><td> BSA (10 mg / ml) </td><td> 2 </td></tr><tr><td> dNTP (her biri 25 mM) </td><td> 1 </td></tr><tr><td> 454 emPCR ileri primer/10uM </td><td> 3 </td></tr><tr><td> 454 emPCR rvs primer/10uM </td><td> 3 </td></tr><tr><td> AmpliTaq Gold DNA polimeraz (5 U / ml) </td><td> 1 </td></tr><tr><td> Yıkandı, yakalama ürün </td><td> 25 </td></tr><tr><td> Toplam </td><td> 100 </td></tr></tbody></table></li><li> Thermo cycler 14 döngüleri amplifikasyonu için aşağıdaki PCR programı kullanın: <ol><li> 95 ° C 12min </li><li> 95 ° C 30s </li><li> 60 ° C (veya istenilen tavlama sıcaklığı) 1 dk. </li><li> 72 ° C 1 dakika </li><li> 2 (x 13) </li><li> 72 ° C 5min </li><li> 10 ° C ∞ </li></ol></li><li> Üreticinin talimatlarına göre bir Qiagen MinElute silika spin kolon üzerinde reaksiyon arındırın. 50 ul EB tampon Zehir. Ürün adım 2.1 başlayan veya sıralama 454 emülsiyon PCR protokolü, doğrudan girdi, ikinci bir tur için yakalama kullanarak. </li></ol> Temsilcisi Sonuçlar: Tavsiye edilen normal karışık bir &#39;pozitif kontrol hedef dizisinin&#39; (1 pikogramdır kolaylıkla yeterli örneğin bir ~ 100bp PCR ürünü) küçük bir miktar önce bir yakalama reaksiyonu gerçekleştirmek için PEC protokolü ile başlatırken kuvvetle tavsiye miktarı (2.1 adım) 454 kütüphane şablon amplifiye ve yakalama pozitif kontrol ürünü yakalamak için tasarlanmış bir tek PEC astar ile yapılır. Hem kontrol özgü olumlu ve 454 adaptörü özgü primer çiftleri ile, bu kontrol reaksiyon yapılmazsa, qPCR saflaştırılmış astar uzatma reaksiyonu (1.3), astar uzatma ürünü (2.3 arka plan karşısında &#39;control hedef&#39; miktarını ölçmek için kullanılan olabilir ) ve yıkandı, boncuk yakalama ürün (3.6). Bu şekilde, hem de etkinliğini deneysel koşullarda arka plan çıkarılması (&quot;özgüllük&quot;) ve hedef kurtarma (&quot;duyarlılık&quot;) verimliliği doğrudan ölçülebilir. Başarılı bir PEC protokolü normalde şu özelliklere sahiptir - Hassasiyet (protokol ile muhafaza kontrolü hedef miktarı ile ölçülen): Yıkandı, boncuk yakalama ürün kontrolü hedefinin toplam miktarı (3.6) = ~ astar uzatma reaksiyonu (2.3) saflaştırılmış toplam miktarın% 1-20. Background (454 emPCR primer çifti ile ölçülen): Yıkandı, boncuk yakalama ürün (3.6), arka plan toplam tutarı &lt;saflaştırılmış astar uzatma reaksiyonu (2.3) toplam miktarı% 0.01 . Nihai ürünün hedef kalan% 1&#39;inden daha azı varsa, astar uzantısı adım başarısız olabilir ve araştırılması gerekir. Nihai ürün arka planda kalan% 0.01 daha fazla varsa, çamaşır adımları yanlış yapılmış olabilir ve araştırılması gerekir.

<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.

PEC yöntem, basit, hızlı, duyarlı ve özgül. Bu nedenle böyle bir RNA kütüphanesi küçük RNA parçaları yakalama, havuzlu bir örnek veya bir metagenomic örnek 16S (ya da diğer loci) çeşitliliği yakalama yapısal farklılıklar sorgulama olarak antik DNA dışında yazarların tasavvur birden çok uygulama,. Bahsetmek bir nokta yakalama duyarlılığı, çok katlı bir yakalama reaksiyon artar PEC primer sayı olarak daha düşük olur. PEC bu nedenle çok büyük (örneğin, bir megabase veya daha fazla) yakalama bölgelerin çekimi için ideal uygun değil, ama son derece iyi, hızlı bir şekilde birçok kişi küçük hedef bölgeler veya hatta SNP pozisyonları yakalamak için çok uygundur.

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<td>AmpliTaq Gold polymerase with GeneAmp PCR buffer II and MgCl2</td>
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<td>N8080241</td>
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<td>QIAGEN MinElute PCR purification kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>28004</td>
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<td>M-270 streptavidin beads</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>653.05</td>
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primer extension capture: targeted sequence retrieval from heavily degraded dna sources

Biz, eski kemiklerin tipik olarak ağır bozulmuş ve mikrobiyal DNA ile kontamine DNA kaynaklardan hedeflenen DNA dizisi alma yöntemi mevcut. Bu yöntem, örnek yıkım ve PCR veya av tüfeği sıralama yaklaşımlar doğrudan göre sıralama talepleri büyük ölçüde azaltır. Biz coğrafi aralığında beş Neandertallerin mitokondriyal DNA (mtDNA) genomların yeniden oluşturmak için bu yöntem kullanılır. MtDNA geç Neandertallerin genetik çeşitlilik, çağdaş insanların daha yaklaşık üç kat daha düşük oldu. MtDNA protein evrim analizi ile birlikte, bu veriler Neandertallerin uzun vadeli etkin nüfus büyüklüğü, modern insan ve günümüze kadar gelen büyük maymunların daha küçük olduğunu öne sürmektedir.

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

Biz beş Neandertal bireylerin tam mitokondriyal genomların yeniden hedeflenen antik DNA dizisi alma, bir yöntem mevcut. Günümüze insanlar bu dizileri Karşılaştırma Neandertallerin uzun vadeli düşük etkili bir nüfus büyüklüğüne sahip olduğunu göstermektedir.

Primer Uzatma Yakalama: Ağır Bozulmuş DNA Kaynakları Hedefli Sıra Alma

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 Görüntüleme.  Max-Planck Institute for Evolutionary Anthropology, Leipzig. Biz beş Neandertal bireylerin tam mitokondriyal genomların yeniden hedeflenen antik DNA dizisi alma, bir yöntem mevcut. Günümüze insanlar bu dizileri Karşılaştırma Neandertallerin uzun vadeli düşük etkili bir nüfus büyüklüğüne sahip olduğunu göstermektedir.

Video: 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.

Fare Oosit alımı

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

Biyoloji

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.

Drosophila burnumun Eklenti Tepkisi (PER) Testi

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

Fare Tails Genomik DNA izolasyonu

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.

Rastgele bir DNA Kütüphane Kullanımı Primer-Aptamer Seçimi

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.

MR Genomik - Sıra Desenler Genomik DNA içinde incelenmesi için Kamu Kaynak

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.

Genomik DNA 5-hydroxymethylcytosine seçici Yakalama

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.

Oranlı metrik Bimoleküler Fenerleri kullanma Yaşam Hücrelerde Tek Engineered RNA Transkriptlerinin Gerçek zamanlı Görüntüleme

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.

Floresan Bazlı Astar Uzatma Tekniği Transkripsiyonal Başlangıç ​​Noktalarını ve RNases yarılması Siteleri belirleme<em&gt; In Vivo</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.

DNA Metilasyon Analizi nesil Dizilimine tarafından hedef

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.

Hücre belirli alt gruplar RNA izolasyonu Hızlı immünoyaftalama ile birlikte Lazer yakalama mikrodiseksiyon kullanma

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