Yazarları Greg Smith, Lisa Pomeranz, Matt Lyman Marlies Eldridge, Halina Staniszewska Goraczniak, Enquist laboratuvar ince ayar bu protokol üyeleri ve katkıları için teşekkür ederiz.

1. İlk Gün: Viral Enfeksiyon ve Tamponlar hazırlanması <ol><li> Doku kültürü enfeksiyon hücreleri 5-10 yemekleri hazırlayın (15 cm çapında), örneğin pseudorabies virüs (PRV) veya herpes Vero hücreleri PK-15 hücreleri virüs simpleks (HSV). </li><li> Hücreleri 95 -% 100 birleşmiş, bir (İçişleri Bakanlığı) 5-10 onları bozar. Bunu yapmak için, plaka, plaka başına 4 ml toplam hacmi virüs stok kullanılarak her aşılamak, daha sonra 37 tabak az 1 saat inkübe ° C Rock plates yavaşça her 15 dakikada bir hücre tek tabaka virüs inokulum tarafından tamamen kaplı kalmasını sağlamak için. Bu arada, bir sonraki adım için ortam sıcak. </li><li> Bir saat sonra enfeksiyon, viral inokulum plakalardan aspire 15 ml sıcak orta ekleyin ve 37 ° C&#39;de 12-20 saat süreyle nemlendirilmiş bir inkübatör. </li><li> LCM tamponlar (bkz. Tablo 1) ve geceleme kaya 4 ° C tam karıştırma için hazırlayın. (Bkz. Tablo 2) viral DNA tabanda için TNE hazırlayın. </li></ol> 2. İkinci Gün, 1. Aşama: Hücre Lizis ve Ultrasantrifügasyon <ol><li> Görme hücre enfeksiyon tekdüze sitopatik etki (CPE) neden oldu onaylamak. Tüm hücreleri yuvarlak var, ama plaka kaldırdı kadar enfeksiyon ilerlemeye izin ver. </li><li> Enfekte olmuş hücreleri ortama Pençe ve 50 ml konik tüpler içine hücreleri ve orta birleştirmek. Bu adım için gerekli konik tüpler sayısı yukarıdaki Adım 1.2 enfeksiyon için kullanılan yemekler sayısına bağlı olacaktır, örneğin bir 50 ml konik tüp maksimum 3 yemekler her Opsiyonel 15 ml orta pelet kullanılır: durulayın 10 ml PBS ile plakaları ve hücreleri ve orta ile birleştirir. </li><li> Santrifüj hücreleri ve 2000 oda sıcaklığında göreceli centifugal kuvveti (RCF) 10 dakika orta, sonra supernatant aspirat ve 10 ml PBS her pelet tekrar süspansiyon haline getirin. Bir viral enfeksiyon pelet Adım 2.2 &#39;de birden fazla konik tüpler içine yayılmış ise, bu adım konik bir tüp içine recombined olabilir. Tekrar santrifüj ve aspirasyon Opsiyonel: Bu pelet -20 ° C&#39;de saklanır ve protokol, daha sonraki bir zamanda devam edilebilir . </li></ol> 3. İkinci Gün, 2. Aşama: Ultrasantrifügasyon Aşağıdaki adımları sırasında hücre pelet ve buz üzerinde LCM tamponlar zaman tutun. <ol><li> 0% gliserol β-mercaptoethanol Ekle - LCM tampon. Ayarlayarak ultrasantrifüjdeki (aşağıda adım 3.9) soğutma başlayın 4 ° C ve vakum açma. </li><li> Uzun clumpy kadar LCM tampon - 0% gliserol 5 ml hücre pelletini tekrar. Gerekirse tüp herhangi bir çözünmemiş hücre pelletini break up, girdap. </li><li> 1.5 ml Freon (1,1,2-trikloro-1 ,2,2-trifluoroethane) ekleyerek ve şiddetle vorteks ayıklayın. Hemen 4 ° C sıcaklıkta 2000 RCF az 10 dakika süreyle santrifüj Geniş çaplı bir pipet (arayüz kaçınarak) ile üst katman toplayın ve yeni bir tüp Opsiyonel transferi: pelet jelatinimsi bir katı ise, yeni bir tüp içine hızlı bir şekilde en üst tabakasını dökmek. </li><li> Freon çıkarma tekrarlayın ve tekrar santrifüj. </li><li> En üst tabakası (~ 5 ml) toplayın ve yeni bir polyallomer ultrasantrifüjdeki tüp transfer. LCM tamponlar kalan iki gliserol β-mercaptoethanol ekleyin. </li><li> LCM (orta tabaka), ve sonra tekrar 2.5 ml% 45 gliserol ile underlaying - - LCM (alt tabaka) 3 ml% 5 gliserol ile Freon ekstraktı (üst tabaka) underlaying polyallomer santrifüj tüpüne bir degrade hazırlayın. Tüp içeriğinin taşma önlemek için ince bir pipet kullanın. </li><li> Gerekirse, LCM tamponlar aynı oranlarda kullanarak eşdeğer bir denge tüp hazırlayın. </li><li> Tüpler ultrasantrifüjdeki kova içine aktarın. LCM tampon üst tabakası (bir kerede ~ 50-100 ul) - hafifçe ekstra% 0 gliserol ekleyerek, 0,1 g birbirlerinin içinde kova Dengesi. </li><li> Yatay veya sallanan bir kova rotor soğuk bir ultrasantrifüjdeki kullanarak, 77,000 RCF (örneğin bir SW41Ti rotor için 25.000 rpm), 4 ° C sıcaklıkta 1 saat süreyle dengeli örnekleri Spin </li><li> Ultrasantrifügasyon sırasında, etanol yağış adım (4.6), kayan DNA yakalamak için bir cam kanca olun. Bir bardak yeniden süspanse her iki ucundan tutun ve bir alev üzerinde orta bölümü askıya. Cam bir bölüm neredeyse erimiş olduğunda, cam germek için nazikçe çekin sıkıca açılı bir kanca (&lt;90 derece) oluşturmak için hafifçe bükün, ve sonunda mühür keskin çekin. Cam, cam kanca ucu açık ise kapatmak için yeterli erir, böylece ateşte ucu tutun. </li><li> Ultrasantrifügasyon sonra, ince bir opak pelet ortasında karanlık bir nokta göreceksiniz. Dikkatle tüpten kenarları boyunca damlayan da dahil olmak üzere sıvı aspire, ancak tüpün alt kısmında pelet kaçınarak. </li><li> Oda sıcaklığında 0,5 ml TNE ekleyin. </li><li> Pelet, pelet hidrasyon sağlamak için en az 10 dakika oturup Optional: 4 tutulması durumunda pelet gecede TNE tekrar süspansiyon ° C </li></ol> 4. Üçüncü Gün: &quot;Ghost&quot; DNA Yağış <ol><li> Küçük çaplı bir pipet ucu (örneğin bir P200 pipet) ile pipetleme pelet kadar Break . Viral DNA, hala bu noktada capsids bulunan olduğundan, bu aşamada kesme hakkında endişelenmeyin. Capsids yok oda sıcaklığında viral pelet, içeren tüp aşağıdaki reaktifler ekleyin: <ul><li> 4.25 ml TNE </li><li> 0.25 ml% 10 SDS </li><li> 2 mg Proteinaz K </li></ul> Bu reaktifler karıştırmak için yavaşça sallayın. Viskozite hafif bir artış olmalıdır. </li></ol> Burada kesme dikkat edin, örneğin büyük çaplı pipetler viral malzeme kullanımı ve vorteks yok <ol start="2"><li> Fenol-kloroform 5 ml ekleyerek viral proteinler ayıklayın ve bir emülsiyon korumak için hemen tersini veya vorteks başlar. 10 saniye boyunca karıştırın, sonra 5 dakika boyunca oda sıcaklığında 2000 RCF emülsiyon santrifüj. , Geniş çaplı bir pipet ucu ile üst tabaka toplama arayüzü kaçınarak, ve yeni bir tüpe transfer İsteğe bağlı: Bu adımda kullanın faz kilidi jel (PLG) tüpler arayüzü kirlenmesini önlemek amacıyla. </li><li> Fenol-kloroform ekstraksiyon tekrarlayın ve tekrar santrifüj. </li><li> Ikinci fenol-kloroform ekstraksiyon sonra, 30 ml cam Corex tüpe üst tabaka toplamak ve tüp -20 ° C&#39;de 10-15 dakika soğuk dondurma olmadığını özen. </li><li> 10 ml buz gibi soğuk etanol, tüp örtüsü, (örneğin Parafilm M üstünden bir parça uzanan) ve hemen karıştırmak için ters. Ince bir vıcık vıcık çökelti oluşur görünür bir DNA &quot;hayalet&quot;, dikkat edin. Hafifçe daha da karıştırmak ve yapışkan DNA birlikte küme çökeltileri neden Tüpü ters çevirin. </li><li> DNA hayalet (ler) yakalamak için bir bardak kanca kullanın. Dikkatlice aşırı sıvı kaldırmak için DNA blot, daha sonra 1.5 ml tüpe kanca (aşağı ucu) yerleştirin ve kurumasını bekleyin. Ekle 0.25 - 0.5 ml TE DNA hayalet çözülür. En az bir saat devam etmek için DNA tabanda İzin. 4 Mağaza DNA ° C İsteğe Bağlı: 1.5 ml tüp içine cam kanca kesiyorum, DNA verimi en üst düzeye çıkarmak için, cam parçaları kapalı böylece tabanda zamanla devam edebilirsiniz. </li></ol> 5. Temsilcisi Sonuçlar Hücreleri ile İçişleri Bakanlığı yeterince yüksek bir, yabani tip PRV suşları için ~ 18 saat sonrası enfeksiyon (HPI) içinde tam CPE ulaşmak, ve yabani tip HSV-1 suşu (Şekil 1) ~ 24 HPI. Bir kez enfekte hücrenin pelet hasat edilir, kalan protokol adımları tam bir uzun gün ya da iki gün (Şekil 2) üzerinden yürütülmektedir. DNA hayalet yakalamak için cam kanca hazırlarken, daha sonra 1.5 ml tüp tabanı (Şekil 3) içine sığabilecek kadar kanca açı 90 dereceden daha az olması, yapmak için önemlidir. Bu kanca ucu Sızdırmazlık pipetlemeyin içindeki DNA kaybını önler. DNA hayaletler yağış adım sırasında formu ve beyaz, vıcık vıcık ipler (Şekil 4) olarak görünecektir. Birbirleriyle ve / veya DNA yakalamak için kullanılan cam kanca Bu agrega ve sopa. Böylece aynı cam kanca yağış çözüm içinde birden çok iş parçacığı DNA toplamak için kullanılabilir. <table border="1"><tbody><tr><td> İstenen hacme dayalı sütunu seçin: </td><td> 10 ml </td><td> 15 ml </td><td> 20 ml </td><td> 40 ml </td></tr><tr><td> 1M KCL </td><td> 1.3 ml </td><td> 1.9 ml </td><td> 2.5 ml </td><td> 5 ml </td></tr><tr><td> 1M Tris (pH, 7.4 &#39;) </td><td> 300 ul </td><td> 450 ul </td><td> 600 ul </td><td> 1.2 ml </td></tr><tr><td> 1M MgCl 2 </td><td> 50 ul </td><td> 75 ul </td><td> 100 ul </td><td> 200 ul </td></tr><tr><td> 0,5 EDTA </td><td> 10 ul </td><td> 15 ul </td><td> 20 ul </td><td> 40 ul </td></tr><tr><td> NP40/IGEPAL </td><td> 50 ul </td><td> 75 ul </td><td> 100 ul </td><td> 200 ul </td></tr><tr><td> β-mercaptoethanol (kullanmadan önce hemen eklemek) </td><td> 4.3 ul </td><td> 6.5 ul </td><td> 8.6 ul </td><td> 17.2 ul </td></tr><tr><td colspan="5"></td></tr><tr><td colspan="5"> İstenen yüzde gliserol (0, 5 veya 45%) oranlar kullanın </td></tr><tr><td> 0% gliserol </td><td> Hiçbiri </td><td> Hiçbiri </td><td> Hiçbiri </td><td> Hiçbiri </td></tr><tr><td> su </td><td> 8.3 ml </td><td> 12.5 ml </td><td> 16.7 ml </td><td> 33.4 ml </td></tr><tr><td> % 5 gliserol </td><td> 0.5 ml </td><td> 0.8 ml </td><td> 1.0 ml </td><td> 2.0 ml </td></tr><tr><td> su </td><td> 7.8 ml </td><td> 11.7 ml </td><td> 15.7 ml </td><td> 31.4 ml </td></tr><tr><td> % 45 glycerol </td><td> 4,5 ml </td><td> 6.8 ml </td><td> 9.0 ml </td><td> 18.0 ml </td></tr><tr><td> su </td><td> 3.8 ml </td><td> 5.7 ml </td><td> 7.7 ml </td><td> 15.4 ml </td></tr></tbody></table> Tablo 1. LCM tamponlar hazırlanması için tarifler <table border="1"><tbody><tr><td> 50 ml hacim için </td></tr><tr><td> 5 ml 1M NaCl </td></tr><tr><td> 2.5 ml 1M Tris, pH 7.5 </td></tr><tr><td> 1 ml 0,5 EDTA </td></tr><tr><td> 41.5 ml H 2 O </td></tr></tbody></table> Tablo 2. TNE (50 mM Tris, pH 7.5, 10 mM EDTA 0,1 M NaCl) hazırlanması <table border="1"><tbody><tr><td> 1 L hacim için </td></tr><tr><td> 0.2 g KCl </td></tr><tr><td> 0.2 g KH 2 PO 4 </td></tr><tr><td> 8 g NaCl </td></tr><tr><td> 1.15g Na 2 HPO 4 </td></tr><tr><td> 1L için H 2 O </td></tr></tbody></table> Tablo 3. PBS (1.5 mM KH 2 PO 4, 137 mM NaCl, 8.1 mM 2 HPO 4 Na; pH 7.0 2.7 mM KCl) hazırlanması <img alt="Şekil 1" src="/files/ftp_upload/3151/3151fig1.jpg" /> Şekil 1: PK15 hücreleri enfeksiyon (A) önce, sonra (B) yüksek bir enfeksiyon çokluğu yarıda enfeksiyon yoluyla ve (C) sitopatik etki (CPE). <img alt="Şekil 2" src="/files/ftp_upload/3151/3151fig2.jpg" /> Şekil 2 protokol bakış. <img alt="Şekil 3" src="/files/ftp_upload/3151/3151fig3.jpg" /> Şekil 3. Üç cam temsilcisi örnekler viral DNA hayaletler toplamak için kullanılan kanca. <img alt="Şekil 4" src="/files/ftp_upload/3151/3151fig4.jpg" /> Şekil 4 iyi biçimlenmiş ve bol miktarda DNA &quot;hayalet&quot; temsili bir örnek .

<ol>
	<li>Szpara, M. L., Parsons, L., Enquist, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence+variability+in+clinical+and+laboratory+isolates+of+herpes+simplex+virus+1+reveals+new+mutations.">Sequence variability in clinical and laboratory isolates of herpes simplex virus 1 reveals new mutations.</a> J Virol. 84, 5303-5313 (2010).</li><li>Banfield, B. W., Kaufman, J. D., Randall, J. A., Pickard, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+pseudorabies+virus+strains+expressing+red+fluorescent+proteins:+new+tools+for+multisynaptic+labeling+applications.">Development of pseudorabies virus strains expressing red fluorescent proteins: new tools for multisynaptic labeling applications.</a> J Virol. 77, 10106-10112 (2003).</li><li>Kobiler, O., Lipman, Y., Therkelsen, K., Daubechies, I., Enquist, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpesviruses+carrying+a+Brainbow+cassette+reveal+replication+and+expression+of+limited+numbers+of+incoming+genomes.">Herpesviruses carrying a Brainbow cassette reveal replication and expression of limited numbers of incoming genomes.</a> Nat Commun. 1, 146-146 (2010).</li><li>Enquist, L. W., Madden, M. J., Schiop-Stanley, P., Vande Woude, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+of+herpes+simplex+type+1+DNA+fragments+in+a+bacteriophage+lambda+vector.">Cloning of herpes simplex type 1 DNA fragments in a bacteriophage lambda vector.</a> Science. 203, 541-544 (1979).</li><li>Smith, G. A., Enquist, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+transposon+mutagenesis+in+Escherichia+coli+of+a+full-length+infectious+clone+of+pseudorabies+virus,+an+alphaherpesvirus.">Construction and transposon mutagenesis in Escherichia coli of a full-length infectious clone of pseudorabies virus, an alphaherpesvirus.</a> J Virol. 73, 6405-6414 (1999).</li><li>Flint, S. J., Enquist, L. W., Racaniello, V. R., Skalka, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles of virology. , (2008).</li><li>Pomeranz, L. E., Reynolds, A. E., Hengartner, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+biology+of+pseudorabies+virus:+impact+on+neurovirology+and+veterinary+medicine.">Molecular biology of pseudorabies virus: impact on neurovirology and veterinary medicine.</a> Microbiol Mol Biol Rev. 69, 462-500 (2005).</li><li>Roizman, B., Pellett, P. E., Knipe, D. M., Howley, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fields Virology. , 2381-2397 (2001).</li><li>Mendez, I. I., Hermann, L. L., Hazelton, P. R., Coombs, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+analysis+of+freon+substitutes+in+the+purification+of+reovirus+and+calicivirus.">A comparative analysis of freon substitutes in the purification of reovirus and calicivirus.</a> J Virol Methods. 90, 59-67 (2000).</li><li>Gharabaghi, F., Aymard, M., Trotemann, P., Gerdil, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+simplified+micromethod+for+subtyping+varicella-zoster+virus.">A rapid and simplified micromethod for subtyping varicella-zoster virus.</a> J Med Virol. 31, 129-134 (1990).</li><li>Granstedt, A. E., Szpara, M. L., Kuhn, B., Wang, S. S., Enquist, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence-based+monitoring+of+in+vivo+neural+activity+using+a+circuit-tracing+pseudorabies+virus.">Fluorescence-based monitoring of in vivo neural activity using a circuit-tracing pseudorabies virus.</a> PLoS One. 4, e6923-e6923 (2009).</li></ol>

Çıkar çatışması ilan etti.

Bu protokol bölümleri başlangıçta BSL4 şartlarda viral DNA izolasyonu için geliştirilmiş, ancak bu eşit olmayan BSL koşulları iyi uyum 4. yaygın DNA genomların olan alfa-herpesvirüslerinin PRV ve HSV-1, DNA izole etmek için bu protokolü kullanan kapsid bir protein ve lipid bir zarf ile çevrili içine 7,8 Bununla beraber, beta ve gama-herpesvirüslerinin ve adenovirüs, büyük olasılıkla diğer büyük DNA virüsleri doğrudan uyarlanabilir. Benzer ekstraksiyon yaygın RNA virü...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> Freon (1,1,2 trikloro-1 ,2,2-trifluoroethane) </td><td> Balıkçı </td><td> T178-4 </td><td> Uygun bertaraf kurallar Freon içeren atıklar için kurum ile kontrol edin, ya da görmek Mendez et al. potansiyel Freon alternatifleri. 9 </td></tr><tr><td> Faz kilit jel tüpler, Ağır 15ml kapasiteli </td><td> 5 PRIME </td><td> 2302850 </td><td> Isteğe bağlı </td></tr><tr><td> Polyallomer ultrasantrifüjdeki borular </td><td> Beckman * </td><td> 331.372 * </td><td> Kendi ultrasantrifüjdeki için uygun Select * tüpleri; bu sadece bir örnek olarak yer almaktadır </td></tr><tr><td> NP-40 / IGEPAL </td><td> Sigma </td><td> I-3021 </td><td></td></tr><tr><td> PK-15 hücre </td><td> ATCC </td><td> CCL-33 </td><td></td></tr><tr><td> Vero hücrelerinde </td><td> ATCC </td><td> CCL-81 </td><td></td></tr><tr><td> PBS </td><td> HyClone </td><td> SH30028.03 </td><td></td></tr></tbody></table>

preparation of viral dna from nucleocapsids

Virüsler zorunlu hücresel parazitler, ve böylece kendi DNA çalışma, konak hücre kirleticiler ve DNA viral malzeme izole gerektirir. Birkaç downstream uygulamalar bu protokolü tarafından sağlanan saf viral DNA, büyük miktarlarda gerektirir. Bu uygulamalar, viral genom konak DNA kaldırılması viral dizileri için veri çıkışı optimize etmek için çok önemlidir sıralama, ve yeni rekombinant virüs suşları üretimi, plazmid ve lineer viral DNA rekombinasyon kolaylaştırır saflaştırılmış co-transfeksiyon 1,2, 3 Bu prosedür, ekstraksiyon ve enfekte hücrelerden doğrusal herpesvirus nucleocapsid saflaştırılmış DNA izole etmek için yoğunluk tabanlı santrifüj bir kombinasyonunu kullanır. 4,5 ilk arıtma adımları ekstraksiyon ve santrifüj adımları sırasında viral DNA içermediğinden ve korumak, saflaştırılmış virüs capsids izole etmek amacı hücresel protein ve DNA kaldırmak. Nucleocapsids, Lizis sonra viral DNA açıklamaları ve son iki fenol-kloroform adımları kalan proteinler kaldırmak. Çözüm yakalanan son DNA, 1.90 ortalama OD 260/280 ile, son derece konsantre ve saf. Kullanılan enfekte hücreler, viral DNA aralığı 150-800 mg veya daha fazla verim miktarına bağlı olarak değişir. Bu DNA saflık 4C uzun süreli depolama sırasında istikrarlı hale getiriyor. Bu DNA böylece ideal, yüksek verimlilik sıralama, yüksek sadakat PCR reaksiyonları ve transfections için çok uygundur. Protokol başlamadan önce, çanak başına hücre (örneğin konfluent 15 cm çanak ortalama 8 x 10 6 PK-15 hücreleri) ve viral stokunun titresi (ortalama sayısını bilmek önemlidir. örneğin 1 x 10 ml başına 8 plak oluşturan birim). Bu protokol için uygun enfeksiyon çokluğu (İçişleri Bakanlığı) hesaplamak için gereklidir. Örneğin 6, 5 bir İçişleri Bakanlığı Yukarıdaki viral stoğu ile PK-15 hücreleri, biri 15 cm çanak bulaştırmak için, 400 ul 3.6 ml (15 cm plaka için toplam aşılama hacmi 4 ml) orta ile viral stok ve sulandırmak. Çoklu viral DNA preparatları aynı zamanda hazırlanabilir. Sadece ultrasantrifüjdeki rotor tarafından düzenlenen tüplerin sayısına göre eş zamanlı olarak hazırlıkları sayısı sınırlıdır (virüs başına bir adım aşağıda 3.9). Burada bir virüs için yapılıyor gibi prosedürü açıklar.

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Preparation of Viral DNA from Nucleocapsids

Biz enfekte hücrelerden yüksek saflıkta herpesvirus nucleocapsid DNA izole süreci tanımlamak. Çözüm yakalanan son DNA ideal, yüksek verimlilik sıralama, yüksek sadakat PCR reaksiyonlar, ve yeni virüs rekombinantlar üretmek için transfections için uygun, yüksek konsantrasyon ve saflık.

Nucleocapsids gelen Viral DNA hazırlanması

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. ...

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JoVE Journal

Immunology and Infection

20.8K Görüntüleme.  Princeton University. Biz enfekte hücrelerden yüksek saflıkta herpesvirus nucleocapsid DNA izole süreci tanımlamak. Çözüm yakalanan son DNA ideal, yüksek verimlilik sıralama, yüksek sadakat PCR reaksiyonlar, ve yeni virüs rekombinantlar üretmek için transfections için uygun, yüksek konsantrasyon ve saflık.

Video: Preparation of Viral DNA from Nucleocapsids

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

Plazmid DNA Rekombinant Grip Virüsü Üretimi

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

İmmünoloji ve Enfeksiyon

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

Viral Patojenler Klinik Örnekler Ekranına bir Pan-Viral Mikroarray Testi (Virochip)

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

Virüs mutasyon Frekans RNA Virüsler ve Karakterizasyonu Fidelity Varyantlar izolasyonu

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2. Viruses modulate host cell abundance and diversity, contribute to the cycling of nutrients, alter host cell phenotype, and influence the evolution of both host cell and viral communities through the lateral transfer of genes 3. Numerous studies have highlighted the staggering genetic diversity of viruses and their functional potential in a variety of natural environments. 
Metagenomic techniques have been used to study the taxonomic diversity and functional potential of complex viral assemblages whose members contain single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and RNA genotypes 4-9. Current library construction protocols used to study environmental DNA-containing or RNA-containing viruses require an initial nuclease treatment in order to remove nontargeted templates 10. However, a comprehensive understanding of the collective gene complement of the virus community and virus diversity requires knowledge of all members regardless of genome composition. Fractionation of purified nucleic acid subtypes provides an effective mechanism by which to study viral assemblages without sacrificing a subset of the community&#x2019;s genetic signature. 
Hydroxyapatite, a crystalline form of calcium phosphate, has been employed in the separation of nucleic acids, as well as proteins and microbes, since the 1960s11. By exploiting the charge interaction between the positively-charged Ca2+ ions of the hydroxyapatite and the negatively charged phosphate backbone of the nucleic acid subtypes, it is possible to preferentially elute each nucleic acid subtype independent of the others. We recently employed this strategy to independently fractionate the genomes of ssDNA, dsDNA and RNA-containing viruses in preparation of DNA sequencing 12. Here, we present a method for the fractionation and recovery of ssDNA, dsDNA and RNA viral nucleic acids from mixed viral assemblages using hydroxyapatite chromotography.

We describe an efficient method to separate single-stranded DNA, double-stranded DNA and RNA molecules from environmental viral communities. Nucleic acids are fractionated using hydroxyapatite chromatography with increasing concentrations of phosphate-containing buffers. This method permits the isolation of all viral nucleic acid types from environmental samples.

Tek iplikli DNA, çift iplikli DNA ve RNA Hidroksiapatit Kromatografisi kullanarak bir Çevre Viral Topluluk ayrılması

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2.  Viruses modulate host cell abundance and diversity, ...

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 &mu;l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 &mu;l was retained while the other 10 &mu;l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.

DNA ekstraksiyonu için A Simple Chelex Protokolü<em&gt; Anofel</em&gt; Spp.

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

Polarize İnsan Havayolu bir sıvı kaplı Kültürünün Oluşturulması Solunum Patojenler için Host Hücre Yanıtı Okumak Epitel Calu-3 Hücreler<em&gt; In vitro</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these glycoprotein &#8216;spikes&#8217; is often technically challenging but important for understanding viral pathogenesis and in drug design. Here, a protocol is presented for viral spike structure determination through computational averaging of electron cryo-tomography data. Electron cryo-tomography is a technique in electron microscopy used to derive three-dimensional tomographic volume reconstructions, or tomograms, of pleomorphic biological specimens such as membrane viruses in a near-native, frozen-hydrated state. These tomograms reveal structures of interest in three dimensions, albeit at low resolution. Computational averaging of sub-volumes, or sub-tomograms, is necessary to obtain higher resolution detail of repeating structural motifs, such as viral glycoprotein spikes. A detailed computational approach for aligning and averaging sub-tomograms using the Jsubtomo software package is outlined. This approach enables visualization of the structure of viral glycoprotein spikes to a resolution in the range of 20-40 &#197; and study of the study of higher order spike-to-spike interactions on the virion membrane. Typical results are presented for Bunyamwera virus, an enveloped virus from the family Bunyaviridae. This family is a structurally diverse group of pathogens posing a threat to human and animal health.

An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

Jsubtomo kullanılarak Elektron Cryotomography Rekonstrüksiyonunda ikinci viral zarf glikoproteini Spikes ortalamasını

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these ...

Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments through the recruitment of viral and cellular proteins to sites occupied by the viral genome. These sites, called replication compartments (RC), can be considered viral-induced nuclear domains where the viral genome is localized and viral and cellular proteins that participate in replication, transcription and post-transcriptional processing are recruited. Moreover, cellular proteins involved in the antiviral response, such as tumor suppressor proteins, DNA damage response (DDR) components and innate immune response factors are also co-opted to RC. Although RC seem to play a crucial role to promote an efficient and productive replication cycle, a detailed analysis of their composition and associated activities has not been made. To facilitate the study of adenoviral RC and potentially those from other DNA viruses that replicate in the cell nucleus, we adapted a simple procedure based on velocity gradients to isolate Ad RC and established a cell-free system amenable to conduct morphological, functional and compositional studies of these virus-induced subnuclear structures, as well as to study their impact on host-cell interactions.

We provide a novel strategy to isolate viral replication compartments (RC) from adenovirus (Ad)-infected human cells. This approach represents a cell-free system that can help to elucidate the molecular mechanisms regulating viral genome replication and expression as well as regulation of viral-host interactions established at the RC.

Adenovirüs ile enfekte Normal İnsan Hücreleri Viral Çoğaltma Bölme zenginleştirilmiş Alt nükleer Kesirler izolasyonu

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 &#176;C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic.

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

tespiti için Liyofilize Döngü aracılı İzotermal Amplifikasyon Reaktifler Gelişimi<em&gt; Coxiella burnetii</em

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

Enzim-linked Lektin Testi tarafından Grip Nöraminidaz İnhibisyon antikor titreleri Ölçme

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...