Bu araştırma, Bilim Ofisi (BER), ABD Enerji Bakanlığı, hayır Anlaşması Kooperatifi tarafından desteklenmiştir. De-FC02-02ER63453, Ulusal Bilim Vakfı Mikrobiyal Genomu Sıralama Programı (ödül numaraları 0.626.826 ve 0.731.916). Biz çevresel örnek toplama ile yaptığı yardım için teknik uzmanlık ve danışmanlık ve K. Eric Wommack John Cam teşekkür ederiz.

1. Çözeltilerin Hazırlanması Hidroksiapatit kromatografi gerçekleştirmeden önce, fosfat tamponlar hazırlıklı olmak gerekir ve hidroksiapatit düzgün sulu olmalıdır. <ol><li> 1M Fosfat Çözüm pH 6,8: 1L şişesi, steril, DEPC ile tedavi edilen H 2 O. 1L sodyum fosfat tekbazlı 119.98g çözülür Steril, DEPC ile tedavi edilen H 2 O. 1L sodyum fosfat dibazik 141.96g eriterek 1L balonuna 1M sodyum fosfat dibazik çözüm hazırlayın 1:1 oranında mono-ve di-çözümleri birleştirin. Bir manyetik karıştırıcı kullanılarak bir heyecan plaka ve karışımı üzerine yerleştirin balonu. PH 6.8 sodyum hidroksit (pH artırmak için) ya da fosforik asit (pH azaltmak için) ekleyerek ayarlayın. </li><li> 0.12M Fosfat Tamponu: 500ml şişesi, 1M Fosfat Çözüm, pH 6.8, 2.5ml% 10 sodyum dodesil sülfat (SDS) ve 0,5 M EDTA, 5ml, 30ml birleştirir. 225ml hacmi steril, DEPC tedavi H2O ekleyin. PH 6.8 ayarlayın. 250ml için steril, DEPC tedavi H2O ekleyin. Otoklav. </li><li> 0.18M Fosfat Tamponu: 500ml şişesi, 1M Fosfat Çözüm, pH 6.8, 2.5ml% 10 sodyum dodesil sülfat (SDS) ve 0,5 M EDTA, 5ml, 45ml birleştirir. 225ml hacmi steril, DEPC ile tedavi edilen H 2 O ekleyin. PH 6.8 ayarlayın. 250ml için steril, DEPC ile tedavi edilen H 2 O ekleyin. Otoklav. </li><li> 0.20M Fosfat Tamponu: 500ml şişesi, 1M Fosfat Çözüm, pH 6.8, 2.5ml% 10 sodyum dodesil sülfat (SDS) ve 0,5 M EDTA, 5ml, 50ml birleştirmek. 225ml hacmi steril, DEPC ile tedavi edilen H 2 O ekleyin. PH 6.8 ayarlayın. 250ml için steril, DEPC ile tedavi edilen H 2 O ekleyin. Otoklav. </li><li> 0.40 M Fosfat Tampon: 1M Fosfat Çözüm, pH 6.8, 2.5ml% 10 sodyum dodesil sülfat (SDS) ve 0,5 M EDTA, 5ml 100 ml, 500 ml cam şişe içinde birleştirir. 225ml hacmi steril, DEPC ile tedavi edilen H 2 O ekleyin. PH 6.8 ayarlayın. 250ml için steril, DEPC ile tedavi edilen H 2 O ekleyin. Otoklav. </li><li> &quot;1.00m Fosfat Tampon&quot; (bu çözüm gerçek fosfat konsantrasyonu 0,91 M): 500ml balon, 30 ml% 10&#39;luk sodyum dodesil sülfat (SDS) 1M Fosfat Çözüm pH6.8, 2.5ml birleştirmek ve 5ml EDTA 0.5M. 225ml hacmi steril, DEPC tedavi H2O ekleyin. PH 6.8 ayarlayın. 250ml için steril, DEPC ile tedavi edilen H 2 O ekleyin. Otoklav. </li><li> Hidroksiapatit hidrasyon: hidroksiapatit 1g dışarı Ağırlık ve 50ml tüp ekleyin. Hidroksiapatit 0.12M fosfat tamponu 6ml ekleyin ve karışımı için ters. Önce 60 sıcaklık getirmek ° C ve iyice karıştırın. 4 ° C uzun süre saklayın </li><li> Kullanmadan önce, 60 ° C&#39;de tüm fosfat tampon ve sulu hidroksiapatit dengelenmesi </li></ol> 2. Econo-Kolon hazırlanması <ol><li> Stopcock kapatın. , Sütun ve sürekli tersini şişeden sütun birkaç kez deiyonize H 2 O ile durulayın . </li><li> Econo-sütun stopcock çıkarın ve sütun otoklavda sterilize etmek. </li><li> Değiştirin ve valfli yakın. Econo-sütun steril ve ceket tüm cam yüzeyler için Sigmacote 1 ml ekleyin. Stopcock ve süzün açın. </li><li> Dolaşan su banyosu sütun takın ve 60 sıcaklığını ayarlamak ° C Ambalaj, alüminyum folyo veya köpük boru izolasyonu gibi bir yalıtım maddesi sütun sütun boyunca ısı kaybını en az miktarda einsure için tavsiye edilir. </li><li> Steril, RNaz-0.12M Fosfat Tampon sonra iki kez steril, RNaz-serbest H 2 O Econo-sütun ile iki kez durulayın. </li></ol> 3. Hydroxypaptite Kromotografisi <ol><li> Emin olun stopcock kapalı, yavaş yavaş 2ml steril bir serolojik pipet kullanarak önceden hazırlanmış, yeniden süspanse hidroksiapatit Econo sütunun alt 2ml ekleyin. </li><li> Hidroksiapatit 30 dakika boyunca yerçekimi kuvveti altında yerleşmek için izin verin. </li><li> Sütun ve yakın stopcock tampon boşaltın. 60 ° C bir ısı bloğu veya su banyosunda 10 dakika için son hacim 500 ul ve inkübe 0.12M fosfat tamponu ile nükleik asitlerin birleştirin </li><li> Sütun rahatsız değil emin olun bir 2ml serolojik pipet kullanarak hidroksiapatit 0.12M fosfat tamponunda hazırlanmış nükleik asitlerin hemen geçerlidir. </li><li> Nükleik asitler, 30 dakika boyunca hidroksiapatit bağlamak için izin verin. </li><li> Sütununun altında 15ml tüp yeri stopcock, açık ve ssDNA içeren ilk örnek toplamak. Stopcock kapatın. </li><li> 0.12M fosfat tamponu, sütun rahatsız stopcock açmak ve önceki adımda 15ml tüp tek iplikli DNA toplamak için değil emin hidroksiapatit 6ml ekleyin. Stopcock kapatın. </li><li> Kloroform:: 1 hacim fenol izoamil alkol (25:25:1 v / v / v) tüp çözüm kuvvetlice karıştırın ve 15 dakika 3500 xg&#39;de santrifüj. Yeni bir 15ml tüp supernatant içeren ssDNA aktarın. </li><li> 6ml e 0.20M fosfat tamponu kullanılarak 3.76 ve 3.87 tekrarlayın.ud ve arındırır RNA sütun koleksiyonu için yeni bir 15ml tüp kullandığınızdan emin olun. </li><li> 0.40M fosfat tamponu 6ml kullanarak yeni koleksiyonu için 15ml tüp kullanmak için emin olun sütun dsDNA Zehir ve arındırmak için 3.76 ve 3.87 tekrarlayın. </li><li> Koleksiyonu için yeni bir 15ml tüp kullanmak için emin olmak için herhangi bir kalıntı dsDNA sütun şerit 1.00m fosfat tamponu 6ml kullanarak 3.76 ve 3.87 tekrarlayın. </li></ol> 4. Nükleik Asit Örnekleri tuzsuzlaştırma <ol><li> Örnek transfer 4-5ml Amicon Ultra-4 Ultracel membran kesilmiş 30.000 molekül ağırlığı ile donatılmış santrifüj filtre cihazı. </li><li> 6.000 xg, 30 iplik &lt;500μl örnek Konsantre ° C, 5 dakika sabit açılı rotor (ya da konsantre hacmi elde edene kadar). Akış atın. </li><li> Örnek (ler) kalan Amicon Ultra-4 santrifüj filtre cihazı ve 4-5ml Rnase ücretsiz 1X TE Tampon ile ses getirmek. </li><li> 6.000 xg, 30 iplik &lt;500μl örnek Konsantre ° C, 5 dakika sabit açılı rotor (ya da konsantre hacmi elde edene kadar). Akış atın. </li><li> Amicon Ultra-4 santrifüj filtre cihazı için 4-5ml Rnase ücretsiz 1X TE Buffer ekleyin. 6.000 xg, 30 santrifüj &lt;500μl örnek Konsantre ° C, 5 dakika sabit açılı rotor (ya da konsantre hacmi elde edene kadar). Akış atın. </li><li> Nükleik asit örnek (ler) tamamen desalt adım 4,5 ek bir 5 kez tekrarlayın. </li><li> Kalan numune (ler), steril bir 1.7ml Eppendorf tüpüne aktarın. 1/10th hacmi 3M sodyum asetat, pH 7.0,% 100 etanol iki cilt ve Glycoblue, 1μl ekleyin. Vorteks ile iyice karıştırın. </li><li> 28.000 xg, 4 ° C, 60 dakika santrifüjleyin. Durusu pelet rahatsız değil emin olun. % 70 etanol 300μl ekle. 28.000 xg, oda sıcaklığında 10 dakika Spin. Durusu, kuru ve Rnase ücretsiz TE tampon uygun bir hacmi her fraksiyonu tekrar süspansiyon haline getirin. </li></ol> 5. Temsilcisi Sonuçlar: Şekil 1, karışık bir viral birleşmesi damıtmak ssDNA, dsDNA ve RNA nükleik asitlerin hidroksiapatit kromatografisi kullanılarak sunulan yöntemleri özetlemektedir . Bu yöntem, nükleik asitlerin negatif yüklü fosfat omurgası ve pozitif yüklü Ca 2 arasındaki ücret etkileşim patlatır + iyonları hidroksiapatit mevcut ve artan fosfat tamponu konsantrasyonları ile nükleik asit alt tiplerinin etkin fraksiyonasyon (ssDNA, dsDNA ve RNA) sağlar 12. Bilinen ssDNA (M13mp18), dsDNA (lambda) ve RNA &quot;topluluk&quot; in vitro bir hidroksiapatit fraksiyonasyon (MS2 ve phi6) viral genomların Şekil 2&#39;de gösterilmiştir. Nükleik asitler, eşit konsantrasyonlarda birlikte hidroksiapatit sütuna uygulanan ve giderek artan konsantrasyon fosfat tampon kullanarak kolonda sürüklenecektir. Her nükleik asit alt tipi,% 9 daha az sonraki bir fraksiyonu üzerinde taşımak diğerlerinden bağımsız elutes. Chesapeake Bay toplanan bir viral toplumdan izole edilen nükleik asitler bu tekniğin uygulama Şekil 3&#39;te gösterilmiştir. Saflaştırılmış virüsleri izole toplam nükleik asit verimi hidroksiapatit sütun uygulandı . Genomik malzeme ssDNA, RNA ve dsDNA oluşan bağımsız sırasıyla 0.12M, 0.20M ve fosfat tampon 0.40M/1.00M konsantrasyonları ile yıkandı. Çift iplikli DNA, 0.40M ve 1.00m fosfat konsantrasyonları yıkandı, ve bu durumda, ssDNA ve RNA genotipler karşılaştırıldığında bu viral bir topluluk hakim. Bu gözlem, geri kazanılabilir nükleik asitlerin çoğunluğu dsDNA 13 beklenen viral deniz toplum kompozisyon ve haliç ortamları ile tutarlı . <img src="/files/ftp_upload/3146/3146fig1.jpg" alt="Şekil 1"/> Şekil 1 nükleik asitlerin fraksiyonasyon hidroksiapatit kromatografi yöntemi akış diyagramı. 0.12M fosfat tamponu içinde hazırlanan ssDNA, dsDNA ve RNA karışımı 60 ° C dereceye kadar ısıtılır ve 60 yaşında bir sabit tutulan bir hidroksiapatit sütun uygulanır ° C su banyosu dolaşan kullanarak. Nükleik asitler (ssDNA, RNA ve dsDNA) fosfat içeren bir tampon artan konsantrasyonları ile hidroksiapatit kolonda sürüklenecektir. Bu rakam Amerikan Mikrobiyoloji Derneği izni ile yeniden ve orijinal Andrews-Pfannkoch ark yer aldı. 2010 12. <img src="/files/ftp_upload/3146/3146fig2.jpg" alt="Şekil 2"/> Şekil 2 hidroksiapatit kromatografisi kullanarak bilinen viral nükleik asitlerin ayrılması. Eşit konsantrasyonda M13mp18 ssDNA, MS2 ssRNA, phi6 dsRNA ve lambda dsDNA (şerit 2-5, sırasıyla) kombine (şeritli 6) ve hidroksiapatit bir sütun uygulanmıştır. Single-iplikli DNA (M13mp18), RNA (MS2/phi6) ve dsDNA (lambda) 0.12M (şerit 8), 0.18M (şerit 9) ve 0.40M/1.00M (10 şeritli) kullanarak hidroksiapatit sütun bağımsız yıkandı, . 1KB merdiveni (şerit 1, 7) genom boyutları onaylamak için kullanılır oldu. Bu rakam Amerikan Mikrobiyoloji Derneği izni ile yeniden ve orijinal Andrews-Pfannkoch ark yer aldı. 2010 12. <img src="/files/ftp_upload/3146/3146fig3.jpg" alt="Şekil 3"/> Şekil 3 Chesapeake Körfezi içinde bir toplumdan izole viral nükleik asitlerin ayrılması. Nükleik asitlerin, izole edilmiş ve hidroksiapatit bir sütun uygulanmıştır. ssDNA (şeritli 0.12M), RNA (şeritli 0.20M) ve dsDNA (şerit 0.40M/1.0M) bağımsız olarak sırasıyla, 0.12M, 0.20M ve 0.40M/1.00M fosfat tampon konsantrasyonu kullanılarak yıkandı,. Hi Kitle DNA Ladder (şeritli L1) ve 1KB Merdiven (şeritli L2), görsel, nükleik asitlerin yaklaşık moleküler ağırlık ve kütle onaylamak için kullanılır. Bu rakam Amerikan Mikrobiyoloji Derneği izni ile yeniden ve orijinal Andrews-Pfannkoch ark yer aldı. 2010 12.

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Çıkar çatışması ilan etti.

Burada sunulan hidroksiapatit kromatografi metodolojisinin amacı, toplumun toplam nükleik asit kompozisyonu okumak için karışık viral toplulukları, nükleik asitlerin fraksiyonasyon için son derece verimli ve güçlü bir araç. Genellikle, ssDNA, RNA ve dsDNA yaklaşık ~ 0and 0.40M sırasıyla daha fazla sütun pho fosfat tamponu konsantrasyonları Zehir. Ancak, bilinen nükleik asitler (örneğin ekili bir virüs hazırlık) kullanarak her bir hazırlık test etmek için önemli böylece hidroksiapat...

<table border="1"><tbody><tr><th> Malzeme Adı </th><th> Şirket </th><th> Katalog Numarası </th><th> Katalog Numarası </th></tr><tr><td> Econo-Kolon </td><td> Bio-Rad </td><td> 737-0717 </td><td> 0.7cm ID paket / 2 </td></tr><tr><td> Hidroksiapatit </td><td> Bio-Rad </td><td> 130-0520 </td><td> DNA Sınıf Bio-Gel HTP Jel, 100g/td&gt; </tr><tr><td> Sodyum Fosfat, mono </td><td> VWR </td><td> VW1497-01 </td><td> Monohidrat, Kristal 500g </td></tr><tr><td> Sodyum Fosfat, Dibazik </td><td> VWR </td><td> VW1496-01 </td><td> Susuz, Toz 500g </td></tr><tr><td> DEPC-tedavi Su </td><td> Invitrogen </td><td> AM9922 </td><td> 1L </td></tr><tr><td> % 10 SDS Çözüm </td><td> Invitrogen </td><td> 24730-020 </td><td> Ultrapure, 1l </td></tr><tr><td> 0,5 M EDTA </td><td> Invitrogen </td><td> AM9262 </td><td> pH 8.0, 1L </td></tr><tr><td> Sigmacote </td><td> Sigma-Aldrich </td><td> SL2-25ml </td><td></td></tr><tr><td> 2ml serolojik pippette </td><td> VWR </td><td> 89130-884 </td><td> Polistiren, Steril </td></tr><tr><td> BD Falcon Santrifüj Tüpleri </td><td> VWR </td><td> 21008-936 </td><td> 15ml, Steril </td></tr><tr><td> Fenol: Kloroform: izoamil Alkol (25:24:1 v / v / v) </td><td> Invitrogen </td><td> 15593-031 </td><td> Ultrapure, 100ml </td></tr><tr><td> Amicon Ultra-4 Santrifüj Aygıt </td><td> Millipore </td><td> UFC803024 </td><td> Ultracel-30 membran </td></tr><tr><td> 20X TE Tampon, Rnase ücretsiz </td><td> Invitrogen </td><td> T11493 </td><td> 100ml </td></tr><tr><td> Glycoblue </td><td> Invitrogen </td><td> AM9516 </td><td> 15mg/ml </td></tr></tbody></table>

separation of single-stranded dna, double-stranded dna and rna from an environmental viral community using hydroxyapatite chromatography

Virüsler, özellikle bakteriyofajlar (faj), Dünya&#39;nın 1,2 en çok sayıda biyolojik varlıklar . Virüsler konak hücre bolluğu ve çeşitliliği, besin bisiklet modüle katkıda hücre fenotipi ev sahipliği değiştirebilir ve lateral transferi yoluyla genleri 3 konak hücre ve viral toplulukların evrim etkisi. Çeşitli çalışmalar, virüsler ve doğal ortamlarında çeşitli fonksiyonel potansiyeli şaşırtıcı genetik çeşitlilik sermiştir. Taksonomik çeşitlilik ve üyeleri, tek iplikli DNA (ssDNA), çift iplikli DNA (dsDNA) ve RNA genotipleri 4-9 içeren karmaşık viral toplulukları fonksiyonel bir potansiyel çalışma Metagenomic teknikleri kullanılmıştır. Şu kütüphane inşaatı çevre DNA içeren virüsler veya RNA içeren çalışmada kullanılan protokolleri nontargeted 10 şablonları kaldırmak için bir başlangıç ​​nukleaz tedavisi gerektirir. Ancak, kapsamlı bir anlayış, virüs topluluk ve virüs çeşitliliği kolektif gen kompleman genom kompozisyonu ne olursa olsun, tüm üyelerinin bilgi gerektirir. Nükleik asit alt tipleri saflaştırılmış Fraksiyonu, toplumun genetik imza bir alt kümesi ödün vermeden viral toplulukları incelemek için etkili bir mekanizma sağlar. Hidroksiapatit, kristal formu, kalsiyum fosfat, 1960&#39;larda 11 yılından bu yana, nükleik asitlerin ayrılması, protein ve mikropların yanı sıra istihdam edilmiştir . + Pozitif yüklü Ca 2 arasındaki ücret etkileşimini exploit hidroksiapatit ve nükleik asit alt tiplerinin negatif yüklü fosfat omurgası iyonlar, tercihen diğerlerinden bağımsız olarak her bir nükleik asit alt tipi Zehir mümkündür. Bu stratejinin istihdam Biz son zamanlarda, DNA dizi analizi 12 hazırlanması ssDNA, dsDNA ve RNA içeren virüsler bağımsız damıtmak genomların . Burada, hidroksiapatit kromatografileri kullanarak karışık viral toplulukları ssDNA, dsDNA ve RNA viral nükleik asitlerin fraksiyonasyon ve kurtarma için bir yöntem mevcut.

Watch this Scientific Journal Video about Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography at JoVE.com

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

Biz çevre viral toplulukların tek iplikli DNA, çift iplikli DNA ve RNA molekülleri ayırmak için etkili bir yöntem açıklanmaktadır. Nükleik asitler, fosfat içeren tamponlar artan konsantrasyonları ile hidroksiapatit kromatografisi kullanılarak fraksiyone. Bu yöntem, tüm çevre örneklerinden viral nükleik asit türleri izolasyonu izin verir.

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, ...

separation-single-stranded-dna-double-stranded-dna-rna-from-an

Research

JoVE Journal

Immunology and Infection

31.4K Görüntüleme.  The J. Craig Venter Institute. Biz çevre viral toplulukların tek iplikli DNA, çift iplikli DNA ve RNA molekülleri ayırmak için etkili bir yöntem açıklanmaktadır. Nükleik asitler, fosfat içeren tamponlar artan konsantrasyonları ile hidroksiapatit kromatografisi kullanılarak fraksiyone. Bu yöntem, tüm çevre örneklerinden viral nükleik asit türleri izolasyonu izin verir.

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

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

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 ...

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering (si)RNAs to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for a variety of diseases including HIV. Many previous reports have shown that novel RNAi-based anti-HIV/AIDS therapeutic strategies have considerable promise; however, a key obstacle to the successful therapeutic application and clinical translation of siRNAs is efficient delivery. Particularly, considering the safety and efficacy of RNAi-based therapeutics, it is highly desirable to develop a targeted intracellular siRNA delivery approach to specific cell populations or tissues. The HIV-1 gp120 protein, a glycoprotein envelope on the surface of HIV-1, plays an important role in viral entry into CD4 cells. The interaction of gp120 and CD4 that triggers HIV-1 entry and initiates cell fusion has been validated as a clinically relevant anti-viral strategy for drug discovery. 
Herein, we firstly discuss the selection and identification of 2'-F modified anti-HIV gp120 RNA aptamers. Using a conventional nitrocellulose filter SELEX method, several new aptamers with nanomolar affinity were isolated from a 50 random nt RNA library. In order to successfully obtain bound species with higher affinity, the selection stringency is carefully controlled by adjusting the conditions. The selected aptamers can specifically bind and be rapidly internalized into cells expressing the HIV-1 envelope protein. Additionally, the aptamers alone can neutralize HIV-1 infectivity. Based upon the best aptamer A-1, we also create a novel dual inhibitory function anti-gp120 aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. Further, we utilize the gp120 aptamer-siRNA chimeras for cell-type specific delivery of the siRNA into HIV-1 infected cells. This dual function chimera shows considerable potential for combining various nucleic acid therapeutic agents (aptamer and siRNA) in suppressing HIV-1 infection, making the aptamer-siRNA chimeras attractive therapeutic candidates for patients failing highly active antiretroviral therapy (HAART).

Several 2&#x2019;-Fluoro RNA aptamers against HIV-1Ba-L gp120 with nanomole affinity are isolated from a RNA library by in vitro SELEX procedure. A new dual inhibitory function anti-gp120 aptamer-siRNA chimera is created and shows considerable promise for systemic anti-HIV therapy.

Hücre tipi siRNA teslimat için özel anti-HIV gp120 aptamers Geliştirme

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering ...

Preparation of Viral DNA from Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.1,2,3 
This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 &mu;g or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. 
Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 106 PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 108 plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.6 For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 &mu;l of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate).
Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.

We describe the process of isolating high purity herpesvirus nucleocapsid DNA from infected cells. The final DNA captured from solution is of high concentration and purity, making it ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections to produce new viral recombinants.

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. ...

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 ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

Ana-Hücre RNA sentezi üzerinde Viral Enfeksiyon Etkisi Tedbir tıklayın Kimya Kullanımı

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

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 ...

Development of an Economical DNA Delivery System by "Acufection" and its Application to Skin Research

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a fascinating approach to investigate immune modulation of cutaneous inflammation in mouse models of human diseases. Here we present a cost-effective protocol that delivered naked DNA in mouse skin and leads to transgene expression. The method is coined &#34;acufection&#34;, denoting acupuncture-mediated DNA transfection. To perform acufection, mouse skin was first infused with DNA in phosphate-buffered saline (PBS) and then pricked lightly with a bundle of acupuncture needles to facilitate the absorption of DNA and transfection into cells. The plasmid DNA is presumably taken up by the keratinocyte and dendritic cells (DCs) in the skin and expressed into protein. Mechanical prick with the needles per se did not cause skin damage or induce keratinocyte activation. The expression of the transfected genes was detected in the skin at both transcriptional and translational levels following acufection for 2 days and maintained up to 7 days. The primary goal for the development of this acufection method was to investigate a previously undefined isoform of IL-15. Using this method, an alternatively spliced IL-15 isoform with partially deleted exon 7 (IL-15&#916;E7) was expressed in the skin and subsequently treated with a Toll-like receptor 7 (TLR7) agonist, imiquimod (IMQ), to induce inflammation. Acufection-delivered IL-15&#916;E7 in skin suppressed keratinocyte proliferation, epidermal thickness and neutrophil recruitment in IMQ-induced cutaneous inflammation. With increasing interest in identifying the regulatory mechanisms of cutaneous inflammation, the protocol described here provides a cost effective and versatile alternative to the gene gun system or microseeding for DNA delivery in vivo. It may potentially allow discovery of the function of a novel gene in the skin or for investigating new treatment for cutaneous diseases.

Development of an Economical DNA Delivery System by &quot;Acufection&quot; and its Application to Skin Research

This protocol is a cost-effective alternative for expressing naked plasmid DNA in mouse skin. The overall goal of the protocol is to deliver immune-related genes into skin tissue to delineate the functional role of a specific gene in cutaneous inflammation.

&quot;Acufection&quot; ve Cilt Araştırmaları onun Uygulaması tarafından bir Ekonomik DNA Yerleştirme Sisteminin Geliştirilmesi

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.