We thank C.C.R. laboratory members for comments and S. Moskowitz for figure preparation. This work was supported by National Institutes of Health Grants F32GM101761 (A.J.H.) and GM54397 (C.C.R.).

NOT: dahil ancak, eldiven, koruyucu gözlük, laboratuvar önlüğü ve uygun akrilik kalkan olarak, sınırlı kişisel koruyucu ekipman kullanımı değil, radyoaktif malzemenin güvenli kullanımı ve bertarafı ile ilgili tüm kurumsal düzenlemelere uyun. Not: standart tampon 40 mM HEPES-KOH, pH 7.5, 50 mM potasyum glutamat, 5 mM DTT, 0.1 mM EDTA (Bu tampon 5x konsantrasyonda önceden yapılır) ve her dNTP 0.3 mM oluşur. T7 kopyalama proteinleri, 10 9, 8 tarif edildiği gibi saflaştırılır. Tek bir T7 primaz tanıma sitesi (5&#39;-GGGTC-3 &#39;, 5&#39;-GTGTC-3&#39;, 5&#39;-TGGTC-3 &#39;) ihtiva eden tek şeritli DNA, DNA sentezi, çeşitli şirketler (uzunluğu satın alınabilir ssDNA T7 primaz için, tercih edilen bir enzim bağlı olabilir, bir pentamer tam uzunlukta bir astar sentezlenmesi için en az bir şablon uzunluğu 1Şifreli Cı esas olarak astar polimerazı ile genişletilmesi için ise 1, ancak, ek nükleotidleri şablonunun 3 &#39;ucunda mevcut olması gerekmektedir. Reaksiyonlar, 10 mM MgCl2 nihai bir konsantrasyona ilave edilmesi ile başlatılır ve 25 ° C sıcaklıkta inkübe edilir. Manuel örnekleme 5 saniye veya daha fazla zaman noktaları için iyi çalışır. daha kısa zaman noktası gerekir, doğru veri elde etmek için bir hızlı soğutma akış aleti kullanmak tavsiye edilir. Manuel Örnekleme yoluyla 1. Çoklu-ciro Primer Sentez Reaksiyonu <ol><li> Deneye başlamadan önce, formamid boya tamponu kısım 2 uL (% 93 (h / h) formamid, 50 mM EDTA,% 0.01 iksilen siyanol, mavi,% 0.01 bromofenol) 8 1.5 ml polipropilen tüplere. Genel olarak, tüp sayısı analiz zaman noktalarının sayısına eşittir. </li><li> , 1 x tampon, 0.1 mM ATP ve CTP, 0.25 mCi / ml [32P α-] CTP oluşan bir 20 uL karışım hazırlayın 0.1 uM SSD(Heksamer konsantrasyon olarak ifade - 0.6 mcM monomer) NA şablon ve 0.1 uM GP4. </li><li> 5 dakika boyunca oda sıcaklığında inkübe karışımı. </li><li> Bir pipet kullanarak karışımı 2 mcL çıkarın ve hiçbir tepki kontrolü (zaman sıfır) için adım 1.1 hazırlanan 1.5 ml tüp içinde 2 mL formamid boya tamponu ile karıştırın. </li><li> (10 mM&#39;lik nihai bir konsantrasyona kadar), reaksiyon karışımına 0.1 M MgCl2 2 uL ekleyin. </li><li> El ile 10 s aralıklarla pipet kullanarak reaksiyon karışımlarının 2 uL örneklerinin alınması ve adım 1.1 hazırlanan 1.5 ml tüpler içinde predispensed formamid boya ile karıştırın. </li><li> ısı bloğu 5 dakika boyunca 95 ° C de bir ısı örnekleri. </li><li> Bir denatüre jel üzerine örneklerin hacimde yükleyin. bisakrilamid T7 primaz tarafından sentezlenen, küçük tetraribonucleotide astar ürünleri 0.4 mm, kalın% 25 akrilamid çözümlenir (19: 1), 3 M üre, 1 x TBE (100 mM Tris-borat, 1mM EDTA). </li><li> Kromatografi üzerinde tüm jel yerleştirinplastik örtü ile phy kağıt ve kapak. Bir jel kurutucu kullanılarak kuru jel. </li><li> Reaksiyon karışımının geri kalan kısmının bir seyreltme serisi hazırlamak ve kromatografi kağıdı üzerine jel üzerine yüklenmiş aynı ses nokta, yani, numunenin 4 uL yüklendiğindekiyle seyreltme 4 ul nokta. NOT: Bu reaksiyon sırasında oluşan ürün konsantrasyonu belirlemek için standart bir eğri olarak hizmet edecektir. Örneğin, TE tamponu (10 mM Tris-HCI, pH 8.0, 1 mM EDTA), bir dizi yayılan iyi iş kullanarak beş kat seyreltme 1:25 ila 1: 15.625 (5 2 -5 6), ama seyreltme olmalıdır aktivitenin seviyesine bağlı olarak ayarlanabilir. </li><li> Bir Phosphorimager ekranını kullanarak kuru jel ve standart Açığa. 12 dilüsyon seri radyoaktivite ölçme ve 8 açıklandığı gibi bir standart eğri oluşturmak Not: Reaksiyon karışımına, T7 DNA polimerazı dahil edilmesi, bir eksa sağlarbenim bu enzim tarafından tetraribonucleotide primerlerin kullanımı. En uygun olarak, polimeraz konsantrasyonu verilerin yorumlanması kolaylaştırmak için doyurarak olmalıdır. 0.4 uM ya da daha yüksek bir polimeraz konsantrasyonu iyi sonuçlar verir. Ek olarak, astar sentezi veya gecikmeli iplikli başlatılması, ya da tek sarmallı DNA bağlayıcı proteinler gibi başka proteinler, etkisi bu şekilde incelenebilir. </li></ol> Bir Rapid-söndürme Enstrüman kullanma 2. Çoklu-ciro Primer Sentez Reaksiyonu <ol><li> Reaksiyon karışımlarının hazırlanması <ol><li> 1x tampon, 0.2 mM ATP ve CTP (CTP [32 P α-] 0.5 mCi / mL olmak üzere 0.2 uM GP4 heksamer, 0.2 uM ssDNA şablonu, 0.6 mM dNTP içeren, çözelti A 400 mcL hazırlayın. </li><li> 1x tamponu ve 20 mM MgCl2 ihtiva eden çözelti, B 400 uL hazırlayın. </li></ol></li><li> Hızlı söndürme akış aracının operasyon <ol><li> rap açınid-söndürme akış enstrüman; Ana menü görünene sonra, cihaz klavyesinde tip 7 ana konumuna şırınga sürücüsü taşımak için. </li><li> &quot;LOAD&quot; Açık tampon şırınga pozisyon. </li></ol></li><li> Yükleme tampon şırıngalar <ol><li> Sürücü şırınga noktasına 10 ml şırınga içeren tampon takın. &quot;LOAD&quot; şırınga topuzu konumunu değiştirin. </li><li> 10 ml şırınga kullanılarak, 10 mM Tris, pH 7.5, 1 mM EDTA ile gösterge tampon hazne A ve B dolum ve söndürme çözeltisi (250 mM EDTA ve% 0.2 SDS) şırınga C doldurun. ve dışarı pistonları iterek hava kabarcıklarını çıkarın. </li><li> &quot;YANGIN&quot; değiştirin topuzu pozisyonları. Tip 2 şırınga sürücü çubuğunun konumunu ayarlamak için. Basın tampon çıkış döngü çıkana kadar şırınga düşürmek için düğmeye &quot;AŞAĞI AYARLA&quot;: (sürekli aşağı 7-gider; 8-büyük bir adım aşağı, 9-küçük bir adım aşağı). Ana menüye dönmek için &quot;ESC&quot; yazın. </li></ol></li><li> yıkama boru <ol><li> Avvakum ach çıkış hattı. Örnek topuzlar için &quot;FLUSH&quot; olarak değiştirin. yatay konuma düğmeleri ayarlayarak yakın tampon şırınga. Vakum açın ve 10 saniye süreyle H 2 O 2 floş döngüler daldırma, daha sonra 10 saniye boyunca MeOH batırın. ~ 15 sn için hava emerek kuru döngüler, veya kuru kadar. </li></ol></li><li> yükleme örnekleri <ol><li> &quot;LOAD&quot; Change örnek topuzu pozisyonu. 1 ml şırınganın içine çözüm A yerleştirin. Numune yük limanlarından birine şırınga takın. Çözelti B için tekrarlayın ve ters örnek yük limanında yerleştirin. </li></ol></li><li> Zaman noktalarında toplanması <ol><li> Ana menüde, söndürme akış çalıştırmak için 1 yazın. Reaksiyon zamanı (ler) ve Enter tuşuna basın, &quot;ENTER&quot;. Reaksiyon döngü numarası gösterilir kullanmak için. Gerekli reaksiyon döngü geçin. Adım 2.2.4 tekrarlayın yıkama işlemi. </li><li> Örnek topuzlar için &quot;LOAD&quot; çevirin. Yük reaksiyonu sadece merkezi karıştırma bölmesinin dışına kadar örnek döngüler içinde karışır. tüm düğmelerini açmak için &quot;FIRE &quot;. Bütün kolları doğru konumda olduğundan emin olun. </li><li> çıkış döngü ucuna 1.5 mL tüp yerleştirin. Basın reaksiyonu çalıştırmak için &quot;GO&quot;. Adım 2.2.4 tekrarlayın yıkama işlemi. Onlar sürücü vurmak kadar tampon şırıngalar A ve B güncelle. </li><li> İstenen her zaman noktası için adımları yineleyin 2.2.6.2-2.2.6.3. İstisna: Başlangıç reaktifi sıfır zaman noktası kontrolünü söndürülmesi (yani, bu durumda MgCl2) içeren solüsyon yüklemeyin. </li><li> örneklerin toplanması bittiğinde, ana menüye dönmek için &quot;ESC&quot; girin. Basın &quot;7&quot; başlangıç ​​konumuna tampon şırınga sürücüsünü dönün. </li></ol></li><li> Enstrüman temizlik <ol><li> Reaksiyon karışımı şırınga çıkarın. örnek yükleme düğmesini &quot;LOAD&quot; pozisyon çevirin. Vakum altında 10 ml 0.3 NH3 PO 4 ve 10 mL MeOH ve ardından 10 ml 0.2 N NaOH ile her bir örnek döngü yıkayın. </li><li> için &quot;LOAD&quot; pozisyon şırınga topuzlarını çevirin. 3 Syr bastırıninge pistonları reaksiyon ve soğutma tamponlar almak için. Yıkama en az iki kez 5 ml H2O ile şırınga. </li><li> 5 ml H2O ile tekrar şırınga doldurun ve &quot;YANGIN&quot; pozisyon topuzlarını çevirin. Topuzu pozisyon değiştirmek ve adım 2.2.4 deki gibi yıkayın H 2 O. çıkarmak için vakum açın. Cihazı kapatın. Not: adımda 1,7-1,12 tarif edildiği gibi ürünler analiz edildi. Buna ek olarak, T7 DNA polimerazı ve kopyalama proteinleri primer sentezi veya uzantı üzerindeki etkisini analiz etmek için, reaksiyon karışımına ilave edilebilir. </li></ol></li></ol> 3. Tek-ciro Primer Sentez Reaksiyonu NOT: Tek ciro primer sentezi / uzatma deneyleri birkaç büyük farklılıklar, çoklu-ciro reaksiyonlar benzer yürütülmektedir: Bu analizler substratların ancak konsantrasyon, astar veya uzatma ürünleri içine radyoaktif işaretli ATP dönüşüm izleyin ve proteinler considera vardırbly kadar yüksek olur. Buna ek olarak, primer sentezi reaksiyon analizine izin milisaniyelik çözünürlük elde etmek amacıyla, tek bir hızlı-soğutma akış makinesi kullanmak gerekir. <ol><li> Reaksiyon karışımlarının hazırlanması. <ol><li> 1x tamponu, 3 uM GP4 heksamer 6 uM ssDNA şablonu, 0.6 mM dNTP, 1 mM CTP ve 2 nM [P32 γ-] ATP ihtiva eden çözelti, 400 uL hazırlayın. 1x tamponu ve 20 mM MgCl2 ihtiva eden çözelti, B 400 uL hazırlayın. </li></ol></li><li> Hızlı-soğutma akış işlemi <ol><li> Adım 2.2 &#39;de açıklandığı gibi reaksiyonlar yürütmek. Eğer arzu edilirse, sırasıyla 15 uM ve 20 uM arasında bir konsantrasyonda T7 DNA polimeraz ya da T7 tek kollu DNA bağlanma proteini ekleyin. Bu durumda, uzantı ürünleri reaksiyonun elle örnekleme izin veren bir zaman rejimi birikirler. </li></ol></li></ol> 4. Veri Analizi <ol><li> doğrusal olmayan, en hafif sığdır ürün ilerleme eğrilerien küçük kareler yetenekli uyan ve / veya sayısal entegrasyon ticari olarak mevcut yazılım kullanarak kareler modelleri. operasyonun ayrıntıları kullanılan yazılım ile değişir, ancak aşağıda verileri montaj için kullanılan denklemlerin genelleştirilmiş biçimleridir olacaktır. </li><li> lineer bir denklem haline eğrileri ilerlemeye uyan birden fazla devir primer sentezi reaksiyonlarının ürün oluşumunun hızını belirler. Önemli bir kavis ise, tek bir üstel fonksiyon verileri uydurularak başlangıç oranı yöntemi 13, 14 kullanımı: burada: Y (e - k t) = y ürün konsantrasyonu, bir tepkime genlik, k mi gözlenen hız sabiti, t saniye cinsinden zamanı. zaman = 0 teğet çizginin eğimi başlangıçtaki oranıdır. NOT: Bir doğrusal denklem ya da önceden kararlı durum patlama denklemine Fit öncesi kararlı durum primer sentezi reaksiyonları y A (e = - k metTepkime genliği ilk t) + oran ss t, k patlama öncesi kararlı durum patlaması için gözlenen oran sabiti, oran ss kararlı durum oranı ss = k cat [Enzim]) &#39;dir, t zamanı , saniyeler içinde. (- K t e) Tek ciro primer sentezi reaksiyonları tek üstel fonksiyon y = A eğrileri ilerleme takın. GP4 durumunda, ticari olarak temin edilebilen yazılımı kullanılarak, her bir ara enzim ile denge içinde olan, üç NTP kondansasyon adımda, bir model için, sayısal entegrasyonu 15 verileri uygun. </li></ol>

<ol>
	<li>Kornberg, A., Baker, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> DNA replication. , (1992).</li><li>Hamdan, S. M., Richardson, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motors,+switches,+and+contacts+in+the+replisome.">Motors, switches, and contacts in the replisome.</a> Annu Rev Biochem. 78, 205-243 (2009).</li><li>Mendelman, L. V., Notarnicola, S. M., Richardson, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+bacteriophage+T7+gene+4+proteins+in+providing+primase+and+helicase+functions+in+vivo.">Roles of bacteriophage T7 gene 4 proteins in providing primase and helicase functions in vivo.</a> Proc Natl Acad Sci U S A. 89 (22), 10638-10642 (1992).</li><li>Qimron, U., Lee, S. J., Hamdan, S. M., Richardson, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primer+initiation+and+extension+by+T7+DNA+primase.">Primer initiation and extension by T7 DNA primase.</a> EMBO J. 25 (10), 2199-2208 (2006).</li><li>Kato, M., Ito, T., Wagner, G., Richardson, C. C., Ellenberger, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modular+architecture+of+the+bacteriophage+T7+primase+couples+RNA+primer+synthesis+to+DNA+synthesis.">Modular architecture of the bacteriophage T7 primase couples RNA primer synthesis to DNA synthesis.</a> Mol Cell. 11 (5), 1349-1360 (2003).</li><li>Kusakabe, T., Richardson, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+4+DNA+primase+of+bacteriophage+T7+mediates+the+annealing+and+extension+of+ribo-oligonucleotides+at+primase+recognition+sites.">Gene 4 DNA primase of bacteriophage T7 mediates the annealing and extension of ribo-oligonucleotides at primase recognition sites.</a> J Biol Chem. 272 (19), 12446-12453 (1997).</li><li>Lee, S. J., Zhu, B., Hamdan, S. M., Richardson, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+sequence-specific+template+binding+by+the+DNA+primase+of+bacteriophage+T7.">Mechanism of sequence-specific template binding by the DNA primase of bacteriophage T7.</a> Nucleic Acids Res. 38 (13), 4372-4383 (2010).</li><li>Hernandez, A. J., Lee, S. J., Richardson, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primer+release+is+the+rate-limiting+event+in+lagging-strand+synthesis+mediated+by+the+T7+replisome.">Primer release is the rate-limiting event in lagging-strand synthesis mediated by the T7 replisome.</a> Proc Natl Acad Sci U S A. 113 (21), 5916-5921 (2016).</li><li>Tabor, S., Richardson, C. 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+single+residue+in+DNA+polymerases+of+the+Escherichia+coli+DNA+polymerase+I+family+is+critical+for+distinguishing+between+deoxy-+and+dideoxyribonucleotides.">A single residue in DNA polymerases of the Escherichia coli DNA polymerase I family is critical for distinguishing between deoxy- and dideoxyribonucleotides.</a> Proc Natl Acad Sci U S A. 92 (14), 6339-6343 (1995).</li><li>Kim, Y. T., Tabor, S., Bortner, C., Griffith, J. D., Richardson, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+characterization+of+the+bacteriophage+T7+gene+2.5+protein.+A+single-stranded+DNA-binding+protein.">Purification and characterization of the bacteriophage T7 gene 2.5 protein. A single-stranded DNA-binding protein.</a> J Biol Chem. 267 (21), 15022-15031 (1992).</li><li>Frick, D. N., Richardson, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+primases.">DNA primases.</a> Annu Rev Biochem. 70, 39-80 (2001).</li><li>Lee, S. J., Richardson, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+lysine+residues+in+the+RNA+polymerase+domain+of+the+gene+4+primase-helicase+of+bacteriophage+T7.">Essential lysine residues in the RNA polymerase domain of the gene 4 primase-helicase of bacteriophage T7.</a> J Biol Chem. 276 (52), 49419-49426 (2001).</li><li>Cao, W., De La Cruz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+full+time+course+analysis+of+nonlinear+enzyme+cycling+kinetics.">Quantitative full time course analysis of nonlinear enzyme cycling kinetics.</a> Sci Rep. 3, 2658 (2013).</li><li>Wang, M. H., Zhao, K. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+determining+kinetic+constants+of+complexing+inactivation+at+identical+enzyme+and+inhibitor+concentrations.">A simple method for determining kinetic constants of complexing inactivation at identical enzyme and inhibitor concentrations.</a> FEBS Lett. 412 (3), 425-428 (1997).</li><li>Johnson, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fitting+enzyme+kinetic+data+with+KinTek+Global+Kinetic+Explorer.">Fitting enzyme kinetic data with KinTek Global Kinetic Explorer.</a> Methods Enzymol. 467, 601-626 (2009).</li><li>Johnson, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+century+of+enzyme+kinetic+analysis,+1913.">A century of enzyme kinetic analysis, 1913.</a> FEBS Lett. 587 (17), 2753-2766 (2013).</li><li>Biswas, T., Resto-Roldan, E., Sawyer, S. K., Artsimovitch, I., Tsodikov, O. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+non-radioactive+primase-pyrophosphatase+activity+assay+and+its+application+to+the+discovery+of+inhibitors+of+Mycobacterium+tuberculosis+primase+DnaG.">A novel non-radioactive primase-pyrophosphatase activity assay and its application to the discovery of inhibitors of Mycobacterium tuberculosis primase DnaG.</a> Nucleic Acids Res. 41 (4), e56 (2013).</li><li>Sanyal, G., Doig, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+DNA+replication+enzymes+as+targets+for+antibacterial+drug+discovery.">Bacterial DNA replication enzymes as targets for antibacterial drug discovery.</a> Expert Opin Drug Discov. 7 (4), 327-339 (2012).</li></ol>

The authors have nothing to disclose.

Bu deneyler en önemli faktör son derece aktif saflaştırılmış enzim mevcudiyetidir. GP4 ile çalışma sırasında, örneğin, -20 ° C&#39;de% 50 gliserol ihtiva eden tampon maddesi içinde saflaştırılmış enzim depolama (20 mM potasyum fosfat, pH 7.4, 0.1 mM EDTA, 1 mM DTT) içinde bulunan bir azalmaya yol açar birkaç ay boyunca preparatın spesifik aktivitesi. Bu nedenle, şu anda, saflaştırılmış 25 mM Tris-HCI, pH 7.5, 50 mM NaCI, 0.1 mM EDTA, 1 mM TCEP içinde GP4 ve% 10 gliserol flaş dondurma, k?...

DNA replikasyon bütün hücresel yaşam korunmuş bir özelliğidir. Çoğaltma bileşenlerinin kimlik ve sayısı çok çeşitlilik gösterirken, genel mekanizmalar evrim 1 arasında paylaşılır. Burada, T7 replizom bileşenleri ile in vitro koşullarında geri kalmış iplikli DNA sentezinin kinetik anlamaya yönelik radyoaktif substratlar kullanılarak jel bazlı, sürekli olmayan kinetik deneyler tarif eder. Bakteriyofaj T7 çoğaltma makineleri sadece dört protein (DNA polimeraz, GP5, ve processivity faktörü, E coli tiyoredoksin, iki fonksiyonlu primaz-sarmal GP4, ve tek sarmallı DNA bağlama proteini, GP2 oluşan çok basittir. 5) 2. Bu özellik, DNA replikasyonu katılan korunmuş biyokimyasal mekanizmaları incelemek için çekici bir model sistem yapar. Özel önem, T7 DNA ile ribonükleotid primerlerin oluşumunun yerleştirilir bir Critica primazDNA sentezinin başlamasından l adımı. Buna ek olarak, bir de, reaksiyon karışımına ekleyerek, T7 DNA polimerazı veya başka kopyalama proteinlerine bu primerlerin kullanımı incelenebilir. T7 primaz-helikaz, GP4 primaz tanıma siteleri ya da PRS olarak adlandırılan özel DNA dizileri DNA sentezi için primerlerin oluşmasını (5&#39;-GTC-3 &#39;) katalize eder. PRS sitozin sitenin tanınması için gerekli olan, şifreli, ancak ürünün 3 kopyalanır değildir. GP4 ile astar sentezlenmesinde birinci basamak, daha sonra bir trimer genişletilmiş ve fonksiyonel olarak tetranukleotıd primerlere pppACCC, pppACCA veya pppACAC şablon 4 sekansına bağlı olan dinükleotidin pppAC, oluşturulmasını içerir. Bu primerler daha sonra da GP4 destekli proses 5, 6, DNA sentezini başlatmak için T7 DNA polimerazı ile kullanılabilmektedir. Bu bağlamda, primazetki, çıkmalarını engelleyen, şablonla son derece kısa tetraribonucleotides stabilize polimeraz aktif bölge 7 içine astar / şablon güvence için elverişli bir şekilde DNA polimeraz yürütmektedir. Bu adımlar (RNA primer sentezi, polimeraz primer yayınım ve uzatma) gecikmeli iplikçik çoğaltmak için birden fazla döngüleri tekrarlanır ve lider sarmalı replikasyonu ile koordine edilmelidir. Burada tarif edilen deneyler, son derece hassas olan ve bir orta seviyede bir süre içinde gerçekleştirilebilir. Ancak, nispeten düşük verimlilik ve büyük bir dikkatle radyoaktif maddelerin kullanımı ve bertaraf olunmalıdır. Reaksiyon, bir sabit veya önceden kararlı durumda ya reaksiyon zamanı derslerin anlamlı analiz için mükellef zaman ölçeklerinde örnekleri ulaşmak için hızlı-söndürme cihazı istihdam olabilir hangi hızına bağlı olarak. Son zamanlarda, biz deneyleri Evidenc sağlamak için burada açıklanan kullanılanOkazaki parçaları başlamasında GP4 ve primaz alanından astar salınımının önemi, e. Ayrıca, verimli astar oluşumu ve kullanımı 8. teşvik, protein, gp2.5 bağlayıcı T7 tek iplikli DNA için bir düzenleyici rolünün kanıt bulunamadı.

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			<td height="63" style="height:63px;width:216px;">(Ethylenedinitrilo) Tetraacetic acid, disodium salt, dihydrate</td>
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			<td>radioactive, take protective measures</td>
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			<td>radioactive, take protective measures</td>
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			<td>custom sequences&#160;</td>
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			<td height="21" style="height:21px;width:216px;">cylene xyanol&#160;</td>
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			<td style="width:119px;">X4126&#160;</td>
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			<td height="63" style="height:63px;width:216px;">Boric acid</td>
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			<td height="42" style="height:42px;width:216px;">Rapid Quench-Flow Instrument&#160;</td>
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			<td height="42" style="height:42px;width:216px;">Kintek Explorer</td>
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			<td height="42" style="height:42px;width:216px;">Kaleidagraph&#160;</td>
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			<td style="width:119px;">N/A</td>
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kinetics of lagging-strand dna synthesis in vitro by the bacteriophage t7 replication proteins

Here we provide protocols for the kinetic examination of lagging-strand DNA synthesis in vitro by the replication proteins of bacteriophage T7. The T7 replisome is one of the simplest replication systems known, composed of only four proteins, which is an attractive feature for biochemical experiments. Special emphasis is placed on the synthesis of ribonucleotide primers by the T7 primase-helicase, which are used by DNA polymerase to initiate DNA synthesis. Because the mechanisms of DNA replication are conserved across evolution, these protocols should be applicable, or useful as a conceptual springboard, to investigators using other model systems. The protocols described here are highly sensitive and an experienced investigator can perform these experiments and obtain data for analysis in about a day. The only specialized piece of equipment required is a rapid-quench flow instrument, but this piece of equipment is relatively common and available from various commercial sources. The major drawbacks of these assays, however, include the use of radioactivity and the relative low throughput.

El ile birden fazla devir koşulları altında GP4 tarafından katalize edilen bir primer sentezi reaksiyonu örnekleme ile, örneğin, bir protokol aşama 1 &#39;de tarif edildiği gibi, şekil 1A&#39;da gösterilen sonuçlar elde edilmiştir. Burada, jel elektroforezinden sonra bir ürün yelpazesi primer sentezi reaksiyonlarının (Şekil 1A) α-konumunda 32 P ile etiketlenmiş CTP kullanılarak gözlemlenebilir. i...

Watch this Scientific Journal Video about Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins at JoVE.com

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

We describe sensitive, gel-based discontinuous assays to examine the kinetics of lagging-strand initiation using the replication proteins of bacteriophage T7.

Kaplama DNA sentezinin kinetiği<em&gt; İn Vitro</em&gt; Bakteriyofaj T7 çoğaltma Proteinler tarafından

Here we provide protocols for the kinetic examination of lagging-strand DNA synthesis in vitro by the replication proteins of bacteriophage T7. The T7 ...

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Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

DNA Replication

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7.3K Görüntüleme.  Harvard Medical School. We describe sensitive, gel-based discontinuous assays to examine the kinetics of lagging-strand initiation using the replication proteins of bacteriophage T7. 

Video: Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

In Vitro Transcription Assays and Their Application in Drug Discovery

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process requires multi-subunit DNA-dependent RNA polymerase (RNAP) and a series of transcription factors that act to modulate the activity of RNAP during gene expression. Sequencing gel electrophoresis of radiolabeled transcripts is used to provide detailed mechanistic information on how transcription proceeds and what parameters can affect it. In this paper we describe the protocol to study how the essential elongation factor NusA regulates transcriptional pausing, as well as a method to identify an antibacterial agent targeting transcription initiation through inhibition of RNAP holoenzyme formation. These methods can be used a as platform for the development of additional approaches to explore the mechanism of action of the transcription factors which still remain unclear, as well as new antibacterial agents targeting transcription which is an underutilized drug target in antibiotic research and development.

In Vitro Transcription Assays and Their Application in Drug Discovery

In this manuscript, we describe a protocol to functionally examine transcription and the inhibitory activity of antibacterial agents targeting bacterial transcription.

İn Vitro Transkripsiyon Tahliller ve İlaç Keşfi Onların Uygulama

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process ...

Genetik

Novel RNA-Binding Proteins Isolation by the RaPID Methodology

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern biology. Only a few in vitro and in vivo methods enable the identification of RBPs associated with a particular target mRNA. However, their main limitations are the identification of RBPs in a non-cellular environment (in vitro) or the low efficiency isolation of RNA of interest (in vivo). An RNA-binding protein purification and identification (RaPID) methodology was designed to overcome these limitations in yeast and enable efficient isolation of proteins that are associated in vivo. To achieve this, the RNA of interest is tagged with MS2 loops, and co-expressed with a fusion protein of an MS2-binding protein and a streptavidin-binding protein (SBP). Cells are then subjected to crosslinking and lysed, and complexes are isolated through streptavidin beads. The proteins that co-purify with the tagged RNA can then be determined by mass spectrometry. We recently used this protocol to identify novel proteins associated with the ER-associated PMP1 mRNA. Here, we provide a detailed protocol of RaPID, and discuss some of its limitations and advantages.

RNA-protein interactions lie at the heart of many cellular processes. Here, we describe an in vivo method to isolate specific RNA and identify novel proteins that are associated with it. This could shed new light on how RNAs are regulated in the cell.

Rapid Metodoloji tarafından Roman RNA Bağlayıcı Proteinler İzolasyon

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern ...

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and transmitted from one generation to the next through the female oocyte is of fundamental importance. Because of the genetic tractability, and the elegant, ordered simplicity by which oocyte development proceeds, Drosophila oogenesis has become an invaluable system for mitochondrial study. An EdU (5-ethynyl-2&#180;-deoxyuridine) labeling method was utilized to detect mitochondrial DNA (mtDNA) replication in Drosophila ovaries. This method is superior to the BrdU (5-bromo-2'-deoxyuridine) labeling method in that it allows for good structural preservation and efficient fluorescent dye penetration of whole-mount tissues.
Here we describe a detailed protocol for labeling replicating mitochondrial DNA in Drosophila adult ovaries with EdU. Some technical solutions are offered to improve the viability of the ovaries, maintain their health during preparation, and ensure high-quality imaging. Visualization of newly synthesized mtDNA in the ovaries not only reveals the striking temporal and spatial pattern of mtDNA replication through oogenesis, but also allows for simple quantification of mtDNA replication under various genetic and pharmacological perturbations.

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

Drosophila oogenesis continues to be exceptionally useful in the study of mitochondrial proliferation and inheritance. This manuscript describes a detailed protocol used to label the replicating mitochondrial DNA (mtDNA) in Drosophila adult ovaries with 5-ethynyl-2&#180;-deoxyuridine (EdU), which facilitates uncovering mechanisms associated with mitochondrial inheritance that were previously debatable.

Edu Boyama tarafından Drosophila Yetişkin Yumurtalıkların Mitokondriyal DNA replikasyonu Yerinde Etiketlenmesi içinde

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and ...

Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic region is replicated at a distinct time during S phase through the simultaneous activation of multiple origins of replication. Time of replication (ToR) correlates with many genomic and epigenetic features and is linked to mutation rates and cancer. Comprehending the full genomic view of the replication program, in health and disease is a major future goal and challenge.
This article describes in detail the &#34;Copy Number Ratio of S/G1 for mapping genomic Time of Replication&#34; method (herein called: CNR-ToR), a simple approach to map the genome wide ToR of mammalian cells. The method is based on the copy number differences between S phase cells and G1 phase cells. The CNR-ToR method is performed in 6 steps: 1. Preparation of cells and staining with propidium iodide (PI); 2. Sorting G1 and S phase cells using fluorescence-activated cell sorting (FACS); 3. DNA purification; 4. Sonication; 5. Library preparation and sequencing; and 6. Bioinformatic analysis. The CNR-ToR method is a fast and easy approach that results in detailed replication maps.

We describe here a relatively fast and simple approach for mapping genome-wide mammalian replication timing, from cell isolation to the basic analysis of the sequencing results. A genomic map of a representative replication program will be provided following the protocol.

DNA İçerik Ölçüm tarafından Memeli Çoğaltma Zamanlama genom Tayini

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic ...

Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing biosynthetic pathways. In this manuscript, the use of a highly effective system for the construction of multigene synthetic operons under the control of an inducible T7 RNA polymerase is described. This system allows many genes to be expressed simultaneously from one plasmid. Here, a set of four related vectors, pMGX-A, pMGX-hisA, pMGX-K, and pMGX-hisK, with either the ampicillin or kanamycin resistance selectable marker (A and K) and either possessing or lacking an N-terminal hexahistidine tag (his) are disclosed. Detailed protocols for the construction of synthetic operons using this vector system are provided along with the corresponding data, showing that a pMGX-based system containing five genes can be readily constructed and used to produce all five encoded proteins in Escherichia coli. This system and protocol enables researchers to routinely express complex multi-component modules and pathways in E. coli.

This study describes methods for the T7-mediated co-expression of multiple genes from a single plasmid in Escherichia coli using the pMGX plasmid system.

İndüklenebilir T7 RNA Polimeraz aracılı Çok Boylu İfade Sistemi, pMGX

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing ...

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

Zebra balığı bir Vivo içinde Model sistemi olarak kullanarak DNA çoğaltma zamanlaması profil oluşturma

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

Methodology for Accurate Detection of Mitochondrial DNA Methylation

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert unmethylated cytosine into uracil, in a single-stranded DNA context. Bisulfite sequencing can be targeted (using PCR) or performed on the whole genome and provides absolute quantification of cytosine methylation at the single base-resolution. Given the distinct nature of nuclear- and mitochondrial DNA, notably in the secondary structure, adaptions of bisulfite sequencing methods for investigating cytosine methylation in mtDNA should be made. Secondary and tertiary structure of mtDNA can indeed lead to bisulfite sequencing artifacts leading to false-positives due to incomplete denaturation poor access of bisulfite to single-stranded DNA. Here, we describe a protocol using an enzymatic digestion of DNA with BamHI coupled with bioinformatic analysis pipeline to allow accurate quantification of cytosine methylation levels in mtDNA. In addition, we provide guidelines for designing the bisulfite sequencing primers specific to mtDNA, in order to avoid targeting undesirable NUclear MiTochondrial segments (NUMTs) inserted into the nuclear genome.

Here, we present a protocol to allow accurate quantification of mitochondrial DNA (mtDNA) methylation. In this protocol, we describe an enzymatic digestion of DNA with BamHI coupled with a bioinformatic analysis pipeline which can be used to avoid overestimation of mtDNA methylation levels caused by the secondary structure of mtDNA.

Mitokondrial DNA metilasyonu doğru algılanması için metodoloji

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert ...

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences is desired. We herein provide a detailed in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) protocol to study the RNA structural change in the presence of an experimental drug for spinal muscular atrophy (SMA), survival of motor neuron (SMN)-C2, and in exon 7 of the pre-mRNA of the SMN2 gene. In in vitro SHAPE, an RNA sequence of 140 nucleotides containing SMN2 exon 7 is transcribed by T7 RNA polymerase, folded in the presence of SMN-C2, and subsequently modified by a mild 2&#39;-OH acylation reagent, 2-methylnicotinic acid imidazolide (NAI). This 2&#39;-OH-NAI adduct is further probed by a 32P-labeled primer extension and resolved by polyacrylamide gel electrophoresis (PAGE). Conversely, 2&#39;-OH acylation in in-cell SHAPE takes place in situ with SMN-C2 bound cellular RNA in living cells. The pre-mRNA sequence of exon 7 in the SMN2 gene, along with SHAPE-induced mutations in the primer extension, was then amplified by PCR and subject to next-generation sequencing. Comparing the two methodologies, in vitro SHAPE is a more cost-effective method and does not require computational power to visualize results. However, the in vitro SHAPE-derived RNA model sometimes deviates from the secondary structure in a cellular context, likely due to the loss of all interactions with RNA-binding proteins. In-cell SHAPE does not need a radioactive material workplace and yields a more accurate RNA secondary structure in the cellular context. Furthermore, in-cell SHAPE is usually applicable for a larger range of RNA sequences (~1,000 nucleotides) by utilizing next-generation sequencing, compared to in vitro SHAPE (~200 nucleotides) that usually relies on PAGE analysis. In case of exon 7 in SMN2 pre-mRNA, the in vitro and in-cell SHAPE derived RNA models are similar to each other.

Detailed protocols for both in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) experiments to determine the secondary structure of pre-mRNA sequences of interest in the presence of an RNA-targeting small molecule are presented in this article.

İn vitro ve hücre içi şeklinde küçük molekül araştırmak için kullanarak Pre-mRNA yapısal değişiklikler indüklenen

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...

High-Density DNA and RNA microarrays - Photolithographic Synthesis, Hybridization and Preparation of Large Nucleic Acid Libraries

Photolithography is a powerful technique for the synthesis of DNA oligonucleotides on glass slides, as it combines the efficiency of phosphoramidite coupling reactions with the precision and density of UV light reflected from micrometer-sized mirrors. Photolithography yields microarrays that can accommodate from hundreds of thousands up to several million different DNA sequences, 100-nt or longer, in only a few hours. With this very large sequence space, microarrays are ideal platforms for exploring the mechanisms of nucleic acid&#183;ligand interactions, which are particularly relevant in the case of RNA. We recently reported on the preparation of a new set of RNA phosphoramidites compatible with in situ photolithography and which were subsequently used to grow RNA oligonucleotides, homopolymers as well as mixed-base sequences. Here, we illustrate in detail the process of RNA microarray fabrication, from the experimental design, to instrumental setup, array synthesis, deprotection and final hybridization assay using a template 25mer sequence containing all four bases as an example. In parallel, we go beyond hybridization-based experiments and exploit microarray photolithography as an inexpensive gateway to complex nucleic acid libraries. To do so, high-density DNA microarrays are fabricated on a base-sensitive monomer that allows the DNA to be conveniently cleaved and retrieved after synthesis and deprotection. The fabrication protocol is optimized so as to limit the number of synthetic errors and to that effect, a layer of &#946;-carotene solution is introduced to absorb UV photons that may otherwise reflect back onto the synthesis substrates. We describe in a step-by-step manner the complete process of library preparation, from design to cleavage and quantification.

In this article, we present and discuss new developments in the synthesis and applications of nucleic acid microarrays fabricated in situ. Specifically, we show how the protocols for DNA synthesis can be extended to RNA and how microarrays can be used to create retrievable nucleic acid libraries.

Yüksek Yoğunluklu DNA ve RNA mikrodizileri - Fotolitografik Sentez, Hibridizasyon ve Büyük Nükleik Asit Kütüphanelerinin Hazırlanması

Photolithography is a powerful technique for the synthesis of DNA oligonucleotides on glass slides, as it combines the efficiency of phosphoramidite ...

Use of Frozen Tissue in the Comet Assay for the Evaluation of DNA Damage

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other environmental stressors. Use of the comet assay in regulatory testing for genotoxic potential in rodents has been driven by adoption of an Organisation for Economic Co-operation and Development (OECD) test guideline in 2014. Comet assay slides are typically prepared from fresh tissue at the time of necropsy; however, freezing tissue samples can avoid logistical challenges associated with simultaneous preparation of slides from multiple organs per animal and from many animals per study. Freezing also enables shipping samples from the exposure facility to a different laboratory for analysis, and storage of frozen tissue facilitates deferring a decision to generate DNA damage data for a given organ. The alkaline comet assay is useful for detecting exposure-related DNA double- and single-strand breaks, alkali-labile lesions, and strand breaks associated with incomplete DNA excision repair. However, DNA damage can also result from mechanical shearing or improper sample processing procedures, confounding the results of the assay. Reproducibility in collection and processing of tissue samples during necropsies may be difficult to control due to fluctuating laboratory personnel with varying levels of experience in harvesting tissues for the comet assay. Enhancing consistency through refresher training or deployment of mobile units staffed with experienced laboratory personnel is costly and may not always be feasible. To optimize consistent generation of high quality samples for comet assay analysis, a method for homogenizing flash frozen cubes of tissue using a customized tissue mincing device was evaluated. Samples prepared for the comet assay by this method compared favorably in quality to fresh and frozen tissue samples prepared by mincing during necropsy. Moreover, low baseline DNA damage was measured in cells from frozen cubes of tissue following prolonged storage.

This protocol describes several procedures for preparing high quality frozen tissue samples at the time of necropsy for use in the comet assay to assess DNA damage: 1) minced tissue, 2) scraped epithelial cells from the gastrointestinal tract, and 3) cubed tissue samples, requiring homogenization using a tissue mincing device.

DNA Hasarının Değerlendirilmesinde Kuyruklu Yıldız Testinde Donmuş Dokunun Kullanımı

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other ...