我们感谢克雷格保罗在他的帮助芯片的核心设施。这项工作得到了NIH / NCI的津贴＃CA118591的iMAT计划。民主左翼联盟也承认一个宾夕法尼亚州的财政支持空间格兰特财团奖学金。本刊物是由美国宾夕法尼亚州立大学材料研究所纳米加工网络和美国国家科学基金会合作协议0335765号，国家纳米技术基础设施网络，与美国康乃尔大学的支持。

1。底漆自由选择协议简介使用相应的寡核苷酸（图1和2，步骤a），其中包含30 NT随机域中央两侧由两个引物区域，PCR构建一个双链DNA库。两种略为不同的“引无”（PF）的协议。在双链DNA库，5&#39; -区域包含内切酶“刻痕”内切酶Nt.BstNBI网站;，这种酶识别双链DNA，但切割DNA底物只有一个链。 3&#39; -地区的双链DNA库包含的内切酶Nt.BbvCI的另一个“刻痕”网站，识别双链DNA，但劈开只有一个链，留在3&#39;end（功率因数为1）， ​​以及作为BspMI完工内切酶酶切位点，其中切割股只剩下3&#39;没有额外的核苷酸（PF 2 ）。我们通常采用功率因数为1协议最初（因为它更容易在遴选过程中产生的独立片段，见下文），然后采用PF 2随后的几轮选择协议。 5&#39; - pN30 - CC - 3“的片段（32 +片段指定）和5&#39; - pN30 - 3”片段（30 +片段）30 NT 32 NT Nt.BbvCI / NT分别产生BstNBI或BspMI / Nt.BstNBI卵裂的双链DNA库，凝胶净化。 32 +片段包含30 NT随机域，与CC在3&#39; -末端侧翼序列（功率因数为1）。 30 +片段（PF 2）只包含30个随机NT域序列。凝胶净化（Nt.BbvCI或Nt.BstNBI削减只有上部的“+”链）获得“自我桥”66 - 片段（含5&#39;和3&#39;侧翼序列的随机N30地区）。获得这种自我的桥梁，直接在PF 1协议，或可以生成和分离，切割后的 PF 2协议（图1和2，步骤b）只有NtBbv.CI或Nt.BstNBI DNA库。 32 + - 32 +片段与纯化蛋白质或培养的黑色素瘤细胞共孵育允许的片段结合的蛋白质或细胞，未绑定的碎片被大水冲走（图1和2，步骤c）。约束或选定的片段用于底漆地区重新生成。 杂交/结扎反应，产生自我的桥梁和纯化在​​同一时间为32 + - 30 +片段纯化; 5&#39;端引物库的建设和3&#39; -末端的引物，取代相同相匹配的引物，还包含一个额外的SP6在3&#39; -末端的转录子（图1和2，步骤d）。杂交/结扎反应的产品，然后用于在体外 RNA转录（图1和2，步骤e）。 RNA的转录，结扎的DNA（包括未被选中的自我桥的DNA片段），由DNA酶消化，我以消除干扰的背景序列。反转录（RT）- PCR数据表明，产品的再生底漆有效reamplified，构成一个“圆”的选择（图1和2，步骤f）。背景在没有实时控制PCR扩增循环次数高，可以完全去除额外的DNA酶I消化后没有检测到较低的周期数字，我们经常聘请。 经过7轮的选择，适体结合特性进行了表征。 Kd值均在10-7 10-8 M的范围（图3）。在结合分析，对各种适配表明添加剂具有约束力，表明他们的目标S100B蛋白上的不同地点。因此，我们在“三明治”结合试验测试适配对，无论在玻璃芯片，采用荧光标记的第二个适配（Codelink幻灯片），第二适配衍生的黄金加上50纳米的黄金纳米粒子（纳米金;图3）纳米线。在这两种情况下，结合特异性高：在Codelink芯片，无夹心约束力适配没有表现出添加剂Kd的裁决具有约束力。随着衍生的纳米线，我们观察到对非目标蛋白质几乎没有任何约束力，适体耦合的纳米金（图3）可以通过观察和个人夹心复合物。 2。材料 2.1。生成的PF的束缚片段的DNA文库和Reamplification 详细的描述在2009年泛和克劳森;潘等人，2008年。 2.2。纯化的蛋白质基础的选择<ol><li> 20毫米的Tris - HCl，pH值7.4 </li><li> 32 +片段和30 +片段</li><li>选择缓冲区（氯化钙2 2.5毫米，5毫米MgCl 2的 1X磷酸盐缓冲液，pH值7.4，GIBCO） </li><li>镍NTA琼脂糖珠（QIAGEN） </li><li>聚丙烯列（QIAGEN） </li><li>结合缓冲液（50毫米的Na 2 HPO 4 </suB&gt; -的NaH 2 PO 4，PH7.2，150 mM氯化钠） </li><li>表达，纯化S100B蛋白结合缓冲液（5微克/μL） </li><li>酚：氯仿：IAA（pH值7.9，Ambion公司） </li><li> 3 M醋酸钠，pH值5.2 </li><li> 100％，70％的乙醇</li></ol> 2.3。 TOPO克隆和测序分析选定的双链DNA库<ol><li> 第 7 次轮选中的PCR产物（可以进行额外的轮继续优化适配子具有约束力） </li><li> pCR2.1 - TOPO载体（Invitrogen公司） </li><li> DH5α，组成电池（Invitrogen公司） </li><li>质粒试剂盒（QIAGEN） </li><li> M13正向引物</li><li> Informax的Vector NTI公司（Invitrogen公司） </li></ol> 2.4。结合特性的适配<ol><li>适体和控制的寡核苷酸（N 30 CC和N 30，IDT） </li><li> T4多聚核苷酸激酶（New England Biolabs公司） </li><li> γ- 32 P - ATP（3000次/毫摩尔; 10μCi/毫升） </li><li>纯净S100B的结合缓冲液（5μg/μL的） </li></ol> 2.5。纯净S100B蛋白和选择适配的“三明治”结合试验<ol><li>第一届适体耦合要么A.）Codelink芯片幻灯片或B）金的纳米线。 </li><li>纯化的S100B蛋白，为稀释或者0.125μM70μL）或1μM50μL为B）。 </li><li> 第二届适体要么与A标记）Alexafluor 546 Codelink阵列幻灯片，或B），加上50 nm的纳米金与衍生金纳米线的研究。 </li></ol> 3。方法 3.1。 PF DNA文库的生成克劳森和潘，2009年Pan 等人 ，2008年中可以找到详细的方法和适体选择的协议。 3.2。核酸适体的选择使用纯净S100B蛋白人力S100钙结合蛋白B（S100B.基因ID＃6285）被用来作为一个目标。 6标签的他S100B蛋白（98个氨基酸）的表达和纯化使用的QIAexpressionist系统。 （QIAGEN公司）。如果需要的话或表示，他的6个标记酶可以去除。在两轮选拔的，从每一步的1μL样品保存液体闪烁计数来确定整体的结合效率。 3.2.1。 PF的图书馆DNA片段的制备<ol><li>再暂停40μL20毫米的Tris - HCl，pH值7.4的32 +和30 +的片段。 </li><li>热3分钟在85 ° C，并降温至37℃，3分钟孵化器在37 ° C。 </li><li>选择缓冲液添加760μL（氯化钙2.5毫米，5毫米MgCl 2的1X磷酸盐缓冲液，pH值7.4，GIBCO公司）和3分钟37 ° C，然后在室温（RT），保持10分钟。 </li><li>穿过一列载镍NTA琼脂糖珠（QIAGEN公司），选择缓冲区预洗，然后在室温下使用，直到。 </li></ol> 3.2.2。镍NTA琼脂糖珠约束S100B在室温下制备<ol><li>离心3秒400μL镍NTA琼脂糖珠，弃上清。 </li><li> 400μL结合缓冲液（50毫米的Na 2 HPO 4的NaH 2 PO 4，PH7.2，氯化钠150毫米）轻轻吹打5次，然后降速和上清液冲洗珠。 </li><li>重复步骤2的两倍。 </li><li>加入400μL纯化S100B（5微克/μL结合缓冲液中悬浮），轻轻地吸液管5次，每3分钟共15分钟。 </li><li>降速和上清液。 </li><li>洗净珠约束S100B的400μL结合缓冲液轻轻吹打3倍，然后降速和上清液。 </li><li>重复步骤6两次。 </li></ol> 3.2.3。选择的S100B的绑定适配<ol><li>转让32 + - +碎片3.2.1和30珠约束S100B。 </li><li>孵育15分钟，轻轻混匀，轻轻吹打，每3分钟。 </li><li>洗净S100B蛋白- DNA复合物，用800μL结合缓冲液轻轻吹打，然后降速和上清液。 </li><li>重复步骤3两次。 </li></ol> 3.2.4。 S100B选择适配的恢复<ol><li>加入200微升20毫米的Tris -盐酸，加热3时85分钟（PH7.4）° C，涡旋混合1分钟和自旋向下，然后将上清转移到一个新的管。 </li><li>一次重复步骤1，并结合上​​清液。 </li><li> 9-12从以前的出版物（11，12）每步净化所选的DNA片段。 </li></ol> 3.2.5。 TOPO克隆和测序鉴定的共识适体序列分析<ol><li>克隆选择PCR产物我的第 10轮不pCR2.1 - TOPO载体（Invitrogen公司）。可以做额外的克隆，进行进一步的选择。 </li><li>序列M13正向引物（我们使用的Hershey医疗中心的分子遗传学研究核心设施）40-50单菌落。 </li><li>使用Informax的Vector NTI公司（Invitrogen）的对齐选择的序列。 </li><li>确定的路线为基础的共识序列。 </li><li>从综合的DNA技术共识适配，然后购买。 </li></ol> 3.3。对选择适配的“三明治”结合试验 3.3.1。芯片格式 ​​使用荧光标记的第二适配。 <ol><li> 1共识ST适配合成5&#39; - amineC6基团。他们在打印缓冲区的终浓度为15微米的悬浮到Codelink激活幻灯片使用一个Apogent发现微网Arrayer在DNA芯片的设施电源，大学园区（GE医疗/生物科技公司Amersham公司）发现），以下Codelink推荐的协议。每张幻灯片都印有12阵列。 </li><li>在一个2 × 8格式的芯片杂交纸盒（数组，TeleChem国际）进行杂交。芯片盒是密封的使用无核酸粘接密封箔（AlumaSeal二，研究产品国际），以防止水分蒸发。 </li><li> S100B蛋白在PBS稀释到适当的浓度在70μL，并应用到芯片盒井。绑定Codelink幻灯片上印的适配是在室温下1小时。录像带井，然后单独洗涤3X用PBS + 5毫米氯化镁2（PBSM），多余的缓冲液洗被抹杀。 PBSM在70μL0.125微米稀释荧光标记的适配，然后加热到85℃，3分钟，3分钟就冰。适配应用的芯片卡式的井，并在室温下1小时孵育。韦尔斯再次单独洗涤PBSM 3X。录像带，然后拆开，广告PBSM冲洗整个幻灯片。离心的幻灯片，然后彻底干燥，并使用一个扫描惠普生物科技ScanArray 4000XL（Perkin Elmer公司）扫描。 </li></ol> 3.3.2。衍生物纳米线的格式，使用2次，再加上50 nm的纳米金适配黄金NWS（〜5微米的长度，直径在320纳米）的合成在多孔氧化铝膜的恒电按照先前公布的协议（13，14）。纳米线多孔膜解散后，悬浮于1 mL无水乙醇。 如上所述，DNA是从综合的DNA技术购买。纳米金（50纳米）均购自特德佩拉。硫醇盐的DNA切割100毫米0.1 M磷酸钠pH值8.3一小时的数码地面电视，然后在离心旋转10柱纯化。 黄金纳米线和纳米粒子的核酸适体附件一位50μLAu的纳米线等分放入0.5 mL的不粘离心管中，并冲洗成10 mM的磷酸盐缓冲液，300毫米氯化钠，pH值7.4。在5&#39;端（在5 10吨间隔“结束）硫醇盐的DNA是在终浓度为0.4μM电线。该样本是振荡30分钟，然后冲洗三次离心（8100克）与10毫米的磷酸盐缓冲液，300毫米氯化钠，pH 7.4和50毫米5毫米氯化镁 2，pH值7.2的磷酸盐缓冲液，三次。悬浮于100μL的缓冲稀释一半的DNA涂层的电线。 衍生的DNA -金纳米粒子的制备，加入50μL100毫米第二届适体（一个10吨的5&#39; -端的间隔）至1 mL，50 nm的金纳米粒子，并加热，在37 °为一小时彗星。加热后，增加了10毫米磷酸钠缓冲液，1M氯化钠，pH值7.4（100μL，150μL和128μL25μL两次）每隔0.5小时。共轭左37 ° C过夜，使用前加热块上。样品50毫米5毫米氯化镁2，pH值7.2的磷酸盐缓冲液，冲洗3次。偶联物悬浮于120μL的缓冲区。 对纳米线的夹心杂交 DNA包覆线，1μL的稀释，添加到缓冲区的PCR管。 S100B蛋白或HtrA1控制的蛋白（1μg/μL的）加入终浓度为50μL缓冲（〜10-12每平方纳米Au的纳米线表面的蛋白质分子添加）1微米。样品振荡1〜2小时。电线50毫米5毫米氯化镁2，pH值7.2的磷酸盐缓冲液，离心（8100克）冲洗三次，分别重悬于20μL金纳米粒子/ DNA的结合物。振荡在2小时的结合物，电线，然后冲洗，离心（1300克）的5倍，5毫米MgCl 2的50 mM磷酸盐缓冲液， pH值7.2除去多余的纳米粒子。对样品进行了重悬于20μL缓冲和干金FE - SEM分析涂层的硅片上。 纳米线的FE - SEM图像，获得使用狮子座1530场发射扫描电子显微镜在5.00千伏工作电压的肖特基场发射电子源。 4。代表性的成果 图1。 5&#39; -引物（功率因数为1）， ​​引物（PF 2）协议。 <img src="/files/ftp_upload/2039/2039fig1a.jpg" alt="图1a" /> （一）功率因数为1的DNA适配子选择协议。库寡核苷酸退火在一起，并受到PCR扩增产生一个双链DNA库（A）。 5&#39;端引无（功率因数为1）从PF的单链DNA库随机地区准备Nt.BstNBI / Nt.BbvCI消化（用红色箭头标记的切割位点）和凝胶纯化，并自凝胶净化（二）桥（黑色箭头）是孤立的。 （三）选择后，选定的序列（PS30 - CC）杂交及其相应的低聚物和自我桥结扎重新生成以前删除的底漆地区。在这个阶段，引物引入包含在3&#39; -末端的SP6启动子（D）。这是包含选定区域（E）用于转录的RNA。最后，RNA是完全重新扩增（模板DNA消化）用RT - PCR技术（F），和选定的子库，准备下一轮的选择。 <img src="/files/ftp_upload/2039/2039fig1b.jpg" alt="图1b" /> （二）的PF 2的DNA适配子选择协议。库寡核苷酸退火和PCR扩增产生一个双链DNA库（A）。无底漆（PF 2）从PF的单链DNA库随机地区准备Nt.BstNBI / BspMI消化（削减网站用箭头表示）和凝胶净化。自我桥是孤立的，无论是从功率因数为1协议和/或开始Nt.BstNBI（二）库单消化，加上凝胶净化。 （三）选择后，选定的序列（指定PS30）杂交及其相应的低聚物和自我的桥梁，并结扎再生先前删除的底漆地区。在功率因数为1，引物引入包含3&#39; -末端的SP6启动子（D ）。这是包含选定区域（E）用于转录的RNA。那么，RNA是专门reamplified用RT - PCR（F），和选定的子库，准备下一轮的选择。 <img src="/files/ftp_upload/2039/2039fig2.jpg" alt="图2" /> 图2。示意图和实验结果/减少到实践的PF选择协议。指定步骤如图1所示的对应。 PCR扩增（步骤一）与限制性内切酶（如表示）兴建的PF库后的消化，消化产品10％的凝胶上分离变性条件下逐页。与PF 1 PF 2选择协议获得相应片段（步骤b）所示。 （步骤三）选择后，势必适配杂交与其相应的低聚物和自我桥，连接再生先前删除的底漆地区。 3&#39; -末端，引物引入包含SP6启动子3&#39; -末端（步骤d）。32 P标记的RNA转录使用结扎产品1，0.5，0.25，0.125 5μL反应液，并6％凝胶上分离变性条件下（步骤e）逐页。笔录，然后用DNase处理，以消除未被选中的随机DNA区域，反转录成cDNA，然后为7，14，21或28个PCR循环reamplified。 8％非变性条件下凝胶的产品是按页分开。 66 NT片段全长的产品，包含选定的PS30 - CC和PS30序列的5&#39;和3&#39;侧翼序列内重新嵌入。迁移PhiX174/HinfI标记显示在左边（步骤六）。控制包括遗漏的反转录（RT）加强。一个少量的产品在这些样品中可以看出在高循环次数;这大概可以完全与第二个（或长期）的DNA酶消化步骤消除。 <img src="/files/ftp_upload/2039/2039fig3a.jpg" alt="图3a" /> 图3。选择适配和其配对使用夹心格式的结合特性 。 A.浓度属32个P -适体结合Kd值测定。使用G - 32的P - ATP（3000Ci /适配是5&#39; -结束标记〜10 MCI）和T4多聚核苷酸激酶（New England Biolabs公司），并具有约束力的毫摩尔，被确定为。 <img src="/files/ftp_upload/2039/2039fig3b.jpg" alt="图3b" /> B. 5&#39; -胺衍生物1 ST适配加上Codelink芯片。纯净S100B蛋白势必会第一届适体，幻灯片彻底冲洗，并AlexaFluor546标记， 第二届适体是必然第一届适体：S100B蛋白复合物。通过冲洗后，荧光定量使用ScanArray扫描仪。 <img src="/files/ftp_upload/2039/2039fig3c.jpg" alt="图3c" /> C. 5&#39; -巯基衍生物第一适配加上金纳米线的使用标准硫醇化学。 第二适配被耦合到50 nm的金纳米粒子以相同的方式。纯净S100B蛋白（左）或纯化HtrA1控制蛋白质（右），那么势必首先衍生的纳米线。彻底冲洗后， 第二届适体的50 nm的金纳米粒子随后被绑定1 日 ，适体纳米线：S100B蛋白复合物。经过清洗后，势必夹心复合物使用场发射扫描电子显微镜的可视化。比例尺= 1微米。

<ol>
	<li>Kulbachinskiy, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+selection+of+aptamers+to+protein+targets.">Methods for selection of aptamers to protein targets.</a> Biochemistry (Moscow). 72, 1505-1518 (2006).</li><li>Mairal, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aptamers:+molecular+tools+for+analytical+applications.">Aptamers: molecular tools for analytical applications.</a> Anal Bioanal Chem. 390, 989-1007 (2008).</li><li>Stoltenburg, R., Reinemann, C., Strehlitz, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SELEX+--+A+(r)evolutionary+method+to+generate+high-affinity+nucleic+acid+ligands.">SELEX -- A (r)evolutionary method to generate high-affinity nucleic acid ligands.</a> Biomolec Engineering. 24, 381-403 (2007).</li><li>Que-Gewirth, N. S., Sullenger, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+progress+and+prospects:+RNA+aptamers.">Gene therapy progress and prospects: RNA aptamers.</a> Gene Ther. 14, 283-2891 (2007).</li><li>Cowperthwaite, M. C., Ellington, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioinformatic+analysis+of+the+contribution+of+primer+sequences+to+aptamer+structures.">Bioinformatic analysis of the contribution of primer sequences to aptamer structures.</a> J Mol Evol. 67, 95-102 (2008).</li><li>Shtatland, T., Gill, S. C., Javornik, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+Escherichia+coli+RNA+with+bacteriophage+MS2+coat+protein:+genomic+SELEX.">Interactions of Escherichia coli RNA with bacteriophage MS2 coat protein: genomic SELEX.</a> Nucleic Acids Res. 28, E93-E93 (2000).</li><li>Jarosch, F., Buchner, K., Klussmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+selection+using+a+dual+RNA+library+that+allows+primerless+selection.">In vitro selection using a dual RNA library that allows primerless selection.</a> Nucleic Acids Res. 34, E86-E86 (2006).</li><li>Vater, A., Jarosch, F., Buchner, K., Klussmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+bioactive+Spiegelmers+to+migraine-associated+calcitonin+gene-related+peptide+rapidly+identified+by+a+novel+approach:+tailored-SELEX.">Short bioactive Spiegelmers to migraine-associated calcitonin gene-related peptide rapidly identified by a novel approach: tailored-SELEX.</a> Nucleic Acids Res. 31, E130-E130 (2003).</li><li>Wen, J. D., Gray, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+genomic+sequences+that+bind+tightly+to+Ff+gene+5+protein:+primer-free+genomic+SELEX.">Selection of genomic sequences that bind tightly to Ff gene 5 protein: primer-free genomic SELEX.</a> Nucleic Acids Res. 32, E182-E182 (2004).</li><li>Musheev, M. U., Krylov, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+aptamers+by+systematic+evolution+of+ligands+by+exponential+enrichment:+addressing+the+polymerase+chain+reaction+issue.">Selection of aptamers by systematic evolution of ligands by exponential enrichment: addressing the polymerase chain reaction issue.</a> Anal Chim Acta. 564, 91-916 (2006).</li><li>Pan, W. H., Clawson, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primer-free+aptamer+selection+using+a+random+DNA+library.">Primer-free aptamer selection using a random DNA library.</a> Methods Mol Biol. , (2009).</li><li>Pan, W., Xin, P., Clawson, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimal-Primer+and+Primer-Free+SELEX+Protocols+for+Selection+of+Aptamers+from+Random+DNA+Libraries.">Minimal-Primer and Primer-Free SELEX Protocols for Selection of Aptamers from Random DNA Libraries.</a> BioTechniques. 44, 351-360 (2008).</li><li>Nicewarner-Pena, S., Freeman, G., Reiss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Submicrometer+metallic+barcodes.">Submicrometer metallic barcodes.</a> Science. 294, 137-141 (2001).</li><li>JC, H. u. l. t. e. e. n., CR, M. a. r. t. i. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+general+template-based+method+for+the+preparation+of+nanomaterials.">A general template-based method for the preparation of nanomaterials.</a> J Materials Chem. 7, 1075-1087 (1997).</li></ol>

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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		<tbody>
		
<tr>
<td>Tris-HCl</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>HotMaster TAQ DNA Polymerase</td>
<td>&nbsp;</td>
<td>5-Prime</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>dNTP Mix</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Acrylamide</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>UltraPure 10X TBE Buffer</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium Persulfate</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>TEMED</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PhiX174 DNA/Hinf I DNA Marker</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Ethidium Bromide</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>&alpha;-32P-dCTP</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Phenol:ChCl3:IAA</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Ethanol</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Restriction Enzymes</td>
<td>&nbsp;</td>
<td>NEB</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Urea</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>&nbsp;</td>
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<td>Photographic Film</td>
<td>&nbsp;</td>
<td>ECE Scientific</td>
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<td>PBS</td>
<td>&nbsp;</td>
<td>Gibco</td>
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<td>CaCl2</td>
<td>&nbsp;</td>
<td>Gibco</td>
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<td>MgCl2</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Ni-NTA Agarose Beads</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Polypropylene Column</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>NaHPO4</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>NaAc</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
<tr>
<td>Detroit-551 cells</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>SK-MEL-31 cells</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>MEM</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>TrypLE Express</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>T4 DNA Ligase</td>
<td>&nbsp;</td>
<td>NEB</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Dimethyl Sulfoxide</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Sp6 RNA Polymerase</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>RNase-Free DNase I</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>RNase Inhibitor</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>SensiScript Reverse Transcriptase</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>pCR-2.1-TOPO Vector</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>DH5&alpha; Compotent Cells</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Plasmid Mini Kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Vector NTI</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>T4 Polynucleotide Kinase</td>
<td>&nbsp;</td>
<td>NEB</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>&gamma;-32P-ATP</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

primer-free aptamer selection using a random dna library

适配是高度结构化的寡核苷酸（DNA或RNA），可以结合与亲和力与抗体 1的目标。他们发现通过体外筛选过程称为指数富集的配体（SELEX） ​​的系统演化，识别目标的种类繁多，从小分子蛋白质和2-4等大分子物质。适配，以及适合在体内诊断和/或治疗应用的属性：除了良好的特异性和亲和力，他们很容易合成，更严格的加工条件下生存，他们是不良的免疫原性，其规模相对较小，可能会导致在轻便渗透组织。 通过标准SELEX过程中确定的适配通常包括〜80个核苷酸（NT），因为它们通常是从〜40 nt的随机地区，再加上每边固定引物位点的〜20 NT的核酸库中选择。固定引物序列，从而可以由近〜50％的库序列，因此可能会产生积极或消极的妥协，在遴选过程中 3适配识别，生物信息学方法虽然建议的固定序列，不作出重大贡献后， 选择 5到适体的结构。为了解决这些潜在的问题，引物序列互补的寡核苷酸被封锁或交换在SELEX 6轮中途不同的序列，或已修整至新台币7，8 6-9。温家宝和灰色9设计了引物基因组SELEX方法，其中引物序列完全不同于前选择库中删除，然后再生，让选定的基因组片段的扩增。然而，聘请技术，一个独特的基因组库将建造，拥有有限的多样性，和轮筛选后的再生依赖线性reamplification步骤。另外，努力规避固定序列的引物，使用高效率的分区造成的问题，会见了 PCR扩增10的问题。 我们已经开发出一个引物（PF）的选择方法，大大简化了SELEX程序，并有效地消除了引物干扰问题，11，12 。协议工作在一个简单的方式。中央库中随机地区是没有多余的侧翼序列和纯化绑定到一个合适的目标（例如纯化的蛋白质或复杂的混合物，如细胞株）。绑定序列得到侧翼序列团聚，并重新放大，生成选定的子库。作为一个例子，在这里我们选择适配S100B蛋白，黑色素瘤的蛋白标记。结合实验显示，在10 -7 Kd的小号- 10 -8 M系列选择了几个回合后 ​​，我们表明，适配功能有效三明治约束力格式。

Watch this Scientific Journal Video about Primer-Free Aptamer Selection Using A Random DNA Library at JoVE.com

Primer-Free Aptamer Selection Using A Random DNA Library

SELEX协议包括多轮选拔，其中每一个需要结合的配体，这反过来又需要固定的引物序列，侧翼的随机库地区的再生。这些固定的引物序列，可以干扰与选择过程（误报和底片）。在这里，我们目前无底漆的协议。

引物的适体的选择，使用一个随机DNA文库

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

primer-free-aptamer-selection-using-a-random-dna-library

Research

JoVE Journal

Biology

24.6K 次观看.  Pennsylvania State University. SELEX协议包括多轮选拔，其中每一个需要结合的配体，这反过来又需要固定的引物序列，侧翼的随机库地区的再生。这些固定的引物序列，可以干扰与选择过程（误报和底片）。在这里，我们目前无底漆的协议。

视频: Primer-Free Aptamer Selection Using A Random DNA Library

Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. Linearized &lambda; DNA is modified to have a biotin on the end of one strand, and a digoxigenin moiety on the other end of the same strand. The biotinylated end is attached to a functionalized glass coverslip and the digoxigeninated end to a small bead. The assembly of these DNA-bead tethers on the surface of a flow cell allows a laminar flow to be applied to exert a drag force on the bead. As a result, the DNA is stretched close to and parallel to the surface of the coverslip at a force that is determined by the flow rate (Figure 1). The length of the DNA is measured by monitoring the position of the bead. Length differences between single- and double-stranded DNA are utilized to obtain real-time information on the activity of the replication proteins at the fork. Measuring the position of the bead allows precise determination of the rates and processivities of DNA unwinding and polymerization (Figure 2).

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system.

使用单分子DNA拉伸含量直接观察DNA复制的酶

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. ...

科研

生物学

DNA Stable-Isotope Probing (DNA-SIP)

DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and nutrients into cellular biomass. As such, this cultivation-independent technique has been an important methodology for assigning metabolic function to the diverse communities inhabiting a wide range of terrestrial and aquatic environments. Following the incubation of an environmental sample with stable-isotope labelled compounds, extracted nucleic acid is subjected to density gradient ultracentrifugation and subsequent gradient fractionation to separate nucleic acids of differing densities. Purification of DNA from cesium chloride retrieves labelled and unlabelled DNA for subsequent molecular characterization (e.g. fingerprinting, microarrays, clone libraries, metagenomics). This JoVE video protocol provides visual step-by-step explanations of the protocol for density gradient ultracentrifugation, gradient fractionation and recovery of labelled DNA. The protocol also includes sample SIP data and highlights important tips and cautions that must be considered to ensure a successful DNA-SIP analysis.

DNA Stable-Isotope Probing DNA-SIP

DNA stable-isotope probing is a cultivation-independent method to identify and characterize active communities of microorganisms that are capable of utilizing specific substrates. Assimilation of substrate enriched in heavy isotope leads to incorporation of labelled atoms into microbial biomass. Density gradient ultracentrifugation retrieves labelled DNA for downstream molecular analyses.

DNA的稳定同位素探测（DNA的SIP）

DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and ...

Single Particle Electron Microscopy Reconstruction of the Exosome Complex Using the Random Conical Tilt Method

Single particle electron microscopy (EM) reconstruction has recently become a popular tool to get the three-dimensional (3D) structure of large macromolecular complexes. Compared to X-ray crystallography, it has some unique advantages. First, single particle EM reconstruction does not need to crystallize the protein sample, which is the bottleneck in X-ray crystallography, especially for large macromolecular complexes. Secondly, it does not need large amounts of protein samples. Compared with milligrams of proteins necessary for crystallization, single particle EM reconstruction only needs several micro-liters of protein solution at nano-molar concentrations, using the negative staining EM method. However, despite a few macromolecular assemblies with high symmetry, single particle EM is limited at relatively low resolution (lower than 1 nm resolution) for many specimens especially those without symmetry. This technique is also limited by the size of the molecules under study, i.e. 100 kDa for negatively stained specimens and 300 kDa for frozen-hydrated specimens in general.
For a new sample of unknown structure, we generally use a heavy metal solution to embed the molecules by negative staining. The specimen is then examined in a transmission electron microscope to take two-dimensional (2D) micrographs of the molecules. Ideally, the protein molecules have a homogeneous 3D structure but exhibit different orientations in the micrographs. These micrographs are digitized and processed in computers as "single particles". Using two-dimensional alignment and classification techniques, homogenous molecules in the same views are clustered into classes. Their averages enhance the signal of the molecule's 2D shapes. After we assign the particles with the proper relative orientation (Euler angles), we will be able to reconstruct the 2D particle images into a 3D virtual volume.
In single particle 3D reconstruction, an essential step is to correctly assign the proper orientation of each single particle. There are several methods to assign the view for each particle, including the angular reconstitution1 and random conical tilt (RCT) method2. In this protocol, we describe our practice in getting the 3D reconstruction of yeast exosome complex using negative staining EM and RCT. It should be noted that our protocol of electron microscopy and image processing follows the basic principle of RCT but is not the only way to perform the method. We first describe how to embed the protein sample into a layer of Uranyl-Formate with a thickness comparable to the protein size, using a holey carbon grid covered with a layer of continuous thin carbon film. Then the specimen is inserted into a transmission electron microscope to collect untilted (0-degree) and tilted (55-degree) pairs of micrographs that will be used later for processing and obtaining an initial 3D model of the yeast exosome. To this end, we perform RCT and then refine the initial 3D model by using the projection matching refinement method3.

This article describes a standard method to get a three-dimensional (3D) reconstruction of biological macromolecules using negative staining electron microscopy (EM). In this protocol, we explain how to get the 3D structure of the Saccharomyces cerevisiae exosome complex at medium resolution using the random conical tilt reconstruction method (RCT).

单粒子电子显微镜外切体综合重建，使用随机的锥形倾斜方法

Single particle electron microscopy (EM) reconstruction has recently become a popular tool to get the three-dimensional (3D) structure of large ...

Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, genome-wide cDNA library cloned into the 3'UTR downstream from the dual-selection fusion protein, thymidine kinase-zeocin (TKzeo). The first round of selection is for stable transformants, followed with introduction of a miRNA of interest, and finally, selecting for cDNAs containing the miRNA's target. Selected cDNAs are identified by sequencing (see Figure 1-3 for Target ID Library Workflow and details).
To ensure broad coverage of the human transcriptome, Target ID Library cDNAs were generated via oligo-dT priming using a pool of total RNA prepared from multiple human tissues and cell lines. Resulting cDNA range from 0.5 to 4 kb, with an average size of 1.2 kb, and were cloned into the p3&#900;TKzeo dual-selection plasmid (see Figure 4 for plasmid map). The gene targets represented in the library can be found on the Sigma-Aldrich webpage. Results from Illumina sequencing (Table 3), show that the library includes 16,922 of the 21,518 unique genes in UCSC RefGene (79%), or 14,000 genes with 10 or more reads (66%).

The Target ID Library is a plasmid-based, genome-wide collection of cloned cDNA used to identify miRNA targets. Here we demonstrate its use and application.

miRNA的目标使用的任务目标ID库的全基因组屏幕

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

随机使用时移视频显微镜细胞迁移的定量分析

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

Improving the Success Rate of Protein Crystallization by Random Microseed Matrix Screening

Random microseed matrix screening (rMMS) is a protein crystallization technique in which seed crystals are added to random screens. By increasing the likelihood that crystals will grow in the metastable zone of a protein's phase diagram, extra crystallization leads are often obtained, the quality of crystals produced may be increased, and a good supply of crystals for data collection and soaking experiments is provided. Here we describe a general method for rMMS that may be applied to either sitting drop or hanging drop vapor diffusion experiments, established either by hand or using liquid handling robotics, in 96-well or 24-well tray format.

Here we describe a general method for random microseed matrix screening. This technique is shown to significantly increase the success rate of protein crystallization screening experiments, reduce the need for optimization, and provide a reliable supply of crystals for data collection and ligand-soaking experiments.

提高蛋白质结晶的成功率随机Microseed矩阵筛选

Random microseed matrix screening  (rMMS) is a protein crystallization technique in which seed crystals are added  to random screens. By increasing the ...

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.

Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.

一个协议，用于噬菌体展示和亲和选择以重组蛋白诱饵

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

单工程的RNA转录物的活细胞实时成像采用比例式双分子信标

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

Reduced-gravity Environment Hardware Demonstrations of a Prototype Miniaturized Flow Cytometer and Companion Microfluidic Mixing Technology

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If humans are to travel beyond low Earth orbit, a transition towards space-ready, point-of-care (POC) testing is required. Such testing needs to be comprehensive, easy to perform in a reduced-gravity environment, and unaffected by the stresses of launch and spaceflight. Countless POC devices have been developed to mimic laboratory scale counterparts, but most have narrow applications and few have demonstrable use in an in-flight, reduced-gravity environment. In fact, demonstrations of biomedical diagnostics in reduced gravity are limited altogether, making component choice and certain logistical challenges difficult to approach when seeking to test new technology. To help fill the void, we are presenting a modular method for the construction and operation of a prototype blood diagnostic device and its associated parabolic flight test rig that meet the standards for flight-testing onboard a parabolic flight, reduced-gravity aircraft. The method first focuses on rig assembly for in-flight, reduced-gravity testing of a flow cytometer and a companion microfluidic mixing chip. Components are adaptable to other designs and some custom components, such as a microvolume sample loader and the micromixer may be of particular interest. The method then shifts focus to flight preparation, by offering guidelines and suggestions to prepare for a successful flight test with regard to user training, development of a standard operating procedure (SOP), and other issues. Finally, in-flight experimental procedures specific to our demonstrations are described.

Spaceflight blood diagnostics need innovation. Few demonstrations have been published illustrating in-flight, reduced-gravity health diagnostic technology. Here we present a method for construction and operation of a parabolic flight test rig for a prototype point-of-care flow-cytometry design, with components and preparation strategies adaptable to other setups.

样机小型化流式细胞仪及配套的混合微流控技术降低重力环境硬件演示

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

逆转录病毒整合位点的扩增，新一代测序和基因组DNA制图

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...