The authors would also like to thank all participants who donated samples for this work. This work was funded by the Office of Justice Program, National Institute of Justice, Department of Justice under award number 2010-DN-BX-K139. The funding agencies had no involvement in the study design, in collection, analysis and interpretation of data, or in the writing of the report. Points of view in this document are those of the authors and do not necessarily represent the official positions of the U.S. Department of Justice.

注:体液从采用经佛罗里达州中部的机构审查委员会的程序大学的志愿者们收集。知情同意书从每个捐赠者获得。 从感动物体和表面1.收集生物粒子的<ol><li>切凝胶膜到期望的大小。确保它被用作固体支持物的显微镜载玻片的适当的大小。例如，对于一个3"×1"的玻璃载片，用2"×0.75"的凝胶膜的大小。 注:长度和宽度的材料可以减少，如果需要的话，但不应超过此尺寸。 </li><li>从凝胶膜去除白背衬和凝胶膜附着到所述显微镜载玻片（ 图1A）。用力按下以确保完整的附件。 注意:有是，不应该被去除，以允许压力被沿片凝胶-FIL施加清晰顶保护层米，而不会污染凝胶膜的表面上。制备的凝胶膜载玻片（附有保护层）可以在​​室温下储存，直到需要。 </li><li>使用无菌镊子时，就可以使用（ 图1B）的凝胶薄膜样品从凝胶膜取下透明顶部保护膜。放置凝胶膜表面中具有所需触摸物体或表面（ 图2）直接接触。取少量的压力，以确保生物颗粒的有效转移到凝胶膜。 注:如果压力太大被施加，载玻片可能破裂。 注意:从同一物体或表面的多个采样可收集到同一块凝胶膜。 </li><li>通过将滑动上的体视显微镜确认生物粒子上凝胶膜转移（ 图3）（反式或反射照明可以使用）。 〜20X使用放大率〜200X的凝胶膜的最佳观看较大的区域，并放大，以取得最佳六尤因个人的生物颗粒。 </li><li>储存在室温下将凝胶薄膜样品玻片在有盖滑动箱或立即用于染色和/或生物粒子集合。 </li></ol>生物粒子的2.可选染色的可视化，以援<ol><li>放置在步骤1中制备到在一个水槽的滑动机架染色凝胶薄膜样品玻片。 </li><li>用一次性移液管，覆盖整个凝胶膜表面用台盼蓝染色（ 图4A）。在室温下孵育1-2分钟。 </li><li>通过倾斜滑动，以允许所述污渍流掉到接收器（ 图4B）除去过量的污点。 注:滑动可以通过轻柔水浸用一次性移液管，如果需要（ 图4C）被用无菌水冲洗。 </li><li>在进行到生物颗粒的隔离之前允许滑动风干。用立体显微镜（20X〜整体观察和查看幻灯片〜200-300X到vIEW个人生物粒子），以确保适当的染色（ 图4D-E）。 注:幻灯片可以存储在室温下在有盖滑动箱。 </li></ol> 3.隔离兴趣分析的生物粒子<ol><li>放置0.2ml的PCR管在机架适当数量并适当标注的管子。 </li><li>准备STR扩增混合液（见材料清单 ）在指定的PCR扩增罩或生物安全柜。涡主拌匀，短暂离心在微型离心机。 <ol><li>每个样品所需要的扩增混合物的体积是3.5微升;制备主混合物的适当的量的样品的期望数量。 </li></ol></li><li>吸取3.5μl的扩增组合成每次0.2毫升管的。帽各管松散。 </li><li>放置一块两面胶带到一个干净的玻璃显微镜载片上或直接到玻璃块。 注意:这将被用于保持该0.2毫升管到位样品采集过程中。 </li><li>将一块双面胶带到一个干净的玻璃显微镜载玻片。将一块水溶性波峰焊磁带上的两面胶带的顶部。 注意:此幻灯片可以存储在干燥器中以供将来使用。 </li><li>放置第0.2毫升管上，在步骤3.4中制备的双面胶带或滑动块。抛开该管，直到样品采集。 </li><li>放置在显微镜下（低倍）水溶性波峰焊胶带幻灯片。使用钨针的尖端，轻轻刮去带的表面，以便收集少量（或"小球"）的粘合剂在针（ 图5A）的末端。 注:该球的大小可以取决于将被收集生物粒子的数量变小或大。 </li><li>小心地从显微镜下除去钨针用粘合剂，而不会干扰针的尖端。针可以在地方，如果在试样放置所需的机架。 </li><li>将制得的凝胶，膜样品（从步骤1和2）在显微镜阶段。调整焦距和放大倍率，直到生物粒子可以很容易地查看。鉴定所述生物颗粒，将被收集。 注:玻璃块可以使用，如果需要支持的幻灯片。 </li><li>检索钨针与粘合剂球，然后将针在凝胶膜表面，其中所关注的生物颗粒的位置。按针（带粘合剂）的前端向下，使其与所述生物粒子（ 图5B）接触。抬起针上，以确保生物粒子被收集。重复此过程，直到生物颗粒的所需数目已收集。 </li><li>放在显微镜阶段的准备0.2毫升PCR管。调整倍率以使含有所述扩增混合物中的管的底部处于焦点。 </li><li>小心地将钨needle取入0.2毫升PCR管，直到针的尖端是在扩增混合物。 注:这是同时通过显微镜观察表现最好的。 </li><li>持在扩增混合物中的针，直到粘合剂溶解和生物粒子被释放到溶液中（ 图5C）。取出针头，将0.2毫升直立松散帽管。 </li><li>重复采集程序的附加样品。清洁钨针与预润湿醇（异丙醇）擦拭在样品之间。 </li></ol> 4.联合微体积核酸分离（直接裂解）和常染色体短串联重复序列（STR）剖析<ol><li>制备的裂解缓冲液（见材料 ）在一个指定的PCR扩增罩或生物安全柜中。添加1.5微升裂解缓冲液，以每管为5微升总反应体积。关闭管盖得严严实实。 注:0.2毫升管已经包含了3.5微升的扩增混合物和收集生物粒子从第3步。 </li><li>在热循环仪的地方的样品，并使用以下循环条件扩增样品:75℃15分钟; 95℃，11分钟; 34个循环的94℃20秒和59℃，3分钟; 60℃，10分钟;和4℃保持。 <ol><li>商店的扩增产物，在4℃下短期储存。 </li></ol></li></ol> 5.产品检测 - 毛细管电泳（CE） <ol><li>在96孔板中，加入10微升的CE运行混合物（9.7微升的去离子甲酰胺和0.3微升尺寸标准，参见材料 ）。加入1μl扩增产物或等位基因梯的每个孔中。 警告:甲酰胺是一种潜在的诱变剂，并应在化学罩处理，而穿着合适的个人防护装备。 </li><li>使用由制造商指定的条件下电泳的扩增试剂盒手册或internallŸ验证条件。 </li><li>分析原始数据，STR分析软件。 </li></ol>

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The authors declare that they have no competing financial interests.

在这里，我们所描述的收集和生物粒子的接触DNA证据的恢复增强的遗传分析方法。发达的方法包括下列步骤:1）通过凝胶膜收集生物材料（ 即，生物粒子）从触摸物体和表面，磨损服装物品或直接由人类皮肤中，回收的生物材料2）显微镜检查，以确保收集的潜在人类的生物材料，并且单或几个生物粒子的3）显微操作使用水溶性粘合剂，以及4）常染色体的DNA的STR使用微量（5微升）一步法裂解/扩增反应所收集的生物粒子的剖面。使用一步法封闭管反应降低了从附加采样操作或转移到其他的反应管的污染和样品损失的可能性。增强的一步法微量（5微升）裂解/ STR扩增反应允许的全部或probativ回收ËSTR单个或几个生物粒子的供体轮廓。这种方法的开发是为了获得单和多源触摸DNA证据（ 如，穿着衣物等生活用品，一摸/处理物体和表面，皮肤/皮肤混合物）单一来源STR配置文件。它已经成功地被用于男性供体模拟物理攻击混合物样品中的检测。 这种方法可以在附加的体液/组织混合物场景进行进一步评估，如受害者刮伤他/她的攻击者，其中，从攻击者只有几个生物粒子将存在之中从受害者压倒性量的生物材料。此外，由于这种方法在目前的研究开发允许分析在单个生物粒子的水平，它可以被用来更好地理解的二次转印14-19的性质和程度，在其中一个中介上surfa转移的DNA轮廓CE，物体或人到另一个表面物体或人20。盲-擦拭方法二次转印的分析可能无法识别从辅助供体材料的痕量。这里开发的方法允许单个或几个生物粒子的分析，因此，其结果不被生物材料的压倒性量从主施主存在混淆。开发这项工作也可能对性攻击案件跟踪生物材料的分析意义的方法，如累及阴道的数字渗透其中，积极识别皮肤细胞的行为微量的可能是至关重要的证明性接触发生。 而从凝胶薄膜样品生物粒子的集合不涉及困难和复杂的操作，也有在此过程中的关键步骤，必须非常小心，以确保苏进行生物粒子的ccessful转移到下游反应器中。生物粒子与水溶性粘合剂的收集必须显微镜观察（ 即，实体显微镜以高倍率（〜200-300X），以确保只有感兴趣的生物粒子被收集。根据所用的粘合剂"球"的大小，有是收集额外的周围材料可包括生物和非生物材料（ 例如，纤维，其他碎片）的潜力。意想不到的附加 ​​生物粒子的收集可能导致混合的DNA轮廓的恢复。非生物材料的收集可以引入抑制剂进入微体积的裂解/ STR反应。如果多个生物粒子被收集具有相同的粘合剂"球"，还有一个潜在的先前采集的生物粒子，以从粘合剂成为独立的，这是不经常观察到，但可以发生当大bioparticl数量ES被收集（ 例如，超过50）配有一个单一的粘合剂"球"。如果大量生物粒子的靶向，建议减少生物粒子的多个集合被提出，但全部转移到同一0.2毫升管。一旦生物粒子已被收集，但必须去除从显微镜阶段凝胶膜滑动并放置0.2毫升管，使得针的尖端不被干扰的过程中采取的。在针到扩增混合物在0.2毫升管的底部的前端的放置，所述针的尖端不应该使与管子的侧面接触，以避免材料在管的侧面损失。而粘合剂的溶解通常是快速（〜30秒或更少取决于该粘合剂"球"的大小），并在显微镜检查能监视时，针的尖端应该慢慢地从扩增混合物除去，以确保完全溶粘合剂的已经实现。如果粘合剂未完全溶解，针可以被返回到液体，以允许完全溶解。 这里所描述的协议已被优化，用于与从法医生物证据生物粒子和人类细胞的使用。选择的裂解缓冲液是与所选择的DNA的STR扩增试剂盒兼容。而有可能是合适的替代裂解缓冲液，其在细胞裂解和兼容下游分析方面效率必须由用户进行验证。此外，在该协议中描述的STR扩增试剂盒和扩增循环数目已被优化为具有单个或几个生物粒子的使用。下一代STR扩增试剂盒用报道增加鲁棒性和灵敏度被选择，并已被证明导致高质量的STR型材从单个或几个生物粒子的回收。虽然许多其他STR套件，具有不同的建议功率放大器阳离子周期数，都可以，他们可能不具备适当的灵敏度，因此，需要在这些协议在使用前进行适当评估。 尽管成功利用从单个或几个生物粒子回收STR配置文件所描述的方法，曲线恢复的成功率也不会100％。因此，对于一些样品在有限的部分DNA轮廓或无简档可以被观察到。这是无法避免的，由于无法决定性地可视地识别最生物粒子如细胞材料，在收集的生物粒子或样品材料从粘合剂损失的核材料转移到反应容器中之前，潜在的降级状态。因此，不建议每个触摸DNA项目单一生物粒子样本的分析。我们经常收集来自个体凝胶薄膜样品20样品组，如果不能获得合适的STR轮廓根据需要将收集额外样品组。牛逼他仅用于集合的限制是生物材料存在于凝胶膜样品（和分析的可能成本的量，虽然微量反应提供的机会，以收集另外的样品进行类似的或更低的成本作为一个单一的标准反应体积（ 例如，25微升）。 这里的开发DNA分析方法描述提供了分子基础的方法来表征，分析和从触摸DNA样品中回收微量生物材料的解释。然而，存在可以从回收的生物粒子，除了测定生物材料的供体的供体（ 即，STR外形）来获得额外的遗传信息。起源组织或体液源（ 例如，皮肤与唾液）可能会提供重要的上下文信息刑事调查。如果没有这样的判定，起源组织来源的模糊性，可以EXPLO资讯科技教育作为犯罪替代的情况下（ 例如，如何体液可能已经沉积），可建议。这里所描述的生物粒子收集和隔离的协议可以用于收集生物粒子或其他细胞（ 例如，口腔，阴道），用于在一个组织源识别使用（mRNA的仿形21-30）的策略，涉及微体积反转录（RT）和扩增反应。随着进一步的优化，可能也有可能开发出的DNA / RNA共分离策略，从同一样品，包括从触摸DNA证据生物粒子允许细胞类型标识（核糖核酸）和STR谱。

触摸的DNA的微量生物证据是细胞物质的直接转移形式从单个对象或另一个人（ 例如，流下的皮肤细胞）中的物理接触1。从各种感动对象获得的DNA图谱的能力（文档，床上用品，鞋，武器，饮料容器，钢笔，皮包手柄）已文献报道2-9。 接触DNA证据的分析，一个关键因素是跟踪生物材料目前已成功恢复。触摸DNA证据通常是通过用无菌棉签擦拭可疑区域收集（称为"盲擦拭"）。使用这种方法，所收集的生物材料的性质是未知的并且执行广义区域的采样。表面沟槽和裂缝的存在可能妨碍往往已经少量BI成功恢复ological物质存在。另外，一个"盲擦拭"的方法将来自不同个体的细胞上存在的项目，即使在个人的细胞位于上项目空间上不同的位置不一定共同样品多孔材料。的混合DNA图谱，这往往是具有挑战性的，低模板DNA样品尤其解决的回收，经常观察到8,10,11和在许多情况下将是擦拭过程本身的相应产物。如果只有少量的材料是存在于从供体中的一个，标准提取和分析技术可能无法从细微贡献者恢复轮廓。此外，或者它是否被用于干或湿的拭子的类型（预润湿用无菌水）可能影响所收集的生物材料的量由于在吸收和吸附和生物材料12释放的效率差异。 STANDARD提取方法可能导致额外的样品损失，由于对样品或样品转移步骤需要物理操纵。 如上所述，简单的擦拭技术生物材料的恢复可能导致生物样品，以及混合的型材的增加发生微量的损失，由于发生故障，以分离单个的生物组分。因此，我们试图开发用于收集目前在感动对象细胞微粒更有选择性和高效的恢复策略。的开发策略从触摸DNA证据生物材料的分析（这里称为"生物粒子"由于这一事实，即一个核并不总是可见的，因为多数的皮肤的外皮层中发现的细胞是死的或濒死角质13）包括以下内容:1）通过凝胶膜FR收集生物材料（ 即生物粒子）的嗡触摸物体和表面，磨损服装物品或直接由人类皮肤回收的生物材料，2）显微镜检查，以确保收集的潜在的人类生物材料，并且单或几个生物粒子3）显微操作使用水溶性粘合剂，以及使用一个微量（5微升）一步法裂解/扩增反应所收集的生物颗粒的4）常染色体DNA的STR谱。 此过程提供了超过传统使用的分析方法具有许多优点。恢复使用凝胶膜生物粒子的允许生物材料本分析前的样品中的显微镜检查。虽然大多数从触摸DNA证据回收的生物粒子的未必有核细胞使之更难以确定哪个或生物粒子多少应选择，这些样品的显微镜检查在分析之前提供给机会来搜寻有核细胞因此maximiz荷兰国际集团DNA分型复苏的可能性。用水溶性胶粘剂辅助显微生物粒子的集合允许直接转移目标的生物粒子进入反应管。此过程在显微镜下可视化，以确保生物材料的成功转移。减少或微量反应增​​加感光度，同时减少每个样品分析的成本。这允许样品的数量增加的来自个体的证据的分析。由于在生物颗粒中触摸DNA证据的遗传状态的范围内，从单个项目的多个采样被推荐。 在这里，我们展示了成功运用的开发协议获得的生物材料在接触DNA证据的捐赠者高度证明的基因图谱。发达的生物粒子收集和DNA分析方法，提供全面的"智能"（ 即具体，可衡量，阿塔inable，现实，及时）的分子基础的方法来表征，分析和跟踪生物材料的解释。而最初开发用于施加到触摸DNA证据的取证分析，这里开发的策略可以应用到生物材料的其它来源的，并提供了一​​个机会，以获得在单细胞水平个人识别信息。

<table><tbody><tr><td>SealRite 1.5 ml natural microcentrifuge tubes, sterile</td><td>USA Scientific</td><td>1615-5510</td><td>numerous alternative suppliers are available, but tubes should be sterile</td></tr><tr><td>0.2 ml PCR tubes with flat cap, natural</td><td>Phenix Research</td><td>MPX-200F</td><td>or equivalent</td></tr><tr><td>Kimwipes</td><td>Fisher Scientific</td><td>06-666 and/or 06-666-11C</td><td></td></tr><tr><td>Alcohol prep pads</td><td>Fisher Scientific</td><td>06-669-62</td><td>alternative is 06-665-24 (Fisher Sci); canister of larger alcohol wipes rather than individual pads</td></tr><tr><td>Sterile water</td><td></td><td></td><td>18.2 M&amp;Omega;, autoclaved</td></tr><tr><td>Glass microscope slides, frosted, 3&quot; x 1&quot;: 1 mm</td><td>Fisher Scientific</td><td>12-544-3</td><td>alternative brands or sizes are acceptable</td></tr><tr><td>Disposable transfer pipet</td><td>Fisher Scientific</td><td>13-711-9D</td><td>alternatives are acceptable</td></tr><tr><td>Trypan blue solution</td><td>Sigma</td><td>T8154</td><td></td></tr><tr><td>Pipets: 0.2-2, 2-20, 10-100, 100-1,000 &amp;mu;l</td><td>various</td><td></td><td></td></tr><tr><td>Sterile, aerosol-resistant pipet tips</td><td>various</td><td></td><td></td></tr><tr><td>Mini-centrifuge</td><td>various</td><td></td><td></td></tr><tr><td>Stereomicroscope</td><td>Leica</td><td>M205C</td><td>others are suitable, but must be capable of magifications up to 350X</td></tr><tr><td>GeneAmp PCR system 9700 thermal cycle (silver or gold block)</td><td>Life Technologies</td><td>N8050200 or 4314878</td><td>other models and manufacturers can be used, but performance with the STR amplification kit must be verified</td></tr><tr><td>3130 Genetic Analyzer</td><td>Life Technologies</td><td>3130-01</td><td>alternatives: 310 or 3500 Genetic Aanlyzers (Life Technologies)</td></tr><tr><td>GeneMapper software</td><td>Life Technologies</td><td>various</td><td>various versions of the software are available and can be used</td></tr><tr><td>WF Gel-Film , x8 retention level</td><td>Gel-Pak</td><td>WF-40-x8-A</td><td>4&quot; x 4&quot; wafer film; can be ordered in other size specifications</td></tr><tr><td>Double sided tape</td><td>various</td><td></td><td></td></tr><tr><td>Glass block, 3&quot; x 1.5&quot; x 3/8&quot;</td><td>McCrone Microscopes and Accessories</td><td>370-2</td><td>or equivalent</td></tr><tr><td colspan="4">[header]</td></tr><tr><td>Tungsten needle with holder</td><td>McCrone Microscopes and Accessories</td><td>106</td><td></td></tr><tr><td>3M water soluble wave solder tape, 5414 transparent</td><td>Allied Electronics</td><td>70113977</td><td></td></tr><tr><td>Lysis buffer (forensicGEM Saliva, Zygem)</td><td>VWR</td><td>95043-992</td><td>Lysis buffer is prepared in 10 &amp;mu;l portions (prepare additional master mix portions as needed to accommodate the desired number of samples). 1.5 &amp;mu;l is used for each sample. To prepare 10 &amp;mu;l of lysis buffer (sufficient for ~6 samples): 6.9 &amp;mu;l sterile water, 2.1 &amp;mu;l 10x buffer - blue, 1 &amp;mu;l forensicGEM.</td></tr><tr><td>STR Amplification kit, AmpFlSTR Identifiler Plus PCR amplification kit</td><td>Life Technologies</td><td>4427368</td><td>Alternative STR amplification kits can be used, but performance would have to be verified by user. The amplification mix is prepared as follows (volumes per sample): 2.2 &amp;mu;l PCR reaction mix, 1.1 &amp;mu;l Primer set, 0.2 &amp;mu;l (1U) AmpliTaq Gold DNA polymerase. A master mix sufficient for the desired number of samples should be prepared. 3.5 &amp;mu;l of the prepared mix is added to the 0.2 ml tube for bioparticle collection.</td></tr><tr><td>AmpliTaq Gold DNA polymerase</td><td>Life Technologies</td><td>N808-0245</td><td></td></tr><tr><td>HiDi formamide</td><td>Life Technologies</td><td>4311320</td><td></td></tr><tr><td>GeneScan 500 LIZ size standard</td><td>Life Technologies</td><td>4322682</td><td></td></tr><tr><td>96 well optical reaction plates</td><td>Life Technologies</td><td>N8010560</td><td></td></tr><tr><td>Plate septa, 96 well</td><td>Life Technologies</td><td>4315933</td><td></td></tr><tr><td>Performance-optimized polymer, POP-7</td><td>Life Technologies</td><td>4363785</td><td>Alternatives such as POP-4 are acceptable</td></tr></tbody></table>

enhanced genetic analysis of single human bioparticles recovered by simplified micromanipulation from forensic &#8216;touch dna&#8217; evidence

DNA图谱可以从&#39;触摸DNA&#39;的证据，其中包括人体生物材料的微观痕迹来获得。目前的方法为痕量DNA的雇用棉签或胶带回收样品的感兴趣的区域。但是，这样的"盲擦拭"的方法将共同的样品多孔材料从不同的个体，即使个人的细胞位于上项目地理上不同的位置。因此，一些触摸DNA样品中所遇到的DNA的混合物被人为由擦拭本身创建的。在一些情况下，在受害者的DNA可以在显著过量中找到因而掩蔽任何潜在行为人的DNA中。 为了规避与标准的回收和分析方法的挑战，我们已经开发出一种成本更低，"智能分析"的方法导致接触DNA证据增强了遗传分析。我们描述了一个优化的用于生物颗粒本触摸DNA样品中的收集，以及涉及一步骤5微升微量裂解/ STR扩增，以允许STR型材从生物粒子施主恢复增强的扩增战略D高效显微恢复策略（多个）。使用个人或少数（ 即 "团块"）生物粒子的结果获得单源配置文件的能力。这些程序代表的替代增强技术的分离和从法医触摸DNA证据单一生物粒子的分析。而在每一个法医调查不是必须的，该方法可以用于在涉及物理攻击（ 例如，绞）情况下，单个源肇事者DNA图谱的回收使用标准的分析技术，可能无法进行非常有益的。此外，这里所开发的策略提供了一个机会，以从各种邻获得在单细胞水平的遗传信息疗法非法医跟踪生物材料。

个人和生物粒子结块的从衬衫领（100％聚酯）所穿男性供体回收该代表性图像示于图6（个体生物粒子）和图7（成群生物粒子）。的生物粒子是从使用此处描述的协议的衬衫领回收:从衬衫领转移生物粒子的凝胶膜通过直接接触来回收的生物粒子用锥虫蓝，收集使用钨针和水溶性粘合剂生物粒子样品的，染色并使用5微升的微体积的裂解/ STR扩增。STR分析图6和7表示将来自个体的DNA的触摸项（20个单/个体生物粒子和20成群生物粒子）进行分析生物粒子的典型采样。收集在每个图像中的生物粒子（S）的标记的长度和宽度的测量（以微米）。在PERC提供每个单独的图像上来自每个采集生物粒子（S）的回收的STR等位基因entage展示可变程度的成功，可预期可从生物粒子与这种类型的证据获得。从触摸DNA样品中回收的生物粒子在很大程度上是死的或濒死的角质形成细胞并因此证明力的STR轮廓不会从收集的每种生物粒子获得，如可在图6和7中可以看出。对于此衬衫领样品的STR型材从14/20（70％）所获得的个人生物粒子和15/20（75％）成群生物粒子。然而，每一个回收的轮廓范围中证明价值:3-97％等位基因恢复单个生物粒子型材和3-100％等位基因恢复为成群生物粒子分布。由于成功率的变化，从每个触摸DNA项目众多的生物粒子收集建议。这确保了更好的机会获得高度probati的已经STR轮廓。 从衬衫衣领的个体生物粒子的样品中的一个获得的STR曲线示于图8中 （从该信息源自的生物粒子被示为左侧）。几乎一个完整的STR轮廓（28的30个等位基因，一个基因座滴出，所获得的由比较参考信息核实信息的准确度），从该个别生物粒子获得的。得到的配置文件是高品质与合理均衡的跨座峰高无等位基因辍学。等位基因辍学和不平衡峰高的文物都经常与低模板DNA样本观察。从衬衫衣领的结块生物粒子的样品中的一个获得的STR曲线示于图9中 ，得到一种完整的STR轮廓（三十○分之三十等位基因，将得到的由比较参考信息核实信息的准确度）。如前面提到的，一个STR配置文件未从不断回收Ÿ生物粒子收集。单一生物粒子（3％等位基因恢复1/30等位基因）的不成功DNA纹的一个例子示于图10（个体生物粒子）。虽然这种个人生物粒子未成功，在这种样品中的生物粒子的供体的高渗透性的STR型材被从同一样品中回收其他生物粒子获得的。这说明了需要从同一对象执行多个单细胞/丛采样。 所开发的"智能"分析这里介绍的触摸DNA证据的方法已成功地用于恢复从单证明的单一来源文件，并从各种"成群"生物粒子碰到物体和衣物（ 如椅子扶手，汽车方向盘，手机，咖啡杯，香烟，笔，衬衫，短裤和针织衫）。但是，重要的是，这种方法也被用于男性D的检测和分析onor DNA（单一来源）模拟身体接触/攻击混合样品（ 如，行为人抓住受害者的手腕，颈部或衣服，或接触受害人的床上用品在性侵犯）。 <img alt="图1" src="/files/ftp_upload/52612/52612fig1.jpg" /> 图1:用于生物粒子收集制备的凝胶膜的幻灯片。 （A）中的白色背面保护罩使用从一块凝胶膜的无菌镊子以暴露粘合剂衬垫除去。凝胶膜，然后附着到与压力牢固的玻璃显微镜载片。（B）的当凝胶膜样品准备好被用于生物粒子收集，顶部透明保护膜使用无菌镊子除去。 <img alt="图2" src="/files/ftp_upload/52612/52612fig2.jpg" /> 图2:生物粒子收集用凝胶从各种基材-film的凝胶膜的幻灯片可以被用来从各种表面收集生物粒子。凝胶膜被放置在与对象或感兴趣的表面直接接触。轻柔的压力，以传输到生物粒子凝胶膜表面施加。收集生物粒子从（A）所穿衣物（衣领），（B）触及的对象（旅行咖啡杯），和（C）直接人体皮肤（男性手腕）显示。 <img alt="图3" src="/files/ftp_upload/52612/52612fig3.jpg" /> 图3:生物粒子上的磨损服装产品（裤腿内侧）生物粒子是通过与对象表面直接接触转移到凝胶膜。生物粒子可以发现为"团块"或个人/单生物粒子（用红​​色箭头表示）。 <img<html> ALT ="图4"SRC ="/文件/ ftp_upload / 52612 / 52612fig4.jpg"/&gt; 图4:在凝胶膜幻灯片生物粒子的可选染色为了更好的可视化的生物粒子，凝胶薄膜样品可染色用锥虫蓝染色。凝胶膜的整个表面覆盖用台盼蓝（A）中，经过染色的1-2分钟，滑动轻轻倾斜，以允许多余的染色剂流掉幻灯片（B）中 。轻柔水浸用无菌水（C）可以用来除去多余的污点。所述载玻片风干后，滑动可以在显微镜下观察，以确保适当的染色（D，E）。注:并非所有的生物粒子会出现染色（ 即蓝色的） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52612/52612fig4large.jpg" target="_blank">请点击这里查看此图的放大版本。</a> OAD / 52612 / 52612fig5.jpg"/&gt; 图5:生物粒子收集和分析。水溶性波峰焊带的（A）中的少量（或"球"）（"粘合剂"）被收集到一个钨针通过小心刮擦的尖端。（B）中的粘合剂，然后触摸到凝胶-膜表面，以收集感兴趣的生物粒子。单个或多个生物粒子可以收集与单个粘合剂球（℃）一旦所需的生物粒子被收集时，针的尖端置于扩增混合物中0.2毫升PCR管。针被保持在液体直到其溶解和生物粒子释放入溶液中观察到。执行在收集过程中的所有步骤，并在显微镜下观察，以确保成功的生物粒子收集和传送。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52612/52612fig5large.jpg" target="_blank">请点击这里查看LAR</a>蒙古包版本这个数字。 <img alt="图6" src="/files/ftp_upload/52612/52612fig6.jpg" /> 图6:衬衫领子样品中单个生物粒子生物粒子是使用凝胶膜从衬衫领所穿男性供体的内部回收。的生物粒子是用台盼蓝和的样品中鉴定20不同的单独生物粒子图像示染色。在每个图像，被收集的生物粒子的圆圈（红色圆圈指示在其中一个配置文件被恢复的生物粒子;黑色圆圈指示生物粒子在其中一个配置文件未恢复）。百分比等位基因恢复（数字观察等位基因走出了一条可能的30），显示生物粒子从一个配置文件被恢复的图像上。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52612/52612fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a><img alt="图7" src="/files/ftp_upload/52612/52612fig7.jpg" /> 图7:生物粒子结块的衬衫的衣领样品中生物粒子是使用凝胶膜从衬衫领所穿男性供体的内部回收。的生物粒子是用台盼蓝和的样品中鉴定20不同成群生物粒子图像示染色。在每个图像，被收集的生物粒子被圈（红色圆圈指示生物粒子，其中的轮廓被回收;黑圆点表示生物粒子，其中，简档未回收的百分比等位基因回收率（观察等位基因数出一个可能的30）。显示每个生物粒子从一个配置文件被恢复。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52612/52612fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图8:从单一的生物粒子获得从一个男性的衬衫领口常染色体STR配置文件的常染色体STR曲线是从一个单一的生物粒子（上左图），使用5微升直接裂解/扩增反应获得。该配置文件的精度是通过比较供体的参考样本来确定。十五常染色体STR基因座和釉原蛋白（性别决定）是共扩增在单个反应并通过毛细管电泳分离。结果将显示在这里作为电泳。等位基因号码每个峰在每个基因座上下面指定。 x轴代表片段大小（碱基对，BP）和Y轴代表信号强度（RFU相对荧光单位;在每个相应的等位基因数提供）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52612/52612fig8highres.jpg" target="_blank">请点击此处查看大图</a><html>这个数字。 <img alt="图9" src="/files/ftp_upload/52612/52612fig9.jpg" /> 图9:从成群的生物粒子获得从一个男性的衬衫领口常染色体STR配置文件的常染色体STR曲线是从成群的生物粒子（上左图），使用5微升直接裂解/扩增反应获得。该配置文件的精度是通过比较供体的参考样本来确定。十五常染色体STR基因座和釉原蛋白（性别决定）是共扩增在单个反应并通过毛细管电泳分离。结果将显示在这里作为电泳。等位基因号码每个峰在每个基因座上下面指定。 x轴表示片段的大小（碱基对，bp）的和Y轴表示信号强度（RFU相对荧光单位;在每个相应的等位基因数目提供）。PG"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图10" src="/files/ftp_upload/52612/52612fig10.jpg" /> 图10:一个不能得到从收集的生物粒子而获得的常染色体的STR轮廓的实施例对左侧中所示的个人生物粒子是从衬衫领凝胶薄膜样品收集。如可从所得的STR轮廓（上右图）中可以看出，只有一个等位基因（在30）可得。等位基因号码每个峰在每个基因座上下面指定。 x轴代表片段大小（碱基对，BP）和Y轴代表信号强度（RFU相对荧光单位;在每个相应的等位基因数提供）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52612/52612fig10highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Enhanced Genetic Analysis of Single Human Bioparticles Recovered by Simplified Micromanipulation from Forensic Ã¢â‚¬ËœTouch DNAÃ¢â‚¬â„¢ Evidence at JoVE.com

Enhanced Genetic Analysis of Single Human Bioparticles Recovered by Simplified Micromanipulation from Forensic &#8216;Touch DNA&#8217; Evidence

在这里，我们描述了用于生物颗粒存在于收集的优化和有效地去除战略"触摸的DNA的样​​品，连同涉及一步法5μl的微体积的裂解/ STR扩增增强扩增，以允许恢复短串联重复序列（STR）的生物颗粒体（多个）的轮廓。

单人的生物粒子从法医"触摸DNA&#39;证据回收简体显微增强遗传分析

DNA profiles can be obtained from &#8216;touch DNA&#8217; evidence, which comprises microscopic traces of human biological material. Current methods for ...

enhanced-genetic-analysis-single-human-bioparticles-recovered

Nanyang Technological University

Research

JoVE Journal

Biology

15.6K 次观看.  University of Central Florida. 在这里，我们描述了用于生物颗粒存在于收集的优化和有效地去除战略"触摸的DNA的样​​品，连同涉及一步法5μl的微体积的裂解/ STR扩增增强扩增，以允许恢复短串联重复序列（STR）的生物颗粒体（多个）的轮廓。 

视频: Enhanced Genetic Analysis of Single Human Bioparticles Recovered by Simplified Micromanipulation from Forensic ‘Touch DNA’ Evidence

The Visual Colorimetric Detection of Multi-nucleotide Polymorphisms on a Pneumatic Droplet Manipulation Platform

A simple and visual method to detect multi-nucleotide polymorphism (MNP) was performed on a pneumatic droplet manipulation platform on an open surface. This approach to colorimetric DNA detection was based on the hybridization-mediated growth of gold nanoparticle probes (AuNP probes). The growth size and configuration of the AuNP are dominated by the number of DNA samples hybridized with the probes. Based on the specific size- and shape-dependent optical properties of the nanoparticles, the number of mismatches in a sample DNA fragment to the probes is able to be discriminated. The tests were conducted via droplets containing reagents and DNA samples respectively, and were transported and mixed on the pneumatic platform with the controlled pneumatic suction of the flexible PDMS-based superhydrophobic membrane. Droplets can be delivered simultaneously and precisely on an open-surface on the proposed pneumatic platform that is highly biocompatible with no side effect of DNA samples inside the droplets. Combining the two proposed methods, the multi-nucleotide polymorphism can be detected at sight on the pneumatic droplet manipulation platform; no additional instrument is required. The procedure from installing the droplets on the platform to the final result takes less than 5 min, much less than with existing methods. Moreover, this combined MNP detection approach requires a sample volume of only 10 &#181;l in each operation, which is remarkably less than that of a macro system.

This work presents a simple and visual method to detect multi-nucleotide polymorphisms on a pneumatic droplet manipulation platform. With the proposed method, the entire experiment, including droplet manipulation and detection of multi-nucleotide polymorphisms, can be performed near 23 &#176;C without the aid of advanced instruments.

在气动操纵液滴平台多核苷酸多态性的视觉比色法检测

A simple and visual method to detect multi-nucleotide polymorphism (MNP) was performed on a pneumatic droplet manipulation platform on an open surface. ...

科研

遗传学

DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the evaluation of DNA-magnetic particles binding via dynamic light scattering (DLS) and electrophoretic light scattering (ELS). Analysis by DLS provides valuable information on the physicochemical properties of particles including particle size, polydispersity, and zeta potential. The latter describes the surface charge of the particle which plays major role in electrostatic binding of materials such as DNA. Here, a comparative analysis exploits three chemical modifications of nanoparticles and microparticles and their effects on DNA binding and elution. Chemical modifications by branched polyethylenimine, tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane are investigated. Since DNA exhibits a negative charge, it is expected that zeta potential of particle surface will decrease upon binding of DNA. Forming of clusters should also affect particle size. In order to investigate the efficiency of these particles in isolation and elution of DNA, the particles are mixed with DNA in low pH (~6), high ionic strength and dehydration environment. Particles are washed on magnet and then DNA is eluted by Tris-HCl buffer (pH = 8). DNA copy number is estimated using quantitative polymerase chain reaction (PCR). Zeta potential, particle size, polydispersity and quantitative PCR data are evaluated and compared. DLS is an insightful and supporting method of analysis that adds a new perspective to the process of screening of particles for DNA isolation.

This protocol describes the synthesis of magnetic particles and evaluation of their DNA-binding properties via dynamic and electrophoretic light scattering. This method focuses on monitoring changes in particle size, their polydispersity and zeta potential of particle surface which play major role in binding of materials such as DNA.

动态和电泳光散射的 DNA-磁性颗粒结合分析

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the ...

化学

Optimized High Quality DNA Extraction from Formalin-Fixed Paraffin-Embedded Human Atherosclerotic Lesions

Formalin-fixed paraffin-embedded (FFPE) tissues represent a valuable source for molecular analyses and clinical genomic studies. These tissues are often poor in cells or difficult to process. Therefore, nucleic acids need to be carefully isolated. In recent years, various methods for DNA isolation have been established for tissues from many diseases, mostly cancer. Unfortunately, genomic DNA extracted from FFPE tissues is highly degraded due to the cross-linking between nucleic acid strands and proteins, as well as random breakings in sequence. Therefore, DNA quality from these samples is markedly reduced, making it a challenge for further molecular downstream analyses. Other problems with difficult tissues are, for example, the lack of cells in calcified human atherosclerotic lesions and fatty tissue, small skin biopsies, and consequently low availability of the desired nucleic acids as it is also the case in old or fixed tissues.
In our laboratories, we have established a method for DNA extraction from formalin-fixed atherosclerotic lesions, using a semi-automated isolation system. We compared this method to other commercially available extraction protocols and focused on further downstream analyses. Purity and concentration of the DNA were measured by spectrometry and fluorometry. The degree of fragmentation and overall quality were assessed.
The highest DNA quantity and quality was obtained with the modified blood DNA protocol for the automated extraction system, instead of the commercial FFPE protocol. With this step-by-step protocol, DNA yields from FFPE samples were in average four times higher and fewer specimens failed the extraction process, which is critical when dealing with small-vessel biopsies. Amplicon sizes from 200&#8211;800 bp could be detected by PCR. This study shows that although DNA obtained from our FFPE tissue is highly fragmented, it can still be used for successful amplification and sequencing of shorter products. In conclusion, in our hands, the automated technology appears to be the best system for DNA extraction, especially for small FFPE tissue specimen.

Here, we present a protocol for semi-automated DNA extraction from formalin-fixed paraffin-embedded lesions of human carotid arteries. The tissue lysis is performed without toxic xylene, which is followed by an automated DNA extraction protocol, including a second lysis step, binding of DNA to paramagnetic particles for cellulose based binding, washing steps, and DNA elution.

Formalin-fixed paraffin-embedded (FFPE) tissues represent a valuable source for molecular analyses and clinical genomic studies. These tissues are often ...

Application of DNA Fingerprinting using the D1S80 Locus in Lab Classes

In biological sciences, DNA fingerprinting has been widely used for paternity testing, forensic applications and phylogenetic studies. Here, we describe a reliable and robust method for genotyping individuals by Variable Number of Tandem Repeat (VNTR) analysis in the context of undergraduate laboratory classes. The human D1S80 VNTR locus is used in this protocol as a highly polymorphic marker based on variation in the number of repetitive sequences.
This simple protocol conveys useful information for teachers and the implementation of DNA fingerprinting in practical laboratory classes. In the presented laboratory exercise, DNA extraction followed by PCR amplification is used to determine genetic variation at the D1S80 VNTR locus. Differences in the fragment size of PCR products are visualized by agarose gel electrophoresis. The fragment sizes and repeat numbers are calculated based on a linear regression of the size and migration distance of a DNA size standard.
Following this guide, students should be able to:
&#8226;&#160; Harvest and extract DNA from buccal mucosa epithelial cells 
&#8226;&#160; Perform a PCR experiment and understand the function of various reaction components 
&#8226;&#160; Analyze the amplicons by agarose gel electrophoresis and interpret the results 
&#8226;&#160; Understand the use of VNTRs in DNA fingerprinting and its application in biological sciences

Application of DNA Fingerprinting using the D1S80 Locus in Lab Classes

Here we describe a simple protocol to generate DNA fingerprinting profiles by amplifying the VNTR locus D1S80 from epithelial cell DNA.

使用 D1S80 轨迹在实验室类中应用 DNA 指纹

In biological sciences, DNA fingerprinting has been widely used for paternity testing, forensic applications and phylogenetic studies. Here, we describe a ...

A Droplet-Based Microfluidic Approach and Microsphere-PCR Amplification for Single-Stranded DNA Amplicons

Droplet-based microfluidics enable the reliable production of homogeneous microspheres in the microfluidic channel, providing controlled size and morphology of the obtained microsphere. A microsphere copolymerized with an acrydite-DNA probe was successfully fabricated. Different methods such as asymmetric PCR, exonuclease digestion, and isolation on streptavidin-coated magnetic beads can be used to synthesize single-stranded DNA (ssDNA). However, these methods cannot efficiently use large amounts of highly purified ssDNA. Here, we describe a microsphere-PCR protocol detailing how ssDNA can be efficiently amplified and separated from dsDNA simply by pipetting from a PCR reaction tube. The amplification of ssDNA can be applied as potential reagents for the DNA microarray and DNA-SELEX (Systematic evolution of ligands by exponential enrichment) processes.

This work provides a method for the fabrication of droplet-based microfluidic platforms and the application of polyacrylamide microspheres for microsphere-PCR amplification. The microsphere-PCR method makes it possible to obtain single-stranded DNA amplicons without separating double-stranded DNA.

一种基于液滴的单链 dna 放大术和微球 pcr 扩增法

Droplet-based microfluidics enable the reliable production of homogeneous microspheres in the microfluidic channel, providing controlled size and ...

生物工程

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using noninvasive sampling techniques to investigate population genetics and demography of wild populations1. This approach has proven to be especially useful when dealing with rare or elusive species2. While a number of these methods have been developed to sample hair, feces and other biological material from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. In this video, we present a novel, inexpensive and noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We describe the general set-up of the hair snare, which consists of strips of packing tape arranged in a web-like fashion and placed along travelling routes in the pikas&#x2019; habitat. We illustrate the efficiency of the snare at collecting a large quantity of hair that can then be collected and brought back to the lab. We then demonstrate the use of the DNA IQ system (Promega) to isolate DNA and showcase the utility of this method to amplify commonly used molecular markers including nuclear microsatellites, amplified fragment length polymorphisms (AFLPs), mitochondrial sequences (800bp) as well as a molecular sexing marker. Overall, we demonstrate the utility of this novel noninvasive hair snare as a sampling technique for wildlife population biologists. We anticipate that this approach will be applicable to a variety of small mammals, opening up areas of investigation within natural populations, while minimizing impact to study organisms.

We present a noninvasive sampling approach to efficiently collect hair samples from elusive small mammals, as shown for the American pika. We demonstrate the utility of this method by extracting DNA from sampled hair and amplifying several types of molecular markers commonly used in studies of wildlife ecology and conservation.

一种无创性的发采样技术，以获得难以捉摸的小型兽类高质量的DNA

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

生物学

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

从基因组DNA选择性捕获的5  - 羟甲基

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

Absorbent Microbiopsy Sampling and RNA Extraction for Minimally Invasive, Simultaneous Blood and Skin Analysis

Conventional skin biopsy limits the clinical research that involves cosmetically sensitive areas or pediatric applications due to its invasiveness. Here, we describe the protocol for using&#160;an absorbent microneedle-based device, absorbent microbiopsy, for minimally invasive sampling of skin and blood mixture. Our goal is to help facilitate rapid progress in clinical research, the establishment of biomarkers for skin disease and reducing the risk for clinical research participants. In contrast to conventional skin biopsy techniques, the absorbent microbiopsy can be performed within seconds and does not require intensive training due to its simple design. In this report, we describe the use of absorbent microbiopsy, including loading and application, on a&#160;volunteer. Then, we show how to isolate RNA from the absorbed sample. Finally, we demonstrate the use of quantitative reverse transcription PCR (RT-qPCR) to quantify mRNA expression levels of both blood (CD3E and CD19) and skin (KRT14 and TYR). The methods that we describe utilize off the shelf kits and reagents. This protocol offers a minimally invasive approach for simultaneous sampling of skin and blood within the same absorbent microbiopsy matrix. We have found human ethics committees, clinicians and volunteers to be supportive of this approach to dermatological research.

In this article, we demonstrate how the absorbent microbiopsy technique is performed and how the sample can be used for RNA extraction for simple and simultaneous sampling of skin and blood in a minimally invasive manner.

吸收性微活检取样和 rna 提取用于微创同时进行血液和皮肤分析

Conventional skin biopsy limits the clinical research that involves cosmetically sensitive areas or pediatric applications due to its invasiveness. Here, ...

医学

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

结合单分子操纵和成像蛋白-DNA相互作用的研究

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable of measuring SSDC in many single DNA molecules simultaneously. Bead-tethered substrate DNAs, each containing a single copy of the target sequence, are prepared in a microfluidic flow channel. An external magnet applies a weak force to the paramagnetic beads. The integrity of up to 1,000 individual DNAs can be monitored by visualizing the microbeads under darkfield imaging using a wide-field, low magnification objective. Injecting of a restriction endonuclease, NdeI, initiates the cleavage reaction. Video microscopy is used to record the exact moment of each DNA cleavage by observing the frame in which the associated bead moves up and out of the focal plane of the objective. Frame-by-frame bead counting quantifies the reaction, and an exponential fit determines the reaction rate. This method allows collection of quantitative and statistically significant data on single molecule SSDC reactions in a single experiment.

A highly parallel method for measuring the site-specific cleavage of DNA at the single molecule level is described. This protocol demonstrates the technique using the restriction endonuclease NdeI. The method can easily be modified to study any process that results in site-specific DNA cleavage.

平行高通量单分子动力学测定，用于现场特异性DNA裂解

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable ...

单人的生物粒子从法医"触摸DNA'证据回收简体显微增强遗传分析

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