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>

<ol>
	<li>Hanson, E. K., Ballantyne, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+blood+from+a+stone':+ultrasensitive+forensic+DNA+profiling+of+microscopic+bio-particles+recovered+from+'touch+DNA'+evidence.">Getting blood from a stone': ultrasensitive forensic DNA profiling of microscopic bio-particles recovered from 'touch DNA' evidence.</a> Methods Mol.Biol. 1039, 3-17 (2013).</li><li>Balogh, M. K., Burger, J., Bender, K., Schneider, P. M., Alt, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STR+genotyping+and+mtDNA+sequencing+of+latent+fingerprint+on+paper.">STR genotyping and mtDNA sequencing of latent fingerprint on paper.</a> Forensic Sci Int. 137 (2-3), 188-195 (2003).</li><li>Barbaro, A., Cormaci, P., Teatino, A., La, M. A., Barbaro, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anonymous+letters?+DNA+and+fingerprints+technologies+combined+to+solve+a+case.">Anonymous letters? DNA and fingerprints technologies combined to solve a case.</a> Forensic Sci Int. 146, S133-S134 (2004).</li><li>Bright, J. A., Petricevic, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+trace+DNA+and+its+application+to+DNA+profiling+of+shoe+insoles.">Recovery of trace DNA and its application to DNA profiling of shoe insoles.</a> Forensic Sci.Int. 145 (1), 7-12 (2004).</li><li>Castello, A., Alvarez, M., Verdu, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+from+a+Computer+Keyboard.">DNA from a Computer Keyboard.</a> Forensic Science Communications. 6 (3), (2004).</li><li>Horsman-Hall, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+STR+profiles+from+firearms+and+fired+cartridge+cases.">Development of STR profiles from firearms and fired cartridge cases.</a> Forensic Sci Int. Genet. 3 (4), 242-250 (2009).</li><li>Petricevic, S. F., Bright, J. A., Cockerton, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+profiling+of+trace+DNA+recovered+from+bedding.">DNA profiling of trace DNA recovered from bedding.</a> Forensic Sci. Int. 159 (1), 21-26 (2006).</li><li>Oorschot, R. A., Jones, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+fingerprints+from+fingerprints.">DNA fingerprints from fingerprints.</a> Nature. 387 (6635), 767 (1997).</li><li>Sewell, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+DNA+and+fingerprints+from+touched+documents.">Recovery of DNA and fingerprints from touched documents.</a> Forensic Sci Int.Genet. 2 (4), 281-285 (2008).</li><li>Raymond, J. J., van Oorschot, R. A., Gunn, P. R., Walsh, S. J., Roux, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trace+evidence+characteristics+of+DNA:+A+preliminary+investigation+of+the+persistence+of+DNA+at+crime+scenes.">Trace evidence characteristics of DNA: A preliminary investigation of the persistence of DNA at crime scenes.</a> Forensic Sci Int Genet. 4 (1), 26-33 (2009).</li><li>Wickenheiser, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trace+DNA:+a+review,+discussion+of+theory,+and+application+of+the+transfer+of+trace+quantities+of+DNA+through+skin+contact.">Trace DNA: a review, discussion of theory, and application of the transfer of trace quantities of DNA through skin contact.</a> J.Forensic Sci. 47 (3), 442-450 (2002).</li><li>Sweet, D., Lorente, M., Lorente, J. A., Valenzuela, A., Villanueva, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+to+recover+saliva+from+human+skin:+the+double+swab+technique.">An improved method to recover saliva from human skin: the double swab technique.</a> J. Forensic Sci. 42 (2), 320-322 (1997).</li><li>Darmon, M. M., Blumenberg, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Biology of the Skin - the Keratinocyte. , (1993).</li><li>Daly, D. J., Murphy, C., McDermott, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transfer+of+touch+DNA+from+hands+to+glass,+fabric+and+wood.">The transfer of touch DNA from hands to glass, fabric and wood.</a> Forensic Sci Int Genet. 6 (1), 41-46 (2012).</li><li>Goray, M., Eken, E., Mitchell, R. J., van Oorschot, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secondary+DNA+transfer+of+biological+substances+under+varying+test+conditions.">Secondary DNA transfer of biological substances under varying test conditions.</a> Forensic Sci Int Genet. 4 (2), 62-67 (2010).</li><li>Goray, M., Mitchell, R. J., van Oorschot, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+secondary+DNA+transfer+of+skin+cells+under+controlled+test+conditions.">Investigation of secondary DNA transfer of skin cells under controlled test conditions.</a> Leg.Med.(Tokyo). 12 (3), 117-120 (2010).</li><li>Lowe, A., Murray, C., Whitaker, J., Tully, G., Gill, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+propensity+of+individuals+to+deposit+DNA+and+secondary+transfer+of+low+level+DNA+from+individuals+to+inert+surfaces.">The propensity of individuals to deposit DNA and secondary transfer of low level DNA from individuals to inert surfaces.</a> Forensic Sci Int. 129 (1), 25-34 (2002).</li><li>Phipps, M., Petricevic, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tendency+of+individuals+to+transfer+DNA+to+handled+items.">The tendency of individuals to transfer DNA to handled items.</a> Forensic Sci Int. (2-3), 168-162 (2007).</li><li>Zoppis, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+fingerprinting+secondary+transfer+from+different+skin+areas:+Morphological+and+genetic+studies.">DNA fingerprinting secondary transfer from different skin areas: Morphological and genetic studies.</a> Forensic Sci Int Genet. 11, 137-143 (2014).</li><li>Gill, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Misleading DNA Evidence: Reasons for Miscarriages of Justice. , (2014).</li><li>Haas, C., Hanson, E., Ballantyne, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+electrophoresis+of+a+multiplex+reverse+transcription-polymerase+chain+reaction+to+target+messenger+RNA+markers+for+body+fluid+identification.">Capillary electrophoresis of a multiplex reverse transcription-polymerase chain reaction to target messenger RNA markers for body fluid identification.</a> Methods Mol. Biol. 830, 169-183 (2012).</li><li>Hanson, E., Ballantyne, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+Profiling+for+the+Identification+of+the+Tissue+Origin+of+Dried+Stains+in+Forenic+Biology.">RNA Profiling for the Identification of the Tissue Origin of Dried Stains in Forenic Biology.</a> Forensic Sci Rev. , 22145-22157 (2010).</li><li>Hanson, E., Haas, C., Jucker, R., Ballantyne, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+and+sensitive+mRNA+biomarkers+for+the+identification+of+skin+in+'touch+DNA'+evidence.">Specific and sensitive mRNA biomarkers for the identification of skin in 'touch DNA' evidence.</a> Forensic Sci Int Genet. 6, 548-558 (2012).</li><li>Hanson, E. K., Ballantyne, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+specific+mRNA+biomarkers+for+the+identification+of+vaginal+secretions+in+sexual+assault+investigations.">Highly specific mRNA biomarkers for the identification of vaginal secretions in sexual assault investigations.</a> Sci Justice. 53 (1), 14-22 (2013).</li><li>Juusola, J., Ballantyne, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Messenger+RNA+profiling:+a+prototype+method+to+supplant+conventional+methods+for+body+fluid+identification.">Messenger RNA profiling: a prototype method to supplant conventional methods for body fluid identification.</a> Forensic Sci Int. 135 (2), 85-96 (2003).</li><li>Juusola, J., Ballantyne, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+mRNA+profiling+for+the+identification+of+body+fluids.">Multiplex mRNA profiling for the identification of body fluids.</a> Forensic Sci Int. 152 (1), 1-12 (2005).</li><li>Fleming, R. I., Harbison, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+a+mRNA+multiplex+RT-PCR+assay+for+the+definitive+identification+of+body+fluids.">The development of a mRNA multiplex RT-PCR assay for the definitive identification of body fluids.</a> Forensic Sci Int Genet. 4 (4), 244-256 (2010).</li><li>Haas, C., Klesser, B., Maake, C., Bar, W., Kratzer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mRNA+profiling+for+body+fluid+identification+by+reverse+transcription+endpoint+PCR+and+realtime+PCR.">mRNA profiling for body fluid identification by reverse transcription endpoint PCR and realtime PCR.</a> Forensic Sci Int Genet. 3 (2), 80-88 (2009).</li><li>Lindenbergh, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multiplex+(m)RNA-profiling+system+for+the+forensic+identification+of+body+fluids+and+contact+traces.">A multiplex (m)RNA-profiling system for the forensic identification of body fluids and contact traces.</a> Forensic Sci Int Genet. 6 (2), 565-577 (2012).</li><li>Richard, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+mRNA+marker+specificity+for+the+identification+of+five+human+body+fluids+by+capillary+electrophoresis.">Evaluation of mRNA marker specificity for the identification of five human body fluids by capillary electrophoresis.</a> Forensic Sci Int Genet. 6 (4), 452-460 (2012).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" width="998">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Name of Material/ Equipment</td>
			<td style="width:147px;">Company</td>
			<td style="width:139px;">Catalog Number</td>
			<td style="width:320px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:395px;">SealRite 1.5 mL natural microcentrifuge tubes, sterile</td>
			<td style="width:147px;">USA Scientific</td>
			<td style="width:139px;">1615-5510</td>
			<td style="width:320px;">numerous altnerative suppliers are available, but tubes should be sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">0.2 mL PCR tubes with flat cap, natural</td>
			<td style="width:147px;">Phenix Research</td>
			<td style="width:139px;">MPX-200F</td>
			<td style="width:320px;">or equivalent</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:395px;">Kimwipes</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:139px;">06-666 and/or 06-666-11C</td>
			<td style="width:320px;"></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:395px;">Alcohol prep pads</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:139px;">06-669-62</td>
			<td style="width:320px;">alternative is 06-665-24 (Fisher Sci); canister of larger alcohol wipes rather than individual pads</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Sterile water</td>
			<td style="width:147px;"></td>
			<td style="width:139px;"></td>
			<td style="width:320px;">18.2 mega-ohm, autoclaved</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Glass microscope slides, frosted, 3&#34; x 1&#34;: 1mm</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:139px;">12-544-3</td>
			<td style="width:320px;">alternative brands or sizes are acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Disposable transfer pipet</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:139px;">13-711-9D</td>
			<td style="width:320px;">alternatives are acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Trypan blue solution</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:139px;">T8154</td>
			<td style="width:320px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Pipets: 0.2-2, 2-20, 10-100, 100-1,1000 mL</td>
			<td style="width:147px;">various</td>
			<td style="width:139px;"></td>
			<td style="width:320px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Sterile, aerosol-resistant pipet tips</td>
			<td style="width:147px;">various</td>
			<td style="width:139px;"></td>
			<td style="width:320px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Mini-centrifuge</td>
			<td style="width:147px;">various</td>
			<td style="width:139px;"></td>
			<td style="width:320px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:395px;">Stereomicroscope</td>
			<td style="width:147px;">Leica</td>
			<td style="width:139px;">M205C</td>
			<td style="width:320px;">others are suitable, but must be capable of magifications up to 350X</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:395px;">GeneAmp PCR system 9700 thermal cycle (silver or gold block)</td>
			<td style="width:147px;">Life Technologies</td>
			<td style="width:139px;">N8050200 or 4314878</td>
			<td style="width:320px;">other models and manufacturers can be used, but performance with the STR amplification kit must be verified</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:395px;">3130 Genetic Analyzer</td>
			<td style="width:147px;">Life Technologies</td>
			<td style="width:139px;">3130-01</td>
			<td style="width:320px;">Alternatives: 310 or 3500 Genetic Aanlyzers (Life Technologies)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:395px;">GeneMapper software</td>
			<td style="width:147px;">Life Technologies</td>
			<td style="width:139px;">various</td>
			<td style="width:320px;">Various versions of the software are available and can be used</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:395px;">WF Gel-Film , x8 retention level</td>
			<td style="width:147px;">Gel-Pak</td>
			<td style="width:139px;">WF-40-x8-A</td>
			<td style="width:320px;">4&#34; x 4&#34; wafer film; can be ordered in other size specifications</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Double sided tape</td>
			<td style="width:147px;">various</td>
			<td style="width:139px;"></td>
			<td style="width:320px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:395px;">Glass block, 3&#34; x 1.5&#34; x 3/8&#34;</td>
			<td style="width:147px;">McCrone Microscopes and Accessories</td>
			<td style="width:139px;">370-2</td>
			<td style="width:320px;">or equivalent</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:395px;">Tungsten needle with holder</td>
			<td style="width:147px;">McCrone Microscopes and Accessories</td>
			<td style="width:139px;">106</td>
			<td style="width:320px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">3M&#160; water soluble wave solder tape, 5414 transparent</td>
			<td style="width:147px;">Allied Electronics</td>
			<td style="width:139px;">70113977</td>
			<td style="width:320px;"></td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:395px;">Lysis buffer (forensicGEM Saliva, Zygem)</td>
			<td style="width:147px;">VWR</td>
			<td style="width:139px;">95043-992</td>
			<td style="width:320px;">Lysis buffer is prepared in 10 ml portions (prepare additional master mix portions as needed to accommodate the desired number of samples). 1.5 ml is used for each sample. To prepare 10 ml of lysis buffer (sufficient for ~6 samples): 6.9 mL sterile water, 2.1 mL 10x buffer - blue, 1 mL forensicGEM.&#160;</td>
		</tr>
		<tr height="210">
			<td height="210" style="height:210px;width:395px;">STR Amplification kit, AmpFlSTR Identifiler Plus PCR amplification kit</td>
			<td style="width:147px;">Life Technologies</td>
			<td style="width:139px;">4427368</td>
			<td style="width:320px;">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 ml PCR reaction mix, 1.1 ml Primer set, 0.2 ml (1U) AmpliTaq Gold DNA polymerase. A master mix sufficient for the desired number of samples should be prepared. 3.5 ml of the prepared mix is added to the 0.2 mL tube for bio-particle collection.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">AmpliTaq Gold DNA polymerase</td>
			<td style="width:147px;">Life Technologies</td>
			<td style="width:139px;">N808-0245</td>
			<td style="width:320px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">HiDi formamide</td>
			<td style="width:147px;">Life Technologies</td>
			<td style="width:139px;">4311320</td>
			<td style="width:320px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">GeneScan 500 LIZ size standard</td>
			<td style="width:147px;">Life Technologies</td>
			<td style="width:139px;">4322682</td>
			<td style="width:320px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">96 well optical reaction plates</td>
			<td style="width:147px;">Life Technologies</td>
			<td style="width:139px;">N8010560</td>
			<td style="width:320px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Plate septa, 96 well</td>
			<td style="width:147px;">Life Technologies</td>
			<td style="width:139px;">4315933</td>
			<td style="width:320px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Performance-optimized polymer, POP-7</td>
			<td style="width:147px;">Life Technologies</td>
			<td style="width:139px;">4363785</td>
			<td style="width:320px;">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/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/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/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/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/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/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 â€˜Touch DNAâ€™ Evidence at JoVE.com

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

Research

JoVE Journal

Biology

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

15.4K 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 &#8216;Touch DNA&#8217; Evidence

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

从成年小鼠海马齿状回的剖析

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

科研

生物学

Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell types exhibit heterogeneous biochemical makeup depending on their physiological conditions and interactions with the environment. Conventional methods of mass spectrometry (MS) used for the analysis of biomolecules in single cells rely on extensive sample preparation. Removing the cells from their natural environment and extensive sample processing could lead to changes in the cellular composition. Ambient ionization methods enable the analysis of samples in their native environment and without extensive sample preparation.1 The techniques based on the mid infrared (mid-IR) laser ablation of biological materials at 2.94 &mu;m wavelength utilize the sudden excitation of water that results in phase explosion.2 Ambient ionization techniques based on mid-IR laser radiation, such as laser ablation electrospray ionization (LAESI) and atmospheric pressure infrared matrix-assisted laser desorption ionization (AP IR-MALDI), have successfully demonstrated the ability to directly analyze water-rich tissues and biofluids at atmospheric pressure.3-11 In LAESI the mid-IR laser ablation plume that mostly consists of neutral particulate matter from the sample coalesces with highly charged electrospray droplets to produce ions. Recently, mid-IR ablation of single cells was performed by delivering the mid-IR radiation through an etched fiber. The plume generated from this ablation was postionized by an electrospray enabling the analysis of diverse metabolites in single cells by LAESI-MS.12 This article describes the detailed protocol for single cell analysis using LAESI-MS. The presented video demonstrates the analysis of a single epidermal cell from the skin of an Allium cepa bulb. The schematic of the system is shown in Figure 1. A representative example of single cell ablation and a LAESI mass spectrum from the cell are provided in Figure 2.

Single cell analysis is performed by mass spectrometry on plant and animal cells at atmospheric pressure by using a sharpened optical fiber to sample the cells for laser ablation electrospray ionization (LAESI) mass spectrometry.

在大气压力质谱直接分析单细胞

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell ...

An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been understood at molecular level. One of the mechanisms mediating the effect of DNA variants on phenotypes is gene expression, which has been shown to be particularly relevant for complex traits1.
This method tests in a cellular context the effect of specific DNA sequences on gene expression. The principle is to measure the relative abundance of transcripts arising from the two alleles of a gene, analysing cells which carry one copy of the DNA sequences associated with disease (the risk variants)2,3. Therefore, the cells used for this method should meet two fundamental genotypic requirements: they have to be heterozygous both for DNA risk variants and for DNA markers, typically coding polymorphisms, which can distinguish transcripts based on their chromosomal origin (Figure 1). DNA risk variants and DNA markers do not need to have the same allele frequency but the phase (haplotypic) relationship of the genetic markers needs to be understood. It is also important to choose cell types which express the gene of interest. This protocol refers specifically to the procedure adopted to extract nucleic acids from fibroblasts but the method is equally applicable to other cells types including primary cells.
DNA and RNA are extracted from the selected cell lines and cDNA is generated. DNA and cDNA are analysed with a primer extension assay, designed to target the coding DNA markers4. The primer extension assay is carried out using the MassARRAY (Sequenom)5 platform according to the manufacturer's specifications. Primer extension products are then analysed by matrix-assisted laser desorption/ionization time of-flight mass spectrometry (MALDI-TOF/MS). Because the selected markers are heterozygous they will generate two peaks on the MS profiles. The area of each peak is proportional to the transcript abundance and can be measured with a function of the MassARRAY Typer software to generate an allelic ratio (allele 1: allele 2) calculation. The allelic ratio obtained for cDNA is normalized using that measured from genomic DNA, where the allelic ratio is expected to be 1:1 to correct for technical artifacts. Markers with a normalised allelic ratio significantly different to 1 indicate that the amount of transcript generated from the two chromosomes in the same cell is different, suggesting that the DNA variants associated with the phenotype have an effect on gene expression. Experimental controls should be used to confirm the results.

Genetic associations often remain unexplained at a functional level. This method aims to assess the effect of phenotype-associated genetic markers on gene expression by analyzing cells heterozygous for transcribed SNPs. The technology allows accurate measurement by MALDI-TOF mass spectrometry to quantify allele-specific primer extension products.

等位基因特异性基因表达检测，测试的遗传协会的功能基础

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been ...

Transduction of Human Cells with Polymer-complexed Ecotropic Lentivirus for Enhanced Biosafety

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for laboratory personnel. Pantropic lentiviruses, such as the commonly used VSV-G pseudotype, are a valuable tool for studying gene function because they can transduce many cell types, including non-dividing cells. However, researchers may wish to avoid production and centrifugation of pantropic viruses encoding oncogenes due to higher biosafety level handling requirements and safety issues. Several potent oncogenes, including c-Myc and SV40 large T antigen, are known to enhance production of induced pluripotent stem cells (iPSC). All other known iPSC-inducing genetic changes (OCT4, SOX2, KLF4, NANOG, LIN28, and p53 loss of function) also have links to cancer, making them of relatively high safety concern as well. 
While these cancer-related viruses are useful in studying cellular reprogramming and pluripotency, they must be used safely. To address these biosafety issues, we demonstrate a method for transduction of human cells with ecotropic lentivirus, with additional emphasis on reduced cost and convenient handling. We have produced ecotropic lentivirus with sufficiently high titer to transduce greater than 90% of receptor-expressing human cells exposed to the virus, validating the efficacy of this approach. 
Lentivirus is often concentrated by ultracentrifugation; however, this process takes several hours and can produce aerosols infectious to human biomedical researchers. As an alternative, viral particles can be more safely sedimented onto cells by complexation with chondroitin sulfate and polybrene (CS/PB). This technique increases the functional viral titer up to 3-fold in cells stably expressing murine retrovirus receptor, with negligible added time and cost. Transduction of human dermal fibroblasts (HDFs) is maximally enhanced using CS/PB concentrations approximately 4-fold lower than the optimal value previously reported for cancer cell lines, suggesting that polymer concentration should be titrated for the target cell type of interest. We therefore describe the use of methylthiazolyldiphenyl-tetrazolium bromide (MTT) to assay for polymer toxicity in a new cell type. We observe equivalent viability of HDFs after viral transduction using either polymer complexation or the standard dose of polybrene (PB, 6 &mu;g/ml), indicating minimal acute toxicity.
In this protocol, we describe the use of ecotropic lentivirus for overexpression of oncogenes in human cells, reducing biosafety risks and increasing the transduction rate. We also demonstrate the use of polymer complexation to enhance transduction while avoiding aerosol-forming centrifugation of viral particles.

Lentiviruses are a valuable research tool for exploring gene function; however, researchers may wish to avoid production of pantropic lentivirus encoding known or suspected oncogenes. As an alternative, we present a safer protocol for use of ecotropic lentivirus on human cells modified to express the ecotropic receptor mSlc7a1.

增强生物安全与聚合物复合物Ecotropic慢病毒的人体细胞的转导

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

生化和高通量的微观评估的脂肪含量<em&gt;秀丽隐杆线虫</em

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

人类心肌细胞的生成：从馈线人类诱导多能干细胞的分化协议

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing

Efforts to detect and investigate key oncogenic mutations have proven valuable to facilitate the appropriate treatment for cancer patients. The establishment of high-throughput, massively parallel "next-generation" sequencing has aided the discovery of many such mutations. To enhance the clinical and translational utility of this technology, platforms must be high-throughput, cost-effective, and compatible with formalin-fixed paraffin embedded (FFPE) tissue samples that may yield small amounts of degraded or damaged DNA. Here, we describe the preparation of barcoded and multiplexed DNA libraries followed by hybridization-based capture of targeted exons for the detection of cancer-associated mutations in fresh frozen and FFPE tumors by massively parallel sequencing. This method enables the identification of sequence mutations, copy number alterations, and select structural rearrangements involving all targeted genes. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus conferring high sensitivity for detecting low frequency mutations.

We describe the preparation of barcoded DNA libraries and subsequent hybridization-based exon capture for detection of key cancer-associated mutations in clinical tumor specimens by massively parallel "next generation" sequencing. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus yielding high sensitivity for detecting low frequency mutations.

由外显子捕获和大规模平行测序检测体细胞遗传学改变在肿瘤标本

Efforts  to detect and investigate key oncogenic mutations have proven valuable to  facilitate the appropriate treatment for cancer patients. The ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

另类文化的人多能干细胞的生产，维护和遗传分析

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糖皮质激素粪分析：在马属动物无创监测肾上腺

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the invasion of non-indigenous species. However, an exact identification of field predator-prey interactions is difficult in many cases. These analyses are often based on a visual evaluation of gut content or the analysis of stable isotope ratios (&#948;15N and &#948;13C). Such methods require comprehensive knowledge about, respectively, morphologic diversity or isotopic signature from individual prey organisms, leading to obstacles in the exact identification of prey organisms. Visual gut content analyses especially underestimate soft bodied prey organisms, because maceration, ingestion and digestion of prey organisms make identification of specific species difficult. Hence, polymerase chain reaction (PCR) based strategies, for example the use of group-specific primer sets, provide a powerful tool for the investigation of food web interactions. Here, we describe detailed protocols to investigate the gut contents of macroinvertebrate consumers from the field using group-specific primer sets for nuclear ribosomal deoxyribonucleic acid (rDNA). DNA can be extracted either from whole specimens (in the case of small taxa) or out of gut contents of specimens collected in the field. Presence and functional efficiency of the DNA templates need to be confirmed directly from the tested individual using universal primer sets targeting the respective subunit of DNA. We also demonstrate that consumed prey can be determined further down to species level via PCR with unmodified group-specific primers combined with subsequent single strand conformation polymorphism (SSCP) analyses using polyacrylamide gels. Furthermore, we show that the use of different fluorescent dyes as labels enables parallel screening for DNA fragments of different prey groups from multiple gut content samples via automated fragment analysis.

Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.

用基团特异 rDNA 引物对水生无脊椎动物进行遗传肠道含量分析的实验室规程

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the ...