Name: improved polymerase chain reaction-restriction fragment length polymorphism genotyping of toxic pufferfish by liquid chromatography/mass spectrometry
Uploaded: 2016-09-20T08:00:00.000+00:00
Duration: 9 min 34 s
Description: Watch this Scientific Journal Video about Improved Polymerase Chain Reaction-restriction Fragment Length Polymorphism Genotyping of Toxic Pufferfish by Liquid Chromatography/Mass Spectrometry at JoVE.

This work was supported by a Grant-in-Aid for Scientific Research by the Japanese Society for the Promotion of Science (15K08060).

1. DNA提取注意:使用DNA提取和PCR后检查如凝胶电泳和LC / ESI-MS一个单独的房间。加乙醇洗涤缓冲液1（含胍盐酸盐），并根据DNA提取试剂盒协议洗涤缓冲液2（不含有盐酸胍）。鱼类批发商和零售市场在日本获得了鱼样品。 <ol><li>将30-50毫克的鱼组织在0.5 ml或1.5 ml离心管。用微型刮勺（除了翅片和皮肤）压扁的组织。 注:在散热片的情况下，用0.5毫升管浸泡。 </li><li>添加裂解缓冲液的180微升和40微升蛋白酶K和旋涡简要的。在56℃下孵育2小时或过夜的块加热器。通过在培养过程中短暂地涡旋，每15分钟混合。 注:完全裂解是没有必要的。 </li><li>离心13,000×g下5分钟。离心后，如果一个小摩UNT的油中存在脂肪酸样品如肝的情况下，表面上，将其丢弃。转移上清液至新的1.5 ml离心管中。 </li><li>加入200μl含有盐酸胍的离液缓冲液中，通过涡旋混合。通过再次涡旋加入200μl的乙醇（99.5％）和组合。 </li><li>将混合物转移到放置在2 ml收集管离心柱。离心以13,000×g的1分钟。丢弃的流量通过。 注意:不要添加漂白剂的浪费，因为胍可与漂白高活性化合物。 </li><li>加入500微升洗涤缓冲液1（含胍盐酸盐）和离心机的对以13,000×g的1分钟。丢弃通过并流的收集管。 </li><li>放置旋转柱中随试剂盒提供新的2ml收集管中。加入500微升洗涤缓冲液2和离心机的用于在20000×g下3分钟。丢弃流通和收集管。离心柱转移到新1.5 ml离心管。 </li><li>吸管200微升的洗脱缓冲液，以旋转柱膜的中央的。 1分钟后，离心在6000×g下1分钟。 </li><li>吸管洗脱液到一次性紫外比色皿的50微升，测量洗脱液的在220-300毫微米的紫外光谱和使用分光光度计在260 nm处的吸光度。 </li><li>验证DNA的UV光谱与260nm处的峰。计算使用简单的方程的近似DNA浓度"DNA浓度（ng /微升）以260纳米×50 =吸收"。 注:从河豚的鳍提取典型的DNA浓度为63±30纳克/微升（N = 20）。 </li><li>如果DNA浓度高于10毫微克/微升，稀释用Tris-EDTA（TE）缓冲液（pH 8）的DNA样品，以5纳克/微升。 </li><li>在-20℃保存样品或进行PCR扩增（第2节）。 </li></ol> 2. PCR <ol><li>成立25微升PCR每个抽充当DNA样品，阳性对照清洁台上于和阴性对照（超纯水代替DNA样品）在冰上按表1和2以避免污染（具有TE缓冲液pH 8.0的靶序列合成的寡核苷酸0.2μM）。 注意:操作简便，使含所有试剂没有模板DNA的鸡尾酒足够数量和分配它。使用的DNA具有聚合酶校对活性，以避免加入3&#39;A突出端向PCR产物。 </li><li>开展使用表3中所示的循环程序热循环仪进行PCR扩增。 </li></ol> 3.酶消化<ol><li>添加2微升含有100mM的Tris-HCl（pH值7.5），100mM的MgCl 2的，10毫二硫苏糖醇及500mM的NaCl至PCR溶液的溶液。 </li><li>将每个限制酶1μL（DRA我和MSP I），轻轻混匀。在37℃下孵育30分钟的第ermal PCR仪。 </li><li>孵育5分钟，在72℃，以激活剩余的DNA聚合酶，它填补了的Msp I.产生的粘性末端</li><li>可选的:使用的3.33（重量％）的15的横连杆比率（％C，双丙烯酰胺的百分比）和聚丙烯酰胺凝胶浓度（％T）分析各2.5微升用聚丙烯酰胺凝胶电泳将反应溶液的（％重量/体积） 38。使用荧光DNA染色染色凝胶使用琥珀色屏幕蓝光透射观察双链DNA。 </li><li>筛选具有0.2-0.5微米的离心过滤装置中的反应溶液，以防止堵塞，这会损坏分析柱。 </li><li>转移滤液到锥形聚丙烯小瓶中并储存在-20℃直至分析。 </li></ol> 4. LC / ESI-MS分析<ol><li>权衡的HFIP 67.2克（ 约 42ml）中，并在1升瓶子的TEA 1.52克（ 约 2.0ml）中。加入955毫升的超纯水（LC / MS GRA德）和搅拌用磁力搅拌器直到试剂完全溶解。取溶液的等分试样，并检查pH值（7.85和7.95之间;理想的pH = 7.9）使用pH计。 注意:不要将等分返回奶瓶中的金属离子，以避免污染。如果pH值不7.9，添加任一HFIP或TEA少量调节pH值，并再次测定pH值。 </li><li>连接到瓶线带采用直冷式冰箱便携式（家用），以防止汽化瓶子冷却液相色谱仪。连接装有甲醇B线另一瓶</li><li>连接保护柱和分析柱（2.0×50毫米，孔大小= 30毫微米）液相色谱仪。开始流动的初始流动相（95％A和5％B）平衡的列。 </li><li>制备0.5毫克/毫升三氟乙酸钠（pH为3.5），其为校准39。 <ol><li>称取三氟交流的50毫克（ 约 34微升）编号中的烧杯中。 </li><li>加入30毫升超纯水和滴定至用10mM氢氧化钠pH值3.5与pH计的援助。 </li><li>填写可达50毫升超纯水。加入50毫升乙腈（LC / MS级），混匀。 </li><li>取工作溶液的一部分，并且它在室温下储存。在4℃保存液的其余部分。 </li></ol></li><li>降低C-陷阱的氮气压力如下。 注意:最近的傅里叶变换质谱仪，其适合于完整蛋白质分析，有控制软件来控制压力。 <ol><li>除去质谱仪的左顶盖。在C-疏水阀是在左侧前景的角落。 </li><li>在控制软件的仪器状态窗口检查高真空的压力值。 注意:值通常，3×10 -8巴。 </li><li>拉动旋钮解锁和旋转旋钮逆时针非常缓慢，以减少高真空新闻URE到1/3（典型地，1×10 -8巴）。所指示的压力应以延迟方式逐渐改变。忽略有关低压警告消息。按下旋钮锁定。 </li><li>恢复安全顶盖。 </li></ol></li><li>校准用三氟乙酸钠39质谱仪。 注意:每日质量校准可以通过该协议被取代，然而，由制造商所建议的建议的校准应该已经在此之前校准完成。 <ol><li>设置扫描参数和调节软件的仪器控制箱的加热源ESI参数如下:扫描类型全MS;扫描范围M / Z 500-4,000;碎片（在源碰撞诱导解离（CID）电压60伏特;极性负;自动增益控制（AGC）靶1×10 6个 ，最大注射时间50毫秒;鞘气流量5;辅助气体流速0;吹扫气流量0;喷雾电压3千伏;毛细管温度320℃; S-镜头射频（RF）级100;加热器温度30℃。 <ol><li>输入的负离子名单为M / Z 792.85908，1064.80870，1336.75832，1608.70794，1880.65755，2152.60717，2424.55679，2696.50640，2968.45602，3376.38045进行监测。 </li></ol></li><li>靠拢加热的ESI探针到质谱仪（位置B）的界面。连接探针和带有管子的注射器。 </li><li>使用嵌入式注射泵不断注入校准物以10微升/分钟的流速。检查在所述定制校准表仅6低级质量离子（M / Z 792.85908-2152.60717）。 <ol><li>与校准继续的信号的强度都稳定之后。校准后，检查仅六个较高质量的离子（M / Z 1880.65755-3376.38045）中，用校准进行。避免使用所有离子同时避免失败校准。 </li></ol></li></ol></li><li>移动探头到appropria为0.4毫升/分钟（位置D）和流速德位置连接到液相色谱仪用惰性金属管。 </li><li>设定在调谐软件的仪器控制箱加热的ESI源参数如下:鞘气流量50;辅助气体流量15;扫气流量1;喷雾电压2.5千伏;毛细管温度350℃; S-镜头RF级100;加热器温度350℃。 </li><li>在工具设置窗口质谱仪设置采集参数如下:方法持续时间8分钟;分流阀，3.5-6分钟质谱仪，0-3.5和6-8分钟浪费;运行时3.5到6分钟;负极性;源CID电压15.0伏特; 3微扫描;标称分辨率功率（在m / z 200）140000; AGC目标1×10 6;最大离子时间50毫秒;扫描1号;扫描范围M / Z 700-3,500;光谱数据类型的配置文件。 </li><li>液相色谱仪的参数设置如下:柱温20℃; SAMPLË盘温度10℃;梯度程序0-0.5分钟5％B，0.5-1分钟5-30％B，1-3.5分钟30-40％B，3.5-5分钟40-98％B，5-6分钟98％B，6- 6.05分钟98-5％的B，6.05-8分钟5％B;流量为0.4毫升/分钟。 </li><li>清除通过点击小瓶的底部气泡。设置样品盘样品瓶，并使用自动进样器开始分析注入1.0微升的样品。 </li><li>打开原始数据文件的浏览器软件，并确认所有的收购都成功完成，包括阳性和阴性对照。 注意:当河豚DNA被成功地扩增和消化典型地，两个或三个峰将在4-5.5分钟的保留时间的总离子流色谱图中观察到。 </li></ol> 5.反卷积与解读<ol><li>启动反卷积软件。选择实验类型的"自动提取物（同位素解决）"。 </li><li>编辑使用以下参数的方法:输出质量M; S / N threshold 3处，相对丰度阈值的0;负电荷是;算算提取离子流色谱（XIC）是; m / z范围700-3,500;充电载体H +;检测出的充电3的最小数量;同位素比值核苷酸;拟合系数80％;其余阈0％;考虑重叠是的;收费范围3-50;最小强度1;预计强度误差3。 <ol><li>另存为一个新的方法和选择它。 </li></ol></li><li>选择其中原始数据文件所在的目录。选择要分析的原始数据文件，然后单击"添加到队列"按钮。 </li><li>点击"运行"按钮，在"运行队列"选项卡来启动反褶积。 </li><li>队列完成后，选择行，并在标题上点击"打开结果"来获取反褶积的结果。 </li><li>解释从峰在使用表4 4-5.5分钟的保留时间导出的单一同位素质量的结果。 </li></ol>

The authors have nothing to disclose.

以提取的DNA，用于提取从血液和组织中的DNA商品化试剂盒在根据与小的修改（蛋白酶K的量和裂解溶液的离心分离）的制造商的协议被使用。然而，任何提取试剂盒可以只要细胞DNA可以用合适的回收率和纯度用于PCR被提取使用。此方法已被使用肌肉，鳍，肝脏，卵巢和皮肤37进行测试。鱼翅是特别适合，因为大的表面积，从而能快速溶解与蛋白酶K鲜，冻，干，煮和烤河豚样品成功地测试了37中。 在PCR引物组（ 见表1）被设计为河豚和放大河豚的线粒体16S核糖体RNA基因。目标DNA扩增为114-115基点（属暗纹 ）和86基点（属Lagocephalus）（ 表5）。为了促进法DEVELOPME核苷酸，被用于参考，而不是收集真实河豚试样具有靶序列合成的寡核苷酸。引物组可以由另一引物响应于所述目的设置来代替，然而，酶消化后DNA终长度应小于100个核苷酸中保持成功反卷积所需的质谱的质量方面。此外，当目标寡核苷酸为大于约75个核苷酸长在C-阱氮气压力应减少和质谱的质量不足以成功去卷积。这些限制可以通过仪器软件的最新发展进行控制，否则在协议中部分描述了机箱内部的C-陷阱阀门必须进行调整。至于样品制备，使用无洗涤剂的试剂为在适当的峰形，足以峰强度和稳定的保留时间31方面随后的LC至关重要。 21,24-33，一个C 18逆相粒状二氧化硅柱23,40,41和疏水相互作用色谱（HILIC）柱42已被用于LC /寡核苷酸的ESI-MS分析。其中，毛细管整料列是优于其他的分离能力方面，但是，在过去的研究中使用的毛细管整料柱在内部制成，以低流速（2微升/分钟），这需要的仪器操作致力于微型LC。为了便于操作方便，市售的柱为长的寡核苷酸在较高流速（100-400微升/分钟）分离评价。三对DNA双链体（26，37和53碱基对）的用上述柱分离，但，将C 18键合性颗粒二氧化硅柱和PS-DVB整料列的循环时间分别为65和70分钟，分别，而认为的C <sub&gt; 18键合性二氧化硅整料柱是8分钟（ 图1）。同时快速分析考虑，选择了-C 18键合性硅胶整体柱对我们而言，尽管有限的分离能力;然而，需要改善的分离时可使用的剩余的两列。从理论上讲，在单块列的情况下，不存在间隙体积，流动相将被迫流过固相的孔隙，同时保持了一致的路径长度，因此能够有效分离27。这样的过程将表现出来，尤其是在生物大分子的分析如寡核苷​​酸，作为大分子的慢扩散。一项所述的单块列的实用优点是该背压比粒状二氧化硅柱27的下部。尽管高流动速率（400微升/分钟）和低塔的温度（20℃），最大后推力系统为12.5兆帕37。这是相当长的寡核苷酸的快速分析为C 18键合性硅石整料列的优点的第一个示范。由于高流速，因此不需要在界面微型LC和精确对准的专用仪器。相反，加热ESI探针需要分解的DNA双链，并如后文所述帮助DNA的电离。 离子对色谱法通常用于寡核苷酸的MS兼容的分离。然而，离子对试剂通常与ESI过程干扰和减小ESI-MS的灵敏度。因此，HFIP经常用于流动相，以提高寡核苷酸的灵敏度。然而，HFIP（沸点59℃）在界面迅速蒸发甲醇（沸点65℃），因此，溶剂这一损失增加的pH并促进离子对试剂的离解（ 即 ，TEA）之前从寡核苷酸。因为本方法使用加热的ESI探针，其nebulizes在350℃的热氮气的洗脱物，这种影响可能被过分强调。取而代之的HFIP-TEA缓冲，ERB和Oberacher推荐cyclohexyldimethylammonium醋酸（CycHDMAA; pH值为8.4）进行基因分型分析，因为在加合物形成的减少与微量金属离子33。作者推断CycHDMAA本身抑制了金属加合物的形成。尽管文献中，显著加合物的形成还没有在本发明的方法观察到的。此外，HFIP-TEA -甲醇系统的值得注意的优点是，与HFIP-TEA -甲醇系统获得的峰面积比分析T的86 bp的扩增时从CycHDMAA -乙腈系统获得更大的17倍poecilonotus（数据未显示）。在HFIP-TEA - 甲醇系统的一个缺点，但是，是相对于CycHDMAA - 乙腈系统的成本增加。的单一同位素质量的计算需要多电荷离子的同位素峰的分离。因此，分辨率是目前的分析至关重要。虽然所需的分辨能力取决于分析物的碱基对长度，常规的TOF分析仪，其具有数万的分辨能力，可以被限制为短寡核苷酸的分析。 在表4所示的理论单同位素质量，从该分析物的相应的碱的组合物计算的。可替代地，Muddiman 等人开发的软件应用程序以从精确质量43计算基础组合物。类似的程序集成到自动化ESI-MS系统，16-18。使用这些算法可以提高本方法的鲁棒性，因为所测量的单一同位素质量并不总是对应于一个独特的基础组合物ö翼3 ppm的从不可避免的测量误差导致的质量偏差。不幸的是，我们无法获得这些软件产品进行本研究。 因为本方法是基于分析基础组合物并没有涉及专门为河豚设计了一个程序本单同位素基于质量的物种确定可以是不仅适于河豚的分化，而且用于检测其它DNA多态性。作为用于检测的DNA多态性的，专用ESI-MS系统是完全自动化的，易于操作，其可以是适合于诊断用途，如检测的病原体15,17,18,20,30。相反地​​，本发明的方法是用共同的研究仪器和设备，因此，适合用于研究用途的可行性。 ESI-MS已被应用到人DNA多态性例如单核苷酸多态性13,28,32，短串联重复26和线粒体DNA analsis 16,19。微小RNA是通过毛细管LC / ESI-MS 44也进行分析。这些公布的申请也可以通过本发明的方法实现的。此外，这种方法可以适用于监测寡核苷酸和低分子量的化合物，如抗生素由于低温分离的相互作用和核糖体RNA 45之间的相互作用。在这样的情况下，寡核苷酸，低分子量的化合物，应同时检测，这是利用LC / ESI-MS的优点。 有一个限制，即该仪器不适合作为常规技术例如Sanger测序和实时PCR进行平行分析。此外，本发明方法只标识基础组合物和在同一分子内的任意碱基置换不能区分。然而，这里描述的基于MS-DNA分析还可能有长期好处与DNA结合染料基技术如凝胶电泳和实时PCR比较精确的第

质谱（MS）是一种公认的技术用于核酸，基质辅助激光解吸/电离（MALDI）和电喷雾电离（ESI）快速和可靠的鉴定被用于电离1,2。在MALDI技术通常具有时间飞行（TOF）分析仪相结合;然而，MALDI-TOF MS中的应用被限制为短的寡核苷酸（〜25个核苷酸（nt）的），由于随后的碎片，加合物的形成和低电离效率1,2。与此相反，ESI是适用于较长寡核苷酸（&gt; 100核苷酸），但多个的许多充电状态电荷的离子（[M-NH〕 正 ）从生物大分子同时产生，因此，分析物的质量可能会超过上光谱仪的m / z范围的限制。这需要卷积谱的解释， 即 ，一个充电状态序列转变成相应的质量通过反褶积。 在小分子的质量测量中，单一同位素质量的峰， 即的情况下，只包含各元素的最常见同位素的分子的质量是最丰富的自然观察3。随着分子量的增加，同位素分布转变到更高米/ z和单一同位素峰变得由基线噪声3-5遮蔽。一旦单一同位素峰值不再是质量大于10 kDa的3检测到，平均分子质量是用于测量，而不是单一同位素质量5。在这样的情况下，其中每个单独的峰是由1达分离同位素分布只能观察到当高分辨率质量分析仪，如TOF，一个傅里叶变换6改性的Kingdon阱分析器，或傅立叶变换离子回旋共振分析器用于分析。然而，最丰富的峰是Sometimes不与均分子量5一致。这些问题可能​​导致困难用于准确地确定分析物。 鉴于天然丰度稳定的同位素的变化，并在所述确定的平均分子质量的困难，单一同位素质量的测量是理想的生物分子3,4-质量表征。在实践中，一个单一同位素峰是否可以观察到与否，单一同位素质量可通过观察到的同位素分布的模式相比较，以从模型分析物4,7-10计算出的理论上的估计。拟合算法8现已并入专有软件。 在ESI-MS的上下文中，都需要一个DNA双链体，纯化和脱盐的离解为直接测量，避免电离的失败是由于离子抑制和加合物形成2,10-14。这些程序troublesome然而，完全自动化的分析系统已经开发商业上涉及聚合酶链反应（PCR），样品制备和ESI-MS用于检测病原体15-20。另一种方法是引入分离液相色谱（LC）。 LC从共存物质提供分析物的上线分离，不需要事先费力的样品制备电离21,22。然而，使用的MS-兼容的移动相的核酸分离是比诸如由于核苷酸的阴离子性质的药物，肽和蛋白质大多数其它化合物更为困难。 LC / ESI-MS的最成功的例子包括使用离子对反相液相色谱。 1,1,1,3,3,3-六氟-2-丙醇的流动相（HFIP）-triethylamine（TEA） -甲醇最初由Apffel 等人提出的。用于分离和检测短寡核苷酸23。 LC / ESI-MS基因分型为differentiatio的应用病原体物种，单核苷酸多态性和短串联重复序列的n个已使用毛细管聚合物整料柱可分开较长的寡核苷酸1,21,24-33报道。 在日本和美国，严重食物中毒了应有的误认和河豚的准备不当发生，这就是尽管河豚的分布和准备是严格食品安全法规34,35控制。此外，也发生在日本36使用河豚提取物的故意杀人罪。因此，河豚物种分化是从公众健康和法医调查所需的立场。此外，由于红鳍东方鲀远比其他种类的河豚的更昂贵，还需要在食品欺诈调查的上下文中分化能力。 这里，用于确定第m的详细方法通过LC / ESI-MS使用反相硅胶整料柱和高分辨率质谱仪的PCR产物的onoisotopic质量进行说明。具体地，该方法已经开发，以允许基于使用在PCR限制性片段长度多态性（RFLP）方法37，这是用于使用MS的动物物种分化的第一个例子的有毒河豚物种分化。

<table><tbody><tr><td>DNeasy Blood &amp;amp; Tissue Kit (50)</td><td>Qiagen</td><td>69504</td><td>DNA extraction kit</td></tr><tr><td>Proteinase K</td><td>Qiagen</td><td>19131</td><td></td></tr><tr><td>Ethanol (99.5+ vol%)</td><td>Wako</td><td>054-07225</td><td>For dilution of wash buffers</td></tr><tr><td>TE buffer (pH 8.0)</td><td>Wako</td><td>310-90023</td><td>For dilution of DNA sample</td></tr><tr><td>PCR primer</td><td>Fasmac</td><td>NA</td><td>Purified with reverse-phase cartridge column by the supplier</td></tr><tr><td>Template DNA</td><td>Eurofins Genomics</td><td>NA</td><td>Purified by HPLC by the supplier</td></tr><tr><td>Ultrapurified water</td><td>NA</td><td>NA</td><td>Generated with a Milli-Q Direct water purification system (Merck Millipore), used for sample preparation</td></tr><tr><td>Detergent Free 5x Phusion HF Buffer</td><td>Thermo Fisher</td><td>F-520L</td><td>Use instead of the provided buffer of DNA polymerase</td></tr><tr><td>Pfu-X DNA polymerase</td><td>Jena Bioscience</td><td>PCR-207S</td><td></td></tr><tr><td>dNTP mix (20 mM each)</td><td>Jena Bioscience</td><td>NA</td><td>Supplied with the DNA polymerase</td></tr><tr><td>10&amp;times; Universal buffer M</td><td>Takara Bio</td><td>NA</td><td>Containing 100 mM Tris-HCl (pH 7.5), 100 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 10 mM dithiothreitol and 500 mM NaCl</td></tr><tr><td>Dra I</td><td>Thermo Fisher</td><td>FD0224</td><td>Restriction enzyme</td></tr><tr><td>Msp I</td><td>Thermo Fisher</td><td>FD0544</td><td>Restriction enzyme</td></tr><tr><td>1,1,1,3,3,3-hexafluoro-2-propanol (HFIP)</td><td>Fluka</td><td>42060-50ML</td><td>Eluent additive for LC-MS grade.</td></tr><tr><td>Triethylamine (TEA)</td><td>Fluka</td><td>65897-50ML</td><td>Eluent additive for LC-MS grade.</td></tr><tr><td>Trifluoroacetic acid</td><td>Sigma-Aldrich</td><td>T6508</td><td>For preparation of calibrant.</td></tr><tr><td>Sodium hydroxide (1.0 M)</td><td>Fluka</td><td>72082</td><td>Dilute to 10 mM with ultrapurified water and use for titration of trifluoroacetic acid.</td></tr><tr><td>Acetonitrile</td><td>Fluka</td><td>34967</td><td>For preparation of calibrant, LC/MS grade.</td></tr><tr><td>Methanol</td><td>Kanto</td><td>25185-76</td><td>For mobile phase, LC/MS grade.</td></tr><tr><td>Water</td><td>Thermo Fisher</td><td>W6-1</td><td>For mobile phase, LC/MS grade.</td></tr><tr><td>Microcentrifuge tube (0.5 ml)</td><td>Eppendorf</td><td>0030123301</td><td>PCR clean grade</td></tr><tr><td>Microcentrifuge tube (1.5 ml)</td><td>Eppendorf</td><td>0030123328</td><td>PCR clean grade</td></tr><tr><td>UVette</td><td>Eppendorf</td><td>0030106300</td><td>Disposable UV cuvette.</td></tr><tr><td>Gel Green</td><td>Biotium</td><td>41004</td><td>Fluorescent DNA stain</td></tr><tr><td>Cosmospin filter G (0.2 &amp;mu;m)</td><td>Nakalai Tesque</td><td>06549-44</td><td>Made of hydrophilic polytetrafluoroethylene (PTFE) membrane. Any centrifugal filter unit (pore size, 0.2&amp;ndash;0.5 &amp;mu;m) made of hydrophilic PTFE or another low-binding membrane is applicable.</td></tr><tr><td>300 &amp;mu;l PP screw vial</td><td>American Chromatography Supplies</td><td>V0309P-1232</td><td></td></tr><tr><td>Preassembled screw cap and septa</td><td>Finneran</td><td>5395-09R</td><td></td></tr><tr><td>Monobis C18 analytical column (2.0&amp;times;50 mm, mesopore size = 30 nm)</td><td>Kyoto Monotech</td><td>2050H30ODS</td><td>Outside Japan, available for purchase from GL Sciences via its distributors. (http://www.glsciences.com/distributors/)</td></tr><tr><td>Monobis C18 guard column</td><td>Kyoto Monotech</td><td>GCSET-ODS3210</td><td>Holder included.</td></tr><tr><td>Cadenza CW-C18 (3.0&amp;times;250 mm)</td><td>Imtakt</td><td>CW036</td><td>C&lt;sub&gt;18&lt;/sub&gt;-bonded particulate silica column</td></tr><tr><td>ProSwift RP-4H (1.0&amp;times;250 mm)</td><td>Thermo Fisher</td><td>066640</td><td>Poly(styrene-divinylbenzene) monolith column</td></tr><tr><td>Themo Mixer C</td><td>Eppendorf</td><td>5382 000.023</td><td>For digestion of fish tissues.</td></tr><tr><td>Spectrophotometer</td><td>Shimadzu</td><td>UV-3150</td><td>Quantification of DNA concentration.</td></tr><tr><td>Thermal cycler</td><td>Bio-Rad</td><td>T100</td><td></td></tr><tr><td>Portable refrigerator</td><td>Twinbird</td><td>D-CUBE</td><td>For aqueous mobile phase to avoid evaporation of HFIP. This can be replaced by an ice box.</td></tr><tr><td>Ultimate 3000 liquid chromatograph</td><td>Thermo Fisher</td><td>NA</td><td></td></tr><tr><td>Q Exactive mass spectrometer</td><td>Thermo Fisher</td><td>NA</td><td>Fourier transform mass spectrometer equipped with the modified Kingdon trap analyzer.</td></tr><tr><td>Protein deconvolution 3.0</td><td>Thermo Fisher</td><td>NA</td><td>Use version 3.0 or higher having an isotopic patter model of nucleotide.</td></tr></tbody></table>

improved polymerase chain reaction-restriction fragment length polymorphism genotyping of toxic pufferfish by liquid chromatography/mass spectrometry

聚合酶链反应（PCR）限制性片段长度多态性（RFLP）通过液相色谱/电喷雾电离质谱（LC / ESI-MS）进行基因分型毒性河豚物种方法的改进版本进行说明。 DNA提取进行使用基于膜的二氧化硅DNA提取试剂盒。用不含去污剂的PCR缓冲液在PCR扩增后，限制酶加入到溶液无需纯化该反应溶液。反相硅胶整料柱和一个傅立叶变换分别采用具有修饰的Kingdon阱分析器高分辨质谱仪分离和检测。流动相由400mM的1,1,1,3,3,3-六氟-2-丙醇，15毫三乙胺（pH值7.9）和甲醇中，以0.4毫升/分钟的流速输送。循环时间为LC / ESI-MS分析是8分钟，包括柱的平衡。其同位素分布模型Ø解卷积软件F中的寡核苷酸被用于从质谱计算出相应的单一同位素质量。对于寡核苷酸（范围26-79个核苷酸）的分析，质量精度为0.62±0.74 ppm的（N = 280）和优异的准确度和精密度均持续180小时不使用锁的质量标准。

三市售列长寡核苷酸在100-400微升/ min的流速分离进行了评价。宽孔径十八碳链（C18）键合性颗粒二氧化硅柱（a）中，使用市售的聚（苯乙烯-二乙烯基苯）（PS-DVB）整料柱（b）和一个C 18键合性硅石整料柱（三）进行了比较（ 图1）。三对DNA双链体（26，37和53碱基对）的用所有列分离，并且在随后的研究中使用的C 18键合性硅石整料柱。 对于一个DNA样品从T的肌肉中提取的分析的代表性结果红鳍东方示于图2。同位素分辨的峰， 如图2a中 ，需要用于成功计算单一同位素质量。理论和MEAST.的被保险单一同位素质量红鳍东方示于图2b。因为与核酸内切酶的消化不经纯化后立即PCR扩增进行，由所述Msp核酸内切酶所产生的3&#39;粘性末端与其余DNA聚合酶填补。根据从合成DNA模板衍生的扩增子的分析，质量精度范围从-2.48到2.40 ppm的（平均0.62±0.74 ppm时，N = 280）。因此，用于区分物种（ 表4）3 ppm的质量偏差。使用表，一切从市场和商店获得的河豚样品进行了适当的区分具体品种37。 根据从T的合成DNA模板得到的样品的定期分析chrysops，分析物的所有单同位素群众±3ppm的内成功地确定为在乐AST 180小时无任何质量校正（ 图3）。这意味着质量校准将每周需要。 <img alt="图1" src="/files/ftp_upload/54402/54402fig1.jpg" /> 图1:从T的合成的DNA模板得到的PCR-RFLP产物的总离子流色谱图 pardalis 使用C 18 键合性颗粒二氧化硅柱（一个，3×250毫米，粒径3微米），聚（苯乙烯-二乙烯基苯）（PS-DVB）整料柱（B，1.0×250毫米），一个C 18 键合性整料二氧化硅柱（三，本发明的方法）的条件（ 一 ）:流速为0.2ml /分钟;甲醇的比率，0-2分钟5％，2-5分钟5％-25％，5-20分钟25％-40％，20-35分钟40％-98％，35-50分钟98％，50- 51分钟98％-5％，51-65分钟5％。条件（B）:流速为0.1毫升/分钟;甲醇的比率，0-2分钟5％，2-5分钟5％-25％，5-35分钟25％-40％，35-40分钟40％-98％，40-55分钟98％，55- 56分钟98％-5％，56-70分钟5％。对于两列的温度是20℃。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54402/54402fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54402/54402fig2.jpg" /> 图2: 用于分析 T 的原始肌肉代表性结果 红鳍东方鲀 （a）中典型的总离子流色谱图和质谱。 （b）由T的合成的DNA模板得到的PCR-RFLP产物的理论和测量单同位素质量红鳍东方鲀 ， <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54402/54402fig2large.jpg" target="_blank">请点击这里查看大</a>- [R版本这个数字。 <img alt="图3" src="/files/ftp_upload/54402/54402fig3.jpg" /> 图 3:从T的合成DNA的稳定性试验酶切扩增子chrysops进行分析，每10小时。在测试过程中没有进行质量校正。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54402/54402fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>名称</td><td>序列</td></tr><tr><td> lago86F </td><td> 5&#39;-CCATGTGGAATGAAAACACC-3&#39; </td></tr><tr><td> taki114F </td><td> 5&#39;-AAAAACAAGAGCCACAGCTCTAA-3&#39; </td></tr><tr><td> fugu86-114R </td><td> 5&#39;-CCCTAGGGTAACTCGGTTCG-3&#39; </td></tr></tbody></table>表1:PCR引物。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> PCR试剂</td><td>使用量</td><td>最终浓度</td></tr><tr><td>超纯水</td><td> 15.75微升</td><td></td></tr><tr><td>洗涤剂无缓冲5倍</td><td> 5.0微升</td><td> 1X </td></tr><tr><td> dNTP混合物（各10毫摩尔） </td><td> 0.5微升</td><td> 0.2毫米</td></tr><tr><td>引物混合物（各lago86F，taki114F和fugu86-114R10μM的TE缓冲液pH 8.0）中</td><td> 1.0微升</td><td>每次0.4μM </td></tr><tr><td> DNA聚合酶（2.5单位/微升） </td><td> 0.25微升</td><td> 0.1单位/微升</td></tr><tr><td>在TE缓冲液模板DNA 8.0（5.0-10纳克/μL的提取样品，0.2μM合成DNA为阳性对照） </td><td> 2.5微升</td><td> 0.5-1.0ñ克/μL对提取的DNA并且20nm合成DNA </td></tr><tr><td></td><td>总计:25微升</td><td></td></tr></tbody></table> 表2:25微升规模PCR的组成部分。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>周期</td><td>条件</td><td>功能</td></tr><tr><td> 1 </td><td> 2分钟，在95℃下</td><td>初始变性</td></tr><tr><td> 2 </td><td> 30秒95℃ </td><td>变性</td></tr><tr><td> 3 </td><td> 30秒56℃ </td><td>退火</td></tr><tr><td> 4 </td><td> 30秒72℃ </td><td>伸长</td></tr><tr><td>五</td><td>重复2-4（共30次） </td><td></td></tr><tr><td> 6 </td><td>在72℃7分钟</td><td>最终延伸</td></tr><tr><td> 7 </td><td>在12℃保持，直到去除样品</td><td></td></tr></tbody></table> 表3:PCR程序。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td rowspan="2">在4.0-5.5分钟保留时间的峰值数</td><td colspan="2">该范围中的第三个峰的单一同位素质量（DA） </td><td colspan="2">范围内的第二峰的单一同位素质量（DA） </td><td rowspan="2">河豚品种</td></tr><tr><td>小型股</td><td>大股</td><td>小型股</td><td>大股</td></tr><tr><td rowspan="5"> 2 </td><td rowspan="5"> → </td><td rowspan="5"> → </td><td> 15890.707-15890.803 </td><td> 16177.531-16177.629 </td><td> L.麦 </td></tr><tr><td> 15921.702-15921.797 </td><td> 16146.537-16146.634 </td><td> L. gloveri </td></tr><tr><td> 15930.713-15930.809 </td><td> 16137.525-16137.622 </td><td> L. lunaris </td></tr><tr><td> 15937.697-15937.792 </td><td> 16131.​​537-16131.​​634 </td><td> L. wheeleri </td></tr><tr><td> 24173.034-24173.179 </td><td> 24566.905-24567.053 </td><td> T. chrysops </td></tr><tr><td rowspan="6"> 3 </td><td rowspan="2"> 16295.706-16295.804 </td><td rowspan="2"> 16467.610-16467.709 </td><td> 11341.851-11341.919 </td><td> 11466.875-11466.944 </td><td> T. pardalis T. snyderi T.红鱼 T. xanthopterus T. stictonotus </td></tr><tr><td> 11654.908-11654.978 </td><td> 11770.920-11770.991 </td><td> T. niphobles </td></tr><tr><td> 16296.701-16296.799 </td><td> 16468.605-16468.704 </td><td> → </td><td> → </td><td> T.红鳍东方鲀 </td></tr><tr><td> 16311.701-16311.799 </td><td> 16452.610-16452.709 </td><td> → </td><td> → </td><td> T. poecilonotus T. exascurus </td></tr><tr><td> 16320.713-16320.811 </td><td> 16443.599-16443.697 </td><td> → </td><td> → </td><td> T. porphyreus T.暗纹 </td></tr><tr><td> 16599.751-16599.851 </td><td> 16780.667-16780.767 </td><td> → </td><td> → </td><td> T. vermicularis </td></tr></tbody></table> 表4:来自LC / ESI-MS衍生单一同位素群众河豚的分化。 <img alt="表5" src="/files/ftp_upload/54402/54402tbl5.jpg" /> 表5:扩增的DNA序列 （a）本序列是相同的。即如表4中所述的其他河豚种类。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54402/54402tbl5large.jpg" target="_blank">请点击这里查看此表的放大版本。</a>

Watch this Scientific Journal Video about Improved Polymerase Chain Reaction-restriction Fragment Length Polymorphism Genotyping of Toxic Pufferfish by Liquid Chromatography/Mass Spectrometry at JoVE.

Improved Polymerase Chain Reaction-restriction Fragment Length Polymorphism Genotyping of Toxic Pufferfish by Liquid Chromatography/Mass Spectrometry

描述了用于通过液相色谱/质谱基因型河豚物种的改进的聚合酶链式反应 - 限制性片段长度多态性方法。反相硅胶整料柱被用于分离消化的扩增子。这种方法可以阐明的寡核苷酸的单同位素质量，其是用于识别基础组合物是有用的。

河豚有毒改进的聚合酶链反应 - 限制性片段长度多态性分型液相色谱/质谱法

An improved version of a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method for genotyping toxic pufferfish species by ...

improved-polymerase-chain-reaction-restriction-fragment-length

Research

JoVE Journal

Genetics

11.1K 次观看.  National Research Institute of Police Science. 描述了用于通过液相色谱/质谱基因型河豚物种的改进的聚合酶链式反应 - 限制性片段长度多态性方法。反相硅胶整料柱被用于分离消化的扩增子。这种方法可以阐明的寡核苷酸的单同位素质量，其是用于识别基础组合物是有用的。 

视频: Improved Polymerase Chain Reaction-restriction Fragment Length Polymorphism Genotyping of Toxic Pufferfish by Liquid Chromatography/Mass Spectrometry

Laboratory Estimation of Net Trophic Transfer Efficiencies of PCB Congeners to Lake Trout (Salvelinus namaycush) from Its Prey

A technique for laboratory estimation of net trophic transfer efficiency (&#947;) of polychlorinated biphenyl (PCB) congeners to piscivorous fish from their prey is described herein. During a 135-day laboratory experiment, we fed bloater (Coregonus hoyi) that had been caught in Lake Michigan to lake trout (Salvelinus namaycush) kept in eight laboratory tanks. Bloater is a natural prey for lake trout. In four of the tanks, a relatively high flow rate was used to ensure relatively high activity by the lake trout, whereas a low flow rate was used in the other four tanks, allowing for low lake trout activity. On a tank-by-tank basis, the amount of food eaten by the lake trout on each day of the experiment was recorded. Each lake trout was weighed at the start and end of the experiment. Four to nine lake trout from each of the eight tanks were sacrificed at the start of the experiment, and all 10 lake trout remaining in each of the tanks were euthanized at the end of the experiment. We determined concentrations of 75 PCB congeners in the lake trout at the start of the experiment, in the lake trout at the end of the experiment, and in bloaters fed to the lake trout during the experiment. Based on these measurements, &#947; was calculated for each of 75 PCB congeners in each of the eight tanks. Mean &#947; was calculated for each of the 75 PCB congeners for both active and inactive lake trout. Because the experiment was replicated in eight tanks, the standard error about mean &#947; could be estimated. Results from this type of experiment are useful in risk assessment models to predict future risk to humans and wildlife eating contaminated fish under various scenarios of environmental contamination.

Laboratory Estimation of Net Trophic Transfer Efficiencies of PCB Congeners to Lake Trout Salvelinus namaycush from Its Prey

A technique for laboratory estimation of net trophic transfer efficiency of polychlorinated biphenyl (PCB) congeners to piscivorous fish from their prey is presented. To maximize applicability of the laboratory results to the field, the piscivorous fish should be fed prey fish that are typically eaten in the field.

多氯联苯同系物净营养转移效率湖鳟鱼实验室估计（<em&gt;红点鲑namaycush</em&gt;），从它的猎物

A technique for laboratory estimation of net trophic transfer efficiency (&#947;) of polychlorinated biphenyl (PCB) congeners to piscivorous fish from ...

科研

环境科学

High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short generation time, are easy to handle, display a high reproductive rate, low cost, and are easily amenable to genetic manipulations by microinjection of DNA in embryos. Recent advances in gene editing tools are enabling precise introduction of mutations and transgenes in zebrafish. Disease modeling in zebrafish often leads to larval phenotypes and early death which can be challenging to interpret if genotypes are unknown. This early identification of genotypes is also needed in experiments requiring sample pooling, such as in gene expression or mass spectrometry studies. However, extensive genotypic screening is limited by traditional methods, which in most labs are performed only on adult zebrafish or in postmortem larvae. We addressed this problem by adapting a method for the isolation of PCR-ready genomic DNA from live zebrafish larvae that can be achieved as early as 72 h post-fertilization (hpf). This time and cost-effective technique, improved from a previously published genotyping protocol, allows the identification of genotypes from microscopic fin biopsies. The fins quickly regenerate as the larvae develop. Researchers are then able to select and raise the desired genotypes to adulthood by utilizing this high-throughput PCR-based genotyping procedure.

Zebrafish have been used as reliable genetic model organisms in biomedical research, especially with the advent of gene-editing technologies. When larval phenotypes are expected, DNA extraction and genotype identification can be challenging. Here, we describe an efficient genotyping procedure for zebrafish larvae, by tail clipping, as early as 72-h post-fertilization.

3dpf 斑马鱼幼虫的高通量 DNA 提取及基因分型研究

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short ...

遗传学

Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium Yersinia ruckeri by Multiplex PCR and Capillary Electrophoresis

Yersinia ruckeri is an important pathogen of farmed salmonids worldwide, but simple tools suitable for epizootiological investigations (infection tracing, etc.) of this bacterium have been lacking. A Multi-Locus Variable-number tandem-repeat Analysis (MLVA) assay was therefore developed as an easily accessible and unambiguous tool for high-resolution genotyping of recovered isolates. For the MLVA assay presented here, DNA is extracted from cultured Y. ruckeri samples by boiling bacterial cells in water, followed by use of supernatant as template for PCR. Primer-pairs targeting ten Variable-number tandem-repeat (VNTR) loci, interspersed throughout the Y. ruckeri genome, are distributed equally amongst two five-plex PCR reactions running under identical cycling conditions. Forward primers are labelled with either of three fluorescent dyes. Following amplicon confirmation by gel electrophoresis, PCR products are diluted and subjected to capillary electrophoresis. From the resulting electropherogram profiles, peaks representing each of the VNTR loci are size-called and employed for calculating VNTR repeat counts in silico. Resulting ten-digit MLVA profiles are then&#160;used to generate Minimum spanning trees enabling epizootiological evaluation by cluster analysis. The highly portable output data, in the form of numerical MLVA profiles, can rapidly be compared across labs and placed in a spatiotemporal context. The entire procedure from cultured colony to epizootiological evaluation may be completed for up to 48 Y. ruckeri isolates within a single working day.

Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium Yersinia ruckeri by Multiplex PCR and Capillary Electrophoresis

The Multi-Locus Variable-number tandem-repeat Analysis (MLVA) assay presented here enables inexpensive, robust and portable high-resolution genotyping of the fish-pathogenic bacterium Yersinia ruckeri. Starting from pure cultures, the assay employs multiplex PCR and capillary electrophoresis to produce ten-loci MLVA profiles for downstream applications.

多点PCR和毛细血管电泳对鱼类致病菌的多点变量数串联重复分析

Yersinia ruckeri is an important pathogen of farmed salmonids worldwide, but simple tools suitable for epizootiological investigations (infection tracing, ...

Development and Testing of Species-specific Quantitative PCR Assays for Environmental DNA Applications

New, non-invasive methods for detecting and monitoring species presence are being developed to aid in fisheries and wildlife conservation management. The use of environmental DNA (eDNA) samples for detecting macrobiota is one such group of methods that is rapidly becoming popular and being implemented in national management programs. Here we focus on the development of species-specific targeted assays for probe-based quantitative PCR (qPCR) applications. Using probe-based qPCR offers greater specificity than is possible with primers alone. Furthermore, the ability to quantify the amount of DNA in a sample can be useful in our understanding of the ecology of eDNA and the interpretation of eDNA detection patterns in the field. Careful consideration is needed in the development and testing of these assays to ensure the sensitivity and specificity of detecting the target species from an environmental sample. In this protocol we will delineate the steps needed to design and test probe-based assays for the detection of a target species; including creation of sequence databases, assay design, assay selection and optimization, testing assay performance, and field validation. Following these steps will help achieve an efficient, sensitive, and specific assay that can be used with confidence. We demonstrate this process with our assay designed for populations of the mucket (Actinonaias ligamentina), a freshwater mussel species found in the Clinch River, USA.

Environmental DNA assays require rigorous design, testing, optimization and validation before the collection of field data can begin. Here, we present a protocol to take users through each step of designing a species-specific, probe-based qPCR assay for the detection and quantification of a target species DNA from environmental samples.

开发和测试针对物种的定量PCR检测环境DNA应用

New, non-invasive methods for detecting and monitoring species presence are being developed to aid in fisheries and wildlife conservation management. The ...

Early Detection of Cyanobacterial Blooms and Associated Cyanotoxins using Fast Detection Strategy

Fast detection of cyanobacteria and cyanotoxins is achieved using a Fast Detection Strategy (FDS). Only 24 h are needed to unravel the presence of cyanobacteria and related cyanotoxins in water samples and in an organic matrix, such as bivalve extracts. FDS combines remote/proximal sensing techniques with analytical/bioinformatics analyses. Sampling spots are chosen through multi-disciplinary, multi-scale, and multi-parametric monitoring in a three-dimensional physical space, including remote sensing. Microscopic observation and taxonomic analysis of the samples are performed in the laboratory setting, which allows for the identification of cyanobacterial species. Samples are then extracted with organic solvents and processed with LC-MS/MS. Data obtained by MS/MS are analyzed using a bioinformatic approach using the online platform Global Natural Products Social (GNPS) to create a network of molecules. These networks are analyzed to detect and identify toxins, comparing data of the fragmentation spectra obtained by mass spectrometry with the GNPS library. This allows for the detection of known toxins and unknown analogues that appear related in the same molecular network.

A fast-multidisciplinarystrategy for early detection of cyanobacterial blooms and associated cyanotoxins is described here. It allows for the detection of cyanobacteria and related cyanotoxins in water samples and in organic matrices, such as bivalve samples, in 24 h.

使用快速检测策略早期检测蓝藻花和相关氰毒素

Fast detection of cyanobacteria and cyanotoxins is achieved using a Fast Detection Strategy (FDS). Only 24 h are needed to unravel the presence of ...

化学

DNA-based Fish Species Identification Protocol

We have developed a fast, simple, and accurate DNA-based screening method to identify the fish species present in fresh and processed seafood samples. This versatile method employs PCR amplification of genomic DNA extracted from fish samples, followed by restriction fragment length polymorphism (RFLP) analysis to generate fragment patterns that can be resolved on the Agilent 2100 Bioanalyzer and matched to the correct species using RFLP pattern matching software.
The fish identification method uses a simple, reliable, spin column- based protocol to isolate DNA from fish samples. The samples are treated with proteinase K to release the nucleic acids into solution. DNA is then isolated by suspending the sample in binding buffer and loading onto a micro- spin cup containing a silica- based fiber matrix. The nucleic acids in the sample bind to the fiber matrix. The immobilized nucleic acids are washed to remove contaminants, and total DNA is recovered in a final volume of 100 &mu;l. The isolated DNA is ready for PCR amplification with the provided primers that bind to sequences found in all fish genomes. The PCR products are then digested with three different restriction enzymes and resolved on the Agilent 2100 Bioanalyzer. The fragment lengths produced in the digestion reactions can be used to determine the species of fish from which the DNA sample was prepared, using the RFLP pattern matching software containing a database of experimentally- derived RFLP patterns from commercially relevant fish species.

This publication describes how to use the Agilent Fish Species Identification System to identify the species of a fish by extracting DNA and performing PCR and RFLP analysis.

以DNA为基础的鱼类物种鉴定协议

We have developed a fast, simple, and accurate DNA-based screening method to identify the fish species present in fresh and processed seafood samples. ...

生物学

Quantification of three DNA Lesions by Mass Spectrometry and Assessment of Their Levels in Tissues of Mice Exposed to Ambient Fine Particulate Matter

DNA adducts and oxidized DNA bases are examples of DNA lesions that are useful biomarkers for the toxicity assessment of substances that are electrophilic, generate reactive electrophiles upon biotransformation, or induce oxidative stress. Among the oxidized nucleobases, the most studied one is 8-oxo-7,8-dihydroguanine (8-oxoGua) or 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxodGuo), a biomarker of oxidatively induced base damage in DNA. Aldehydes and epoxyaldehydes resulting from the lipid peroxidation process are electrophilic molecules able to form mutagenic exocyclic DNA adducts, such as the etheno adducts 1,N2-etheno-2&#39;-deoxyguanosine (1,N2-&#949;dGuo) and 1,N6-etheno-2&#39;-deoxyadenosine (1,N6-&#949;dAdo), which have been suggested as potential biomarkers in the pathophysiology of inflammation. Selective and sensitive methods for their quantification in DNA are necessary for the development of preventive strategies to slow down cell mutation rates and chronic disease development (e.g., cancer, neurodegenerative diseases). Among the sensitive methods available for their detection (high performance liquid chromatography coupled to electrochemical or tandem mass spectrometry detectors, comet assay, immunoassays, 32P-postlabeling), the most selective are those based on high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-ESI-MS/MS). Selectivity is an essential advantage when analyzing complex biological samples and HPLC-ESI-MS/MS evolved as the gold standard for quantification of modified nucleosides in biological matrices, such as DNA, urine, plasma and saliva. The use of isotopically labeled internal standards adds the advantage of corrections for molecule losses during the DNA hydrolysis and analyte enrichment steps, as well as for differences of the analyte ionization between samples. It also aids in the identification of the correct chromatographic peak when more than one peak is present.
We present here validated sensitive, accurate and precise HPLC-ESI-MS/MS methods that were successfully applied for the quantification of 8-oxodGuo, 1,N6-dAdo and 1,N2-dGuo in lung, liver and kidney DNA of A/J mice for the assessment of the effects of ambient PM2.5 exposure.

We describe here methods for sensitive and accurate quantification of the lesions 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxodGuo), 1,N6-etheno-2&#39;-deoxyadenosine (1,N6-dAdo) and 1,N2-etheno-2&#39;-deoxyguanosine (1,N2-dGuo) in DNA. The methods were applied to the assessment of the effects of ambient fine particulate matter (PM2.5) in tissues (lung, liver and kidney) of exposed A/J mice.

环境细颗粒物小鼠组织中三种 DNA 损伤的质谱定量及水平评价

DNA adducts and oxidized DNA bases are examples of DNA lesions that are useful biomarkers for the toxicity assessment of substances that are ...

免疫与感染

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

快速，高效的斑马鱼基因分型采用PCR与高分辨率熔体分析

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

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 ...

DNA Extraction from Tail Clip: A Method Used in Zebrafish Genotyping

Source: Xing L., et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/51138/" target="_blank">Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis</a>. J. Vis. Exp. (2014).
This article describes DNA extraction from zebrafish tail clip. The protocol demonstrates a rapid and efficient method to analyze zebrafish genotypes.

从尾夹中提取 DNA:一种用于斑马鱼基因分型的方法

Source: Xing L., et al. Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis. J. Vis. Exp. (2014).
This article describes ...