Name: the lambda select cii mutation detection system
Uploaded: 2018-04-26T15:00:00.000+00:00
Duration: 7 min 8 s
Description: Watch this Scientific Journal Video about The Lambda Select cII Mutation Detection System at JoVE.com

我们要感谢所有同事和合作者对我们最初的研究所作的贡献, 这份手稿中的结果已经在本稿中提到 (为了说明目的)。作者的工作得到了国家卫生研究院牙科和颅面研究研究所 (1R01DE026043) 和加州大学烟草相关疾病研究计划 (ab) 的资助 (TRDRP-26IR-0015) 和 ST (TRDRP-25IP-0001)。研究的发起者在研究设计、数据收集、数据分析、数据解释、报告撰写或提交出版物的决定中都没有作用。

1. 小鼠胚胎成纤维细胞的基因组 DNA 分离
注意: 根据发布的协议53, 原代小鼠胚胎成纤维细胞与 C57BL/6 遗传背景的 BB 转基因小鼠胚胎分离。此协议的起始材料由 1 x 106到 1 x 107胚胎成纤维细胞, 由测试复合与控制处理。使用标准方法收集和计数这些单元格在引用10、54、55中描述。
<ol><li>准备缓冲 A (0.3 米蔗糖, 60 毫米氯化钾, 15 毫米氯化钠, 60 毫米三盐酸, ph 值 8.0, 0.5 毫米胺, 0.15 毫米胺, 和2毫米 edta), 缓冲 B (150 毫米氯化钠和5毫米 edta, ph 7.8) 和缓冲 C (20 毫米三盐酸, ph 8。0, 20 毫米氯化钠, 20 毫米 EDTA, 1% SDS), 并保持室温 (RT), 长达一年54,55。</li>
	<li>并用重悬细胞在2–4毫升缓冲区 a 在15毫升锥形离心管通过反复吹打上和下使用宽口径1000µL 吸管提示。</li>
	<li>使用玻璃血清吸管添加一个包含 1% octylphenoxypolyethoxyethanol (例如、Nonidet P40) 的卷 (2–4 mL) 缓冲区。</li>
	<li>将管子在冰上孵化5分钟。</li>
	<li>离心管在 1000 x g在 RT 5 分钟。</li>
	<li>使用玻璃血清学吸管, 用10–15毫升缓冲液将上清, 并清洗颗粒。</li>
	<li>并用重悬2–4毫升缓冲 B 中的颗粒。</li>
	<li>添加一个体积 (2–4毫升) 缓冲 C, 含有600µg/毫升蛋白酶 K。</li>
	<li>在37摄氏度孵育3小时的管子。</li>
	<li>添加 RNase a 到最终浓度为100µg/毫升。</li>
	<li>在37摄氏度的1小时内孵化试管。</li>
	<li>添加一个体积 (2–4毫升) 苯酚 (饱和在0.1 米三盐酸, pH 值 8.0): 氯仿: 异戊醇 (25:24:1 卷/卷)。 注: 苯酚可能造成严重的健康危害, 必须极其谨慎地处理。苯酚对皮肤具有高度腐蚀性, 易于吸收, 并具有其他功效。处理苯酚时, 要经常使用双手套, 戴上防护眼镜, 在化学油烟机上工作。</li>
	<li>在4摄氏度的管转子上反转管5分钟, 混合良好。</li>
	<li>离心管在 1000 x g 5 分钟在4摄氏度。</li>
	<li>使用玻璃血清学吸管将水相 (顶层) 移到新管上。</li>
	<li>重复 phenol:chloroform:isoamyl 乙醇萃取1至3次, 直到水相清晰, 界面不再混浊。</li>
	<li>添加1/10 体积 (200–400µL) 3M 醋酸钠, pH 5.2。</li>
	<li>添加2.5 体积 (5–10毫升) 100% 乙醇 (冷), 并通过手轻轻地翻转管2–3分钟。</li>
	<li>将 DNA 与玻璃挂钩, 将其转移到含有1–5毫升70% 乙醇的新管, 并彻底清洗。或者, 离心管在高速 (3500 x g) 在4°c, 以颗粒的 DNA 和彻底清洗它与1–5毫升70% 乙醇。</li>
	<li>在 RT 上离心管高速 (3500 x g), 取出上清, 并将 DNA 风干10-15 分钟。</li>
	<li>将 DNA 溶解在10–100µL TE 缓冲器 (10 毫米三盐酸, 1 毫米 EDTA, pH 7.5), 并贮存在4摄氏度 (用于短储存一个月) 或在-20 摄氏度 (更长的贮存期)。λ DNA 的最佳浓度选择cII检测0.5–1.0 µg/µL (在 TE 缓冲区中)56。</li>
</ol>2.体外包装反应
<ol><li>每一个包装反应, 添加〜5µg 基因组 dna (最终体积: 8–12µL, 基因组 dna 被隔离在1 节从小鼠胚胎成纤维细胞治疗体外与测试化合物或控制) 到一个离心管, 其中包含第一个反应组合 (~ 10 µL)。 注: 内部准备或商业可用的λ包装提取物用于不同的实验室。在这里使用的商用包装提取套件中56 (参见材料表), 红色管包含第一反应组合 (~ 10 µL) 和蓝色管包含第二反应组合 (~ 70 µL 为至少5反应)。</li>
	<li>在30摄氏度孵化管90分钟。</li>
	<li>将第二反应混合物所需的体积 (~ 12 µL) 添加到管中。</li>
	<li>在30摄氏度的90分钟内孵化试管。</li>
	<li>
将1.1 毫升的 SM 缓冲器添加到管中。 注: 本解决方案的1毫升 (即, 包装反应混合物) 将用于筛选λ cII突变体。其余的将用于 titering。<ol>
<li>通过混合5.8 克氯化钠、2.0 克 MgSO47H2O、50毫升 1 M 三盐酸 (pH 7.5) 和5毫升 2% (w/v) 明胶, 准备SM 缓冲。将 dH2O 添加到最终体积为1升的蒸压釜, 其温度为30分钟, 在室温下保存达1年。</li>
	</ol>
</li>
	<li>涡流管包含包装的 DNA 样本十年代在 RT (有力的涡流)。</li>
	<li>脉冲旋转管在离心和储存在冰上, 直到准备使用。如果样品不将在同一天内使用的包装, 添加50µL 氯仿每毫升的包装 DNA 样本, 涡旋轻轻, 并存储在4°c 长达2周。</li>
</ol>3. 制备大肠杆菌G1250 细菌培养
<ol><li>
在电镀前至少两天, 在 TB1-kanamycin 琼脂板上制作一些从大肠杆菌 ( 大肠杆菌) G1250 的细菌条纹板。
	<ol>
<li>准备 TB1-kanamycin 琼脂板如下。混合5.0 克氯化钠, 10.0 克酪蛋白蛋白胨, 12.0 克琼脂。添加800毫升 dH2o 添加1毫升0.1% 硫胺盐酸盐。用氢氧化钠或 HCl 将 pH 值调整为 7.0. 将 dH2O 添加到最终卷1公升。</li>
		<li>搅拌好, 蒸压釜30分钟, 使冷却到55摄氏度。添加50.0 毫克卡那霉素和混合。倒入无菌的100毫米培养皿 (每道20–25毫升)。贮存盘在摄氏4摄氏度, 长达两周。</li>
	</ol>
</li>
	<li>在固定的30°c 孵化器中孵化细菌条纹板, 至少24小时。</li>
	<li>
在电镀前一天, 将10毫升的 TB1 液体介质与100µL 20% (瓦特/v) maltose-1 M MgSO4溶液结合在一个无菌的50毫升螺帽锥管中。
	<ol>
<li>准备 TB1 液体培养基如下。混合5.0 克氯化钠和10.0 克酪蛋白蛋白胨, 然后添加800毫升 dH2O, 1 ml 0.1% 毫升盐酸硫胺, 并调整 pH 值为7.0 与氢氧化钠或 HCl。然后, 将 dH2O 添加到最终卷1升, 混合好, 和高压釜30分钟. 将培养基在 RT 中储存多达三月。</li>
		<li>准备 20% (w/v) Maltose-1 M MgSO4 , 如下所示。混合20.0 克麦芽糖和24.6 克 MgSO4。7H2o, 然后将 dH2o 添加到最终卷100毫升。过滤消毒和贮存在4摄氏度, 长达6月。</li>
	</ol>
</li>
	<li>使用无菌接种循环56从细菌条纹板上接种多个菌落的液体培养基。</li>
	<li>在30摄氏度摇晃的孵化器中一夜之间孵化液体培养 (250–300 rpm)。</li>
	<li>在电镀当天, 离心机的圆锥管含有 G1250 液体培养在 1500 x g 10 分钟的 RT, 以颗粒的细菌细胞。</li>
	<li>丢弃上清, 并并用重悬10毫升10毫米MgSO4 中的细胞颗粒。</li>
	<li>使用紫外-可见光分光光度计 (例如、100µL 细胞悬浮 + 900 µL 10 mM MgSO4)56, 测量1:10 波长 600 nm 的细胞悬浮液稀释的吸光度。</li>
	<li>将电池悬浮液稀释至最终 OD600 , 以10毫米 MgSO4为0.5。G1250大肠杆菌的准备悬浮与 OD600 = 0.5 被称为 ' G1250 电镀文化 '。</li>
	<li>将 G1250 电镀文化储存在冰上, 并在1-2 小时内使用。</li>
</ol>4. 电镀包装的 DNA 样品
<ol><li>
每一个包装的 DNA 样本, 准备十六无菌 14 x 100 毫米2圆底管和十六 TB1 琼脂板。十从每套将用于筛选和六 titering (三的效价20和三的效价 100)。
	<ol>
<li>准备 TB1 琼脂板如下。混合5.0 克氯化钠, 10.0 克酪蛋白蛋白胨和12.0 克琼脂, 然后添加800毫升 dH2O 和1毫升0.1% 硫胺盐酸盐。用氢氧化钠或 HCl 将 pH 值调整为 7.0, 并将 dH2O 添加到最终卷1升。</li>
		<li>搅拌好, 蒸气釜30分钟, 使混合物冷却到55摄氏度, 然后倒入无菌100毫米培养皿 (每道20–25毫升)。将盘子存放在4摄氏度, 最多两周。 注: TB1 琼脂板应在使用前至少准备24小时。</li>
	</ol>
</li>
	<li>整除200µL 的 G1250 电镀培养成每一个圆底管。</li>
	<li>
对于 titering, 对已打包的 dna 样本进行1:100 稀释, 并通过涡流 (即、10µL 封装的 dna 样本 + 990 µL SM 缓冲器) 进行混合。</li>
	<li>添加20µL 的1:100 稀释到每一个三滴度20管。</li>
	<li>添加100µL 的1:100 稀释到每一个三滴度100管。</li>
	<li>
用于筛选, 将100µL 的 "未稀释的"打包的 DNA 样本添加到十筛选管中的每一个。</li>
	<li>处理所有 titering 和筛选管 (2 x 3 + 10 = 16 管), 如下所示: 混合涡流大约十年代, 然后在室温下孵化30分钟, 以允许宿主大肠杆菌吸附噬菌体。</li>
	<li>
加入2.5 毫升微波熔融 TB1 顶琼脂 (冷却到55°c) 到每个滴度或筛选管, 立即混合涡流 (轻轻), 并倒入适当的效价或筛选 TB1 琼脂板。
	<ol>
<li>准备 TB1 顶琼脂如下。混合5.0 克氯化钠, 10.0 克酪蛋白蛋白胨和7.0 克琼脂, 然后添加800毫升 dH2o 添加1毫升0.1% 硫胺盐酸盐, 然后用氢氧化钠或 HCl 将 pH 值调整为7.0。最后, 将 dH2O 添加到最终卷1升。</li>
		<li>在室温下存放三个月的20–25和高压釜。</li>
		<li>在使用之前, 将准备好的 TB1 琼脂在微波炉中熔化, 混合均匀, 并允许在水浴中冷却到55摄氏度。 注: 熔融和冷却 TB1 顶部添加到每个滴度或筛选管的内容, 并在混合后, 倒入适当的效价或筛选 TB1 琼脂板。</li>
	</ol>
</li>
	<li>在室温下, 让盘子代表15–30分钟, 盖子半开以防止凝结。</li>
	<li>反转板, 并将十筛板放置在固定的24°c 孵化器和孵化为 46–48 h (即, 选择性条件) 和六滴度板在固定37°c 孵化器和孵化为24小时/隔夜 (即,非选择性条件)。 注: 为了质量保证和标准化的结果, 商业上可用的控制噬菌体解决方案含有混合的 wildtype 和突变体cII与已知突变频率被镀在包装的 DNA 样本和包括在所有检测运行56。</li>
</ol>5. 检查的效价和筛板确定cII突变频率
<ol><li>在24小时/隔夜孵化在37°c, 计数形成的斑块的数量在三滴度20板材和效价100板材。要更容易地识别斑块, 请将盘子放在白色灯箱旁边, 并在黑色背景上取下盖子 (参见图 2)。</li>
	<li>平均 (平均) 计数的斑块数量在每组的效价20板和滴度100板 (每三个)。</li>
	<li>从最接近每片50–200斑块范围的滴度板中选择平均数。</li>
	<li>计算十筛板上筛查的斑块总数, 如下所示: 筛查的总斑块 = (在所选的滴度板上的平均斑块数÷µL 的稀释系数) x 稀释因子 x. 每筛板上的包装 DNA 样品µL 数量 [100 µL/盘] x 筛板的编号 [10]。 注: 例如, 如果每板的平均斑块数量在20版的128和相应的数量在一组的效价100板块是 478, 数字128将用于计算斑块的总数量被筛如下: (128 ÷ 20) x 100 [稀释因子] x 100 [µL/盘] x 10 [板材] = 64万 这相当于将平均斑块的数量乘以5000或 1000, 如果平均值是基于计数从滴度20板或滴度100板, 分别。</li>
	<li>以下 46–48 h 孵化24摄氏度, 计算十筛板形成的斑块总数 (按照5.1 节中提供的说明)。 注意: cII突变频率是通过将筛板中形成的斑块的总数除以所筛选的斑块总数来计算的。使用上面的示例, 如果在十筛选板中计数的斑块总数为 112, 则此 DNA 样本的cII突变频率为112÷ 64万 = 17.5 x 10-5。</li>
</ol>6. 验证假定的λ cII突变体、PCR 扩增和 DNA 测序
<ol><li>核心的问题与无菌, 宽孔吸管尖端的牌匾, 并驱逐它 (通过吹打上下) 成一个不育的离心管, 含有500µL 的无菌 SM 缓冲。</li>
	<li>在室温下孵化至少2小时, 或在4摄氏度过夜, 使噬菌体微粒从琼脂插头洗脱。</li>
	<li>在不育的 14 x 100 毫米2圆底管, 混合200µL 的 G1250 电镀文化与1µL 的药芯噬菌体溶液, 并孵化为30分钟在 RT。</li>
	<li>该样品使用2.5 毫升55°c 熔融 TB1 顶琼脂和孵化板在24°c 为 46–48 h (选择性条件), 如4.8–4.10 节所述。</li>
	<li>一旦次生斑块形成, 使用吸管尖端仔细挑选一个独立的经验证的λ cII突变斑块 (避免接触下琼脂)。 注: Replating 假定的cII突变斑块有两个用途: (一) 电镀工艺品有时可能被误认为是小尺寸的斑块;(ii) 从筛板上取出的琼脂糖芯可能含有非突变噬菌体和变种噬菌体。低密度 replating 的次生斑块将为 PCR 和后续 DNA 序列分析提供无污染的突变模板。</li>
	<li>将牌匾转移到含有25µL 双蒸馏水的离心管上, 吹打上下。</li>
	<li>把管子放在沸水中5分钟。</li>
	<li>离心机以最大速度 (1.8万 x g) 在 RT 上3分钟。</li>
	<li>将上清的10µL 立即转移到一个新的离心管, 其中含有40µL 的 pcr mastermix, 其中试剂的最终浓度为 1 x Taq PCR 缓冲器, 10 pmol 每个向前和反向底漆, 12.5 nmol 每 dNTP和 2.5 U 的 Taq DNA 聚合酶。 注: 正向和反向引物如下: 5 '-CCACACCTATGGTGTATG-3 ' (相对于cII启动密码子) 和 5 '-CCTCTGCCGAAGTTGAGTAT-3 ' (位置 +345 到 +365 相对于cII起始密码子), 分别是-68 到-50。</li>
	<li>使用以下循环参数放大模板: 3 分钟变性在95°c, 其次是30周期三十年代在95°c, 1 分钟在60°c, 并且1分钟在72°c, 与最后的延长10分钟在72°c。</li>
	<li>根据制造商的说明, 使用商业上可用的 pcr 纯化试剂盒纯化含有cII基因和侧翼区域的 432 bp pcr 产品。</li>
	<li>使用适当的测序平台进行 dna 测序, 并分析生成的 dna 序列, 以检测cII转基因中的突变 (见下面的说明)。 注意: 最好通过使用软件 (如基于 web 的 T-咖啡序列对齐服务器) 与引用cII序列对齐来实现这一点。有关如何使用程序的说明, 请访问: <a href="http://tcoffee.crg.cat/">http://tcoffee.crg.cat/</a>
</li>
</ol>

所有的作者声明没有利益冲突。

λ选择cII检测用于检测从 BB 啮齿动物的器官/组织中提取的细胞基因组 DNA 中回收的cII转基因中的突变 (3)。这些转基因动物的基因组包含多个串联拷贝的染色体集成λLIZ 穿梭载体, 运载cII (294 bp) 和lacI (1080 bp) 转基因, 作为突变的报告基因1,2,25. λ选择cII检测的基础是从转基因动?...

开发了多种转基因动物模型和突变检测系统, 用于哺乳动物细胞致癌物质的诱变检测。其中, 转基因大蓝 (简称 BB) 小鼠和λ选择cII突变检测系统是由该组和许多其他研究组在世界范围内的致突变实验所使用的1,2, 3,4,5,6,7,8,9。在过去的16年中, 我们研究了各种化学和/或物理剂的诱变效应, 这些转基因动物或其相应的胚胎成纤维细胞培养的试验化合物处理, 并随后分析了cII转基因的表型和基因型由λ选择cII化验和 DNA 测序, 分别为10,11,12,13,14,15,16,17,18,19,20,21,22,23,24. 这些转基因动物的基因组含有噬菌体λ梭向量 (λLIZ), 它集成在4号染色体上, 作为多拷贝头到尾 concatemer1,2,25。λLIZ 梭向量携带两个突变的报告基因, 即lacI和cII转基因1,2,25,26,27, 28,29,30,31,32,33,34,35,36, 37,38,39,40,41,42,43,44,45,46,47. λ选择cII检测是基于从转基因动物器官/组织的细胞基因组 DNA 中恢复λLIZ 梭向量的方法1,2,25.回收的λLIZ 梭载体然后被包装成λ噬菌体头, 能够感染指标宿主大肠杆菌. 随后, 受感染的细菌在选择性条件下生长, 以便在cII转基因1、3中对突变进行评分和分析。
在这里, 我们描述了λ选择cII分析的详细协议, 该方法包括从转基因动物细胞/器官中分离出的基因组 DNA, 从体外处理体内/与一个测试化合物, 检索λLIZ 梭向量从基因组 DNA, 载体的包装到传染性的λ噬菌体, 感染的宿主大肠杆菌与噬菌体, 鉴定的cII突变体在选择性条件下确定cII突变频率, 并DNA 序列分析建立了cII突变谱。该协议可应用于转基因鼠/大鼠细胞培养处理体外与化学/物理剂的兴趣, 或组织/器官的相应动物处理体内与测试化学/代理1, 2,4,48,49,50,51,52。λ选择cII分析的示意图演示显示在图 1中。

<table><tbody><tr><td>Agar</td><td>MO Bio Laboratories, Inc.</td><td>12112-05</td><td>Bacteriological grade</td></tr><tr><td>BigDye Terminator v3.1 Cycle Sequencing Kit</td><td>Thermo Fisher Scientific</td><td>4337455</td><td>None</td></tr><tr><td>Casein Peptone</td><td>Alfa Aesar</td><td>H26557</td><td>None</td></tr><tr><td>Gelatine</td><td>J. T. Baker</td><td>2124-01</td><td>Powder</td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>BP 229-1 / M-13750</td><td>None</td></tr><tr><td>LB Agar</td><td>Fisher Scientific</td><td>BP 9724-500</td><td>None</td></tr><tr><td>QIAquick PCR purification kit</td><td>Qiagen</td><td>8104</td><td>50 PCR purification reactions</td></tr><tr><td>Sodium Acetate Trihydrate</td><td>Fisher Scientific</td><td>M-15756</td><td>None</td></tr><tr><td>&lt;em&gt;Taq5000&lt;/em&gt; DNA Polymerase&amp;nbsp;</td><td>Qiagen</td><td>201207</td><td>None</td></tr><tr><td>Thiamine Hydrochloride</td><td>Macron Fine Chemicals</td><td>2722-57</td><td>None</td></tr><tr><td>Transpack Packaging Extract</td><td>Stratagene Corp., Acquired by BioReliance | Sigma-Aldrich Corp.</td><td>200223</td><td>50 packaging reactions</td></tr><tr><td>Tris Base</td><td>Fisher Scientific</td><td>BP 152-1 / EC 201-064-4</td><td>None</td></tr><tr><td>Trypton</td><td>Biosciences</td><td>RC-110</td><td>None</td></tr></tbody></table>

the lambda select cii mutation detection system

开发了多种转基因动物模型和突变检测系统, 用于哺乳动物细胞致癌物质的诱变检测。其中, 转基因小鼠和 Lambda (λ) 选择cII突变检测系统已被全球许多研究团体用于致突变实验。在这里, 我们描述了 Lambda 选择cII突变检测的详细协议, 该方法可应用于转基因小鼠/大鼠的培养细胞或与化学/物理制剂相关的动物。该协议包括以下步骤: (1) 从转基因动物的细胞或器官/组织中分离出基因组 DNA, 分别处理体外或体内, 并用一个测试化合物;(2) 从基因组 DNA 中回收携带突变报告基因 (即、 cII转基因) 的 lambda 梭载体;(3) 将获救的载体包装成传染性噬菌体;(4) 感染宿主细菌并在选择性条件下培养, 以允许传播诱导的cII突变;(5) 评分的cII突变体和 DNA 序列分析, 以确定cII突变频率和变异谱, 分别。

根据数据分布情况, 参数化或非参数测试用于确定治疗和控制组之间的cII突变频率差异的意义 (即、诱导与自发突变频率).不同治疗组诱导的cII突变频率的比较是由各种 (配对的) 统计试验 (如适用) 进行的。Adams 和 Skopek 的超几何测试通常用于比较整个诱导和自发突变谱57, 尽管其他测试 (如χ2测试或方差分析) 也可用于比较频率?...

Watch this Scientific Journal Video about The Lambda Select cII Mutation Detection System at JoVE.com

The Lambda Select cII Mutation Detection System

Lambda 选择cII突变检测系统

我们描述了一个详细的协议, 为 Lambda 选择cII突变试验的培养细胞的转基因啮齿动物或相应的动物处理的化学/物理剂的利益。该方法已广泛用于哺乳动物细胞致癌物质的诱变检测。

A number of transgenic animal models and mutation detection systems have been developed for mutagenicity testing of carcinogens in mammalian cells. Of ...

the-lambda-select-cii-mutation-detection-system

Research

JoVE Journal

Immunology and Infection

The Lambda Select cII Mutation Detection System

7.8K 次观看.  University of Southern California. 我们描述了一个详细的协议, 为 Lambda 选择cII突变试验的培养细胞的转基因啮齿动物或相应的动物处理的化学/物理剂的利益。该方法已广泛用于哺乳动物细胞致癌物质的诱变检测。

视频: The Lambda Select cII Mutation Detection System

Wild-type Blocking PCR Combined with Direct Sequencing as a Highly Sensitive Method for Detection of Low-Frequency Somatic Mutations

Accurate detection and identification of low frequency mutations can be problematic when assessing residual disease after therapy, screening for emerging resistance mutations during therapy, or when patients have few circulating tumor cells. Wild-type blocking PCR followed by sequencing analysis offers high sensitivity, flexibility, and simplicity as a methodology for detecting these low frequency mutations. By adding a custom designed locked nucleic acid oligonucleotide to a new or previously established conventional PCR based sequencing assay, sensitivities of approximately 1 mutant allele in a background of 1,000 WT alleles can be achieved (1:1,000). Sequencing artifacts associated with deamination events commonly found in formalin fixed paraffin embedded tissues can be partially remedied by the use of uracil DNA glycosylase during extraction steps. The optimized protocol here is specific for detecting MYD88 mutation, but can serve as a template to design any WTB-PCR assay. Advantages of the WTB-PCR assay over other commonly utilized assays for the detection of low frequency mutations including allele specific PCR and real-time quantitative PCR include fewer occurrences of false positives, greater flexibility and ease of implementation, and the ability to detect both known and unknown mutations.

Wild-type blocking PCR followed by direct sequencing offers a highly sensitive method of detection for low frequency somatic mutations in a variety of sample types.

野生类型阻止PCR结合直接测序作为低频体细胞突变的检测的高灵敏度的方法

Accurate detection and identification of low frequency mutations can be problematic when assessing residual disease after therapy, screening for emerging ...

科研

遗传学

Detection of Rare Mutations in CtDNA Using Next Generation Sequencing

The analysis of circulating tumor DNA (ctDNA) using next-generation sequencing (NGS) has become a valuable tool for the development of clinical oncology. However, the application of this method is challenging due to its low sensitivity in analyzing the trace amount of ctDNA in the blood. Furthermore, the method may generate false positive and negative results from this sequencing and subsequent analysis. To improve the feasibility and reliability of ctDNA detection in the clinic, here we present a technique which enriches rare mutations for sequencing, Enrich Rare Mutation Sequencing (ER-Seq). ER-Seq can distinguish a single mutation out of 1 x 107 wild-type nucleotides, which makes it a promising tool to detect extremely low frequency genetic alterations and thus will be very useful in studying disease heterogenicity. By virtue of the unique sequencing adapter&#39;s ligation, this method enables an efficient recovery of ctDNA molecules, while at the same time correcting for errors bidirectionally (sense and antisense). Our selection of 1021 kb probes enriches the measurement of target regions that cover over 95% of the tumor-related driver mutations in 12 tumors. This cost-effective and universal method enables a uniquely successful accumulation of genetic data. After efficiently filtering out background error, ER-seq can precisely detect rare mutations. Using a case study, we present a detailed protocol demonstrating probe design, library construction, and target DNA capture methodologies, while also including the data analysis workflow. The process to carry out this method typically takes 1-2 days.

This manuscript describes a technique for detecting mutations of low frequency in ctDNA, ER-Seq. This method is differentiated by its unique use of two-directional error correction, a special background filter, and efficient molecular acquirement.

利用下一代测序检测 CtDNA 中的稀有突变

The analysis of circulating tumor DNA (ctDNA) using next-generation sequencing (NGS) has become a valuable tool for the development of clinical oncology. ...

癌症研究

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been used to analyze the spectrum of clonal mutations in malignancy. Though far more efficient than traditional Sanger methods, NGS struggles with identifying rare clonal and subclonal mutations due to its high error rate of ~0.5&#8211;2.0%. Thus, standard NGS has a limit of detection for mutations that are &#62;0.02 variant allele fraction (VAF). While the clinical significance for mutations this rare in patients without known disease remains unclear, patients treated for leukemia have significantly improved outcomes when residual disease is &#60;0.0001 by flow cytometry. In order to mitigate this artefactual background of NGS, numerous methods have been developed. Here we describe a method for Error-corrected DNA and RNA Sequencing (ECS), which involves tagging individual molecules with both a 16 bp random index for error-correction and an 8 bp patient-specific index for multiplexing. Our method can detect and track clonal mutations at variant allele fractions (VAFs) two orders of magnitude lower than the detection limit of NGS and as rare as 0.0001 VAF.

Next-generation sequencing (NGS) is a powerful tool for genomic characterization that is limited by the high error rate of the platform (~0.5&#8211;2.0%). We describe our methods of error-corrected sequencing that allow us to obviate the NGS error rate and detect mutations at variant allele fractions as rare as 0.0001.

基于纠错 DNA 和 RNA 测序的稀有事件检测方法

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation into thousands of nano-sized water-in-oil individual reactions. Recently, ddPCR has become one of the most accurate and sensitive tools for circulating tumor DNA (ctDNA) detection. One of the major limitations of the standard ddPCR technique is the restricted number of mutations that can be screened per reaction, as specific hydrolysis probes recognizing each possible allelic version are required. An alternative methodology, the drop-off ddPCR, increases throughput, since it requires only a single pair of probes to detect and quantify potentially all genetic alterations in the targeted region. Drop-off ddPCR displays comparable sensitivity to conventional ddPCR assays with the advantage of detecting a greater number of mutations in a single reaction. It is cost-effective, conserves precious sample material, and can also be used as a discovery tool when mutations are not known a priori.

Here, we present a protocol to accurately quantify multiple genetic alterations of a target region in a single reaction using drop-off ddPCR and a unique pair of hydrolysis probes.

单液滴数字聚合酶链反应对热点地区突变的综合和同步检测

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation ...

Efficient Purification and LC-MS/MS-based Assay Development for Ten-Eleven Translocation-2 5-Methylcytosine Dioxygenase

The epigenetic transcription regulation mediated by 5-methylcytosine (5mC) has played a critical role in eukaryotic development. Demethylation of these epigenetic marks is accomplished by sequential oxidation by ten-eleven translocation dioxygenases (TET1-3), followed by the thymine-DNA glycosylase-dependent base excision repair. Inactivation of the TET2 gene due to genetic mutations or by other epigenetic mechanisms is associated with a poor prognosis in patients with diverse cancers, especially hematopoietic malignancies. Here, we describe an efficient single step purification of enzymatically active untagged human TET2 dioxygenase using cation exchange chromatography. We further provide a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach that can separate and quantify the four normal DNA bases (A, T, G, and C), as well as the four modified cytosine bases (5-methyl, 5-hydroxymethyl, 5-formyl, and 5-carboxyl). This assay can be used to evaluate the activity of wild type and mutant TET2 dioxygenases.

Here, we present a protocol for an efficient single step purification of the active untagged human ten-eleven translocation-2 (TET2) 5-methylcytosine dioxygenase using ion-exchange chromatography and its assay using a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach.

高效纯化和基于 LC ms/ms 的十-十一 Translocation-2 5-甲基胞嘧啶酶的检测开发

The epigenetic transcription regulation mediated by 5-methylcytosine (5mC) has played a critical role in eukaryotic development. Demethylation of these ...

生物化学

qKAT: Quantitative Semi-automated Typing of Killer-cell Immunoglobulin-like Receptor Genes

Killer cell immunoglobulin-like receptors (KIRs) are a set of inhibitory and activating immune receptors, on natural killer (NK) and T cells, encoded by a polymorphic cluster of genes on chromosome 19. Their best-characterized ligands are the human leukocyte antigen (HLA) molecules that are encoded within the major histocompatibility complex (MHC) locus on chromosome 6. There is substantial evidence that they play a significant role in immunity, reproduction, and transplantation, making it crucial to have techniques that can accurately genotype them. However, high-sequence homology, as well as allelic and copy number variation, make it difficult to design methods that can accurately and efficiently genotype all KIR genes. Traditional methods are usually limited in the resolution of data obtained, throughput, cost-effectiveness, and the time taken for setting up and running the experiments. We describe a method called quantitative KIR semi-automated typing (qKAT), which is a high-throughput multiplex real-time polymerase chain reaction method that can determine the gene copy numbers for all genes in the KIR locus. qKAT is a simple high-throughput method that can provide high-resolution KIR copy number data, which can be further used to infer the variations in the structurally polymorphic haplotypes that encompass them. This copy number and haplotype data can be beneficial for studies on large-scale disease associations, population genetics, as well as investigations on expression and functional interactions between KIR and HLA.

Quantitative killer cell immunoglobulin-like receptor (KIR) semi-automated typing (qKAT) is a simple, high-throughput, and cost-effective method to copy number type KIR genes for their application in population and disease association studies.

qkat: 杀手细胞免疫球蛋白样受体基因的定量半自动分型

Killer cell immunoglobulin-like receptors (KIRs) are a set of inhibitory and activating immune receptors, on natural killer (NK) and T cells, encoded by a ...

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in&#160;patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

患者生物流体中肿瘤相关循环DNA的检测与监测

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

在纯化造血干/祖细胞识别DNA突变

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

免疫与感染

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

多路复用讯问癌症相关的信号转导在培养的细胞中的荧光素酶的筛选平台

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

医学

Naked-Eye Detection of Rare Point Mutations in DNA

The protocol describes a naked-eye colorimetric test for the detection of somatic point mutations in an excess of wild type DNA. The future foreseen application of the method is the identification of rare mutations in circulating cell-free DNA from liquid biopsies, with a relevance in cancer diagnostics and stratification of oncological patients for personalized therapy. As a proof of concept, the test has been designed to detect the BRAFV600E mutation in the BRAF gene, which is important to identify the sub-group of melanoma patients that can benefit from targeted therapies with BRAF inhibitors. However, this colorimetric test can be easily generalized to other somatic mutations of clinical relevance due to the use of universal detection probes, thus providing strong potential in oncological diagnostics.
The test detects 0.5% of BRAFV600E in an excess of BRAFWT DNA, which matches the sensitivity of some commercial instrumental assays. Such sensitivity is clinically relevant for diagnostic purposes, allowing the early identification of drug-sensitive patients. In contrast to commercial assays based on real-time PCR, this test requires minimal instrumentation and processing, as it can be performed on DNA amplified with a standard PCR (or isothermal techniques) and provides a naked-eye readout with a one-tube reaction of a few steps in only one hour. At present, the test has been used only on synthetic DNA samples. However, the latter have been designed to mimic a real sample amplified from circulating cell-free DNA, to favor the translation of the test to clinical diagnostics.

This protocol permits the naked-eye identification of point mutated DNA in a 200-fold excess of wild type DNA molecules, by exploiting gold nanoparticles and paramagnetic microparticles.

The protocol describes a naked-eye colorimetric test for the detection of somatic point mutations in an excess of wild type DNA. The future foreseen ...