这项工作的经费部分是由 NSF 植物基因组研究 (IOS-1127155) 的赠款提供的。

注意: 图 1 显示了数组计算全息协议的五步骤。它们是: 1) 制备植物材料;2) 分离高质量的 DNA 样本;3) DNA 样品的标记和纯化;4) 对整个基因组阵列进行杂交、冲洗和扫描;和 5) 计算全息数据分析。 m 蒺藜 整个基因组平铺阵列包含总共971041种独特的低聚体探针, 其目标是基因组中超过5万基因或基因模型 (参见 材料表 )。独特的探针间隔约每150基对 (bp) 在 exonic 地区和 261 bp 在子地区的 m 蒺藜 基因组. 
 1. 植物材料的制备 <ol> <li> 划破野生类型 (重量; m 蒺藜 cv Jemalong A17) 和 FN6191 突变种子, 浓缩硫酸为8分钟 (参见 材料表 )。将硫酸移至废液容器中. </li>
	<li> 用蒸压去离子水冲洗种子三次. </li>
	<li> 表面消毒20% 漂液的种子10分钟 </li>
	<li> 用蒸压去离子水冲洗种子三次. </li>
	<li> 将种子储存在4和 #176; C 为3天。在春后, 在土壤中将种子在 16 h/8 h 光/暗循环和150和 #181 的生长腔中迁移和发芽一周; 电子/m 2 /秒的光照强度. </li>
	<li> 将幼苗移植到1加仑的花盆中, 在一个温室中生长 16 h/8 h 光/暗循环, 150 和 #181; 电子/m 2 /s 光强度为三周至四星期。收集来自植物的幼叶样本进行 DNA 分离. </li>
</ol> 2。高质量 DNA 样品的分离 <ol> <li> 从一株植物中提取1克幼叶组织, 并立即将其冷冻在液氮中. </li>
	<li> 在液氮中用砂浆和杵将冷冻叶组织研磨成细粉. </li>
	<li> 使用 dna 隔离套件分离基因组 dna (请参阅 材料表 ). </li>
	<li> 重50和 #181 纯化的 DNA 样品; 高压蒸双去离子 H 2 O (ddH 2 o) 或 1x TE 缓冲器 (10 毫米三盐酸, 1 毫米 EDTA, pH 7.0). </li>
	<li> 用分光光度计测量 DNA 浓度 (请参阅 材料表 ). </li>
	<li> 评估 260 nm/280 nm 和 260 nm/230 纳米 DNA 样本的比值. 
	注: 高质量的 DNA 样品应具有 260 nm/280 纳米比和 #8805; 1.8 和 260 nm/230 nm 比值和 #8805; 2.0. </li>
	<li> 通过在1% 琼脂糖凝胶中运行, 进一步评估 DNA 样本. 
	注: 高质量的 DNA 样品应具有较高的分子量, 不应被 RNA 污染。理想的情况下, DNA 样本的最终浓度应该是 150 ng/和 #181; L. </li>
</ol> 3。DNA 标记和纯化 <ol> <li> 稀释1和 #181; g 基因组 DNA 样品与 ddH 2 O 到最后的容量20和 #181; l 在500和 #181; l 离心管。
	<li> 添加5和 #181; L 随机入门 (请参阅 材料表 ) 到管道. </li>
	<li> 涡流管短暂, 并把它放入一个 thermocycler 孵化和变性 DNA 在98和 #176; C 为 10 min. </li>
	<li> 把管子从 thermocycler 中取出, 立即放在冰水上, 冷却5分钟. </li>
	<li> 准备两个标记混合, 如下所示 表 1 . </li>
	<li> 添加25和 #181; 标记混合1和 2, 分别引用 dna 和取样 dna 管. </li>
	<li> 将标签混合和 DNA 混合移三倍, 然后简单地旋转试管. </li>
	<li> 将管子放入 thermocycler, 在37和 #176 孵育; c 为2小时, 然后在65和 #176; c 为20分钟, 使外克莱诺酶钝化 (请参阅 材料表 ). </li>
	<li> 添加430和 #181; 1x 和 #160 的 L; 每根管子的缓冲器. </li>
	<li> 混合内容并简单地旋转管子. </li>
	<li> 将管道中的溶液转换为2毫升收集管的净化柱 (请参阅 材料表 ). </li>
	<li> 离心纯化柱在 1.4万 x g 为10分钟. </li>
	<li> 放弃传递解决方案. </li>
	<li> 在每列中添加480和 #181; L 1x 和 #160; TE 缓冲区. </li>
	<li> 在 1.4万 x g 上再次离心该列10分钟. </li>
	<li> 丢弃 pass-through 解决方案. </li>
	<li> 将残留在柱底部的标记 DNA 样本转移到新的离心管上. </li>
	<li> 使用吸管测量标记的 DNA 样本的体积, 并将最后的体积带到80和 #181; L 带有 1x TE 缓冲区. </li>
	<li> 使用分光光度计测量标记的 DNA 样本的浓度 (请参见 材料表 ). </li>
	<li> 混合等量的标记样本 dna 和参考 dna, 并将最终的体积带到160和 #181; L 与 #160; 1x TE 缓冲区. </li>
	<li> 添加256和 #181; l 2x 杂交缓冲, 50 和 #181; 人类 Cot-1 DNA 和50和 #181; l 10 和 #215; aCGH 阻塞代理 (请参阅 材料表 ), 并将它们移为三次. </li>
	<li> 旋转导管并将其放入 thermocycler, 在98和 #176 中孵育; C 为 10 min. </li>
	<li> 在37和 #176 孵育试管; C 为20分钟. </li>
</ol> 4。基因组阵列的杂交、洗涤和扫描 <ol> <li> 将杂交烤箱预热到67和 #176; 在杂交开始前至少4小时. </li>
	<li> 将垫片滑动到杂交室的底部. </li>
	<li> 从步骤3.23 中加载490和 #181; L 的杂交溶液到垫片滑动. </li>
	<li> 用一条 苜蓿蒺藜 基因芯片芯片来覆盖衬垫滑动, 形成杂交室. </li>
	<li> 投入杂交室盖子和紧固用夹子的房间. </li>
	<li> 将组装的杂交室放入杂交烤箱, 孵育67和 #176; C 为 40-48 h. </li>
	<li> 准备三滑动洗碗碟: 两个与250毫升洗涤缓冲区, 我保持在室温下, 一个与洗涤缓冲器 II 保持在37和 #176; C. </li>
	<li> 准备两个滑动洗涤罐: 一个带有70毫升乙腈, 一个带有70毫升稳定和干燥溶液; 在室温下保持通风罩 (请参阅 材料表 ). </li>
	<li> 将杂交室从烤箱中取出, 取出箱夹和盖子. </li>
	<li> 将微阵列芯片和垫片滑动到带洗涤缓冲器的滑动洗涤盘中, 并将芯片与盖板分开. </li>
	<li> 将微阵列芯片移动到第二个带洗涤缓冲的滑动洗涤盘中, 然后在室温下用搅拌板轻轻地将溶液循环到5分钟. </li>
	<li> 将微阵列芯片转移到第三个滑动洗涤盘上, 用5ml 洗涤缓冲器 II, 在37和 #176 的磁性搅拌板上轻轻地用搅拌棒冲洗; C 为 1 min. </li>
	<li> 将微阵列芯片转移到一个通风柜中, 并将其洗净三十年代. </li>
	<li> 将微阵列芯片转移到带有稳定和干燥溶液的滑动洗涤罐中, 并将其洗净三十年代. </li>
	<li> 慢慢取出芯片, 晾干1分钟. </li>
	<li> 使用扫描仪扫描微阵列芯片 (请参阅 2 和 #181; m 分辨率下的材料表 )。设置参数以保存多个图像和整个幻灯片扫描。将通道1和2的增益设置为 100%, 并取消自动增益. </li>
</ol> 5。计算全息数据分析 <ol> <li> 提取 Cy3 和 Cy5 荧光使用扫描软件对阵列上每个探测器的强度 (请参见 材料表 ). </li>
	<li> 使用软件的分段分析来检测样本 dna 和参考 dna 之间的拷贝数变化 (cnv)。使用段的阈值意味着日志 2 采样/参考比和 #177; 2.5 标准偏差用于确定 cnv. </li>
	<li> 使用信号映射软件 (请参阅 材料表 ) 在整个基因组中检查日志的分布情况 2 样本/参考比率. </li>
</ol>

<ol>
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E., Finkel, S. E., Tavare, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+expression+arrays+for+copy+number+detection:+an+example+from+E.+coli.">Using expression arrays for copy number detection: an example from E. coli.</a> BMC Bioinformatics. 8, 203 (2007).</li><li>Werner, J. D., 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=Quantitative+trait+locus+mapping+and+DNA+array+hybridization+identify+an+FLM+deletion+as+a+cause+for+natural+flowering-time+variation.">Quantitative trait locus mapping and DNA array hybridization identify an FLM deletion as a cause for natural flowering-time variation.</a> Proc Natl Acad Sci U S A. 102 (7), 2460-2465 (2005).</li><li>Xi, J., Chen, Y., Nakashima, J., Wang, S. M., Chen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medicago+truncatula+esn1+defines+a+genetic+locus+involved+in+nodule+senescence+and+symbiotic+nitrogen+fixation.">Medicago truncatula esn1 defines a genetic locus involved in nodule senescence and symbiotic nitrogen fixation.</a> Mol Plant Microbe Interact. 26 (8), 893-902 (2013).</li><li>Schnabel, E., Journet, E. P., de Carvalho-Niebel, F., Duc, G., Frugoli, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Medicago+truncatula+SUNN+gene+encodes+a+CLV1-like+leucine-rich+repeat+receptor+kinase+that+regulates+nodule+number+and+root+length.">The Medicago truncatula SUNN gene encodes a CLV1-like leucine-rich repeat receptor kinase that regulates nodule number and root length.</a> Plant Mol Biol. 58 (6), 809-822 (2005).</li><li>Horvath, B., 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=Loss+of+the+nodule-specific+cysteine+rich+peptide,+NCR169,+abolishes+symbiotic+nitrogen+fixation+in+the+Medicago+truncatula+dnf7+mutant.">Loss of the nodule-specific cysteine rich peptide, NCR169, abolishes symbiotic nitrogen fixation in the Medicago truncatula dnf7 mutant.</a> Proc Natl Acad Sci U S A. 112 (49), 15232-15237 (2015).</li><li>Kim, 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=An+antimicrobial+peptide+essential+for+bacterial+survival+in+the+nitrogen-fixing+symbiosis.">An antimicrobial peptide essential for bacterial survival in the nitrogen-fixing symbiosis.</a> Proc Natl Acad Sci U S A. 112 (49), 15238-15243 (2015).</li><li>. Medicago truncatula Mutant Database Available from: <a target="_blank" href="https://medicago-mutant.noble.org/mutant/FNB.php">https://medicago-mutant.noble.org/mutant/FNB.php</a> (2017)</li><li>Burton, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SNP+genotyping+with+the+next+generation+of+CGH+microarray.">SNP genotyping with the next generation of CGH microarray.</a> MLO Med Lab Obs. 45 (7), (2013).</li></ol>

作者声明没有竞争的金融利益。

我们已经开发了一个基于阵列的计算全息平台, 用于检测和表征快中子轰击 (FNB) 诱发突变体的m 蒺藜cv Jemalong A17。为了证明阵列全息计算方法在检测基因突变中的应用, 我们对突变 FN6191 进行了 aCGH 分析, 并与野生型植物形成了高结瘤表型, 并与S. 根瘤菌 Sm1021进行了接种。对于分段分析, 如果段中的探测器的日志2比率的平均值高于上限阈值或低于该给定数组比较的下限阈值, 则?...

豆类 (豆科) 是第三大家族的开花植物, 与许多经济上重要的物种, 如大豆 (甘氨酸 max) 和苜蓿 (苜蓿)。豆科植物可以与固氮土壤细菌相互作用, 通常称为根瘤菌来发展根结节, 将大气氮减少到氨供寄主植物使用。因此, 种植豆类作物需要很少的氮肥投入, 从而有助于可持续农业。豆类作物生产的叶子和种子的高蛋白含量, 作为优良的饲料和谷物作物。然而, 种植豆科植物通常有复杂的基因组结构, 使功能研究的基因发挥关键作用在豆类特定的过程繁琐。苜蓿蒺藜已被广泛采用为豆科植物的模型, 主要是因为 (1) 它有一个相对较小的单倍体基因组大小 (~ 550) 的二倍体基因组;(2) 植物可稳定转化为基因功能研究;和 (3) 它与紫花苜蓿 (m), 牧草皇后, 和许多其他经济重要的农作物的转化研究密切相关。最近, m 蒺藜cv Jemalong A17 的基因组序列已被释放12。对基因组的诠释显示, 基因组中有超过5万预测基因或基因模型。要确定m 蒺藜基因组中大多数基因的功能是一项具有挑战性的任务。为了促进基因的功能研究, 在 15万 M1线范围内, 利用快中子轰击 (FNB) 诱变, 在m. 蒺藜cv Jemalong A173中生成了一组突变体的综合集合.,4。快中子, 高能电离诱变, 已被用于在许多植物物种中生成突变体, 包括拟南芥5,6, 大米 (水稻品种)7, 西红柿 (茄汁番茄), 黄豆 (甘氨酸大豆;G. max)8,9, 大麦 (大麦大麦), 和莲花日本人10。从 FNB 诱变衍生出的很大一部分突变是由于 DNA 的缺失, 其范围从几个碱基对到巨型碱基对9,11。许多表型相关基因已被成功识别并具有特征4、12、13、14、15、16、17,18,19. 以前, FNB 突变体的潜在基因的分子克隆依赖于地图的方法, 这是时间消耗和限制分子水平的突变体数量。最近, 一些免费的方法, 包括 transcript-based 方法, 基因组平铺阵列的比较基因组杂交 (计算全息) 的 DNA 拷贝数变异检测, 和整个基因组测序, 已被用来促进不同生物体 (包括动植物) 中缺失突变体的特征20,21,22,2324,25 26,27,28,29,30,31。
为了便于在m. 蒺藜中对 FNB 突变体的定性, 开发并验证了基于基因组的整体比较基因组杂交 (计算全息) 平台。正如在动物系统中报告的那样, 基于阵列的全息计算平台允许在m. 蒺藜FNB 突变体的整个基因组水平检测拷贝数变体 (cnv)。此外, 病变可以通过 PCR 确认和删除边界可以确定的排序。总的来说, 阵列全息计算平台是一个有效的工具, 以识别病变的m 蒺藜FNB 突变体。本文对m 蒺藜FNB 突变体的阵列全息程序和删除边界的 PCR 特征进行了说明。
以下协议提供了实验步骤和有关试剂、设备和分析工具的信息, 这些研究人员有兴趣执行整个基因组阵列的比较基因组杂交 (计算全息) 拷贝数分析植物的变异。例如,苜蓿蒺藜FN6191 突变体用于识别与突变表型相关的缺失区域和候选基因。m 蒺藜FN6191 突变体, 最初是从快中子轰击引发的删除突变的集合中分离出来的32 (参见材料表), 在接种后用土壤展示了一个超级结瘤表型细菌, Sihorhizobium 根瘤菌Sm1021, 与野生型植物形成对比。

<table><tbody><tr><td>&lt;em&gt;Medicago truncatula&lt;/em&gt; genome array, 1 x 1 M</td><td>Agilent</td><td>G4123A</td><td></td></tr><tr><td>&lt;em&gt;Medicago truncatula&lt;/em&gt; FN6191 (mutant)</td><td>In house</td><td>FN6191</td><td></td></tr><tr><td>&lt;em&gt;Medicago truncatula&lt;/em&gt; Jemalong A17 (reference)</td><td>In house</td><td>A17</td><td></td></tr><tr><td>Sulfuric acid</td><td>Sigma-Aldrich</td><td>320501</td><td></td></tr><tr><td>DNeasy Plant Mini Kit</td><td>Qiagen</td><td>69104</td><td></td></tr><tr><td>Nanodrop Spectrophotometer</td><td>Thermo Scientific</td><td>1000D</td><td></td></tr><tr><td>SureTag DNA Labeling Kit</td><td>Agilent</td><td>5190-3400</td><td></td></tr><tr><td>Random primer</td><td>Agilent</td><td>5190-3399</td><td></td></tr><tr><td>Acetonitrile</td><td>Sigma-Aldrich</td><td>271004-1L</td><td></td></tr><tr><td>Thermocycler</td><td>MJ research</td><td>PTC-200</td><td></td></tr><tr><td>Centrifuge</td><td>Labnet international Inc</td><td>Spectrafuge 24D</td><td></td></tr><tr><td>Stabilization and Drying Solution</td><td>Agilent</td><td>5185-5979</td><td></td></tr><tr><td>Oligo aCGH/ChIP-on-chip Hybridization Kit</td><td>Agilent</td><td>5188-5380</td><td></td></tr><tr><td>Hybridization Chamber gasket slides</td><td>Agilent</td><td>G2505</td><td></td></tr><tr><td>Human Cot-1 DNA</td><td>Agilent</td><td>5190-3393</td><td></td></tr><tr><td>Oligo aCGH/ChIP-on-chip Wash Buffer 1 and 2</td><td>Agilent</td><td>5188-5221</td><td></td></tr><tr><td>Hybridization Chamber, stainless</td><td>Agilent</td><td>G2534A</td><td></td></tr><tr><td>Hybridization oven</td><td>Agilent</td><td>G2545A</td><td></td></tr><tr><td>Purification Columns</td><td>Agilent</td><td>5190-3391</td><td></td></tr><tr><td>Laser scanner</td><td>Roche</td><td>MS200</td><td></td></tr><tr><td>NimbleScan 2.6</td><td>Roche Nimblegen</td><td>5225035001</td><td></td></tr><tr><td>Signal Map 1.9</td><td>Roche Nimblegen</td><td>Signalmap1.9</td><td></td></tr></tbody></table>

an array-based comparative genomic hybridization platform for efficient detection of copy number variations in fast neutron-induced medicago truncatula mutants

突变体是基因功能研究的宝贵的遗传资源。为了产生突变体集合, 可利用三种诱变, 包括生物如 T-脱氧核糖核酸或转, 化学物质如甲基磺酸乙酯 (EMS), 或物理如电离辐射。所观察到的突变类型因所使用的诱变而异。对于电离辐射诱发突变体, 突变包括删除, 重复, 或重排。虽然 T-DNA 或 transposon-based 诱变仅限于易受转化的物种, 但化学或物理诱变可应用于种类繁多的物种。然而, 从化学或物理诱变衍生的突变的定性传统上依赖于地图的克隆方法, 这是劳动密集型和耗时。在这里, 我们表明, 一个高密度基因组阵列的比较基因组杂交 (aCGH) 平台可用于有效地检测和表征拷贝数变异 (cnv) 的突变从快中子轰击 (FNB) 突变中获得的苜蓿蒺藜, 豆科植物。整个基因组序列分析显示, 在m 蒺藜中有超过5万基因或基因模型。目前, FNB 诱发突变体的m. 蒺藜是从超过 15万 M1 线, 代表了宝贵的基因资源的功能研究的基因组。这里描述的 aCGH 平台是一个有效的工具, 用于表征 FNB 诱导突变体在m 蒺藜。

图 2显示了在整个基因组中, 突变体与重量信号的归一化对数2比值的分布。对计算全息数据的分析显示, 4 号染色体上大约有 22 kb 的删除, 它包含了整个SUNN基因33和 FN6191 突变体中的其他一些注释基因 (图 2,图 3)。候选删除区域被73连续探针覆盖, 该阵列的平均正常化日志2...

Watch this Scientific Journal Video about An Array-based Comparative Genomic Hybridization Platform for Efficient Detection of Copy Number Variations in Fast Neutron-induced Medicago truncatula Mutant

An Array-based Comparative Genomic Hybridization Platform for Efficient Detection of Copy Number Variations in Fast Neutron-induced Medicago truncatula Mutants

该协议提供了实验步骤和有关试剂、设备和分析工具的信息, 这些研究人员有兴趣执行整个基因组阵列的比较基因组杂交 (计算全息) 对拷贝数变化的分析植物.

一种基于阵列的比较基因组杂交平台, 有效检测快中子诱导的苜蓿蒺藜突变体的拷贝数变化

Mutants are invaluable genetic resources for gene function studies. To generate mutant collections, three types of mutagens can be utilized, including ...

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An Array-based Comparative Genomic Hybridization Platform for Efficient Detection of Copy Number Variations in Fast Neutron-induced Medicago truncatula Mutants

Unit 2

Genomes and Evolution

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7.7K 次观看.  Noble Research Institute. 该协议提供了实验步骤和有关试剂、设备和分析工具的信息, 这些研究人员有兴趣执行整个基因组阵列的比较基因组杂交 (计算全息) 对拷贝数变化的分析植物.

视频: An Array-based Comparative Genomic Hybridization Platform for Efficient Detection of Copy Number Variations in Fast Neutron-induced Medicago truncatula Mutants

Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play in the functioning of cells and whole organisms. This cause has been tremendously aided by the recent introduction of the CRISPR/Cas9 system-a versatile tool that allows researchers to manipulate the genome and transcriptome in order to, among other things, knock out, knock down, or knock in genes in a targeted manner. For the purpose of knocking out a gene, CRISPR/Cas9-mediated double-strand breaks recruit the non-homologous end-joining DNA repair pathway to introduce the frameshift-causing insertion or deletion of nucleotides at the break site. However, an individual guide RNA may cause undesirable off-target effects, and to rule these out, the use of multiple guide RNAs is necessary. This multiplicity of targets also means that a high-volume screening of clones is required, which in turn begs the use of an efficient high-throughput technique to genotype the knockout clones. Current genotyping techniques either suffer from inherent limitations or incur high cost, hence rendering them unsuitable for high-throughput purposes. Here, we detail the protocol for using fluorescent PCR, which uses genomic DNA from crude cell lysate as a template, and then resolving the PCR fragments via capillary gel electrophoresis. This technique is accurate enough to differentiate one base-pair difference between fragments and hence is adequate in indicating the presence or absence of a frameshift in the coding sequence of the targeted gene. This precise knowledge effectively precludes the need for a confirmatory sequencing step and allows users to save time and cost in the process. Moreover, this technique has proven to be versatile in genotyping various mammalian cells of various tissue origins targeted by guide RNAs against numerous genes, as shown here and elsewhere.

The genotyping technique described here, which couples fluorescent polymerase chain reaction (PCR) to capillary gel electrophoresis, allows for high-throughput genotyping of nuclease-mediated knockout clones. It circumvents limitations faced by other genotyping techniques and is more cost effective than sequencing methods.

用荧光PCR毛细管电泳技术在基因型CRISPR / Cas9介导的敲除突变体的高通量形式

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play ...

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遗传学

Generating Transgenic Plants with Single-copy Insertions Using BIBAC-GW Binary Vector

When generating transgenic plants, generally the objective is to have stable expression of a transgene. This requires a single, intact integration of the transgene, as multi-copy integrations are often subjected to gene silencing. The Gateway-compatible binary vector based on bacterial artificial chromosomes (pBIBAC-GW), like other pBIBAC derivatives, allows the insertion of single-copy transgenes with high efficiency. As an improvement to the original pBIBAC, a Gateway cassette has been cloned into pBIBAC-GW, so that the sequences of interest can now be easily incorporated into the vector transfer DNA (T-DNA) by Gateway cloning. Commonly, the transformation with pBIBAC-GW results in an efficiency of 0.2&#8211;0.5%, whereby half of the transgenics carry an intact single-copy integration of the T-DNA. The pBIBAC-GW vectors are available with resistance to Glufosinate-ammonium or DsRed fluorescence in seed coats for selection in plants, and with resistance to kanamycin as a selection in bacteria. Here, a series of protocols is presented that guide the reader through the process of generating transgenic plants using pBIBAC-GW: starting from recombining the sequences of interest into the pBIBAC-GW vector of choice, to plant transformation with Agrobacterium, selection of the transgenics, and testing the plants for intactness and copy number of the inserts using DNA blotting. Attention is given to designing a DNA blotting strategy to recognize single- and multi-copy integrations at single and multiple loci.

Using a pBIBAC-GW binary vector makes generating transgenic plants with intact single-copy insertions, an easy process. Here, a series of protocols is presented that guide the reader through the process of generating transgenic Arabidopsis plants, and testing the plants for intactness and copy number of the inserts.

用 BIBAC 二元矢量生成单拷贝插入的转基因植物

When generating transgenic plants, generally the objective is to have stable expression of a transgene. This requires a single, intact integration of the ...

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been co-opted as a powerful tool for precise eukaryotic genome engineering. Here, we present a rapid and simple method using chimeric single guide RNAs (sgRNA) and CRISPR-Cas9 Ribonucleoproteins (RNPs) for the efficient and precise generation of genomic point mutations in C. elegans. We describe a pipeline for sgRNA target selection, homology-directed repair (HDR) template design, CRISPR-Cas9-RNP complexing and delivery, and a genotyping strategy that enables the robust and rapid identification of correctly edited animals. Our approach not only permits the facile generation and identification of desired genomic point mutant animals, but also facilitates the detection of other complex indel alleles in approximately 4 - 5 days with high efficiency and a reduced screening workload.

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

Here, we present a method to engineer the genome of C. elegans using CRISPR-Cas9 ribonucleoproteins and homology dependent repair templates.

利用 CRISPR/Cas9 Ribonucleoproteins 快速简便地生成线虫基因组点突变体的管道

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been ...

Identifying Mutations by High Resolution Melting in a TILLING Population of Rice

Target Induced Local Lesions In Genomes (TILLING) is a strategy of reverse genetics for the high-throughput screening of induced mutations. However, the TILLING system has less applicability for insertion/deletion (Indel) detection and traditional TILLING needs more complex steps, like CEL I nuclease digestion and gel electrophoresis. To improve the throughput and selection efficiency, and to make the screening of both Indels and single base substitions (SBSs) possible, a new high-resolution melting (HRM)-based TILLING system is developed. Here, we present a detailed HRM-TILLING protocol and show its application in mutation screening. This method can analyze the mutations of PCR amplicons by measuring the denaturation of double-stranded DNA at high temperatures. HRM analysis is directly performed post-PCR without additional processing. Moreover, a simple, safe and fast (SSF) DNA extraction method is integrated with HRM-TILLING to identify both Indels and SBSs. Its simplicity, robustness and high throughput make it potentially useful for mutation scanning in rice and other crops.

In this article, we present the protocol that is described as high-resolution melting analysis (HRM)-based Target Induced Local Lesions In Genomes (TILLING). This method utilizes fluorescence changes during the melting of the DNA duplex and is suitable for high-throughput screening of both insertion/deletion (Indel) and single base substition (SBS).

通过高分辨率熔融在水稻种群中识别突变

Target Induced Local Lesions In Genomes (TILLING) is a strategy of reverse genetics for the high-throughput screening of induced mutations. However, the ...

Development of Targeting Induced Local Lesions IN Genomes (TILLING) Populations in Small Grain Crops by Ethyl Methanesulfonate Mutagenesis

Targeting Induced Local Lesions IN Genomes (TILLING) is a powerful reverse genetics tool that includes chemical mutagenesis and detection of sequence variation in target genes. TILLING is a highly valuable functional genomics tool for gene validation, especially in small grains in which transformation-based approaches hold serious limitations. Developing a robust mutagenized population is key to determining the efficiency of a TILLING-based gene validation study. A TILLING population with a low overall mutation frequency indicates that an impractically large population must be screened to find desired mutations, whereas a high mutagen concentration leads to high mortality in the population, leading to an insufficient number of mutagenized individuals. Once an effective population is developed, there are multiple ways to detect mutations in a gene of interest, and the choice of platform depends upon the experimental scale and availability of resources. The Cel-1 assay and agarose gel-based approach for mutant identification is convenient, reproducible, and a less resource-intensive platform. It is advantageous in that it is simple, requiring no computational knowledge, and it is especially suitable for validation of a small number of genes with basic lab equipment. In the present article, described are the methods for development of a good TILLING population, including preparation of the dosage curve, mutagenesis and maintenance of the mutant population, and screening of the mutant population using the PCR-based Cel-1 assay.

Development of Targeting Induced Local Lesions IN Genomes TILLING Populations in Small Grain Crops by Ethyl Methanesulfonate Mutagenesis

Described is a protocol for developing a Targeting Induced Local Lesions IN Genomes (TILLING) population in small grain crops with use of ethyl methanesulfonate (EMS) as a mutagen. Also provided is a protocol for mutation detection using the Cel-1 assay.

通过乙基甲烷硫素突变在小谷物作物中靶向诱导局部病变(TILLING)种群的发展

Targeting Induced Local Lesions IN Genomes (TILLING) is a powerful reverse genetics tool that includes chemical mutagenesis and detection of sequence ...

发育生物学

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

Similar to Agrobacterium tumerfaciens, Agrobacterium rhizogenes can transfer foreign DNAs into plant cells based on the autonomous root-inducing (Ri) plasmid. A. rhizogenes can cause hairy root formation on plant tissues and form composite plants after transformation. On these composite plants, some of the regenerated roots are transgenic, carrying the wild type T-DNA and the engineered binary vector; while the shoots are still non-transgenic, serving to provide energy and growth support. These hairy root composite plants will not produce transgenic seeds, but there are a number of important features that make these composite plants very useful in plant research. First, with a broad host range,A. rhizogenes can transform many plant species, especially dicots, allowing genetic engineering in a variety of species. Second, A. rhizogenes infect tissues and explants directly; no tissue cultures prior to transformation is necessary to obtain composite plants, making them ideal for transforming recalcitrant plant species. Moreover, transgenic root tissues can be generated in a matter of weeks. For Medicago truncatula, we can obtain transgenic roots in as short as three weeks, faster than normal floral dip Arabidopsis transformation. Overall, the hairy root composite plant technology is a versatile and useful tool to study gene functions and root related-phenotypes. Here we demonstrate how hairy root composite plants can be used to study plant-rhizobium interactions and nodulation in the difficult-to-transform species M. truncatula.

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

We demonstrate how hairy root composite plants can be used to study plant-rhizobium interactions and nodulation in the difficult-to-transform species Medicago truncatula.

复合植物代苜蓿truncatula</em用于检测结瘤

Similar to Agrobacterium tumerfaciens, Agrobacterium rhizogenes can transfer foreign DNAs into plant cells based on the autonomous root-inducing (Ri) ...

生物学

Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the subcellular distribution of a specific tagged fluorescent marker in the secretory pathway. Arabidopsis is a powerful biological model for genetic studies because of its genome size, generation time, and conservation of molecular mechanisms among kingdoms. The array genotyping as an approach to map the mutation in alternative to the traditional method based on molecular markers is advantageous because it is relatively faster and may allow the mapping of several mutants in a really short time frame. This method allows the identification of proteins that can influence the integrity of any organelle in plants. Here, as an example, we propose a screen to map genes important for the integrity of the endoplasmic reticulum (ER). Our approach, however, can be easily extended to other plant cell organelles (for example see1,2), and thus represents an important step toward understanding the molecular basis governing other subcellular structures.

A fundamental quest in cell biology is to define the mechanisms that underlie the identity of the organelles that make eukaryotic cells. Here we propose a method to identify the genes responsible for the morphological and functional integrity of plant organelles using fluorescence microscopy and next-generation sequencing tools.

荧光显微镜筛选和新一代测序：参与细胞器完整性的基因鉴定的有用工具

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the ...

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 ...

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the homogenization of the tissue of interest that could potentially cause changes in the metabolic pathways being studied1. Mass spectrometric imaging (MSI) is a powerful analytical tool that can provide spatial information of analytes within intact slices of biological tissue samples1-5. This technique has been used extensively to study various types of compounds including proteins, peptides, lipids, and small molecules such as endogenous metabolites. With matrix-assisted laser desorption/ionization (MALDI)-MSI, spatial distributions of multiple metabolites can be simultaneously detected. Herein, a method developed specifically for conducting untargeted metabolomics MSI experiments on legume roots and root nodules is presented which could reveal insights into the biological processes taking place. The method presented here shows a typical MSI workflow, from sample preparation to image acquisition, and focuses on the matrix application step, demonstrating several matrix application techniques that are useful for detecting small molecules. Once the MS images are generated, the analysis and identification of metabolites of interest is discussed and demonstrated. The standard workflow presented here can be easily modified for different tissue types, molecular species, and instrumentation.

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Mass spectrometric imaging (MSI) is a powerful tool that can be used to discover and identify various chemical species in intact tissues, preserving the compounds in their native environments, which can provide new insights into biological processes. Herein a MSI method developed for the analysis of small molecules is described.&#160;

MALDI-质谱成像代谢物的研究<em&gt;苜蓿</em&gt;根瘤

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the ...

Array Comparative Genomic Hybridization (Array CGH) for Detection of Genomic Copy Number Variants

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a clinical diagnostic service laboratory. DNA extracted from a patient&#8217;s sample (blood, saliva or other tissue types) is labeled with a fluorochrome (either cyanine 5 or cyanine 3). A reference DNA sample is labeled with the opposite fluorochrome. There follows a cleanup step to remove unincorporated nucleotides before the labeled DNAs are mixed and resuspended in a hybridization buffer and applied to an array comprising ~60,000 oligonucleotide probes from loci across the genome, with high probe density in clinically important areas. Following hybridization, the arrays are washed, then scanned and the resulting images are analyzed to measure the red and green fluorescence for each probe. Software is used to assess the quality of each probe measurement, calculate the ratio of red to green fluorescence and detect potential copy number variants.

Array Comparative Genomic Hybridization Array CGH for Detection of Genomic Copy Number Variants

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a diagnostic service laboratory.

一种基于阵列的比较基因组杂交平台, 有效检测快中子诱导的苜蓿蒺藜突变体的拷贝数变化

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