Name: a converging strategy for the generation of a virtually sequenced cdna library from unreferenced pacific oysters
Uploaded: 2019-06-13T13:30:00
Description: Watch this Scientific Journal Video about A Converging Strategy for the Generation of a Virtually Sequenced cDNA Library from Unreferenced Pacific Oysters at JoVE.com

这项工作部分得到了中国自然科学基金(赠款号31471703、A0201300537和31671854给J.V.和L.L.,授予G.Y.编号31470435)和100名外国人才计划(授予编号JSB2014012到J.V.)的支持。

注:图1显示了原理图概述。 
1. 样本收集
<ol><li>获取牡蛎标本。在收获后、运输和实验室使用前,在购买后4-7天内将牡蛎留在冰上。 注:根据本协议,牡蛎是从南京中彩批发市场(原产于中国福建宁德和中国江苏连云港)、青岛海杰水产品公司(原产于中国山东青岛)、居城购买牡蛎。烟台水产品公司(原产于中国山东烟台)和青岛金秀水产品公司(原产于中国山?...

<ol>
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通过将COX1和ND基因与公开的牡蛎DNA基因组数据库进行比较,允许对区域海鲜市场具有类似表型的无配表型牡蛎标本进行基因鉴定。该方法的意义在于其简单性,因为评估虚拟cDNA库只需要一个PCR反应。两个保守的线粒体COX1和ND基因从cDNA库被放大,该库由每个牡蛎的RNA提取物的逆转转录产生。通过直接在液氮中研磨牡蛎组织,简化了RNA分离方法(步骤2.1)。对每个试样的COX1和ND基因进行测序后,序列排列表明,...

由于交货时间长、创业推理或针对具体国家的海关法规,获取活的参考生物材料可能具有挑战性。作为替代方案,也可以从表象相同的标本中收集所需的生物材料。然而,这些样本在基因型水平上可能有很大差异,因此,由于新来源的材料与同一物种的数字化存储参考基因组的比较,往往变得困难,甚至徒劳。现有的DNA扩增方法。测序单个样本的高度保守的基因是识别物种1的广泛使用和强大的工具,例如常用的线粒体基因,这些基因经常用作cDNA库的质量评估的参考基因2 ,3,4,5,6.本文提出的方法的基本原理是,与参考基因组的相应序列相比,单个匿名牡蛎样本中的线粒体基因序列高度保存表明,其他基因也可能显示低发分,因为线粒体DNA进化速度比核DNA7要快,只需公开使用,即可对各种科学和工业相关的基因进行扩增和分离可作为参考的排序数据。
本文所述方法的总体目标是提供一个优化的工作流程,用于生成虚拟序列化的牡蛎 cDNA 库,该库可用作牡蛎基因克隆的模板 DNA。在虚拟测序中,绕过了新基因组测序;相反,一个已知的、数字存储的参考序列直接用于利用或设计用于生产cDNA的引物,最终构成一个库(或添加到预先存在的库)。其目的是生成收敛的 cDNA 库,这意味着生成的 cDNA 序列和参考序列之间的相似性可以从低到高背离排列。使用细胞色素 C 氧化酶亚单位 1 (COX1) 和 NADH 脱氢酶 (ND) 作为参考基因的一个关键优势是,由于这些线粒体基因的高度保护,即使高度地理分离的牡蛎标本也可以被分析。在证明了这些成熟的标记方法后,我们证明了其应用于两种酶候选体,它们涉及糖核苷酸生物合成,可能具有工业相关性8、9、10.太平洋牡蛎的生物技术潜力尚未开发。因此,我们认为,这种制备虚拟序列cDNA库的收敛方法也适合希望从这种相关生物材料中生成cDNA的非专家研究人员。

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			<td height="21" style="height:21px;width:261px;">Chemicals:</td>
			<td style="width:199px;"></td>
			<td style="width:131px;"></td>
			<td style="width:236px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;">1% Triton X-100</td>
			<td>Solarbio</td>
			<td>9002-93-1</td>
			<td>*Alternative distributors possible</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:261px;">2,5-Dihydroxybenzoic acid</td>
			<td>Alfa Aesar</td>
			<td>490-79-9</td>
			<td>*Alternative distributors possible</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetonitrile</td>
			<td>Merck</td>
			<td>75-05-8</td>
			<td>*Alternative distributors possible</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agarose for molecular biology</td>
			<td>Biowest Chemicals</td>
			<td>111860</td>
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		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ampicilin</td>
			<td>Solarbio</td>
			<td>69-52-3</td>
			<td>*Alternative distributors possible</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chloroform</td>
			<td>Lingfeng, Shanghai</td>
			<td>67-66-3</td>
			<td>*Alternative distributors possible</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DEPC water</td>
			<td>Thermo Scientific</td>
			<td>R0601</td>
			<td></td>
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			<td height="20" style="height:20px;">Ethanol</td>
			<td>Jinhuada, Guangzhou</td>
			<td>64-17-5</td>
			<td>*Alternative distributors possible</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Guanidinium thiocyanate-phenol reagent</td>
			<td>Invitrogen</td>
			<td>15596018</td>
			<td>TRIzol reagent</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Imidazole</td>
			<td>Energy Chemical</td>
			<td>288-32-4</td>
			<td>*Alternative distributors possible</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Isopropyl alcohol</td>
			<td>Nanjing Chemical Reagent</td>
			<td>67-63-0</td>
			<td>*Alternative distributors possible</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Isopropyl &#946;-D-thiogalactopyranoside</td>
			<td>Solarbio</td>
			<td>367-93-1</td>
			<td>*Alternative distributors possible</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Kanamycin</td>
			<td>Solarbio</td>
			<td>25389-94-0</td>
			<td>*Alternative distributors possible</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LB Agar</td>
			<td>Thermo Fisher</td>
			<td>22700025</td>
			<td>*Alternative distributors possible</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LB Broth</td>
			<td>Thermo Fisher</td>
			<td>10855021</td>
			<td>*Alternative distributors possible</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Methanol</td>
			<td>Jinhuada, Guangzhou</td>
			<td>67-56-1</td>
			<td>*Alternative distributors possible</td>
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			<td height="24" style="height:24px;">MgCl2 hexahydrate</td>
			<td>Xilong Huagong</td>
			<td>7791-18-6</td>
			<td>*Alternative distributors possible</td>
		</tr>
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			<td height="20" style="height:20px;">NaCl</td>
			<td>Xilong Huagong</td>
			<td>7647-14-5</td>
			<td>*Alternative distributors possible</td>
		</tr>
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			<td height="23" style="height:23px;">NAD+</td>
			<td>Duly Biotech</td>
			<td>53-84-9</td>
			<td>*Alternative distributors possible</td>
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			<td height="20" style="height:20px;">Phenyl-methylsulfonyl fluoride</td>
			<td>Macklin</td>
			<td>329-98-6</td>
			<td>*Alternative distributors possible</td>
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			<td height="20" style="height:20px;">Tris</td>
			<td>Solarbio</td>
			<td>77-86-1</td>
			<td>*Alternative distributors possible</td>
		</tr>
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			<td height="20" style="height:20px;">UDP-glucose</td>
			<td>Wuhu Nuowei Chemicals</td>
			<td>28053-08-9</td>
			<td>*Alternative distributors possible</td>
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			<td height="21" style="height:21px;">UDP-glucuronic acid</td>
			<td>SIGMA</td>
			<td>63700-19-6</td>
			<td>*Alternative distributors possible</td>
		</tr>
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			<td height="21" style="height:21px;">Tools/Instruments:</td>
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			<td></td>
			<td></td>
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			<td height="20" style="height:20px;">MALDI-TOF mass spectrometer</td>
			<td>Bruker</td>
			<td>Autoflex</td>
			<td>*Alternative distributors possible</td>
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			<td height="20" style="height:20px;">Metal block heater</td>
			<td>Long Yang Scientific Instruments</td>
			<td>Thermoshaker HB20</td>
			<td>*Alternative distributors possible</td>
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		<tr height="21">
			<td height="21" style="height:21px;">PCR thermocycler</td>
			<td>Hema</td>
			<td>9600</td>
			<td>*Alternative distributors possible</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Enzyme and Kits:</td>
			<td></td>
			<td></td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">10&#215;Ligation buffer</td>
			<td>Thermo Scientific</td>
			<td>B69</td>
			<td>*Alternative distributors possible</td>
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		<tr height="20">
			<td height="20" style="height:20px;">5&#215;PrimeSTAR buffer</td>
			<td>Takara</td>
			<td>9158A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alkaline phosphatase</td>
			<td>ThermoFisher FastAP</td>
			<td>EF0654</td>
			<td>*Alternative distributors possible</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">COX forward primer</td>
			<td>Genscript</td>
			<td>ATGTCAACAAATCATTTAGACATTG</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">COX reverse primer</td>
			<td>Genscript</td>
			<td>ACTTGACCAAAAACATAAGACATG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cutsmart Buffer</td>
			<td>NEB</td>
			<td>B7204S</td>
			<td>*Alternative distributors possible</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">dNTP mix</td>
			<td>Invitrogen</td>
			<td>18427088</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">MgUGD forward primer</td>
			<td>Genscript</td>
			<td>ACATATGACCCTGTCCAAGATCTGTTGT</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">MgUGD reverse primer</td>
			<td>Genscript</td>
			<td>ACTCGAGACTCTGTGAGGCGGTGGAG</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">MgUXS forward primer</td>
			<td>Genscript</td>
			<td>CCATATGGCAGAATCCTCACAATCAC</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">MgUXS reverse primer</td>
			<td>Genscript</td>
			<td>ACTCGAGCACATTTTTGAATTTGCAGACGT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ND forward primer</td>
			<td>Genscript</td>
			<td>ATGAGATGGCAATTATTTTTTAAT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ND reverse primer</td>
			<td>Genscript</td>
			<td>ATGTATTTTGGAAAAATCTCCAC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PCR Cleanup Kit</td>
			<td>AxyGen</td>
			<td>AP-PCR-250</td>
			<td>*Alternative distributors possible</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pET-30a(+) vector</td>
			<td>Merck Millipore</td>
			<td>69909</td>
			<td></td>
		</tr>
	</tbody>
</table>

a converging strategy for the generation of a virtually sequenced cdna library from unreferenced pacific oysters

以前在开发新型细胞系或基因组测序项目等关键实验中使用参考物种的生物材料,往往难以为进一步研究或第三方提供。样品的消耗性质。虽然现在广泛分布于亚洲、澳大利亚和北美的太平洋沿岸,但个别太平洋牡蛎标本在基因上相当多样化,因此不能直接作为基因库的起始材料。在本文中,我们演示了使用从区域海鲜市场获得的未引用的太平洋牡蛎标本来生成 cDNA 库。然后将这些库与公开提供的牡蛎基因组进行比较,并使用线粒体参考基因细胞色素C氧化酶亚单位I(COX1)和NADH脱氢酶(ND)选择最接近的相关库。生成的 cDNA 库的适用性也通过克隆和表达两个基因来证明编码酶 UDP-葡曲酸脱氢酶 (UGD) 和 UDP-xylose 合成酶 (UXS),它们负责 UDP-xylose 的生物合成。UDP-葡萄糖。

图1显示了从太平洋牡蛎个体中提取的收敛cDNA库所述制备方法的原理图概述。图2显示了与参考材料的COX1和ND基因序列高度背离的远亲牡蛎标本的COX1和ND基因序列。图3显示了与参考材料的COX1和ND基因序列有低差异的密切相关的牡蛎标本的COX1和ND基因序列。图4显示了cDNA库成功克隆工业相关基因MgUGD和MgUXS。
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Watch this Scientific Journal Video about A Converging Strategy for the Generation of a Virtually Sequenced cDNA Library from Unreferenced Pacific Oysters at JoVE.com

A Converging Strategy for the Generation of a Virtually Sequenced cDNA Library from Unreferenced Pacific Oysters

我们描述了如何使用未引用的太平洋牡蛎标本的RNA样本的策略,并通过与公开提供的基因组数据进行比较来评估遗传物质,以生成几乎序列化的cDNA库。

从未引用的太平洋牡蛎生成虚拟序列的 cDNA 库的聚合策略

The access to biological material of reference species, which were used previously in key experiments such as in the development of novel cell lines or ...

我们要用这种方法解决的关键问题是，我们如何使用当地食品市场采购的材料中的RNA样本来克隆与生物技术相关的基因？在这里，我们使用太平洋牡蛎作为案例研究，以演示这种方法和潜在的进一步应用。这项技术的主要优点是可以绕过牡蛎样品的传统基因组或cDNA测序。相反，将细胞色素c氧化酶亚基一和NADH脱氢酶的序列与太平洋牡蛎公开提供的参考序列进行比较，以选择最密切相关的标本。从该样本中编写的cDNA可以直接用于克隆与生物技术相关的基因，这些基因可以使用公共序列信息进行选择。展示这个程序的将是南京农业大学糖学和糖生物工程研究中心的两名研究生李永梅和李玉泉。要准备牡蛎组织样本，请使用灭菌手术刀从每个牡蛎标本的近似几何中心切出约100毫克的均匀软组织。将样品转移到装满50毫升液氮的砂浆中。将闪冻的牡蛎组织研磨成细粉。将每个标本的冷冻组织中的75毫克称重到无菌的1.5毫升离心管中，并混合一毫升硫化酸酯-酚提取试剂。此过程最关键的步骤是 RNA 提取步骤。为了最大限度地减少RNA降解，必须缩短收获牡蛎组织和RNA提取之间的时间。将样品在14，000倍g和4摄氏度下离心15分钟。将上清液转移到新的1.5毫升离心管中，加入200微升氯仿，在涡流混合器上彻底混合10至15秒，直到混合物变成乳白色。接下来，像之前一样，离心机15分钟。使用 200 微升移液器，小心地将上水层（而不会干扰相间）转移到新的 1.5 毫升离心管中。将500微升异丙醇加入离心管，轻轻反转以混合样品。然后将样品留在冰上20分钟。在14，000倍的g和4摄氏度下离心8分钟，并取出上光。将颗粒重新浓缩在一毫升75%乙醇中，在14，000倍的g和4摄氏度下离心5分钟。取出所有上继剂，然后用乙醇重复洗涤。在室温下干燥颗粒6分钟。然后，将干燥的RNA颗粒溶解在25微升DEPC处理的水中，并保持管在冰上。在24小时内使用RNA样本。要生成 cDNA 库，首先根据手稿添加溶液，为 300 微升 PCR 管中的每个 RNA 样本制备反应混合物。将提取的RNA样品的微升加入管中。在42摄氏度下在PCR热循环器中孵育混合物60分钟，然后将温度升高至95摄氏度5分钟。在零下 20 摄氏度下存储生成的 cDNA 库长达 12 个月。首先，在含有PCR混合物的管子中加入一个微升的cDNA库，根据手稿准备。将 PCR 管放入 PCR 热循环器中，然后执行 PCR 放大，初始变性步骤和 35 个 PCR 反应周期，包括退火步、伸长步和变性步骤（根据手稿）。循环后，执行一个最终的伸长步骤五分钟。使用 PCR 产品的五微升通过红糖凝胶电泳验证质量。分别在759或748个碱基对上将扩增的COX1或ND基因作为单波段观察。为了净化PCR产品的其余部分，在每个样品中加入100微升含有高浓度的超热带盐的DNA结合缓冲液。漩涡短暂地混合内容。将净化柱放入两毫升离心管中。将反应混合物移入柱中。将装有装置的柱管以14，000倍g将装有的柱子放在离心机中，在室温下放置一分钟。在丢弃两毫升离心管中的滤液后，用 700 微升和 400 微升的洗涤溶液 W2 以 14，000 倍 g 离心洗涤两次。接下来，在金属块加热器中加热一毫升去电水至65摄氏度。将柱转移到新的1.5毫升离心管中，将25微升的预热去电化水移液器转移到白柱膜的中心。让膜在室温下浸泡一分钟。然后，在室温下将带柱的管以14，000倍g离心一分钟。丢弃柱，在零下 20 摄氏度下储存纯化 PCR 产品长达 12 个月。可选地，COX1 或 ND 反向底转也可用于双向测序。对于桑格测序，请使用相关的 COX1 或 ND 前底。检索测序结果后，使用NCBI核苷酸 BLAST 在线工具将序列与太平洋牡蛎参考菌株的基因组序列进行比较。使用MgUGD和MgUXS基因的分别前向和反向底因，像以前一样，通过PCR进行放大和纯化。使用消化缓冲液孵育纯化PCR产品后，在1.5毫升离心管中溶解500毫微克的pET-30a，并加入去电化水以补充16微升，来准备预消化的pET-30a载体。然后，在管中加入两个10倍浓缩消化缓冲液和一个微升，每个20个单位限制内接性Nde1和Xho1。在37摄氏度下孵育混合物3小时。之后，加入一单位碱性磷酸酶的微升，在37摄氏度下再孵育一小时。然后，将管子放在预热金属块加热器中，在 75 摄氏度下放置 10 分钟，以灭活碱性磷酸酶。培养带有MgUGD和MgUXS基因的大肠杆菌BL21细胞后，在两升摇瓶中将培养成400毫升LB介质，并在37摄氏度的温度下以200转/小时将其放在摇摇器上。三小时后，检查波长为 600 纳米的光度计上的光学密度，并确保其吸收量达到约 0.5。然后将摇床温度降至 20 摄氏度。加入400微升的一摩尔IPTG，诱导重组蛋白的表达3小时。收获细胞后，在4摄氏度下通过声波干扰细胞20分钟。在14000倍的g和4摄氏度下离心20分钟，并在新管中收集上一代，用于活动测试。活动测定后，在MgUXS和MgUGD混合物中加入20微升甲醇和40微升氯仿，从而抑制反应。涡流样品混合物，并在14000倍g下离心6分钟和4摄氏度。收集新管中每个管的上水层，用于 MALDI-TOF 质谱法。在本次实验中，将高度发散标本的COX1和ND基因序列的序列排列与参考太平洋牡蛎菌株进行比较。红色箭头显示参考序列与获得的cDNA样本序列之间的核苷酸差异。密切相关的牡蛎标本的COX1和ND基因序列显示，与参考太平洋牡蛎菌株的背离较低。MALDI-TOF质谱表明cDNA库成功应用，克隆了与工业相关的基因MgUGD和MgUXS。凝胶图像还识别了由连续表达和纯化的MgUGD和MgUXS的酶作用产生的UDP-葡萄糖酸和UDP-xylose。此方法不需要参考序列与获得的标记序列的完美匹配，应给予研究人员处理未引用牡蛎的信心。原则上，此方法可应用于任何未引用的生物样本，这些样本中来自密切相关的参考物种的序列是公开提供的。这种方法允许非专家研究人员轻松获取具有潜在生物学重要性的遗传材料，并为更多的科学家使用易于获取但未完全鉴定的生物样本铺平了道路。

Research

JoVE Journal

Bioengineering

Synthetic Spider Silk Production on a Laboratory Scale

实验室规模的人工合成蜘蛛丝生产

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation ...

科研

生物工程

Designing a Bio-responsive Robot from DNA Origami

DNA折纸设计生物反应灵敏的机器人

Nucleic acids are astonishingly versatile. In addition to their natural role as storage medium for biological information1, they can be utilized in ...

An Improved Method for the Preparation of Type I Collagen From Skin

对于I型胶原蛋白的制备皮肤方法的改进

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

脂质双层囊泡代使用微流控喷射

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

三维全层皮肤的生成等效和自动伤人

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry

从藻类水热液化使用二维气相色谱时间飞行质谱法水提取部分的定性鉴定

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in ...

A Microcontroller Operated Device for the Generation of Liquid Extracts from Conventional Cigarette Smoke and Electronic Cigarette Aerosol

一种单片机操作装置, 用于生成传统卷烟烟雾和电子烟气溶胶中的液体萃取物

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among ...

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

人类多能干细胞和免疫损伤的成熟小脑有机体可伸缩生成

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...

Production of Membrane-Filtered Phase-Shift Decafluorobutane Nanodroplets from Preformed Microbubbles

从预成型微气泡生产膜过滤相移十氟丁烷纳米滴

There are many methods that can be used for the production of vaporizable phase-shift droplets for imaging and therapy. Each method utilizes different ...

Generation of a Simplified Three-Dimensional Skin-on-a-chip Model in a Micromachined Microfluidic Platform

在微加工微流体平台中生成简化的三维芯片皮肤模型

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of ...