CN113186256A - 生物样本的空间区别、多重核酸分析 - Google Patents
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Abstract
本申请涉及生物样本的空间区别、多重核酸分析，包括一种空间标注生物样本的核酸的方法，其包括以下步骤：(a)提供固体载体，其包含随机定位在所述固体载体上的不同核酸探针，其中所述不同核酸探针各自包括与所述固体载体上的其它随机定位探针的条形码序列不同的条形码序列；(b)在所述固体载体上进行核酸检测反应以定位所述固体载体上的所述条形码序列；(c)使生物样本与具有所述随机定位探针的所述固体载体接触；(d)使所述随机定位探针与来自所述生物样本的部分的靶核酸杂交；和(e)修饰与所述靶核酸杂交的所述随机定位探针，由此产生包括所述条形码序列和靶特异性修饰的经修饰探针，由此空间标注所述生物样本的所述核酸。
Description
本申请是申请日为2016年4月4日、申请号为201680020206.8、发明名称为“生物样本的空间区别、多重核酸分析”的发明专利申请的分案申请。
技术领域
本发明涉及生物样本的空间区别、多重核酸分析，包括以空间方式为生物样本的核酸加标签(tag)的方法。
背景技术
每四名男性将有一个死于癌症。来自美国癌症学会(American Cancer Society)的进一步统计预测，每五名女性将有一个将遭受相同命运。多种癌症可获得治疗。然而，成功大多依赖于早期检测。
现在癌症被称为基因组疾病。许多肿瘤学家和癌症研究者希望，基因组分析工具的进步将提供早期检测和治疗路径。然而，这些工具更多用在研究实验室中，还未成熟到可容易地供大多数肿瘤学家使用的程度。需要改良。
据称，在诊断时，所有癌症患者都是嵌合体。其为嵌合体是因为其具有至少两个不同的基因组：其生来就具有的基因组，以及其由于癌症不情愿地获得的基因组。此外，随着肿瘤生长，癌细胞的不同群体变得明显。导致在肿瘤内甚至更复杂的嵌合体。这种癌细胞异质性通常导致对癌症疗法反应不同的细胞子群。最终结果通常是一个细胞子群的初始阳性反应，导致观察到患者肿瘤缩小，但之后肿瘤组织会再生长，并且在一些情形中会转移。尽管早期检测到癌症，但无法鉴别对治疗具有抗性的细胞子群可导致损失治疗侵袭性癌症所需的时间。这对于患者在情感上和身体上都会产生不利后果。
需要可区别肿瘤中的癌细胞子群的基因组工具。本公开满足这种需要且还提供其它优点。
发明内容
本公开提供以空间方式为生物样本的核酸加标签(tag)的方法。所述方法可包括以下步骤：(a)提供包含随机定位在固体载体上的多个不同核酸探针的固体载体，其中所述不同核酸探针各自包括不同于固体载体上的其它随机定位探针的条形码序列的条形码序列；(b)在固体载体上进行核酸检测反应以定位固体载体上的条形码序列；(c)使生物样本与具有随机定位探针的固体载体接触；(d)使随机定位探针与来自生物样本中靠近随机定位探针的部分的靶核酸杂交；和(e)修饰与靶核酸杂交的随机定位探针，由此产生包括条形码序列和靶特异性修饰的经修饰探针，由此以空间方式为生物样本的核酸加标签。
本公开进一步提供以空间方式为生物样本的核酸加标签的方法，所述方法包括以下步骤：(a)将不同核酸探针附接至固体载体以产生固体载体上的随机定位探针，其中所述不同核酸探针各自包括条形码序列，且其中随机定位探针各自包括与固体载体上的其它随机定位探针不同的条形码序列；(b)在固体载体上进行核酸检测反应以测定固体载体上的随机定位探针的条形码序列；(c)使生物样本与具有随机定位探针的固体载体接触；(d)使随机定位探针与来自生物样本中靠近随机定位探针的部分的靶核酸杂交；和(e)延伸随机定位探针以产生包括条形码序列和来自靶核酸的序列的延伸探针，由此以空间方式为生物样本的核酸加标签。
还提供以空间方式为生物样本的核酸加标签的方法，所述方法包括以下步骤：(a)提供多个附接至固体载体的核酸引物，其中所述多个中的核酸引物包括为所述多个中的核酸引物共有的通用引物序列；(b)使核酸探针群体与所述多个核酸引物结合，其中核酸探针包括与通用引物序列杂交的通用引物结合序列、靶捕获序列和与群体中其它核酸探针的条形码序列不同的条形码序列，由此将不同核酸探针附接在固体载体上的随机定位位置；(c)通过延伸核酸引物来扩增不同核酸探针，由此在固体载体上的随机定位位置产生具有条形码序列和靶捕获序列的拷贝的核酸簇；(d)进行测序反应以测定固体载体上的随机定位位置的条形码序列；(e)使生物样本与固体载体上的核酸簇接触；(f)使所述簇的靶捕获序列与来自生物样本中靠近所述簇的部分的靶核酸杂交；和(g)延伸靶捕获序列以产生包括来自靶核酸的序列和条形码序列的拷贝的延伸探针，由此为生物样本的核酸加标签。
本公开进一步提供以空间方式为生物样本的核酸加标签的方法，所述方法包括以下步骤：(a)在固体载体上提供珠粒阵列，其中将不同核酸探针附接至阵列中的不同珠粒，其中不同核酸探针各自包括条形码序列，其中各珠粒包括与固体载体上的其它珠粒不同的条形码序列，且其中不同核酸探针各自包括靶捕获序列；(b)在固体载体上进行解码器探针杂交反应以测定固体载体上的随机定位探针的条形码序列；(c)使生物样本与珠粒阵列接触；(d)使不同核酸探针与来自生物样本中靠近珠粒的部分的靶核酸杂交；和(e)延伸不同核酸探针以产生包括来自靶核酸的序列和条形码序列的延伸探针，由此为生物样本的核酸加标签。
附图说明
图1显示可用于在伊路米那(Illumina)流动槽上生成有条形码寡dT探针，产生具有mRNA序列的延伸有条形码探针，并从流动槽释放延伸探针的步骤和试剂的图解代表。
图2显示指示在探针的桥式扩增和用BspH1进行限制性酶消化以移除一个用于桥式扩增的引物结合位点之后，探针上寡dT捕获序列的可用性的数据。
图3显示实例1中所述且图2中所示流动槽的测序度量法。
图4显示实例1中所述且图2中所示的流动槽的21个瓦片(tile)中测定的独特条形码数目。
图5显示在图案化流动槽上捕获的细胞的图像(图A)和细胞计数数据(图B)。
图6显示在不同条件下保持粘附至流动槽的细胞。
图7显示用于产生附接至凝胶的探针的步骤和试剂的图解代表(图A)，用于使用凝胶附接探针捕获靶核酸并对探针进行荧光标记(label)的步骤和试剂的图解代表(图B)，以及在用探针捕获且从凝胶移除组织后，由荧光标记的靶核酸产生的图像。
图8显示用于使用BeadArrayTM附接探针捕获靶核酸并对探针进行荧光标记的步骤和试剂的图解代表(图A)，以及在用探针捕获并从BeadArrayTM移除组织后，由荧光标记的靶核酸产生的图像。
具体实施方式
本公开提供用于在进行生物样本的多重核酸分析时保存空间信息的组合物、设备和方法。多种工具可用于多重核酸分析，包括例如核酸微阵列和所谓的“下一代”测序平台。所述工具容许平行检测极大且复杂的核酸集合，包括例如代表生物体的全部或几乎全部遗传物质的DNA集合(即‘基因组’)、代表生物体的所表达基因的所有或几乎所有补体的RNA(或cDNA)集合(即‘转录组’)，并且在一些情形中集合可包括来自若干个不同生物体的若干个基因组和/或转录组(例如来自群落或生态系的代谢物组或生物群系)。尽管这些工具提供大量关于所评估生物样本中存在何种核酸序列的信息，其固有地无法区别任何特定核酸驻留于生物样本中的何处。实际上，应用于多重核酸分析工具的大多数样品是源自生物样本中多种不同细胞的混合物的匀浆物。因此，空间信息丢失并且从这些工具获得的结果构成样本的平均转录组或平均基因组，个别细胞之间的重要差异被丢失。
在特定实施例中，本公开向现有多重核酸分析工具提供新的有用的修改以容许保存从其获得核酸的生物样本的空间信息。例如，通常用于多重合成测序(SBS)技术的固体载体可经修改用于捕获来自生物样本的核酸和以空间方式为其加标签。在替代性实例中，珠粒阵列(例如用于基因分型或基因表达分析的那些)可用于捕获来自生物样本的核酸和以空间方式为其加标签。如下文实例中所述，用于由伊路米那(圣地亚哥，加利福尼亚州)商业化的SBS或BeadArrayTM平台的固体载体可经修改用于以空间方式加标签。然而，应理解，多种固体载体中的任一个可根据本文中的教示制造并使用。可将以空间方式加标签的核酸从固体载体移除，汇集在一起并附接至第二固体载体用于在多种多重核酸分析***中的任一种中检测，包括例如本文所述的测序平台或微阵列平台。
通过本文中的方法、组合物或设备提供的空间信息可包括例如一或多种细胞在组织(或其它样本)中的定位，所述细胞在一或多个基因座具有特定等位基因(例如基因分型)，在基因组中具有特定结构变异(例如融合、***、缺失、重排等)，具有特定后生印记(例如甲基化)，表达特定基因，表达基因的特定等位基因，表达基因的特定剪接变体或诸如此类。除了根据核酸在生物样本中的空间定位来鉴别所述核酸以外，本公开的方法、组合物或设备可用于根据空间定位定量一或多种核酸。例如，组织(或其它样本)中的一或多种细胞的空间信息可包括基因组中特定等位基因或染色体区域的量(例如倍数性)；遗传基因座中后生修饰(例如甲基化)的量；特定基因、等位基因或剪接变体的表达水平；或诸如此类。所述量可为绝对量或根据业内针对混合或未以空间方式加标签样品获得的类似测量的相对量。
本文所述方法可用于定位检测生物样本中的核酸。在一些实施例中，可使用一方法来鉴别或表征生物样本的所有转录组或基因组。或者，可使用一方法来鉴别或表征仅一部分样本的转录组或基因组。本文方法中评估的转录物或基因的亚组可关于特定疾病或病状。
本文所述方法可用于定位或空间检测生物样本中的核酸(DNA或RNA)。因此，一或多个RNA或DNA分子可相对于其天然位置或定位定位于细胞或组织或其它生物样本内。例如，可将一或多种核酸定位至细胞或相邻细胞群或一类细胞，或定位至组织样品内的区域的特定区中。个别RNA或DNA分子的天然定位或位置可使用本公开的方法、设备或组合物来测定。
除非另外指明，否则本文所用术语应理解为取其在相关技术中的普通含义。本文所用若干术语和其含义陈述于下文中。
如本文所用术语“扩增子”在关于核酸使用时意指复制核酸的产物，其中所述产物具有与所述核酸的核苷酸序列的至少一部分相同或互补的核苷酸序列。扩增子可通过多种使用核酸或其扩增子作为模板的扩增方法中的任一种来产生，包括例如聚合酶延伸、聚合酶链式反应(PCR)、滚环式扩增(RCA)、多重置换扩增(MDA)、连接延伸或连接链式反应。扩增子可为具有单拷贝的特定核苷酸序列(例如PCR产物)或多拷贝核苷酸序列(例如RCA的多联体(concatameric)产物)的核酸分子。靶核酸的第一扩增子通常为互补拷贝。后续扩增子是在生成第一扩增子后从靶核酸或从第一扩增子产生的拷贝。后续扩增子可具有与靶核酸基本上互补或与靶核酸基本上一致的序列。
如本文所用术语“阵列”是指可根据相对定位彼此区分的特征或位点群体。在阵列的不同位点的不同分子可根据阵列中所述位点的定位彼此区分。阵列的个别位点可包括特定类型的一或多个分子。例如，位点可包括具有特定序列的单一靶核酸分子，或位点可包括若干个具有相同序列(和/或其互补序列)的核酸分子。阵列的位点可为定位于相同衬底上的不同特征。实例性特征包括但不限于衬底中的孔、衬底中或衬底上的珠粒(或其它颗粒)、衬底的突出物、衬底上的***物或衬底中的沟道。阵列的位点可为各自带有不同分子的分离衬底。附接至分离衬底的不同分子可根据衬底在与衬底结合的表面上的定位或根据衬底在液体或凝胶中的定位来鉴别。其中分离衬底定位于表面上的实例性阵列包括但不限于孔中具有珠粒的阵列。
如本文所用术语“附接”是指两个物体彼此接合、紧固、粘附、连接或结合的状态。例如，分析物(例如核酸)可通过共价键或非共价键附接至材料(例如凝胶或固体载体)。共价键的特征为原子之间共享电子对。非共价键是不涉及电子对共享的化学键并且可包括例如氢键、离子键、范德华力(van der Waals force)、亲水相互作用和疏水相互作用。
如本文所用术语“条形码序列”打算意指核酸中的一系列核苷酸，其可用于鉴别核酸、核酸的特征或已对核酸实施的操作。条形码序列可为天然序列或在从其获得有条形码核酸的生物体中并非天然存在的序列。条形码序列可为群体中单一核酸种类所独有，或条形码序列可为群体中若干种不同核酸种类所共享。例如，群体中的每一核酸探针可包括来自群体中所有其它核酸探针的不同条形码序列。或者，群体中的每一核酸探针可包括来自群体中的一些或大多数其它核酸探针的不同条形码序列。例如，即使具有共有条形码的探针在沿其长度的其它序列区域彼此不同，群体中的每一探针可具有存在于群体中的若干个不同探针中的条形码。在特定实施例中，用于生物样本的一或多个条形码序列不存在于所述生物样本的基因组、转录组或其它核酸中。例如，条形码序列可与特定生物样本中的核酸序列具有少于80％、70％、60％、50％或40％序列一致性。
如本文所用术语“生物样本”打算意指一或多种细胞、组织、生物体或其部分。生物样本可从多种生物体中的任一种获得。实例性生物体包括但不限于哺乳动物，例如啮齿类动物、小鼠、大鼠、兔、荷兰猪、有蹄类动物、马、绵羊、猪、山羊、牛、猫、狗、灵长类动物(即人类或非人灵长类动物)；植物，例如拟南芥(Arabidopsis thaliana)、玉米、高粱、燕麦、小麦、稻、芸苔或大豆；藻类，例如莱茵衣藻(Chlamydomonas reinhardtii)；线虫，例如秀丽隐杆线虫(Caenorhabditis elegans)；昆虫，例如黑腹果蝇(Drosophila melanogaster)、蚊子、果蝇、蜜蜂或蜘蛛；鱼，例如斑马鱼；爬行动物；两栖动物，例如青蛙或光滑爪蟾(Xenopuslaevis)；盘基网柄菌(Dictyostelium discoideum)；真菌，例如卡氏肺囊虫(Pneumocystiscarinii)、红鳍东方鲀(Takifugu rubripes)；酵母，酿酒酵母(Saccharamoycescerevisiae)或粟酒裂殖酵母(Schizosaccharomyces pombe)；或恶性疟原虫(Plasmodiumfalciparum)。靶核酸还可源自原核生物，例如细菌、大肠杆菌(Escherichia coli)、肺炎葡萄球菌(Staphylococci pneumoniae)或肺炎支原体(Mycoplasma pneumoniae)；古细菌；病毒，例如C型肝炎病毒或人免疫缺陷病毒；或类病毒。样本可源自上述生物体的同质培养或群体或另一选择为来自例如群落或生态系中的若干种不同生物体的集合。
如本文所用术语“裂解位点”打算意指核酸分子中易发生键断裂的定位。所述定位可对导致键断裂的特定化学、酶或物理过程具有特异性。例如，所述定位可为无碱基核苷酸或具有易于移除以产生无碱基位点的碱基的核苷酸。易于移除的核苷酸的实例包括如下文中更详细地陈述的尿嘧啶和8-氧代-鸟嘌呤。所述定位还可在限制性内切核酸酶(例如切口酶)的识别序列处或所述识别序列附近。
如本文所用术语“簇”在关于核酸使用时是指附接至固体载体以形成特征或位点的核酸群体。所述核酸通常为单一种类的成员，由此形成单克隆簇。核酸的“单克隆群体”是关于特定核苷酸序列同源的群体。簇无需为单克隆。倒不如说，对于一些应用，簇可主要以来自第一核酸的扩增子群体化，并且还可具有低水平的来自第二核酸的污染扩增子。例如，当要在检测应用中使用簇阵列时，污染的可接受水平可为不以不可接受的方式影响检测技术的信噪比或分辨率的水平。因此，明显克隆性通常将与通过本文所述方法制造的阵列的特定使用或应用相关。在个别簇可接受的实例性污染水平包括但不限于至多0.1％、0.5％、1％、5％、10％、5 25％或35％污染扩增子。簇中的核酸通常例如通过其5'端共价附接至固体载体，但在一些情形中其它附接方式是可能的。簇中的核酸可为单链或双链。在一些但并非所有实施例中，簇是通过称为桥式扩增的固相扩增方法来制造。簇和其产生方法的实例性配置陈述于例如以下文献中：美国专利第5,641,658号、美国专利公开第2002/0055100号、美国专利第7,115,400号、美国专利公开第2004/0096853号、美国专利公开第2004/0002090号、美国专利公开第2007/0128624号和美国专利公开第2008/0009420号，其各自通过引用并入本文中。
如本文所用术语“不同的”在关于核酸使用时意指，核酸具有彼此不相同的核苷酸序列。两个或更多个核酸可具有沿其整个长度不同的核苷酸序列。或者，两个或更多个核酸可具有沿其长度的大部分不同的核苷酸序列。例如，两个或更多个核酸可具有对于两个或更多个分子不同的靶核苷酸序列部分，同时还具有在两个或更多个分子上相同的通用序列部分。两个珠粒可因附接至不同核酸而彼此不同。
如本文所用术语“每一”在关于项目的集合使用时打算标识集合中的个别项目，但不一定是指集合中的每一项目。如果明确公开或上下文明确指示其它含义，则存在例外。
如本文所用术语“延伸”在关于核酸使用时打算意指将至少一个核苷酸或寡核苷酸添加至核酸。在特定实施例中，可例如通过聚合酶催化(例如DNA聚合酶、RNA聚合酶或逆转录酶)将一或多个核苷酸添加至核酸3'端。可使用化学或酶促方法将一或多个核苷酸添加至核酸的3’或5'端。可例如通过化学或酶促(例如连接酶催化)方法将一或多个寡核苷酸添加至核酸的3’或5'端。核酸可以模板引导方式延伸，借此使延伸产物与杂交至所延伸核酸的模板核酸互补。
如本文所用术语“特征”意指特定种类的分子在阵列中的定位。特征可仅含单一分子或其可含有相同种类的若干分子的群体。阵列的特征通常是离散的。离散特征可邻接或其可在彼此之间具有间隔。特征的大小和/或特征之间的间隔可变，使得阵列可具有高密度、中等密度或较低密度。高密度阵列的特征为位点相离小于约15μm。中等密度阵列的位点相离约15至30μm，而低密度阵列的位点相离大于30μm。本文可用阵列的位点可例如相离小于100μm、50μm、10μm、5μm、1μm或0.5μm。本公开的设备或方法可用于以足以区别具有上文密度或密度范围的位点的分辨率检测阵列。
如本文所用术语“流体混合物”打算意指同时存在于溶液中的两种或更多种不同项目。通常，所述两种或更多种项目可在溶液中自由扩散。所述两种或更多种项目可为不同类型的项目(例如核酸和蛋白质，其为不同类型的分子)，或其可为相同类型项目的不同种类(例如两个具有不同序列的核酸分子)。可在流体混合物中的实例性项目包括但不限于分子、细胞或珠粒。
如本文所用术语“流动槽”打算意指容器，其具有可进行反应的腔室、用于将试剂递送到腔室的入口和用于从腔室移除试剂的出口。在一些实施例中，腔室经配置用于检测在所述腔室中发生的反应。例如，腔室可包括一或多个透明表面，容许对腔室中的生物样本、光学标记分子或诸如此类进行光学检测。实例性流动槽包括但不限于用于核酸测序设备中的那些，例如用于由伊路米那公司(圣地亚哥，加利福尼亚州)商业化的Genome
或
平台的流动槽；或用于由生命技术(LifeTechnologies，卡尔斯巴德，加利福尼亚州)商业化的SOLiDTM或艾恩托伦特(Ion Torrent)TM测序平台的流动槽。实例性流动槽以及其制造和使用方法还描述于例如以下文献中：WO2014/142841 A1；美国专利申请公开案第2010/0111768 A1号和美国专利第8,951,781号，其各自通过引用并入本文中。
如本文所用术语“凝胶”打算意指可渗透液体和气体的半刚性材料。通常，凝胶材料可在吸收液体时膨胀并且可在通过干燥移除液体时收缩。实例性凝胶包括但不限于具有胶体结构的那些，例如琼脂糖；聚合物网状结构，例如明胶；或交联聚合物结构，例如聚丙烯酰胺、SFA(参见例如美国专利申请公开第2011/0059865 A1号，其是通过引用并入本文中)或PAZAM(参见例如美国专利申请公开第2014/0079923 A1号，其是通过引用并入本文中)。特别有用的凝胶材料将与其所驻留的孔或其它凹陷特征的形状一致。
如本文所用术语“核酸”和“核苷酸”打算与其在本领域中的使用一致并且要包括天然种类或其功能类似物。核酸的特别有用的功能类似物能以序列特异性方式与核酸杂交或能用作复制特定核苷酸序列的模板。天然核酸一般具有含有磷酸二酯键的主链。类似物结构可具有替代主链链接，包括业内已知多种链接中的任一种。天然核酸一般具有脱氧核糖(例如在脱氧核糖核酸(DNA)中所发现)或核糖(例如在核糖核酸(RNA)中所发现)。核酸可含有具有业内已知的这些糖部分的多种类似物中的任一种的核苷酸。核酸可包括天然或非天然核苷酸。就此来说，天然脱氧核糖核酸可具有一或多个选自由腺嘌呤、胸腺嘧啶、胞嘧啶或鸟嘌呤组成的群组的碱基，并且核糖核酸可具有一或多个选自由尿嘧啶、腺嘌呤、胞嘧啶或鸟嘌呤组成的群组的碱基。可包括在核酸或核苷酸中的有用的非天然碱基为业内已知。术语“探针”或“靶”在关于核酸或核酸序列使用时打算在本文所述方法或组合物的情况下作为核酸或序列的语义标识符，并且不一定限制在另有明确指示者以外的核酸或序列的结构或功能。术语“探针”和“靶”可类似地适用于其它分析物，例如蛋白质、小分子、细胞或诸如此类。
如本文所用术语“节距”在关于阵列特征使用时打算指相邻特征的中心-中心间距。可根据平均节距来表征特征的图案。图案可为有序的，使得平均节距附近的变异系数较小，或图案可为随机的，在这种情形中变异系数可相对较大。在任一情形中，平均节距可为例如至少约10nm、0.1μm、0.5μm、1μm、5μm、10μm、100μm或更大。或者或另外，平均节距可为例如至多约100μm、10μm、5μm、1μm、0.5μm、0.1μm或更小。当然，特定特征图案的平均节距可介于选自上文范围的较低值之一与较高值之一之间。
如本文所用术语“多T或多A”在关于核酸序列使用时打算分别意指一系列两个或更多个胸腺嘧啶(T)或腺嘌呤(A)碱基。多T或多A可分别包括至少约2、5、8、10、12、15、18、20或更多个T或A碱基。或者或另外，多T或多A可分别包括至多约30、20、18、15、12、10、8、5或2个T或A碱基。
如本文所用术语“随机”可用于指空间排列或在表面上的定位的组成。例如，本文所述阵列有至少两种类型的顺序，第一种类型是关于特征(也称为“位点”)的间隔和相对定位，并且第二种类型是关于存在于特定特征处的特定种类的分子的属性或预先确定的了解。因此，阵列特征可随机间隔，使得最近的相邻特征在彼此之间具有可变间隔。或者，特征之间的间隔可为有序的，例如形成规则图案，例如直线网格或六边形网格。在另一方面中，阵列特征可关于占据每一特征的分析物的种类(例如特定序列的核酸)的属性或预先确定的了解是随机的，与间隔产生随机图案或是有序图案无关。本文所述阵列可在一个方面是有序的且在另一方面是随机的。例如，在本文所述的一些实施例中，在其中核酸附接于位点的条件下使表面与核酸群体接触，所述位点关于其相对定位是有序的，但关于在任一特定位点存在的核酸种类的序列的了解‘随机定位’。在提到在表面上的定位“随机分布”的核酸时，打算指不了解或没有预先确定关于在所述定位将捕获何种核酸(不管所述定位是否按有序图案排列)。
如本文所用术语“固体载体”是指不溶于水性液体的刚性衬底。衬底可无孔或多孔。衬底可任选地能吸收液体(例如由于多孔性)，但通常将具有足够刚性使得衬底在吸收液体时不会显著膨胀并且在通过干燥移除液体时不会显著收缩。无孔固体载体通常不可渗透液体或气体。实例性固体载体包括但不限于玻璃和经改性或功能化玻璃、塑料(包括丙烯酸酯类、聚苯乙烯以及苯乙烯与其它材料的共聚物、聚丙烯、聚乙烯、聚丁烯、聚氨基甲酸酯、TeflonTM、环烯烃、聚酰亚胺等)、尼龙、陶瓷、树脂、Zeonor、硅石或基于硅石的材料(包括硅和改性硅)、碳、金属、无机玻璃、光学纤维束和聚合物。对于一些实施例特别有用的固体载***于流动槽设备内。实例性流动槽更详细地陈述于本文中。
如本文所用术语“以空间方式加标签”打算意指具有指示定位的序列的核酸。通常，核酸是具有在一或多个将用于核酸的生物样本中未发现的序列的合成分子。然而，在一些实施例中，核酸分子可为天然来源或核酸序列可天然存在于例如用于核酸的生物样本中。空间标签指示的定位可为在生物样本中或生物样本上、固体载体中或固体载体上或其组合的定位。条形码序列可用作空间标签。
如本文所用术语“组织”打算意指细胞和任选地细胞间物质的聚集体。通常，组织中的细胞在溶液中不能自由漂浮，而是彼此附接形成多细胞结构。实例性组织类型包括肌肉、神经、表皮和***。
如本文所用术语“通用序列”是指为两个或更多个核酸分子所共有的一系列核苷酸，即使所述分子还具有彼此不同的序列区域。存在于分子集合的不同成员中的通用序列可容许使用与通用序列互补的通用捕获核酸群体来捕获多种不同核酸。类似地，存在于分子集合的不同成员中的通用序列可容许使用与通用序列互补的通用引物群体复制或扩增多种不同核酸。因此，通用捕获核酸或通用引物包括可与通用序列特异性杂交的序列。靶核酸分子可经修饰以附接例如不同靶序列的一个或两个末端的通用接头。
下文所述和权利要求书中所列举的实施例可根据上文定义来理解。
本公开提供以空间方式为生物样本的核酸加标签的方法。所述方法可包括以下步骤：(a)将不同核酸探针附接至固体载体以产生固体载体上的随机定位探针，其中所述不同核酸探针各自包括条形码序列，且其中随机定位探针各自包括与固体载体上的其它随机定位探针不同的条形码序列；(b)在固体载体上进行核酸检测反应以测定固体载体上的随机定位探针的条形码序列；(c)使生物样本与具有随机定位探针的固体载体接触；(d)使随机定位探针与来自生物样本中靠近随机定位探针的部分的靶核酸杂交；和(e)延伸随机定位探针以产生包括条形码序列和来自靶核酸的序列的延伸探针，由此以空间方式为生物样本的核酸加标签。
多种固体载体中的任一种可用于本公开的方法、组合物或设备中。特别有用的固体载体是用于核酸阵列的那些。实例包括玻璃、经改性玻璃、功能化玻璃、无机玻璃、微球体(例如惰性和/或磁性颗粒)、塑料、多糖、尼龙、硝酸纤维素、陶瓷、树脂、硅石、基于硅石的材料、碳、金属、光学纤维或光学纤维束、聚合物和多孔(例如微量滴定)板。实例性塑料包括丙烯酸酯类、聚苯乙烯、苯乙烯与其它材料的共聚物、聚丙烯、聚乙烯、聚丁烯、聚氨基甲酸酯和TeflonTM。实例性的基于硅石的材料包括硅和各种形式的改性硅。
在特定实施例中，固体载体可在容器内或为容器的一部分，所述容器为例如孔、管、沟道、比色皿、陪替式培养皿(Petri plate)、瓶或诸如此类。特别有用的容器是流动槽，例如如以下文献中所述：WO 2014/142841 A1；美国专利申请公开第2010/0111768 A1号和美国专利第8,951,781号或本特利(Bentley)等人，自然(Nature)456:53-59(2008)，其各自通过引用并入本文中。实例性流动槽是可从伊路米那公司(圣地亚哥，加利福尼亚州)购得的用于测序平台(例如Genome
或
平台)的那些。另一种特别有用的容器是多孔板或微量滴定板中的孔。
任选地，固体载体可包括凝胶涂层。通过凝胶将核酸附接至固体载体通过可从伊路米那公司(圣地亚哥，加利福尼亚州)购得或描述于以下文献中的流动槽例示：美国专利申请公开第2011/0059865 A1号、第2014/0079923 A1号或第2015/0005447 A1号；或PCT公开第WO 2008/093098号，其各自通过引用并入本文中。可用于本文所述方法和设备中的实例性凝胶包括但不限于具有胶体结构的凝胶，例如琼脂糖；聚合物网状结构，例如明胶；或交联聚合物结构，例如聚丙烯酰胺、SFA(参见例如美国专利申请公开第2011/0059865 A1号，其是通过引用并入本文中)或PAZAM(参见例如美国专利申请公开第2014/0079923 A1号或第2015/0005447 A1号，其各自是通过引用并入本文中)。
在一些实施例中，固体载体可经配置为核酸可附接的特征的阵列。特征可以多种所需格式中的任一种存在。例如，特征可为孔、凹部、沟道、***物、凸起区域、桩、柱或诸如此类。在一些实施例中，特征可含有珠粒。然而，在特定实施例中，特征无需含有珠粒或颗粒。实例性特征包括存在于用于由454生命科学(454LifeSciences，罗氏(Roche)子公司，巴塞尔，瑞士)或艾恩托伦特(生命技术的子公司，卡尔斯巴德，加利福尼亚)出售的市售测序平台的衬底中的孔。具有孔的其它衬底包括例如蚀刻纤维光学衬底和其它衬底，描述于以下文献中：美国专利第6,266,459号、第6,355,431号、第6,770,441号、第6,859,570号、第6,210,891号、第6,258,568号、第6,274,320号、美国专利申请公开第2009/0026082 A1号、第2009/0127589 A1号、第2010/0137143 A1号、第2010/0282617 A1号或PCT公开第WO 00/63437号，其各自通过引用并入本文中。在一些实施例中，衬底的孔可包括凝胶材料(具有或不具有珠粒)，如美国专利申请公开第2014/0243224 A1号中所述，其是通过引用并入本文中。
固体载体上的特征可为在非金属表面(例如玻璃、塑料或上文所例示的其它材料)上的金属特征。可使用业内已知方法将金属层沉积于表面上，例如湿法等离子体蚀刻、干法等离子体蚀刻、原子层沉积、离子束蚀刻、化学气相沉积、真空溅镀或诸如此类。如果适当，可使用多种市售仪器中的任一种，包括例如
Ionfab
或Optofab
***(牛津仪器(Oxford Instruments)，英国)。还可通过电子束蒸发或溅镀来沉积金属层，如桑顿(Thornton)，材料科学年度评论(Ann.Rev.Mater.Sci.)7:239-60(1977)中所述，其是通过引用并入本文中。金属层沉积技术(例如上文所例示的那些)可与光刻技术组合以在表面上产生金属区域或补片。组合金属层沉积技术和光刻技术的实例性方法提供于美国专利第8,895,249号或美国专利申请公开第2014/0243224A1号中，其各自通过引用并入本文中。
特征可在固体载体上表现为斑块或补片的网格。特征可以重复图案或无规则的非重复图案来定位。特别有用的重复图案是六边形图案、直线图案、网格图案、具有反射对称的图案、具有旋转对称的图案或诸如此类。也可使用不对称图案。节距在不同的最近相邻特征对之间可相同，或节距在不同的最近相邻特征对之间可变。
高密度阵列的特征为平均节距小于约15μm。中等密度阵列的平均节距为约15至30μm，而低密度阵列的平均节距大于30μm。本发明中可用阵列的平均节距可小于100μm、50μm、10μm、5μm、1μm或0.5μm。上文和本文中其它地方陈述的平均节距值和范围打算适用于有序阵列或随机阵列。
在特定实施例中，固体载体上的特征可各自具有大于约100nm2、250nm2、500nm2、1μm2、2.5μm2、5μm2、10μm2、100μm2或500μm2的面积。或者或另外，特征可各自具有小于约1mm2、500μm2、100μm2、25μm2、10μm2、5μm2、1μm2、500nm2或100nm2的面积。上述范围可描述，在从上方查看或成像时，固体载体上的珠粒或其它颗粒的表观面积。
在特定实施例中，固体载体可包括珠粒或其它颗粒的集合。可将颗粒悬浮于溶液中或可将其定位于衬底表面上。具有定位于表面上的珠粒的阵列的实例包括其中珠粒定位于孔中的那些阵列，例如BeadChip阵列(伊路米那公司，圣地亚哥，CA)、用于来自454生命科学(罗氏子公司，巴塞尔，瑞士)的测序平台的衬底或用于来自艾恩托伦特(生命技术的子公司，卡尔斯巴德，加利福尼亚)的测序平台的衬底。其它具有定位于表面上的珠粒的固体载体描述于以下文献中：美国专利第6,266,459号、第6,355,431号、第6,770,441号、第6,859,570号、第6,210,891号、第6,258,568号或第6,274,320号；美国专利申请公开第2009/0026082 A1号、第2009/0127589 A1号、第2010/0137143 A1号或第2010/0282617 A1号或PCT公开第WO 00/63437号，其各自通过引用并入本文中。上文引用文献中的若干文献描述在固体载体中或固体载体上加载珠粒之前将核酸探针附接至珠粒的方法。因此，珠粒集合可包括各自附接有独特探针的不同珠粒。然而，应理解，可使珠粒包括通用引物，且随后可将珠粒加载至阵列上，由此形成用于本文所述方法中的通用阵列。如前文所述，通常用于珠粒阵列的固体载体可在无珠粒情况下使用。例如，可将核酸(例如探针或引物)直接附接至孔或附接至孔中的凝胶材料。因此，上文参考文献阐明可经修改用于本文所述方法和组合物中的材料、组合物或设备。
因此，用于本文所述方法中的固体载体可包括珠粒阵列，其中将不同核酸探针附接至阵列中的不同珠粒。在此实施例中，可将每一珠粒附接至不同核酸探针，且珠粒可随机分布于固体载体上以将不同核酸探针有效附接至固体载体。任选地，固体载体可包括尺寸容纳不超过单一珠粒的孔。在此一配置中，可通过珠粒在孔中的配合产生的力将珠粒附接至孔。还可能使用附接化学物质或粘着剂将珠粒固持于孔中。
附接至珠粒的核酸探针可包括条形码序列。珠粒群体可经配置使得每一珠粒附接至仅一种类型的条形码且所述群体中存在各自具有不同条形码的多种不同珠粒。在此实施例中，将珠粒随机分布至固体载体将导致将核酸探针(和其相应条形码序列)随机定位于固体载体上。在一些情形中，可有多个珠粒具有相同条形码序列，使得群体中存在冗余。将珠粒的冗余群体随机分布在容量大于珠粒群体中的独特条形码数目的固体载体上将导致固体载体上条形码的冗余。或者，珠粒群体中不同条形码的数目可超过固体载体的容量，以产生关于固体载体上的条形码群体不冗余的阵列。在一些实施例中，固体载体的容量将依照附接或以其它方式容纳珠粒的特征(例如单一珠粒占据孔)的数目来确定。
固体载体可包括多个不同核酸探针，或可通过本文所述方法制造以附接多个不同核酸探针。例如，固体载体可包括至少10、100、1×103、1×104、1×105、1×106、1×107、1×108、1×109或更多不同探针。或者或另外，固体载体可包括至多1×109、1×108、1×107、1×106、1×105、1×104、1×103、100或更少不同探针。应理解，例如在探针已经扩增以形成簇时，不同探针可各自以若干个拷贝存在。因此，上文范围可描述固体载体上的不同核酸簇的数目。还将理解，上文范围可描述本文陈述为特定核酸探针所独有的不同条形码、靶捕获序列或其它序列元件的数目。或者或另外，所述范围可描述使用本文所述方法在固体载体上产生的延伸探针或经修饰探针的数目。
在使固体载体与核酸探针接触之前可在固体载体上存在特征。例如，在其中探针通过与引物杂交附接至载体的实施例中，引物可附接于特征，而特征外的空隙区域基本上没有任何引物。核酸探针可使用以下文献中陈述的方法被捕获在固体载体上的预成型特征处，并任选地在固体载体上扩增：美国专利第8,895,249号、美国专利第8,778,849号或美国专利申请公开第2014/0243224 A1号，其各自通过引用并入本文中。或者，固体载体可具有引物苔(lawn)或可另外缺少特征。在这种情形中，可借助在固体载体上附接核酸探针来形成特征。任选地，所捕获的核酸探针可在固体载体上扩增，使得所得簇成为特征。尽管附接在上文例示为在引物与探针的互补部分之间的捕获，应理解，并非引物的捕获部分可存在于预成型特征处或作为苔存在。其它实例性捕获部分包括但不限于能与核酸探针反应形成共价键的化学部分或能非共价结合至核酸探针上的配体的受体。
将核酸探针附接至固体载体的步骤可通过提供含有不同核酸探针的混合物的流体和使此流体混合物与固体载体接触来实施。所述接触可导致流体混合物与表面接触，来自流体混合物的多种不同核酸探针将附接至所述表面。因此，探针随机到达所述表面(所述表面具有经配置以附接探针的预成型特征或经配置用于附接的均匀表面)。因此，探针可被随机定位在固体载体上。
最终附接至表面的不同探针的总数和类别可针对特定应用或用途来选择。例如，在其中出于将探针附接至载体的目的使不同核酸探针的流体混合物与固体载体接触的实施例中，不同探针种类的数目可超过固体载体的探针占据数。因此，附接至固体载体的不同探针的数目和类别可相当于固体载体的探针占据数。或者，固体载体上的不同探针种类的数目和类别可小于占据数(即探针种类将存在冗余，使得固体载体可含有多个具有相同探针种类的特征)。所述冗余可例如通过使固体载体与含有显著低于固体载体的探针占据数的数目和类别的探针种类的流体混合物接触来实现。
核酸探针的附接可通过以下方式来调介：核酸探针与附接至固体载体的互补引物杂交、核酸探针上的反应性部分与固体载体之间的化学键形成(实例陈述于美国专利第8,895,249号、美国专利第8,778,849号或美国专利申请公开第2014/0243224 A1号中，其各自是通过引用并入本文中)、核酸探针上的部分与固体载体结合部分的亲和性相互作用(例如已知受体-配体对之间，例如抗生蛋白链菌素-生物素、抗体-表位、凝集素-碳水化合物等)、核酸探针与固体载体的物理相互作用(例如氢键结、离子力、范德华力等)或业内已知将核酸附接至表面的其它相互作用。
在一些实施例中，核酸探针的附接关于核酸探针与已附接至或将附接至固体载体的其它核酸探针之间的任何序列差异是非特异性的。例如，不同探针可具有与表面附接的引物互补的通用序列，或不同探针可具有调介与表面的附接的共有部分。或者，不同探针(或不同探针的子群)可各自具有与固体载体上的独特引物互补的独特序列，或其可具有与固体载体上的一或多种不同反应性部分相互作用的独特部分。在所述情形中，独特引物或独特部分可任选地附接在预先界定的定位，以在相应的预先界定的定位选择性地捕获特定探针或特定类型的探针。
固体载体上的一或多个特征可各自包括特定探针的单一分子。在一些实施例中，特征可经配置以容纳不超过单一核酸探针分子。然而，不论特征是否可容纳多于一个核酸探针分子，但特征都仅可包括单一核酸探针分子。或者，个别特征可包括多个核酸探针分子，例如彼此具有相同序列的核酸探针分子的系综。在特定实施例中，所述系综可通过从单一核酸探针模板扩增以产生例如呈附接至表面的簇的扩增子来产生。
本文所述方法可使用多种扩增技术中的任一种。可使用的实例性技术包括但不限于聚合酶链式反应(PCR)、滚环式扩增(RCA)、多重置换扩增(MDA)或随机引物扩增(RPA)。在一些实施例中，例如阵列的特征能在具有所需容量的体积中含有扩增子时，扩增可在溶液中实施。优选地，用于本公开的方法中的扩增技术将在固相上实施。例如，可将一或多个引物种类(例如用于一或多个存在于核酸探针中的通用引物结合位点的通用引物)附接至固体载体。在PCR实施例中，可将一种或两种用于扩增的引物附接至固体载体(例如通过凝胶)。利用两个附接至固体载体的引物种类的格式通常称为桥式扩增，因为双链扩增子在位于已复制模板序列侧翼的两个表面附接的引物之间形成桥状结构。可用于桥式扩增的实例性试剂和条件描述于例如以下文献中：美国专利第5,641,658号、第7,115,400号或第8,895,249号；或美国专利公开第2002/0055100 A1号、第2004/0096853 A1号、第2004/0002090 A1号、第2007/0128624 A1号或第2008/0009420 A1号，其各自通过引用并入本文中。还可使用附接至固体载体的一种扩增引物和溶液中的第二引物来实施固相PCR扩增。使用表面附接引物与可溶引物的组合的实例性格式是用于乳液PCR中的格式，例如如以下文献中所述：德莱斯曼(Dressman)等人，美国国家科学院院刊(Proc.Natl.Acad.Sci.USA)100:8817-8822(2003)；WO 05/010145；或美国专利申请公开第2005/0130173 A1号或第2005/0064460 A1号，其各自通过引用并入本文中。乳液PCR阐明格式并且应理解，出于本文所述方法的目的，乳液的使用是可选的并且实际上对于若干实施例来说，不使用乳液。
RCA技术可经修改用于本公开方法。可用于RCA反应中的实例性组分和RCA据以产生扩增子的原理描述于例如以下文献中：利萨尔迪(Lizardi)等人，自然遗传学(Nat.Genet.)19:225-232(1998)和美国专利申请公开第2007/0099208 A1号，其各自通过引用并入本文中。用于RCA的引物可在溶液中或附接至固体载体。引物可为本文所述通用引物中的一或多种。
MDA技术可经修改用于本公开方法中。MDA的一些基础原理和可用条件描述于例如以下文献中：迪恩(Dean)等人，美国国家科学院院刊99:5261-66(2002)；拉赫(Lage)等人，基因组研究(Genome Research)13:294-307(2003)；瓦尔克(Walker)等人，病毒检测的分子方法(Molecular Methods for Virus Detection),学术出版公司(Academic Press,Inc.),1995；瓦尔克等人，核酸研究(Nucl.Acids Res.)20:1691-96(1992)；US 5,455,166；US 5,130,238；和US 6,214,587，其各自通过引用并入本文中。用于MDA的引物可在溶液中或在扩增位点附接至固体载体。同样，引物可为本文所述通用引物中的一或多种。
在特定实施例中，可使用上文例示的扩增技术的组合。例如，RCA和MDA可组合使用，其中使用RCA来生成溶液中的多联体扩增子(例如使用溶液相引物)。随后可使用所述扩增子作为使用附接至固体载体的引物(例如通用引物)的MDA的模板。在此实例中，在组合的RCA和MDA步骤后产生的扩增子将被附接至固体载体。
在本文所述方法中使用或在本公开的设备或组合物中存在的核酸探针可包括条形码序列，并且对于包括多个不同核酸探针的实施例，探针可各自包括与所述多个探针中的其它探针不同的条形码序列。条形码序列可具有多种长度中的任一长度。对于群体，较长序列通常可容纳较大数目和类别的条形码。通常，多个探针中的所有探针都将具有相同长度的条形码(虽然具有不同序列)，但也可能对于不同探针使用不同长度的条形码。条形码序列的长度可为至少2、4、6、8、10、12、15、20个或更多核苷酸。或者或另外，条形码序列的长度可为至多20、15、12、10、8、6、4个或更少核苷酸。可使用的条形码序列的实例陈述于例如美国专利申请公开第2014/0342921 A1号和美国专利第8,460,865号中，其各自通过引用并入本文中。
本公开方法可包括在固体载体上进行核酸检测反应以测定定位于固体载体上的核酸探针的条形码序列的步骤。在多个实施例中，将探针随机定位在固体载体上并且核酸检测反应提供定位每一不同探针的信息。实例性核酸检测方法包括但不限于探针的核酸测序、核酸与探针的杂交、与探针杂交的核酸的连接、与探针杂交的核酸的延伸、延伸与探针杂交的第一核酸之后将经延伸核酸连接至与探针杂交的第二核酸、或业内已知的其它方法，例如美国专利第8,288,103号或第8,486,625号中陈述的方法，其各自通过引用并入本文中。
测序技术(例如合成测序(SBS)技术)是用于测定条形码序列的特别有用的方法。SBS可如下文来实施。为起始第一SBS循环，可使一或多种经标记核苷酸、DNA聚合酶、SBS引物等与固体载体上的一或多个特征(例如其中核酸探针附接至固体载体的特征)接触。可检测那些其中SBS引物延伸使经标记核苷酸被纳入的特征。任选地，核苷酸可包括可逆终止部分，所述部分一旦在核苷酸被添加至SBS引物后立即终止进一步引物延伸。例如，可将具有可逆终止子部分的核苷酸类似物添加至引物，使得在递送解封剂以移除部分以前不会发生后续延伸。因此，对于使用可逆终止的实施例，可将解封试剂递送至固体载体(在进行检测之前或之后)。可在多个递送步骤之间实施洗涤。然后可将所述循环重复n次以将引物延伸n个核苷酸，由此检测长度n的序列。可容易地针对本公开的组合物、设备或方法加以调整的实例性SBS程序、流体***和检测平台描述于例如以下文献中：本特利等人，自然456:53-59(2008),PCT公开第WO 91/06678号、第WO 04/018497号或第WO 07/123744号；美国专利第7,057,026号、第7,329,492号、第7,211,414号、第7,315,019号或第7,405,281号，以及美国专利申请公开第2008/0108082号，其各自通过引用并入本文中。
可使用使用循环反应的其它测序程序，例如焦磷酸测序。焦磷酸测序检测在将特定核苷酸纳入新生核酸链时无机焦磷酸盐的释放(PPi)(罗纳吉(Ronaghi)等人，分析生物化学(Analytical Biochemistry)242(1),84-9(1996)；罗纳吉,基因组研究(Genome Res.)11(1),3-11(2001)；罗纳吉等人，科学(Science)281(5375),363(1998)；或美国专利第6,210,891号、第6,258,568号或第6,274,320号，其各自是通过引用并入本文中)。在焦磷酸测序中，所释放的PPi可通过立即由ATP硫酸化酶转化为腺苷三磷酸(ATP)来检测，并且所生成的ATP水平可通过荧光素酶产生的光子来检测。因此，测序反应可通过发光检测***来监测。用于荧光基检测***的激发辐射源对于焦磷酸测序程序来说不是必需的。可用于将焦磷酸测序应用于本公开的设备、组合物或方法的可用流体***、检测器和程序描述于例如以下文献中：PCT专利申请公开第WO2012/058096号、美国专利申请公开第2005/0191698 A1号或美国专利第7,595,883号或第7,244,559号，其各自通过引用并入本文中。
也可使用连接测序反应，包括例如以下文献中所述的那些：森杜尔(Shendure)等人，科学309:1728-1732(2005)；或美国专利第5,599,675号或第5,750,341号，其各自通过引用并入本文中。一些实施例可包括杂交测序程序，例如如以下文献中所述：贝恩斯(Bains)等人，理论生物学杂志(Journal of Theoretical Biology)135(3),303-7(1988)；多玛纳(Drmanac)等人，自然生物技术(Nature Biotechnology)16,54-58(1998)；福多(Fodor)等人，科学251(4995),767-773(1995)；或PCT专利申请公开第WO 1989/10977号，其各自通过引用并入本文中。在连接测序和杂交测序程序二者中，存在于阵列位点处的靶核酸(或其扩增子)经历寡核苷酸递送和检测的重复循环。本文或本文引用的参考文献中所述的组合物、设备或方法可容易地针对连接测序或杂交测序程序加以调整。通常，寡核苷酸经荧光标记并且可使用荧光检测器来检测，所述荧光检测器类似于关于本文中或本文所引用的参考文献中的SBS程序描述的那些。
一些测序实施例可利用涉及DNA聚合酶活性的实时监测的方法。例如，核苷酸纳入可通过带有荧光团的聚合酶与γ-磷酸盐标记的核苷酸之间的荧光共振能量转移(FRET)相互作用或用零模波导(ZMW)来检测。用于基于FRET的测序的技术和试剂描述于例如以下文献中：莱文(Levene)等人，科学299,682–686(2003)；伦德奎斯特(Lundquist)等人，光学快报(Opt.Lett.)33,1026–1028(2008)；克拉克(Korlach)等人，美国国家科学院院刊105,1176–1181(2008)，其各自通过引用并入本文中。
一些测序实施例包括检测在将核苷酸纳入延伸产物中时释放的质子。例如，基于所释放质子的检测的测序可使用可从艾恩托伦特(吉尔福德，康涅狄格州，生命技术和赛默飞世尔科技(Thermo Fisher)子公司)购得的电检测器和相关技术或以下文献中所述的测序方法和***：美国专利申请公开第2009/0026082 A1号；第2009/0127589 A1号；第2010/0137143 A1号；或US 2010/0282617 A1，其各自通过引用并入本文中。
核酸杂交技术也是可用于测定条形码序列的方法。在一些情形中，可使用组合杂交方法，例如用于解码多重珠粒阵列的那些(参见例如美国专利第8,460,865号，其是通过引用并入本文中)。所述方法利用经标记核酸解码器探针，其与条形码序列的至少一部分互补。杂交反应可使用具有已知标记的解码器探针来实施，使得标记最终在固体载体上的定位根据核酸互补性原理鉴别核酸探针。在一些情形中，使用多个具有可区别标记的不同探针的汇集物，由此容许多重解码操作。在解码操作中测定的不同条形码的数目可超过用于解码操作的标记数。例如，解码可在若干个阶段中实施，其中每一阶段构成与解码器探针的不同汇集物的杂交。不同汇集物中可存在相同解码器探针，但存在于每一解码器探针上的标记可在汇集物之间不同(即每一解码器探针在不同汇集物中时处于不同“状态”)。可使用这些状态和阶段的不同组合将可解码的条形码数目扩大到远远超过可用于解码的不同标记数。所述组合方法更详细地陈述于以下文献中：美国专利第8,460,865号或冈德森(Gunderson)等人，基因组研究14:870-877(2004)，其各自通过引用并入本文中。
本公开方法可包括使生物样本与附接有核酸探针的固体载体接触的步骤。在一些实施例中，核酸探针是随机定位在固体载体上。可在使生物样本与固体载体接触之前已解码核酸探针的属性和定位。或者，核酸探针的属性和定位可在使固体载体与生物样本接触后确定。
在一些实施例中，生物样本是一或多个细胞。细胞在与固体载体接触时可为个别细胞且不含任何组织或多细胞结构。例如，细胞可存在于流体中(例如在存在多个不同细胞时，流体可为不同细胞的流体混合物)并且可使流体与附接有不同探针的固体载体接触。可使用多种细胞中的任一种，包括例如来自原核生物、古细菌或真核生物的细胞。用于本公开的方法、组合物或设备中的一或多种细胞可为单细胞生物体或来自多细胞生物体。可自其获得一或多种细胞的实例性生物体包括但不限于哺乳动物、植物、藻类、线虫、昆虫、鱼、爬行动物、两栖动物、真菌或恶性疟原虫。实例性种类陈述于前文中或为业内已知。
本公开实施例还可使用一或多种亚细胞组分作为生物样本。例如，流体混合物可包括一或多个核、高尔基体(golgi apparatus)、线粒体、叶绿体、膜部分、泡囊、内质网或业内已知的其它组分。生物样本的其它可用类型是一或多种病毒或类病毒。
应理解，生物样本可为上文细胞、亚细胞组分、病毒或类病毒的同质培养或群体。或者，生物样本可为例如源自群落或生态系中若干种不同生物体的细胞、亚细胞组分、病毒或类病毒的非同质集合。实例性群落是存在于多细胞生物体(例如哺乳动物)的消化***、肺或其它器官中的细菌的集合。
在本文所述方法中与固体载体接触的一或多种细胞、亚细胞组分、病毒或类病毒可附接至固体载体。附接可使用业内已知方法来实现，例如本文中关于将核酸附接至固体载体所例示的那些方法。在一些实施例中，附接对于特殊类型的细胞、亚细胞组分、病毒或类病毒具有选择性。例如，固体载体可包括抗体或其它受体，其对存在于流体混合物中所存在的不同细胞、亚细胞组分、病毒或类病毒中的一个或亚组上的表位或配体具有选择性。在其它实施例中，细胞、亚细胞组分、病毒或类病毒的附接可通过非选择性部分来调介，例如具有广泛反应性的化学部分。
在特定实施例中，可裂解一或多种已与固体载体接触的细胞、亚细胞组分、病毒或类病毒以释放靶核酸。裂解可使用业内已知方法来实施，例如采用化学处理、酶处理、电穿孔、加热、低渗处理、超声处理或诸如此类中的一或多种的方法。实例性裂解技术陈述于以下文献中：桑布鲁克(Sambrook)等人，分子克隆：实验室手册第三版(Molecular Cloning:ALaboratory Manual,Third Ed.)，冷泉港实验室(Cold Spring Harbor Laboratory)，纽约(2001)；和奥苏贝尔(Ansubel)等人，分子生物学实验手册(Current Protocols inMolecular Biology)，约翰威利父子公司(John Wiley and Sons)，巴尔的摩，马里兰州(1999)。
在一些实施例中，生物样本是组织切片。组织可源自多细胞生物体，例如上文关于细胞所例示的那些。可使组织切片与固体载体接触，例如通过将组织敷设于固体载体表面上来进行。组织可为刚从生物体切除的，或其可预先例如通过以下方式来保存：冷冻、包埋于诸如石蜡等材料中(例如***固定的石蜡包埋样品)、***固定、浸润、脱水或诸如此类。任选地，可例如使用本文中关于将核酸、细胞、病毒、珠粒或诸如此类附接至固体载体所例示的技术和组合物将组织切片附接至固体载体。作为另一选择，组织可经渗透化处理并且组织中的细胞在组织与固体载体接触时裂解。可使用多种处理中的任一种，例如上文关于裂解细胞所述的那些。从经渗透化处理的组织释放的靶核酸可由表面上的核酸探针捕获。
组织可以任何对于其在本文中的方法、组合物或设备中的使用便捷或所需的方式来制备。可使用新鲜、冷冻、固定或未固定的组织。可使用本文所述或业内已知的方法来固定或包埋组织。
用于本文中的组织样品可通过在适于维持或保存组织结构完整性的温度(例如低于-20℃)下深度冷冻来固定。在另一实例中，组织可使用业内已知的***固定和石蜡包埋(FFPE)方法来制备。可根据需要使用其它固定剂和/或包埋材料。可使用已知方法将经固定或包埋的组织样品切片，即切成薄片。例如，可使用设定在适于维持组织样品的结构完整性和样品中核酸的化学性质二者的温度下的冷冻切片机或低温恒温器将组织样品切片。
在一些实施例中，将在核酸的释放、捕获或修饰前处理组织样品以从样品移除包埋材料(例如移除石蜡或***)。这可通过使样品与适当溶剂(例如二甲苯和乙醇洗涤液)接触来实现。处理可在使组织样品与本文所述的固体载体接触之前进行，或处理可在组织样品位于固体载体上时进行。用于操作与附接有核酸的固体载体一起使用的组织的实例性方法陈述于美国专利申请公开第2014/0066318A1号中，其是通过引用并入本文中。
在本文所述方法、组合物或设备中与固体载体接触的组织样品或其它生物样本的厚度可为需要的任何适宜厚度。在代表性实施例中，厚度将为至少0.1μm、0.25μm、0.5μm、0.75μm、1μm、5μm、10μm、50μm、100μm或更厚。或者或另外，与固体载体接触的生物样本的厚度将不大于100μm、50μm、10μm、5μm、1μm、0.5μm、0.25μm、0.1μm或更薄。
生物样本的特别相关的来源是人类。样本可源自器官，包括例如肌肉骨骼***的器官，例如肌肉、骨骼、腱或韧带；消化***的器官，例如唾液腺、咽、食管、胃、小肠、大肠、肝、胆囊或胰脏；呼吸***的器官，例如喉、气管、支气管、肺或膈；泌尿***的器官，例如肾、输尿管、膀胱或尿道；生殖器官，例如卵巢、输卵管、子宫、***、胎盘、睾丸、附睾、输精管、精囊、***、***或阴囊；内分泌***的器官，例如垂体、松果腺、甲状腺、甲状旁腺或肾上腺；循环***的器官，例如心脏、动脉、静脉或毛细管；淋巴***的器官，例如***、***、骨髓、胸腺或脾；中枢神经***的器官，例如脑、脑干、小脑、脊髓、脑神经或脊神经；感觉器官，例如眼、耳、鼻或舌；或皮肤器官，例如皮肤、皮下组织或乳腺。在一些实施例中，生物样本是从体液或***物获得，例如血液、淋巴液、眼泪、汗液、唾液、***、***分泌物、耳垢、粪便物或尿液。
来自人类的样本可在使用时被视为(或怀疑)健康或患病的。在一些情形中，可使用两个样本：第一样本被视为患病的，并且第二样本被视为健康的(例如用作健康对照)。可评估多种病状中的任一种，包括但不限于自体免疫疾病、癌症、囊性纤维化、非整倍性、病原感染、心理病状、肝炎、糖尿病、性传播疾病、心脏病、卒中、心血管疾病、多发性硬化症或肌营养不良症。特别相关的病状是遗传病状或与具有可鉴别遗传印记的病原体相关的病状。
如上文所述，流动槽提供用于本文所述方法中的便捷设备。例如，流动槽是用于容纳固体载体的便捷设备，所述固体载体将以用于一些核酸测序方案或一些核酸杂交方案的多种流体试剂(例如重复流体递送)来处理。在一些实施例中，例如在将细胞、亚细胞组分、病毒或类病毒的流体混合物递送至固体载体时，可将生物样本递送至流动槽中的固体载体。在一些实施例中，可优选地打开流动槽以暴露内部的固体载体或从流动槽移除固体载体以容许将生物样本便捷地递送至固体载体。例如，打开流动槽或移除固体载体可容许使用者或机器人装置将组织切片敷设于固体载体上。打开流动槽或从流动槽移除固体载体可为暂时的。因此，随后可关闭流动槽或使固体载体返回流动槽以继续进行本文所述方法的一或多个后续步骤。
在一些实施例中，流动槽可具有容许将其打开或拆开的构造。例如，在进行测序反应以例如解码条形码时，流动槽可处于关闭状态。随后可拆开流动槽使得可将组织放置于流动槽表面上。可通过粘着剂将流动槽固持在一起，使得可移除一或多个表面以打开流动槽。例如，流动槽可具有间隔体，所述间隔体在顶部或底部上具有粘着剂表面(类似于单侧或双侧粘性胶带)并且此间隔体可存在于两个固体载体之间。一个或两个固体载体可经配置以附接核酸并支撑如本文所述的生物样本。间隔体可具有开放区(例如通过激光切割间隔体材料来产生)，其产生以两个固体载体和间隔体为边界的流体沟道。因此，一个或两个固体载体可非永久性地粘附至间隔体以容许移除其一个或两个，以在将组织或其它样本放置于其上时容许到达表面。
用于本文所述组合物、设备或方法中的核酸探针可包括靶捕获部分。在特定实施例中，靶捕获部分是靶捕获序列。靶捕获序列通常与靶序列互补，使得通过形成探针-靶杂合复合物来进行靶捕获。靶捕获序列可具有多种长度中的任一种，包括例如上文在条形码序列情况下所例示的长度。
在多个实施例中，多个不同核酸探针可包括与来自生物样本的不同靶核酸序列杂交的不同靶捕获序列。可使用不同靶捕获序列选择性地结合至来自生物样本的一或多个所需靶核酸。在一些情形中，不同核酸探针可包括为固体载体上的所有探针或探针亚组共有的靶捕获序列。例如，固体载体上的核酸探针可具有多A或多T序列。所述探针或其扩增子可与具有多A或多T尾的mRNA分子、cDNA分子或其扩增子杂交。尽管mRNA或cDNA种类将具有不同靶序列，捕获将通过共有多A或多T序列区域来调介。
可在本文所述方法中捕获并分析多种靶核酸中的任一种，包括但不限于信使RNA(mRNA)、复制DNA(cDNA)、基因组DNA(gDNA)、核糖体RNA(rRNA)或转移RNA(tRNA)。特定靶序列可选自数据库和使用业内已知的技术和数据库设计的适当捕获序列。
可用的其它靶捕获部分包括例如本文陈述为可用于将核酸探针附接至固体载体的部分。
本文所述方法可包括使固体载体上的核酸探针与来自生物样本中靠近探针的部分的靶核酸杂交的步骤。通常，靶核酸将从生物样本区域扩散至靠近样本中该区域的固体载体的区域。此处，靶核酸将与靠近样本中释放所述靶核酸的区域的核酸探针相互作用。可形成靶-探针杂合复合物，其中靶核酸遇到核酸探针上的互补靶捕获序列。靶-探针杂合复合物的定位通常将与生物样本中产生所述靶核酸的区域相关联。在多个实施例中，固体载体将包括多个核酸探针，生物样本将释放多个靶核酸，并且将在固体载体上形成多个靶-探针杂合体。靶核酸的序列和其在载体上的定位将提供关于生物样本的核酸含量的空间信息。尽管上文实例是在从生物样本释放的靶核酸的情况下描述的，但应理解，靶核酸无需被释放。相反，例如在将靶核酸以所述靶核酸还可结合至固体载体上的适当核酸探针的方式附接至生物样本的暴露表面时，所述靶核酸可保持与生物样本接触。
本公开方法可包括延伸与靶核酸杂交的固体载体附接探针的步骤。在探针包括条形码序列的实施例中，所得延伸探针将包括条形码序列和来自靶核酸的序列(即使呈互补形式)。延伸探针是来自生物样本的靶核酸的由此以空间方式加标签的形式。延伸探针的序列鉴别生物样本中存在何种核酸以及靶核酸定位于生物样本中的何处。应理解，在延伸探针中还可包括存在于核酸探针中的其它序列元件。所述元件包括例如引物结合位点、裂解位点、其它标签序列(例如样品鉴别标签)、捕获序列、核酸结合蛋白或核酸酶的识别位点或诸如此类。
探针的延伸可使用本文所例示的方法或业内已知用于核酸扩增或核酸测序的其它方法来实施。在特定实施例中，可例如通过聚合酶催化(例如DNA聚合酶、RNA聚合酶或逆转录酶)将一或多个核苷酸添加至核酸3'端。可使用化学或酶方法将一或多个核苷酸添加至核酸的3’或5'端。可例如通过化学或酶(例如连接酶催化)方法将一或多个寡核苷酸添加至核酸的3’或5'端。核酸可以模板引导方式延伸，借此延伸产物与杂交至所延伸核酸的模板核酸互补。在一些实施例中，DNA引物是通过逆转录酶使用RNA模板来延伸，由此产生cDNA。因此，在本文所述方法中制造的延伸探针可为逆转录DNA分子。用于延伸核酸的实例性方法陈述于美国专利申请公开第US 2005/0037393 A1号或美国专利第8,288,103号或第8,486,625号中，其各自通过引用并入本文中。
与核酸探针杂交的靶核酸的全部或部分可通过延伸来复制。例如，延伸探针可包括至少1、2、5、10、25、50、100、200、500、1000或更多个从靶核酸复制的核苷酸。延伸产物的长度可通过例如在延伸反应中使用可逆终止的核苷酸和运行有限数目的延伸循环来控制。循环可如针对SBS技术所例示来运行，并且不需要使用经标记核苷酸。因此，在本文所述方法产生的延伸探针可包括不多于1000、500、200、100、50、25、10、5、2或1个从靶核酸复制的核苷酸。当然，延伸探针可具有在上文所述范围内或所述范围外的任何长度。
尽管本公开方法例示为延伸与靶核酸杂交的探针以复制靶核酸的至少一部分的实施例，应理解，探针可以替代性方式经修饰。与靶核酸杂交的探针可经历产生探针的靶特异性修饰的反应。靶特异性修饰将仅在探针例如通过基于互补的杂交与靶核酸相互作用时才得到。在多个实施例中，靶特异性修饰将对与探针相互作用的特定靶核酸的序列具有特异性。可用靶特异性修饰的实例包括但不限于通过连接或移位***或添加序列(参见例如美国专利申请公开第2010/0120098 A1号，通过引用并入本文中)、化学修饰(例如补骨脂素交联或添加可检测标签部分)、核酸酶修饰、连接发夹状连接体或美国专利申请公开第US2005/0037393 A1号或美国专利第8,288,103号或第8,486,625号的核酸分析中陈述的其它修饰，其各自通过引用并入本文中。
应理解，在本文所述方法、组合物或设备中使用的探针无需为核酸。可使用其它分子，例如蛋白质、碳水化合物、小分子、颗粒或诸如此类。探针可为核酸组分(例如具有条形码、引物结合位点、裂解位点和/或本文所述的其它序列元件)和另一部分(例如捕获或修饰靶核酸的部分)的组合。
本文所述方法可进一步包括获取与固体载体接触的生物样本的图像的步骤。固体载体可处于本文所述多种状态中的任一种。例如，固体载体可包括附接的核酸探针或源自所附接核酸探针的簇。或者，固体载体可不包括核酸探针，而是处于在附接核酸探针之前的状态或处于从固体载体移除核酸探针之后的状态。因此，可在本文所述方法中的多个点中的任一点获得图像。
图像可使用业内已知的检测装置获得。实例包括经配置用于光、明视野、暗视野、相位差、荧光、反射、干涉或共焦成像的显微镜。生物样本可在成像前染色以提供不同区域或细胞之间的对比。在一些实施例中，可使用多于一种染色剂来使样本的不同方面成像(例如组织的不同区域、不同细胞、特定亚细胞组分或诸如此类)。在其它实施例中，生物样本可在不染色的情况下成像。
在特定实施例中，可使用荧光显微镜(例如共焦荧光显微镜)来检测例如借助荧光标记发荧光的生物样本。荧光样本还可使用具有荧光检测光学装置的核酸测序装置来成像，例如由伊路米那公司(圣地亚哥，加利福尼亚州)商业化的Genome
或
平台装置；或由生命技术(卡尔斯巴德，加利福尼亚州)商业化的SOLiDTM测序平台。可使用的其它成像光学装置包括在以下文献中所述的检测装置中发现的光学装置：本特利等人，自然456:53-59(2008)；PCT公开第WO 91/06678号、第WO 04/018497或WO 07/123744号；美国专利第7,057,026号、第7,329,492号、第7,211,414号、第7,315,019号或第7,405,281号；和美国专利申请公开第2008/0108082号，其各自通过引用并入本文中。
生物样本的图像可以所需分辨率获得，例如以区别组织、细胞或亚细胞组分。因此，分辨率可足以区别生物样本的相隔至少0.5μm、1μm、5μm、10μm、50μm、100μm、500μm、1mm或更远的组分。或者或另外，分辨率可经设置以区别生物样本的相隔至少1mm、500μm、100μm、50μm、10μm、5μm、1μm、0.5μm或更近的组分。
本文所述方法可包括将生物样本的图像中的定位与附接至生物样本正在、曾经或将要与其接触的表面的核酸探针的条形码序列相关联的步骤。因此，可将图像中可鉴别的生物样本的特征与被发现存在于其附近的核酸相关联。在此一关联中可使用多种形态特征中的任一种，包括例如细胞形状、细胞大小、组织形状、染色图案、特定蛋白质的存在(例如如通过免疫组织化学染色所检测)或在病理学或研究应用中常规评估的其它特征。因此，可将如通过目测观察测定的组织或其组分的生物学状态与如通过空间分辨核酸分析所测定的分子生物学特征相关联。
生物样本在其上成像的固体载体可包括基准标记物以帮助确定样本或其图像相对于附接至固体载体的探针的取向。实例性基准包括但不限于珠粒(具有或不具有荧光部分或例如经标记探针可结合至其的核酸的部分)、在已知或可测定特征附接的荧光分子或组合形态形状与荧光部分的结构。实例性基准陈述于美国专利申请公开第2002/0150909A1号或美国专利申请第14/530,299号中，其各自通过引用并入本文中。一或多个基准优选地在获得生物样本的图像时可见。优选地，固体载体包括至少2、3、4、5、10、25、50、100或更多个基准标记物。基准可按图案提供，例如沿固体载体的外边缘或生物样本驻留的定位的周边提供。在优选实施例中，使用与用于可视化生物样本相同的成像条件来检测一或多个基准。然而，如果需要，可获得单独图像(例如一个生物样本的图像和另一个基准图像)并且所述图像可彼此对齐。
任选地，生物样本可在获得图像后和在固体载体上的核酸探针捕获靶核酸后从固体载体移除。因此，本公开方法可包括洗涤固体载体以移除来自生物样本的细胞、组织或其它材料的步骤。样本的移除可使用任何适宜技术来进行，并且将取决于组织样品。在一些情形中，固体载体可用水洗涤。水可含有各种添加剂，例如表面活性剂(例如去污剂)、酶(例如蛋白酶和胶原酶)、裂解试剂或诸如此类，以帮助移除样本。在一些实施例中，用包含蛋白酶的溶液处理固体载体。或者或另外，溶液可包括纤维素酶、半纤维素酶或壳多糖酶(例如如果需要从植物或真菌来源移除组织样品)。在一些情形中，洗涤溶液的温度将为至少30℃、35℃、50℃、60℃或90℃。条件可经选择以在不使靶核酸与固体载体附接的核酸探针之间形成的杂合复合物变性的同时移除生物样本。
本公开方法可进一步包括从固体载体移除一或多个延伸探针的步骤。在特定实施例中，探针将已包括裂解位点使得延伸探针的产物也将包括所述裂解位点。或者，可在修饰步骤期间将裂解位点引入探针中。例如，可在延伸步骤期间将裂解位点引入延伸探针中。
实例性裂解位点包括但不限于对导致键断裂的化学、酶或物理过程敏感的部分。例如，定位可为由内切核酸酶识别的核苷酸序列。适宜的内切核酸酶和其识别序列为业内所熟知并且在多种情形中甚至是市售的(例如来自新英格兰生物实验室(New EnglandBiolabs)，贝弗利，马萨诸塞州；赛默飞世尔(ThermoFisher)，沃尔瑟姆，马萨诸塞州；或西格玛奥德里奇(Sigma Aldrich)，圣路易斯，密苏里州)。特别有用的内切核酸酶将断裂核酸链中3’远离其在核酸中的结合位点的位点的键，其实例包括II型或IIs型限制性内切核酸酶。在一些实施例中，内切核酸酶将切割二倍体核酸中的仅一条链(例如切口酶)。仅裂解一条链的内切核酸酶的实例包括Nt.BstNBI和Nt.AlwI。
在一些实施例中，裂解位点是无碱基位点或易被移除而产生无碱基位点的具有碱基的核苷酸。易被移除而产生无碱基位点的核苷酸的实例包括尿嘧啶和8-氧代-鸟嘌呤。无碱基位点可通过使用化学或酶试剂水解核苷酸残基来产生。一旦形成，可裂解无碱基位点(例如通过用内切核酸酶或其它单链裂解酶处理、暴露于热或碱)，提供核酸的位点特异性裂解的方式。可在核酸的一条链上的尿嘧啶核苷酸产生无碱基位点。可使用尿嘧啶DNA糖苷酶(UDG)移除尿嘧啶碱基，在所述链上生成无碱基位点。随后可通过用内切核酸酶(例如EndoIV内切核酸酶、AP裂解酶、FPG糖苷酶/AP裂解酶、EndoVIII糖苷酶/AP裂解酶)、热或碱处理在无碱基位点裂解具有无碱基位点的核酸链。在特定实施例中，使用可从新英格兰生物实验室获得的USERTM试剂在核酸中的尿嘧啶碱基产生单一核苷酸空位。
无碱基位点还可在并非尿嘧啶的非天然/经修饰脱氧核糖核苷酸生成，并通过用内切核酸酶、热或碱处理以类似方式裂解。例如，可通过暴露于FPG糖苷酶将8-氧代-鸟嘌呤转化为无碱基位点。可通过暴露于AlkA糖苷酶将脱氧次黄苷转化为无碱基位点。随后可通常通过用适宜内切核酸酶(例如EndoIV或AP裂解酶)处理来裂解由此生成的无碱基位点。
可用于裂解核酸的裂解位点和方法的其它实例陈述于例如美国专利第7,960,120号中，其是通过引用并入本文中。
可汇集从固体载体释放的经修饰核酸探针(例如延伸核酸探针)以形成流体混合物。所述混合物可包括例如至少10、100、1×103、1×104、1×105、1×106、1×107、1×108、1×109个或更多不同的经修饰探针。或者或另外，流体混合物可包括至多1×109、1×108、1×107、1×106、1×105、1×104、1×103、100、10个或更少不同的经修饰探针。可操作流体混合物以容许检测经修饰核酸探针。例如，经修饰核酸探针可在第二固体载体(即不同于在已与生物样本接触并经修饰后从其释放核酸探针的固体载体)上在空间上分离，或探针可在流体流中在时间上分离。
在一般用于基于微阵列的技术或核酸测序技术的捕获或检测方法(例如前文中陈述的那些)中，经修饰核酸探针(例如延伸核酸探针)可在固体载体上分离。例如，经修饰探针可通过与互补核酸杂交附接至微阵列。经修饰探针可附接至珠粒或附接至流动槽表面并且任选地扩增，如在多个核酸测序平台中所实施。经修饰探针可使用微流体装置、液滴操作装置或流式细胞仪在流体流中分离。通常，在这些分离装置上实施检测，但检测并非在所有实施例中都是必需的。
特别有用的液滴操作装置是液滴致动器，如例如以下文献中所述：美国专利第8,637,242号、美国专利第6,911,132号，标题为“通过基于电润湿的技术操作液滴的设备(Apparatus for Manipulating Droplets by Electrowetting-Based Techniques)”，2005年6月28日授予；柏姆拉(Pamula)等人，美国专利公开第20060194331号，标题为“在印刷电路板上操作液滴的设备和方法(Apparatuses and Methods for ManipulatingDroplets on a Printed Circuit Board)”，2006年8月31日公开；波拉克(Pollack)等人，国际专利公开第WO/2007/120241号，标题为“基于液滴的生物化学”，2007年10月25日公开；森德拉夫(Shenderov)，美国专利第6,773,566号，标题为“用于微流体的静电致动器和其使用方法(Electrostatic Actuators for Microfluidics and Methods for UsingSame)”，2004年8月10日授予；森德拉夫，美国专利第25 6,565,727号，标题为“不具有移动部分的微流体致动器(Actuators for Microfluidics Without Moving Parts)”，2003年5月20日授予；金(Kim)等人，美国专利公开第20030205632号，标题为“电润湿驱动的微泵送(Electrowettingdriven Micropumping)”，2003年11月6日公开；金等人，美国专利公开第20060164490号，标题为“促进液滴从喷嘴完全转移的方法和设备(Method and Apparatusfor Promoting the Complete Transfer of Liquid Drops from a Nozzle)”，2006年7月27日公开；金等人，美国专利公开第20070023292号，标题为“在印刷电路板上移动的小物体(Small Object Moving on Printed Circuit Board)”，2007年2月1日公开；沙阿(Shah)等人，美国专利公开第20090283407号，标题为“在液滴微流体中使用磁性颗粒的方法(Methodfor Using Magnetic Particles in Droplet Microfluidics)”，2009年11月19日公开；金等人，美国专利公开第20100096266号，标题为“实施反馈控制芯片上的液滴电操作的方法和设备(Method and Apparatus for Real-time Feedback Control of ElectricalManipulation of Droplets on Chip)”，2010年4月22日公开；韦列夫(Velev)，美国专利第7,547,380号，标题为“具有流体表面的液滴输送装置和方法(Droplet TransportationDevices and Methods Having a Fluid Surface)”，2009年6月16日授予；施特林(Sterling)等人，美国专利第7,163,612号，标题为“用于化学、生物化学和生物学分析等的用于通过电润湿进行微流体控制的方法、设备和物品(Method,Apparatus and Articlefor Microfluidic Control via Electrowetting,for Chemical,Biochemical andBiological Assays and the Like)”，2007年1月16日授予；贝克尔(Becker)等人，美国专利第7,641,779号，标题为“用于可编程流体处理的方法和设备(Method and Apparatusfor Programmable Fluidic Processing)”，2010年1月5日授予；贝克尔等人，美国专利第6,977,033号，标题为“用于可编程流体处理的方法和设备”，2005年12月20日授予；德克利(Decre)等人，美国专利第7,328,979号，标题为“流体实体的操作***(System forManipulation of a Body of Fluid)”，2008年2月12日授予；山河(Yamakawa)等人，美国专利公开第15 20060039823号，标题为“化学分析设备(Chemical Analysis Apparatus)”，2006年2月23日公开；吴(Wu)，美国专利公开第20110048951号，标题为“用于热交换化学过程的基于数字微流体的设备(Digital Microfluidics Based Apparatus for Heat-exchanging Chemical Processes)”，2011年3月3日公开；富耶尔(Fouillet)等人，美国专利公开第20090192044号，标题为“电极寻址方法(Electrode Addressing Method)”，2009年7月30日公开；富耶尔等人，美国专利第7,052,244号，标题为“通过静电力使小液体体积沿微链线位移的装置(Device for Displacement of Small Liquid Volumes Along aMicro-catenary Line by Electrostatic Forces)”，2006年5月30日授予；马尚(Marchand)等人，美国专利公开第20080124252号，标题为“液滴微反应器(DropletMicroreactor)”，2008年5月29日公开；安达(Adachi)等人，美国专利公开第20090321262号，标题为“液体转移装置(Liquid Transfer Device)”，2009年12月31日公开；鲁(Roux)等人，美国专利公开第20050179746号，标题为“用于控制液滴在两个或若干个固体衬底之间位移的装置(Device for Controlling the Displacement of a Drop Between Two orSeveral Solid Substrates)”，2005年8月18日公开；和丁德萨(Dhindsa)等人，“虚拟电润湿沟道：具有连续沟道功能性的电子液体输送(Virtual Electrowetting Channels:Electronic Liquid Transport with Continuous Channel Functionality)”，实验室芯片(Lab Chip)，10:832–836(2010)，其各自通过引用并入本文中。
经修饰探针(例如延伸核酸探针)可例如在从流体混合物分离后使用上文所述或业内已知的方法来检测。在特定实施例中，在第二固体载体(即与其中在探针与生物样本之间进行接触的第一固体载体不同的固体载体)上分离的经修饰探针可使用基于微阵列的技术或核酸测序技术来检测，例如前文所述的那些。在流体流中分离的探针可使用配备于已知微流体装置、液滴操作装置或流式细胞仪中的光学、电或其它检测器来检测。可使用检测方法来测定延伸探针的靶核酸序列、条形码序列或其它序列区域。
关于从其中产生探针的固体载体移除经修饰探针，已例示若干个实施例。然而，应理解，可使固体载体上的探针与生物样本接触，在固体载体上在来自样本的靶核酸存在下修饰，随后可在固体载体上检测经修饰探针。在此一实施例中，可在检测步骤之前从固体载体移除生物样本。
在特定实施例中，本公开提供以空间方式为生物样本的核酸加标签的方法，其包括以下步骤：(a)提供多个附接至固体载体的核酸引物，其中所述多个中的核酸引物包括为所述多个中的核酸引物共有的通用引物序列；(b)使核酸探针群体与所述多个核酸引物结合，其中核酸探针包括与通用引物序列杂交的通用引物结合序列、靶捕获序列和与群体中其它核酸探针的条形码序列不同的条形码序列，由此将不同核酸探针附接在固体载体上的随机定位位置；(c)通过延伸核酸引物来扩增不同核酸探针，由此在固体载体上的随机定位位置产生具有条形码序列和靶捕获序列的拷贝的核酸簇；(d)进行测序反应以测定固体载体上的随机定位位置的条形码序列；(e)使生物样本与固体载体上的核酸簇接触；(f)使所述簇的靶捕获序列与来自生物样本中靠近所述簇的部分的靶核酸杂交；和(g)延伸靶捕获序列以产生包括来自靶核酸的序列和条形码序列的拷贝的延伸探针，由此为生物样本的核酸加标签。
如前文所例示，可将多个核酸引物附接至固体载体，其中所述多个中的核酸引物包括为所述多个中的核酸引物共有的通用引物序列。在此实施例中，可将第二多个核酸引物附接至固体载体，并且所述第二多个中的核酸引物可具有为所述第二多个中的核酸引物共有的第二通用引物序列。在此实施例中，与载体接触的多个不同核酸探针可包括与固体载体上的通用引物杂交的通用引物结合序列，如上文所述，并且不同核酸探针还可包括与第二通用引物序列杂交的第二通用引物结合序列。通用引物和通用引物结合位点的此配置对于通过桥式扩增扩增不同核酸探针可特别有用，其中所述第一和第二多个中的核酸引物被延伸。
通常，在核酸探针含有第一和第二通用引物结合位点时，其将被定位在探针末端。在一些实施例中，可能需要从核酸探针或从自探针产生的扩增子移除至少一个引物结合位点。因此，核酸探针可任选地包括在靶捕获序列与一个通用引物结合序列之间的裂解位点。在这种情形中，可进行裂解反应以分离通用引物结合位点与靶捕获序列。通常，探针(或其扩增子)中含有靶捕获序列的部分将可被附接至固体载体，导致从固体载体移除引物结合位点并保留靶捕获序列。因此，经裂解探针可用于杂交靶核酸并且经裂解探针可使用前文所述方法来延伸。
在一些实施例中，核酸探针将包括两个不同裂解位点。第一裂解位点将被定位于第一引物结合位点与探针的一或多个其它序列元件之间。第二裂解位点可被定位于第二引物结合位点与探针的一或多个其它序列元件之间。裂解位点可对不同裂解反应具有反应性，使得可选择性裂解每一位点而无需裂解其他位点。因此，第一裂解位点可在修饰探针之前(例如，在产生延伸探针之前)被裂解，由此将第一引物结合位点与保持附接至固体载体的一或多个其它序列元件分离。第二裂解位点可在修饰探针之后(例如，在产生延伸探针之后)裂解，由此释放经修饰探针用于后续检测。
或者，核酸探针可包括第一裂解位点，并且用于捕获或扩增核酸探针的引物可包括第二裂解位点。在此配置中，第一裂解位点可定位于第一引物结合位点与探针的一或多个其它序列元件之间，使得裂解将第一引物结合位点与探针的保持附接至固体载体的一或多个其它序列元件分离。同样，这个第一裂解步骤通常将在修饰探针之前(例如，在产生延伸探针之前)实施。可实施第二裂解步骤以在修饰探针之后(例如，在产生延伸探针之后)裂解第二裂解位点，由此释放经修饰探针用于后续检测。
上文两个实施例例示定位在核酸探针(或经修饰核酸探针)的附接点与探针(或经修饰探针)的含有信息的一或多个序列(例如空间条形码或靶序列)之间的裂解位点。因此，这个裂解位点可用于释放经修饰探针(例如延伸探针)以检测序列信息并测定生物样本中存在何种序列以及所述序列存在于样本中的何处。
在一些实施例中，在本文所述方法中与固体载体接触的一或多个探针可包括测序引物结合位点。因此，经修饰探针(例如延伸探针)可在测序技术中检测，所述测序技术包括使测序引物与测序引物结合位点杂交的步骤。测序引物结合位点可定位在探针中，使得探针的经修饰形式(例如延伸探针)的裂解将产生包括测序引物结合位点的经释放探针。测序引物结合位点可为通用测序引物结合位点，使得多个不同探针(例如具有不同条形码和/或靶序列)将具有相同测序引物结合位点。
本公开进一步提供以空间方式为生物样本的核酸加标签的方法，所述方法包括以下步骤：(a)在固体载体上提供珠粒阵列，其中将不同核酸探针附接至阵列中的不同珠粒，其中不同核酸探针各自包括条形码序列，其中各珠粒包括与固体载体上的其它珠粒不同的条形码序列，且其中不同核酸探针各自包括靶捕获序列；(b)在固体载体上进行解码器探针杂交反应以测定固体载体上的随机定位探针的条形码序列；(c)使生物样本与珠粒阵列接触；(d)使不同核酸探针与来自生物样本中靠近珠粒的部分的靶核酸杂交；和(e)延伸不同核酸探针以产生包括来自靶核酸的序列和条形码序列的延伸探针，由此为生物样本的核酸加标签。
应理解，固体载体或附接至固体载体的核酸的操作可使用珠粒作为固体载体来实施。可在实施所述操作之前或之后将珠粒附接至表面(例如孔阵列，如在来自伊路米那的BeadArrayTM中)。例如，核酸探针可在将珠粒分布于阵列上之前或之后捕获于珠粒上，核酸探针可在将珠粒分布于阵列上之前或之后经扩增以产生珠粒上的扩增子，等等。
实例I
使用伊路米那流动槽以空间方式为来自组织样品的mRNA加标签
生成含有有条形码寡-dT的簇、然后通过限制性酶消化之后测序揭示有条形码寡-dT的方法描述于图1中。通过寡聚物合成(集成DNA技术(Integrated DNA Technologies))制备含有单链有条形码寡-dA、P5’、P7、SBS3测序引物结合位点和BspHI限制性酶位点(显示于图1的上图中)的片段的库。条形码为27聚体并且是在合成期间随机生成。包括SBS3测序引物的结合位点用于通过测序来解码条形码。包括寡-dA段以在成簇和线性化之后生成寡dT位点。桥式扩增和成簇是根据标准簇化学(伊路米那TruSeq PE簇试剂盒v3 cBot P/N:15037931)在伊路米那GA流动槽上使用制造商推荐方案来实施。
在桥式扩增和成簇后，通过使用提供于TruSeq PE簇试剂盒中的甲酰胺基嘧啶DNA糖苷酶(Fpg)酶裂解P7引物中的8-氧代-G来使簇线性化。在这之后用200单位/mL BspH1(NEB目录号R0517L)在37℃下15min进行限制性酶消化，以从簇的P5接头锚定链移除P7’，以显露寡-dT段用于随后在mRNA存在下延伸。已测试在100-400U/mL范围内的酶浓度15或30min。条形码的解码是用SBS3测序引物来起始。
如图1的下图中所示，在条形码解码后使用簇中的寡-dT序列来捕获多A+RNA。有条形码cDNA是通过使用TruSeq RNA样品制备试剂盒(伊路米那P/N:15012997)和MMLV逆转录酶第1链cDNA合成试剂盒(挨皮森特(Epicentre)P/N:MM070150)根据制造商推荐的条件延伸簇的寡-dT链来产生。使用所捕获RNA作为模板。使用伊路米那的尿嘧啶特异性切除试剂(USER)(伊路米那的TruSeq PE簇试剂盒)从流动槽的P5序列释放有条形码cDNA，从而释放有条形码cDNA库用于在第二伊路米那流动槽上测序。
通过使线性化簇与Cy5标记的多A(24聚体)杂交来确认在用BspH1进行限制性酶消化后寡dT捕获序列的可用性，如图2的图A中所图解。简单来说，在限制性酶消化后，用0.1NNaOH处理簇并用HT2低盐缓冲液洗涤以移除流动槽上的第二链。随后，使500nM Cy5寡-dA(24聚体)以30μl/min速率流过线性化且变性的簇并在40℃下培育5min，随后成像。在其中存在含有寡dT的BODT-1库的GA流动槽的泳道2-7中检测Cy5标记的多A与寡dT的杂交(参见图2的图B中所示流动槽的图像)。如自流动槽图像(图B)和棒图(图C)可见，显示对照PhiX库(流动槽的泳道1和8)在Cy5信号中具有极低荧光。这些结果显示，可在簇中产生寡-dT位点，所述簇在线性化后可特异性结合至Cy5多A(24聚体)。
上述流动槽的使用3.2pM BODT-1库的测序度量法给出于图3中所示表中。在来自GA测序的21个瓦片中检测数百万个读数。在测序后，测定独特条形码的数目，如图4中所绘示。这是通过假定每个通滤器(passing filter，PF)读数都是条形码并测定每个泳道中的独特读数(条形码)数目来实施。在与PhiX对照库相比测序瓦片后，检测到介于5百万与11百万之间的独特有条形码簇。这些结果显示，有条形码寡-dT序列库的序列解码是可行的并生成数百万个独特条形码。
实例II
在伊路米那流动槽上的细胞粘附
在图案化流动槽(HiSeq X10流动槽，伊路米那)上捕获单细胞。所有试剂流动步骤都是使用蠕动泵或cBOT簇生成仪器(伊路米那)来进行。简单来说，使不含核酸酶的水在图案化流动槽的所有泳道上流动，之后使30-70K多D赖氨酸溶液(100μg/ml和20μg/ml)以100μl/min的流速流动8min。还测试热灭活胎牛血清(生命技术编号10082-139)作为粘着剂。将粘着剂在流动槽泳道上培育1hr，之后用1X PBS+0.5％普流尼克(Pluronic)F-68(生命技术编号24040-032)洗涤。然后，通过以下方式将细胞粘附至经涂布流动槽：使5至50个细胞/μl或约100–1000个细胞/泳道以100μl/min的速率流动，之后进行60min的培育步骤来结合细胞。用1X PBS/0.5％普流尼克以75μl/min的速率洗涤流动槽。如果细胞固定在流动槽上，在如上所述使细胞流动后使1％多聚甲醛(PFA)在流动槽上流动并培育15min，之后进行1XPBS/0.5％普流尼克洗涤步骤。移除流动槽并使用显微镜计数每个泳道的细胞数。
图5的图A显示在图案化流动槽上捕获的细胞的图像。图5的图B中显示的细胞计数数据确认，与BSA涂布的对照或未经粘着剂处理的对照相比，多D赖氨酸涂布的流动槽有助于细胞粘附。如图6中所示，粘附细胞可用1％PFA成功固定。
实例III
通过附接至凝胶表面的探针空间定位捕获靶mRNA
此实例描述在凝胶涂布的载玻片上产生多T探针的苔，将组织切片放置于多T探针的苔顶部上，从组织切片释放RNA，用多T探针捕获所释放的mRNA，逆转录以用Cy3标记多T探针，移除组织并对载玻片成像。
图7的图A显示用于产生附接至凝胶的探针的步骤和试剂的图解代表。简单来说，用无硅烷丙稀酰胺(SFA)涂布显微镜载玻片，附接P5和P7引物(参见美国专利申请公开第2011/0059865 A1号，其是通过引用并入本文中)，使具有多A序列和P5或P7互补序列的探针分别与P5和P7引物杂交，并延伸P5和P7引物以产生多T序列延伸物。通过使Cy5标记的多A寡核苷酸与经延伸引物杂交并使用Axon成像器使表面成像来进行质量控制步骤。
如图7的图B中所示，将组织切片放置于具有多T延伸引物的凝胶上。处理组织以释放mRNA并使所释放mRNA的多A尾与延伸引物的多T序列杂交。使用所捕获mRNA作为模板延伸多T序列并用Cy3选择性标记经延伸引物。从凝胶移除组织并对表面成像以检测Cy3荧光。
如图7的图像中所示，凝胶中靠近释放mRNA种类的组织区域的区域显现荧光，而未释放mRNA的区域在图像中显现较暗。由此，所捕获mRNA产生组织的指纹样图像。
实例IV
通过附接至BeadArrayTM表面的探针空间定位捕获靶mRNA
此实例描述将组织切片放置在具有多T探针的BeadArrayTM的顶部上，从组织切片释放RNA，用多T探针捕获所释放的mRNA，逆转录以用Cy5标记多T探针，移除组织并对BeadArrayTM成像。
如图8的图A中所示，将小鼠嗅觉器官组织切片放置在具有多T探针的BeadArrayTM上。处理组织以释放mRNA并且将所释放mRNA的多A尾与探针的多T序列杂交。使用所捕获的mRNA作为模板延伸多T序列并且用Cy5选择性标记延伸引物。从BeadArrayTM移除组织并对BeadArrayTM成像以检测Cy5荧光。
如图7的图B中所示，BeadArrayTM中靠近释放mRNA种类的组织区域的区域显现荧光，而未释放mRNA的区域在图像中显现较暗。由此，所捕获的mRNA产生组织的指纹样图像。
在本申请通篇中，已提到多个出版物、专利或专利申请。在本申请中这些出版物的全文的公开是通过引用并入本文中。
术语“包含”在本文中旨在为开放式的，不仅包括所列举的要素，而且进一步涵盖任何额外要素。
本文已描述多个实施例。但应理解，可进行多种修改。因此，其它实施例在以下权利要求书的范围内。
Claims (10)
1.一种以空间方式为生物样本的核酸加标签的方法，其包含：
(a)将不同核酸探针附接至固体载体以产生所述固体载体上的随机定位探针，其中所述不同核酸探针各自包含条形码序列，且其中所述随机定位探针各自包含与所述固体载体上的其它随机定位探针不同的条形码序列；
(b)在所述固体载体上进行核酸检测反应以测定所述固体载体上的所述随机定位探针的所述条形码序列；
(c)使生物样本与包含所述随机定位探针的所述固体载体接触；
(d)使所述随机定位探针与来自所述生物样本中靠近所述随机定位探针的部分的靶核酸杂交；和
(e)延伸所述随机定位探针以产生延伸探针，所述延伸探针包含所述条形码序列和来自所述靶核酸的序列，由此以空间方式为所述生物样本的所述核酸加标签。
2.根据权利要求1所述的方法，其中步骤(a)进一步包含扩增所述固体载体上的所述随机定位探针的每一个，以形成包含所述固体载体上的所述核酸探针的多个扩增子的簇。
3.根据权利要求2所述的方法，其中所述固体载体包含多个所述簇，并且所述多个簇的平均节距小于10微米。
4.根据权利要求2或3所述的方法，其中所述固体载体包含多个所述簇并且所述簇的平均面积小于100μm2。
5.根据前述权利要求中任一权利要求所述的方法，其中所述核酸检测反应包含测序反应。
6.根据权利要求1所述的方法，其中所述核酸检测反应包含解码器探针杂交反应。
7.根据前述权利要求中任一权利要求所述的方法，其中所述固体载体包含珠粒阵列，其中将所述不同核酸探针附接至所述阵列中的不同珠粒。
8.根据权利要求7所述的方法，其中步骤(a)包含将所述不同珠粒随机分布在所述固体载体上。
9.根据权利要求7所述的方法，其中所述固体载体包含尺寸容纳不超过所述多个珠粒中的单一珠粒的孔。
10.根据权利要求9所述的方法，其中将所述不同珠粒附接至不同条形码序列并且其中不同条形码序列的数目超过孔的数目。
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