限制性酶切位点被设计成的扩增引物可用于将限制性核酸内切酶识别位点引入扩增基因的末端(Scharf et al., 1986)。Medlin 等人 (1988) 扩增引物中的限制性位点可用于在扩增基因的末端引入限制性内切酶识别位点(Scharf 等人,1986 年)。Medlin 等人(1988 年 使用这种方法从低等真核生物中克隆 18 个 S rRNA 基因。通常每个引物中都包含不同的限制性位点,这允许使用强制克隆程序。强制克隆具有明显的优势,即载体的多接头在两个不同的识别位点被切割后,不能在分子内反应中重新连接到自身身上。因此,高比例的转化体将包含可预测方向的克隆 PCR 产物。 限制性酶切位点被设计成的扩增引物可用于将限制性核酸内切酶识别位点引入扩增基因的末端(Scharf et al.,1986)。Medlin 等人(1988)使用这种方法从低等真核生物中克隆 18 个 S rRNA 基因。通常每个引物中都包含不同的限制性位点,这允许使用强制克隆程序。使用这种方法从低等真核生物中克隆 18 个 S rRNA 基因。通常每个引物中都包含不同的限制性位点,这允许使用强制克隆程序。强制克隆具有明显的优势,即载体的多接头在两个不同的识别位点被切割后,不能在分子内反应中重新连接到自身身上。因此,高比例的转化体将包含可预测方向的克隆 PCR 产物。 “强制克隆”技术的缺点是扩增的基因可能在内部限制性位点被切割。如果内部限制性位点离末端足够远,则从电泳凝胶上的迁移率变化或两个条带的出现中可以明显看出内部切割。如果多个条带很明显,则可以克隆每个条带以获得完整的基因。 "强制克隆 "技术的缺点是扩增的基因可能在内部限制性位点被切割。如果内部限制性位点离末端足够远,则从电泳凝胶上的迁移率变化或两个条带的出现中可以明显看出内部切割。如果多个条带很明显,则可以克隆每个条带以获得完整的基因。
强制克隆技术的第二个缺点是,位于 DNA 分子末端附近的限制性位点与限制性核酸内切酶的反应效率不如内部位点。例如,HpaII 和 MnoI 需要至少一个碱基位于5^(')5^{\prime}裂解识别序列的结束 (Baumstark等人,1979)。 强制克隆技术的第二个缺点是,位于 DNA 分子末端附近的限制性位点与限制性核酸内切酶的反应效率不如内部位点。例如,HpaII 和 MnoI 需要至少一个碱基位于 5^(')5^{\prime} 末端的识别序列进行裂解(Baumstark 等人,1979 年)。由于涉及的长度变化很小,因此不能使用迁移率偏移直接监测末端限制性位点消化的效率。然而,在这些情况下,连接测定可用于确定消化效率。在该检测中,消化的 PCR 产物与标准 DNA(例如噬菌体 lambda DNA)的相容限制性内切物混合,并使用 T4 DNA 连接酶连接。如果存在适当的粘性末端,则扩增的 DNA 分子将转化为异质大小的高分子量连接产物。否则,扩增产物的迁移率将不受连接的影响。 裂解识别序列的结束 (Baumstark 等人,1979)。由于涉及的长度变化很小,因此不能使用迁移率偏移直接监测末端限制性位点消化的效率。然而,在这些情况下,连接测定可用于确定消化效率。在该检测中,消化的 PCR 产物与标准 DNA(例如噬菌体 lambda DNA)的相容限制性内切物混合,并使用 T4 DNA 连接酶。如果存在适当的粘性末端,则扩增的 DNA 分子将转化为异质大小的高分子量连接产物。 避免内部限制性位点困难的一种方法是将“罕见”限制性位点掺入引物中。不幸的是,只有一种合适的酶 Not I 可用。这种方法尚未被证明是可行的,因为它与平末端连接相比没有优势,并且在消化 Not I 位点时遇到了困难(Tom Schmidt,个人通信)。 避免内部限制性位点困难的一种方法是将 "罕见 "限制性位点掺入引物中。不幸的是,只有一种合适的酶 Not I 可用。这种方法尚未被证明是可行的,因为它与平末端连接相比没有优势,并且在消化 Not I 位点时遇到了困难(Tom Schmidt,个人通信)。
2. 平末端克隆技术 2.平末端克隆技术
平末端连接程序是目前克隆 PCR 反应产物最实用的方法。平淡的双链 DNA 分子5^(')5^{\prime}磷酸盐和 3' 羟基可以通过 T4 DNA 连接酶连接。该反应不如“粘性末端”连接有效,可能是因为底物的非共价氢键使“粘性末端”反应基本上是一级的。 平淡的双链 DNA 分子 5^(')5^{\prime} 磷酸盐和 3' 羟基可以通过 T4 DNA 连接酶连接。该反应不如 "粘性末端 "连接有效,可能是因为底物的非共价氢键使 "粘性末端 "反应基本上是一级的。
尽管双链 PCR 产物可以作为直接测序的模板(Wrischnik等人,1987 年;Wong et al., 1987;Saiki等人,1988b),它们通常不会产生从双链质粒或单链DNA分子的测序中获得的那么多的可用序列信息(Engelke等人,1988;Gyllensten 和 Erlich,1988 年;Stoflet et al., 1988;Higuchi 和 Ochman,1989 年;Mitchell 和 Merrill,1989 年)。这显然是由于线性双链模板的快速再结合引起的,其中排除了测序引物。拓扑约束显然起到了减慢变性质粒 DNA 的重新结合的作用,因此使其比线性双链模板更容易测序。 尽管双链 PCR 产物可以作为直接测序的模板(Wrischnik 等人,1987 年;Wong et al.,1987 年;Saiki 等人,1988 年),它们通常不会产生从双链质粒或单链 DNA 分子的测序中获得的那么多的可用序列信息(Engelke 等人,1988 年;Gyllensten 和 Erlich,1988 年;Stoflet et al.拓扑约束显然起到了减慢变性质粒 DNA 的重新结合的作用,因此使其比线性双链模板更容易测序。
单链 DNA 产物可以使用“不对称引发”方法通过聚合酶链反应合成。在“不对称引物”中,其中一个寡核苷酸引物的浓度显著降低,结果是该引物在早期复制周期中耗尽。在仅存在一种引物的情况下,扩增将不再呈指数级进行。相反,单链副本将由过量提供的引物制成。 单链 DNA 产物可以使用 "不对称引发 "方法通过聚合酶链反应合成。在 "不对称引物 "中,其中一个寡核苷酸引物的浓度显著降低,结果是该引物在早期复制周期中耗尽。在仅存在一种引物的情况下,扩增将不再呈指数级进行。相反,单链副本将由过量提供的引物制成。
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杰弗里斯,AJ,威尔逊,V.,诺伊曼,R.和凯特,J.(1988)。通过聚合酶链反应扩增人类小卫星:实现单细胞的 DNA 指纹图谱。核酸研究 16:10953-10971。 Kwoz, S. 和 Higuchi, R. (1989)。避免 PCR 假阳性。自然 339:237。 李,H.,吉伦斯滕,UB,崔,X.,Saiki,RK,Erlich,H.和Arnheim,N.(1988)。扩增和分析单个人类精子和二倍体细胞中的 DNA 序列。自然 335:14-417。 Lo, Y., Mehal, WZ 和 Fleming, KA (1988)。假阳性结果和聚合酶链反应。柳叶刀 2:679。 李,H.,吉伦斯滕,UB,崔,X.,Saiki,RK,Erlich,H.和Arnheim,N.(1988)。扩增和分析单个人类精子和二倍体细胞中的 DNA 序列。自然 335:14-417。 Lo,Y.,Mehal,WZ 和 Fleming,KA (1988)。假阳性结果和聚合酶链反应。柳叶刀 2:679。 Medlin, L., Elwood, HJ, Stickel, S. 和 Sogin, ML(1988 年)。酶促扩增的真核生物 16S 样 rRNA 编码区的表征。基因 71:491-499。梅辛,J.(1983 年)。用于克隆的新 M13 载体。方法 Enzymol,101:20-78。 米切尔,LG 和梅里尔,CR(1989 年)。聚合酶链反应后用于双脱氧测序的单链 DNA 的亲和生成。肛门。生物化学。178: 239-242. Mullis, KB和Faloona, F.(1987)。通过聚合酶催化的链式反应在体外特异性合成 DNA。方法 Enzymol。155: 335-350. 米切尔,LG 和梅里尔,CR(1989 年)。聚合酶链式反应后用于双脱氧测序的单链 DNA 的亲和生成。Mullis, KB 和 Faloona, F.(1987)。通过聚合酶催化的链式反应在体外特异性合成 DNA。方法 Enzymol。 奥尔森,GJ(1988 年)。使用核糖体 RNA 进行系统发育分析。方法 Enzymol。164:793812 Olsen, GJ、Lane, DJ、Giovannoni, SJ 和 Pace, NR (1986)。微生物生态学和进化:核糖体 RNA 方法。微生物学年鉴。40: 337-365. 奥尔森,GJ(1988 年)。使用核糖体 RNA 进行系统发育分析。方法 Enzymol。164:793812 Olsen,GJ、Lane,DJ、Giovannoni,SJ 和 Pace,NR (1986 年)。微生物生态学和进化:核糖体 RNA 方法。微生物学年鉴。40: 337-365. 佩斯,NR,斯塔尔,DA,莱恩,DJ 和奥尔森,GJ(1986 年)。通过核糖体 RNA 序列分析天然微生物种群。Adv. Microb.生态学 9:1-55。 Saiki,RK,Scharf,S.,Faloona,F.,Mullis,KB,Horn,GT,Erlich,HA和Arnheim,NA(1985)。β-珠蛋白基因组序列的酶促扩增和限制性位点分析用于诊断镰状细胞性贫血。科学 230:1350-1354。 佩斯,NR,斯塔尔,DA,莱恩,DJ 和奥尔森,GJ(1986 年)。通过核糖体 RNA 序列分析天然微生物种群。Adv. Microb.生态学 9:1-55。 Saiki,RK,Scharf,S.,Faloona,F.,Mullis,KB,Horn,GT,Erlich,HA 和Arheim,NA(1985 年)。Mullis,KB,Horn,GT,Erlich,HA和Arnheim,NA(1985)。β-珠蛋白基因组序列的酶促扩增和限制性位点分析用于诊断镰状细胞性贫血。 Saiki, RK, Bugawan, TL, Mullis, KB和Erlich, HA (1986)。使用等位基因特异性寡核苷酸探针分析酶促扩增的 β-珠蛋白和 HLA-DQalpha DNA。自然 324:163-166。 Saiki,RK,Gelfand,DH,Stoffel,S.,Scharf,SJ,Higuchi,R.,Horn,GT,Mullis,KB和Erich,HA(1988a)。使用热稳定 DNA 聚合酶对 DNA 进行引物定向酶促扩增。科学 239:487-491。 Saiki,RK,Bugawan,TL,Mullis,KB和Erlich,HA (1986)。使用等位基因特异性寡核苷酸探针分析酶促扩增的β-珠蛋白和HLA-DQalpha DNA。自然 324:163-166。Scharf,SJ,Higuchi,R.,Horn,GT,Mullis,KB和Erich,HA(1988a)。使用热稳定 DNA 聚合酶对 DNA 进行引物定向酶促扩增。科学 239:487-491。 Saiki, R. K., Gyllensten, U. B. 和 Erlich, H.A. (1988b)。聚合酶链反应。在:基因组分析-一种实用方法(Davies, KE,编辑),第 141-152 页。华盛顿特区:IRL Press. Sanger, F.、Nicklen, S. 和 Coulson, AR (1977)。使用链终止抑制剂进行 DNA 测序。美国国家科学院院刊 74:5463-5467。 Saiki, R. K., Gyllensten, U. B. 和 Erlich, H.A. (1988B)。聚合酶链反应。在:基因组分析-一种实用方法(Davies, KE,编辑),第 141-152 页。华盛顿特区:IRL Press.Sanger, F.、Nicklen, S. 和 Coulson, AR (1977)。使用链终止抑制剂进行 DNA 测序。美国国家科学院院刊 74:5463-5467。 Scharf, SJ, Horn, GT 和 Erlich, HA (1986)。扩增基因组序列的直接克隆和序列分析。科学 233:1076-1078。 Shuldiner, AR, Nirula, A. 和 Roth, J. (1989)。来自密切相关靶序列的 PCR 的杂交 DNA 伪影。核酸研究17:4409。 Scharf, SJ, Horn, GT 和 Erlich, HA (1986)。扩增基因组序列的直接克隆和序列分析。科学 233:1076-1078。 Shuldiner, AR, Nirula, A. 和 Roth, J. (1989)。来自密切相关靶序列的 PCR 的杂交 DNA 伪影。核酸研究 17:4409。 Stackebrandt, E., Ludwig, W., Schubert, W., Klink, F., Schesner, H., Roggentin, T. 和 Hirsch, P. (1984)。出芽无肽聚糖真细菌早期进化起源的分子遗传学证据。自然 307:735-737。无肽聚糖真细菌。自然 307:735-737。 Stackebrandt, E., Ludwig, W., Schubert, W., Klink, F., Schesner, H., Roggentin, T. 和 Hirsch, P. (1984)。出芽无肽聚糖真细菌早期进化起源的分子遗传学证据。自然 307:735-737。 斯塔尔,DA,莱恩,DJ,奥尔森,GJ 和佩斯,NR (1985). 通过 55 个 rRNA 序列表征黄石温泉微生物群落。Appl. Environ.微生物学。45:1379-1384 斯塔尔,DA,弗莱舍,B.,曼斯菲尔德,HW 和蒙哥马利,L.(1988 年)。使用基于系统发育的杂交探针进行反刍动物微生物生态学研究。Appl. Environ.Microbid 54:1079-1084 斯塔尔,DA,莱恩,DJ,奥尔森,GJ 和佩斯,NR (1985).通过 55 个 rRNA 序列表征黄石温泉微生物群落。Appl. Environ.微生物学。45:1379-1384 斯塔尔,DA,弗莱舍,B.,曼斯菲尔德,HW 和蒙哥马利,L.(1988 年)。使用基于系统发育的杂交探针进行反刍动物微生物生态学研究。Appl. Environ.Microbid 54:1079-1084。 Stoflet,ES,Koeberl,DD,Sarker,G.和Sommer,SS(1988)。使用转录本测序进行基因组扩增。科学 239:490-494。 Vogelstein, B. 和 Gillespie, D. (1979)。从琼脂糖中制备和分析纯化 DNA。国家科学院院刊76:615-619。 Wang, AM 和 Mark, DF (1990)。定量 PCR。在:PCR 方案-方法和应用指南(Innis, MA, Gelfand, DH, Sninsky, JJ 和 White, TJ 编辑),第 70-75 页。圣地亚哥:学术出版社。Wayne, LG, Brenner, DJ, Colwell, RR, Grimont, P. A. D., Kandler, O., Krichevsky, Wang, AM 和 Mark, DF (1990)。定量 PCR。在:PCR 方案-方法和应用指南(Innis, MA, Gelfand, DH, Sninsky, JJ 和 White, TJ 编辑),第 70-75 页。圣地亚哥:学术出版社。Wayne, LG, Brenner, DJ, Colwell, RR, Grimont, P. MI, Moore, LH, Moore, W. E. C., Murray, R. G. E., Stackebrandt, E., Starr, MP 和 Truper, HG (1987)。关于 Whatley, J. M. 和 Watley, Rolution 的方法 和解特设委员会的报告。微生物学。修订版 51:221-271 Woese, CR, Guttell, RR, Gupta, R. 和 Knoler, H. Fibe 酸。微生物学。修订版 47:621-669。Saiki, R. K., Higuchi, R. G., Erlich, H. A. 和 Kazazian, H. H., Jr Nong, C., Dowling, C. E., Saiki, R. K., Higuchi, RG, Ers 使用直接基因组测序 (1987)。β-地中海贫血的特征化扩增了单拷贝 DNA。自然 330:384 (1987)。人类基因组 DNA 中的长度突变:酶扩增 DNA 的直接测序。核酸研究15:529-542。 Woese, CR, Guttell, RR, Gupta, R. 和 Knoler, H. Fibe 酸。微生物学。修订版 47:621-669。Higuchi, RG, Ers 使用直接基因组测序 (1987)。β-地中海贫血的特征化扩增了单拷贝 DNA。自然 330:384 (1987)。人类基因组 DNA 中的长度突变:酶扩增 DNA 的直接测序。核酸研究 15:529-542。 Zehr, J. 和 McRenolds, LA (1989)。使用简并寡核苷酸扩增来自海洋蓝细菌 Trichodesmium thiebautii 的 nifH 基因。Appl. Environ.微生物学。55: 2522-2526. Zehr, J. 和 McRenolds, LA (1989)。使用简并寡核苷酸扩增来自海洋蓝细菌 Trichodesmium thiebautii 的 nifH 基因。Appl. Environ.微生物学。
8
核酸探针的开发与应用
David A. Stahl 和 Rudolf Amann伊利诺伊大学兽医病理生物学系,2001 South Lincoln Avenue, Urbana, IL 61801, USA 伊利诺伊大学兽医病理生物学系,2001 South Lincoln Avenue,Urbana, IL 61801, USA
Another aspect of nucleic acid probe technology concerns detection systems. Although all early studies used (and most contemporary studies still use) radioactive probes, concerns of safety and versatility have fostered the development of a variety of non-radioactive detection systems. These techniques are essential adjuncts to hybridization. However, it would require an entire volume to describe and properly elaborate upon the various existing and evolving approaches for the detection of nucleic acid hybrids; certainly beyond the scope of a single chapter. As a com- 核酸探针技术的另一个方面涉及检测系统。虽然所有早期研究都使用(而且大多数当代研究仍在使用)放射性探针,但对安全性和多功能性的关注促进了各种非放射性检测系统的发展。这些技术是杂交的重要辅助手段。然而,要描述并适当阐述现有的和不断发展的各种核酸杂交检测方法,需要整整一卷书的篇幅;当然,这也超出了一章的范围。作为一
B. GENERAL CONSIDERATIONS FOR NUCLEIC ACID HYBRIDIZATION B.核酸杂交的一般注意事项
The use of determinative nucleic acid hybridization (nucleic acid probes) must take into account the characteristic features of formation and breakdown of a specific double-helix. Most important is the balance between specificity of the reaction and detection of reaction product. The latter is largely dependent upon the detection system used (see below). Specificity and sensitivity are usually competing entities and must be balanced according to the determinative requirements. As an introduction to the applied sections, an overview of fundamental and practical aspects of nucleic acid hybridization is offered. The following is a collection of common terms and definitions applied to studies and descriptions of hybridization reactions (adapted from Lathe, 1985). 使用确定性核酸杂交(核酸探针)必须考虑到特定双螺旋形成和分解的特征。最重要的是在反应的特异性和反应产物的检测之间取得平衡。后者在很大程度上取决于所使用的检测系统(见下文)。特异性和灵敏度通常是相互竞争的,必须根据决定性要求加以平衡。作为应用部分的引言,本文概述了核酸杂交的基本和实用方面。以下是应用于杂交反应研究和描述的常用术语和定义集(改编自 Lathe, 1985)。
Complement. With reference to the Watson-Crick canonical base pairs, A pairs with T (or U ) and G pairs with C . The canonical base pairs are also referred to as complementary or as complements of each other (i.e. A is complementary to T). 互补。关于沃森-克里克碱基对,A 与 T(或 U)成对,G 与 C 成对。典型碱基对也被称为互补碱基对或互补碱基对(如 A 与 T 互补)。
Probe. A single strand of DNA or RNA, intended to hybridize with a complementary sequence (e.g. the coding sequence for a particular protein or stable RNA) in order to detect that sequence. Probes are synthesized both enzymatically and chemically (see below). 探针。DNA 或 RNA 单链,用于与互补序列(如特定蛋白质或稳定 RNA 的编码序列)杂交,以检测该序列。探针可通过酶法和化学法合成(见下文)。
Target. The target sequence for a probe is the complementary sequence to which the probe is designed to hybridize. 目标。探针的靶序列是指探针与之杂交的互补序列。
Match. ‘Match’ is generally used to refer to canonical pairing between probe and target at a specified position. ‘Match’ is also used to describe complete complementarity between probe and target sequences. 匹配。匹配 "一般指探针和目标物在指定位置上的典型配对。匹配 "也用于描述探针和目标序列之间的完全互补性。
Mismatch. A mismatch at a particular position occurs when the nucleotide in the probe does not complement the nucleotide at the same position in the target sequence (non-canonical pair). 错配。当探针中的核苷酸与目标序列中同一位置的核苷酸不互补(非经典配对)时,就会出现特定位置的错配。
Homology. The percentage homology ( hh ) between two sequences of identical length ( nn ) and containing a given number of mismatches ( mm ) is given by the expression h=100(n-m)//nh=100(n-m) / n. Similarity is now considered the semantically correct term for this value. However, for the purposes of this chapter the terms homology and similarity are used interchangeably. 同源性。两个长度相同( nn )且包含一定数量错配( mm )的序列之间的同源性百分比( hh )由表达式 h=100(n-m)//nh=100(n-m) / n 给出。相似性现在被认为是该值在语义上的正确术语。不过,在本章中,同源性和相似性这两个术语可以互换使用。
Stringency. Stringency describes the conditions of hybridization or the hybridization wash step. The greater the stringency of the hybridization or subsequent wash step (see below), the fewer, if any, mismatches remain in the duplex structure. 严格度。严格程度是指杂交或杂交洗涤步骤的条件。杂交或后续清洗步骤(见下文)的严格程度越高,双链结构中残留的错配(如果有的话)就越少。
Oligonucleotide. A short single strand of DNA (oligodeoxyribonucleotide) usually 6-50 nucleotides in length. 寡核苷酸。通常长度为 6-50 个核苷酸的 DNA 短单链(寡脱氧核苷酸)。
Duplex structure. Double-helix structure composed of antiparallel strands of DNA-DNA, DNA-RNA or RNA-RNA held in association by specific hydrogen bonding between the nucleotides comprising each strand. 双链结构。由 DNA-DNA、DNA-RNA 或 RNA-RNA 的反平行链组成的双螺旋结构,每条链上的核苷酸之间通过特定的氢键结合在一起。
(i) Melting point (i) 熔点
For determinative studies the single most important feature defining a given double-helix structure is its melting point (T_(m))\left(T_{m}\right). In this respect, the double helix structure has been likened to a one-dimensional crystalline lattice (Marmur and Doty, 1959). The breakdown of the helix (or crystal) occurs at a specific melting temperature and propagation of the helix (or growth of the crystal) follows a nucleation event (formation of a seed crystal or short duplex structure). However, with the exception of homopolymers (e.g. poly U-poly A), the double-helix structure does not dissociate (melt) completely at a specific temperature. Rather, denaturation proceeds over a temperature range. 对于确定性研究而言,定义特定双螺旋结构的唯一最重要特征是其熔点 (T_(m))\left(T_{m}\right) 。在这方面,双螺旋结构被比作一维晶格(Marmur 和 Doty,1959 年)。螺旋体(或晶体)在特定的熔化温度下分解,螺旋体的传播(或晶体的生长)遵循成核事件(种子晶体或短双链结构的形成)。然而,除均聚物(如聚 U 聚 A)外,双螺旋结构不会在特定温度下完全解离(熔化)。相反,变性是在一定温度范围内进行的。
The T_(m)T_{\mathrm{m}} is defined as the temperature corresponding to the mid-point in the transition from helix to random coil (frequently measured optically by monitoring the characteristic hyperchromatic shift of denaturation). In addition, the temperature range over which transition from helix to T_(m)T_{\mathrm{m}} 被定义为从螺旋过渡到无规线圈的中点所对应的温度(通常通过监测变性的特征性高色移进行光学测量)。此外,从螺旋过渡到无规线圈的温度范围为
random coil occurs varies significantly, depending upon length, nucleotide composition and ionic strength. The zipping (renaturation) or unzipping (denaturation) of the helix is influenced by nucleotide composition in the vicinity of base-pair formation or denaturation (GC-rich versus AT-rich regions). This effect and its influence on the observed transition temperature is referred to as cooperativity. At low ionic strength local compositional differences are suggested to contribute to a broadening of the transition range (Dove and Davidson, 1962). 由于长度、核苷酸组成和离子强度的不同,随机螺旋的形成也有很大差异。螺旋的拉链(再饱和)或解压缩(变性)会受到碱基对形成或变性附近核苷酸组成(富含 GC 与富含 AT 的区域)的影响。这种效应及其对观察到的转变温度的影响被称为合作性。在低离子强度下,局部成分差异被认为有助于扩大转变范围(Dove 和 Davidson,1962 年)。
(ii) T_(m)T_{m} versus T_(d)T_{d} versus T_(w)T_{w} (ii) T_(m)T_{m} 对 T_(d)T_{d} 对 T_(w)T_{w}
To this point, discussion has been restricted to long double-helix structures (greater than about 500 nucleotides in length). The temperature mid-point of the helix-to-random coil transition is the same for the formation or the breakdown of long duplexes. This is not true for oligonucleotides in duplex structure (e.g. bound to immobilized DNA). The dissociation temperatures T_(d)T_{\mathrm{d}} of short oligonucleotide hybrids, unlike longer complementary structures, is concentration dependent. Thus, under non-equilibrium conditions (such as temperature-dependent dissociation of membrane-bound probe) the observed mid-point in dissociation may be lower than a more thermodynamically rigorous determination of T_(m)T_{\mathrm{m}}. Another practical convention is the use of T_(w)T_{w}. This refers to the optimal temperature for washing at a specified monovalent cation concentration. In practice the T_(m)T_{\mathrm{m}} (or T_(d)T_{\mathrm{d}} ) and T_(w)T_{w} are frequently treated as equivalent. 到目前为止,讨论仅限于长双螺旋结构(长度超过约 500 个核苷酸)。从螺旋到随机线圈转变的温度中点对于长双链的形成或分解都是相同的。但双链结构中的寡核苷酸(如与固定 DNA 结合)则不然。与较长的互补结构不同,短寡核苷酸杂交体的解离温度 T_(d)T_{\mathrm{d}} 与浓度有关。因此,在非平衡条件下(如膜结合探针的解离与温度有关),观察到的解离中点可能低于更严格的热力学测定 T_(m)T_{\mathrm{m}} 。另一个实用惯例是使用 T_(w)T_{w} 。这是指在特定单价阳离子浓度下的最佳洗涤温度。在实践中, T_(m)T_{\mathrm{m}} (或 T_(d)T_{\mathrm{d}} )和 T_(w)T_{w} 经常被视为等价物。
(iii) Estimation of T_(m)T_{m} (iii) T_(m)T_{m} 的估算
1. DNA probes of greater than 50 nucleotides 1.超过 50 个核苷酸的 DNA 探针
The basic parameters for estimating the T_(m)T_{\mathrm{m}} of a given double-helix structure were established in the early 1960s and 1970s. Empirical relationships describing their contributions to T_(m)T_{\mathrm{m}} are summarized below. These relationships were derived from relatively large double-helix structures and so are of less value in predicting the behavior of short duplexes. However, slight modifications offer reasonable approximations of shorter duplex structure stability (see below). The following provides the reader with a rationalization for the equations used for estimating T_(m)T_{\mathrm{m}} and also a better feeling for the relative importance of those parameters that are routinely adjusted in nucleic acid hybridization reactions. 用于估算给定双螺旋结构的 T_(m)T_{\mathrm{m}} 的基本参数是在 20 世纪 60 年代初和 70 年代确立的。下文总结了描述它们对 T_(m)T_{\mathrm{m}} 贡献的经验关系。这些关系是从相对较大的双螺旋结构中推导出来的,因此对预测短双链的行为价值较低。不过,稍加修改就可以合理地近似预测较短双链结构的稳定性(见下文)。以下内容为读者提供了估算 T_(m)T_{\mathrm{m}} 所用方程的合理性,同时也让读者更好地了解核酸杂交反应中常规调整参数的相对重要性。
Ionic strength. The T_(m)T_{\mathrm{m}} (in degrees Celsius) of DNA increases linearly with the logarithm of the ionic strength and is independent of the base ratio of the DNA (Dove and Davidson, 1962; Schildkraut and Lifson, 1965): 离子强度。DNA 的 T_(m)T_{\mathrm{m}} (摄氏度)随离子强度的对数线性增加,与 DNA 的碱基比率无关(Dove 和 Davidson,1962 年;Schildkraut 和 Lifson,1965 年):
where M_(1)M_{1} and M_(2)M_{2} are the respective ionic strengths of the two solutions. 其中 M_(1)M_{1} 和 M_(2)M_{2} 分别是两种溶液的离子强度。 G+CG+C content. The effect of base composition on the thermal denaturation of DNA was early observed and has served as the basis for determining the %(G+C)\%(\mathrm{G}+\mathrm{C}) content of microorganisms (Marmur and Doty, 1959): G+CG+C 含量。人们很早就观察到碱基组成对 DNA 热变性的影响,并以此为基础确定微生物的 %(G+C)\%(\mathrm{G}+\mathrm{C}) 含量(Marmur 和 Doty,1959 年):
The above relationship was derived for solutions of DNA in 1xx1 \times SSC (standard saline citrate (SSC) contains 0.15 M sodium chloride and 0.015 M trisodium citrate). 上述关系是针对 DNA 在 1xx1 \times SSC(标准柠檬酸盐(SSC)含有 0.15 M 氯化钠和 0.015 M 柠檬酸三钠)中的溶液得出的。
The first equation combining the observed dependence of T_(m)T_{m} on both the salt concentration (M)(M) and the %(G+C)\%(G+C) was formulated by Schildkraut and Lifson (1965): Schildkraut 和 Lifson(1965 年)提出了第一个方程,将观察到的 T_(m)T_{m} 对盐浓度 (M)(M) 和 %(G+C)\%(G+C) 的依赖性结合起来:
Percentage of mismatched base pairs. A 1%1 \% base pairing mismatch corresponds to about a 1-1.5^(@)C1-1.5^{\circ} \mathrm{C} decrease in T_(m)T_{\mathrm{m}} (Bonner et al., 1973; Ausubel et al., 1987). The rate of DNA reassociation is approximately halved (at the optimum temperature for reassociation) for every 10^(@)C10^{\circ} \mathrm{C} reduction in T_(m)T_{\mathrm{m}} (Bonner et al., 1973). 错配碱基对的百分比。 1%1 \% 碱基配对错配大约相当于 T_(m)T_{\mathrm{m}} 减少 1-1.5^(@)C1-1.5^{\circ} \mathrm{C} (Bonner 等人,1973 年;Ausubel 等人,1987 年)。 T_(m)T_{\mathrm{m}} 每减少 10^(@)C10^{\circ} \mathrm{C} ,DNA 的重新结合率大约减半(在重新结合的最佳温度下)(Bonner 等人,1973 年)。
Influence of formamide. The inclusion of formamide lowers the melting point of the double helix. For every 1%1 \% increase in the concentration 甲酰胺的影响。甲酰胺会降低双螺旋的熔点。甲酰胺浓度每增加 1%1 \% 1^(')1^{\prime} of formamide the T_(m)T_{\mathrm{m}} is reduced by about 0.7^(@)C0.7^{\circ} \mathrm{C} (McConaughy et al., 1969). Therefore, by adding formamide to the hybridization solution it is possible to perform the hybridization at a lower temperature without loss of high stringency (mismatch discrimination). This not only increases the life of nucleic acid by eliminating degradation (e.g. strand scission, depurination) but also avoids the loss of membrane-bound nucleic acid at high temperatures. 如果加入甲酰胺 1^(')1^{\prime} , T_(m)T_{\mathrm{m}} 会降低约 0.7^(@)C0.7^{\circ} \mathrm{C} (McConaughy 等人,1969 年)。因此,通过在杂交溶液中加入甲酰胺,可以在较低温度下进行杂交,而不会失去高严格性(错配识别)。这不仅通过消除降解(如链裂解、去质化)延长了核酸的寿命,还避免了膜结合核酸在高温下的损失。
Effect of chain length. A correction for probe length nn must be subtracted from the estimation of T_(m)T_{\mathrm{m}} for probes less than about 500 nucleotides in length (Crothers et al., 1965). The following corrections have been used. 链长的影响。对于长度小于 500 个核苷酸的探针,在估算 T_(m)T_{\mathrm{m}} 时必须减去探针长度 nn 的校正(Crothers et al.)我们采用了以下校正方法。
no correction for n > 500n>500 nucleotides; 不修正 n > 500n>500 核苷酸;
reduction by 500//n500 / n or 600//n600 / n for probes 50-50050-500 nucleotides in length (Meinkoth and Wahl, 1984; Ausubel et al., 1987); 对于长度为 50-50050-500 核苷酸的探针,减少 500//n500 / n 或 600//n600 / n (Meinkoth 和 Wahl,1984 年;Ausubel 等人,1987 年);
reduction by 675/n for probes 50-100 nucleotides in length (Davis et al., 1986). 对于长度为 50-100 个核苷酸的探针来说,该值减少了 675/n(Davis 等人,1986 年)。
These length corrections are commonly included in equations used for estimating the T_(m)T_{\mathrm{m}} of shorter probes (Crothers et al., 1965; Britten et al., 1974). 这些长度修正通常包含在用于估计较短探针的 T_(m)T_{\mathrm{m}} 的公式中(Crothers 等人,1965 年;Britten 等人,1974 年)。
On the basis of the above primarily empirical observations, a number of equations have been formulated for approximating T_(m)T_{m}. Only those in reasonably general use will be presented here. The first relationship was formulated by Frank-Kamenetskii (1971) by simplifying an equation of Owen et al. (1969). 在上述主要是经验观察的基础上,我们提出了许多用于逼近 T_(m)T_{m} 的方程。这里只介绍那些比较常用的方程。第一个关系式是 Frank-Kamenetskii(1971 年)通过简化 Owen 等人(1969 年)的方程式而提出的。
This formula incorporates the %(G+C)\%(\mathrm{G}+\mathrm{C}) of a DNA probe and the concentration of monovalent cations (usually Na^(+)\mathrm{Na}^{+}) for estimating the melting point of the duplex. It has no correction for the average length of the probe and is only valid for probes of about 500 nucleotides (an average length of nick-translated or sheared DNA). 该公式包含 DNA 探针的 %(G+C)\%(\mathrm{G}+\mathrm{C}) 和单价阳离子的浓度(通常为 Na^(+)\mathrm{Na}^{+} ),用于估算双链的熔点。它没有对探针的平均长度进行修正,只适用于大约 500 个核苷酸的探针(缺口翻译或剪切 DNA 的平均长度)。
Equation (5) (below) has been used for probes longer than 50 nucleotides and takes into account the %(G+C)\%(\mathrm{G}+\mathrm{C}) content, concentration MM of monovalent cations, length nn of the probe and the percent formamide ( w//v\mathrm{w} / \mathrm{v} ) in the hybridization solution (Meinkoth and Wahl, 1984). 等式 (5) (如下)适用于长度超过 50 个核苷酸的探针,并考虑了杂交溶液中的 %(G+C)\%(\mathrm{G}+\mathrm{C}) 含量、单价阳离子浓度 MM 、探针长度 nn 和甲酰胺 ( w//v\mathrm{w} / \mathrm{v} ) 百分比(Meinkoth 和 Wahl,1984 年)。 T_(m)=81.5+16.6 log M+0.41[%(G+C)]-(500)/(n-0.61(%" formamide "))T_{\mathrm{m}}=81.5+16.6 \log M+0.41[\%(\mathrm{G}+\mathrm{C})]-\frac{500}{n-0.61(\% \text { formamide })}
Figure 8.1 ( T_(m)T_{\mathrm{m}} versus salt versus GC plot) compares the melting points predicted by these equations for DNA probes of 35,50 and 65%(G+C)65 \%(\mathrm{G}+\mathrm{C}) in varying concentrations of monovalent cations (average probe length of 500 nucleotides and in the absence of formamide). 图 8.1( T_(m)T_{\mathrm{m}} 与盐和 GC 的关系图)比较了在不同浓度的一价阳离子中(探针平均长度为 500 个核苷酸,且不含甲酰胺),这些方程预测的 35、50 和 65%(G+C)65 \%(\mathrm{G}+\mathrm{C}) DNA 探针的熔点。
The following examples are calculations of T_(m)T_{\mathrm{m}} for several different hybridization reactions. The first example includes a mismatch correction. 以下示例计算了几种不同杂交反应的 T_(m)T_{\mathrm{m}} 值。第一个例子包括错配校正。
EXAMPLE 1. A cloned gene fragment of Escherichia coli is labeled by nick translation (average chain length of 250 nucleotides) and used to detect a heterologous gene (about 65%65 \% similarity). For a hybridization buffer containing 6xx6 \times SSC and no formamide, the T_(m)T_{m} estimation includes the following considerations. 例 1.用缺口翻译法标记大肠杆菌克隆的基因片段(平均链长为 250 个核苷酸),并用来检测异源基因(相似度约为 65%65 \% )。对于含有 6xx6 \times SSC 且不含甲酰胺的杂交缓冲液, T_(m)T_{m} 估计值包括以下考虑因素。
1xx1 \times SSC contains 0.15 M sodium chloride and 0.015 M trisodium citrate. Therefore the molar concentration of monovalent cations in 6xx6 \times SSC equals 1xx1 \times SSC 中含有 0.15 M 氯化钠和 0.015 M 柠檬酸三钠。因此, 6xx6 \times SSC 中的单价阳离子摩尔浓度等于
The G+C content of an average Escherichia coli gene is 50%50 \%. 大肠杆菌基因的平均 G+C 含量为 50%50 \% 。
Fig. 8.1 Relationship between T_(m)T_{\mathrm{m}}, salt concentration and %(G+C)\%(\mathrm{G}+\mathrm{C}). Two different equations describing the relationship between T_(m)T_{\mathrm{m}} and salt concentration are plotted. The equation of Schildkraut and Lifson (1965) is shown by solid lines and that of Frank-Kamenetskii (1971) by broken lines. The relationship between salt concentration ( 0.01-1.2M0.01-1.2 \mathrm{M} ) and T_(m)T_{\mathrm{m}} is displayed for three values of %(G+C)(35%\%(\mathrm{G}+\mathrm{C})(35 \%, 50%50 \%, and 65%65 \% ). These relationships approximate values for probes of an average length greater than 500 nucleotides and in the absence of formamide. 图 8.1 T_(m)T_{\mathrm{m}} 、盐浓度和 %(G+C)\%(\mathrm{G}+\mathrm{C}) 之间的关系。图中绘制了描述 T_(m)T_{\mathrm{m}} 与盐浓度之间关系的两个不同方程。实线表示 Schildkraut 和 Lifson(1965 年)的方程,断线表示 Frank-Kamenetskii (1971 年)的方程。盐浓度 ( 0.01-1.2M0.01-1.2 \mathrm{M} ) 和 T_(m)T_{\mathrm{m}} 之间的关系显示在 %(G+C)(35%\%(\mathrm{G}+\mathrm{C})(35 \% 、 50%50 \% 和 65%65 \% 三个值上。)这些关系是平均长度大于 500 个核苷酸的探针在没有甲酰胺的情况下的近似值。
The T_(m)T_{\mathrm{m}} for a perfect duplex is as estimated by Meinkoth and Wahl (1984) (eq. (5)): 完美双工的 T_(m)T_{\mathrm{m}} 是由 Meinkoth 和 Wahl(1984 年)估算得出的(公式 (5)):
Including 35%35 \% mismatch destabilization (Bonner et al., 1973), the melting point is reduced by 35^(@)C35^{\circ} \mathrm{C} : 包括 35%35 \% 错配失稳(Bonner 等人,1973 年)在内,熔点降低了 35^(@)C35^{\circ} \mathrm{C} :
However, the above calculation does not take into account the rate of hybridization. In general, for long probes, the rate of hybridization is optimal at about 25^(@)C25^{\circ} \mathrm{C} below the T_(m)T_{\mathrm{m}} (Britten and Kohne, 1966; Wetmur and Davidson, 1968). Therefore, hybridizations are generally performed at temperatures considerably below the melting point. The above estimated melting point would not be the kinetically optimal temperature for formation of a duplex containing 35% mismatched base pairs. Keep in mind that stringency is adjusted both by the conditions of hybridization and the wash conditions following hybridization. Thus, in this example the 66.1^(@)C66.1^{\circ} \mathrm{C} temperature would better serve as an estimate of the wash 不过,上述计算并未考虑杂交率。一般来说,对于长探针,最佳杂交速率约为 25^(@)C25^{\circ} \mathrm{C} 低于 T_(m)T_{\mathrm{m}} (Britten 和 Kohne,1966 年;Wetmur 和 Davidson,1968 年)。因此,杂交通常在大大低于熔点的温度下进行。上述估计的熔点并不是形成含有 35% 错配碱基对的双链体的最佳动力学温度。请记住,严格程度可通过杂交条件和杂交后的洗涤条件进行调整。因此,在本例中, 66.1^(@)C66.1^{\circ} \mathrm{C} 温度最好作为洗涤条件的估计值。
temperature necessary to achieve the desired stringency. All practical examples given in this chapter concern immobilized (as opposed to solution) hybridization, and are amenable to controlling stringency at a posthybridization wash step. In practice the important point is the relationship between signal intensity and the specificity of the hybridization. Hybridization and wash conditions are always adjusted to fit the needs of the study. 达到所需严格度所需的温度。本章给出的所有实际例子都涉及固定杂交(而非溶液杂交),并可在杂交后的洗涤步骤中 控制严格程度。在实践中,重要的一点是信号强度与杂交特异性之间的关系。杂交和洗涤条件总是根据研究的需要进行调整。
Other considerations. The contribution of mismatch to hybrid destabilization was derived from duplex structures of relatively uniform mismatch distribution. For hybridization involving patchy distribution (for example between genes containing highly conserved domains) these relationships would not be expected to apply strictly. 其他考虑因素。错配对杂交不稳定性的影响是根据错配分布相对均匀的双链结构得出的。对于涉及斑块分布的杂交(例如含有高度保守结构域的基因之间的杂交),预计这些关系不会严格适用。
(iv) Oligonucleotide probes (iv) 寡核苷酸探针
With increasing availability of chemically synthesized DNA, synthetic oligonucleotides should be considered part of the general working arsenal of both the microbial systematist and molecular biologist. Under appropriate conditions of stringency, oligonucleotide probes often discriminate between targets that differ in a single nucleotide. For example, oligonucleotide probes have been used to detect single-base-pair differences in sequences as complex as the human genome (Conner et al., 1983; Miyada and Wallace, 1987). Thus, they also offer exquisite specificity (with appropriate design) to the microbial systematist. 随着化学合成 DNA 的日益普及,合成寡核苷酸应被视为微生物系统学家和分子生物学家常用的工作武器之一。在适当的严格条件下,寡核苷酸探针通常可以区分单个核苷酸不同的目标物。例如,寡核苷酸探针已被用于检测人类基因组等复杂序列中的单碱基对差异(Conner 等人,1983 年;Miyada 和 Wallace,1987 年)。因此,它们也为微生物系统学家提供了精湛的特异性(通过适当的设计)。
Probe length. The specificity of a probe is determined by its length and the complexity of the target sequence. A minimum probe size of about 9 (with 4 G-C pairs) is required for stable hybridization (Szostak et al., 1979). The frequency of a random occurrence of a target sequence of length nn within a DNA sequence of length LL, assuming random and representative nucleotide occurrence is (0.25)^(n)xx2L(0.25)^{n} \times 2 L ( 2L2 L because DNA is double stranded). This relationship should be used as a starting point for probe design. Direct experimental observations include the following. A 12-nucleotide oligonucleotide with one mismatch will specifically hybridize to a single band within a restriction digest of lambda DNA ( 50 kb ). An oligonucleotide of 13-15 nucleotides is sufficient to identify a unique gene in a restriction digest of total yeast DNA (Szostak et al., 1979). 探针长度。探针的特异性取决于其长度和目标序列的复杂程度。要实现稳定的杂交,探针的最小长度约为 9(含 4 个 G-C 对)(Szostak 等人,1979 年)。长度为 nn 的目标序列在长度为 LL 的 DNA 序列中随机出现的频率,假设随机出现的代表性核苷酸为 (0.25)^(n)xx2L(0.25)^{n} \times 2 L ( 2L2 L ,因为 DNA 是双链的)。这种关系应作为探针设计的出发点。直接实验观察结果如下。有一个错配的 12 核苷酸寡核苷酸会特异性杂交到λ DNA 限制性消化液(50 kb)中的一个条带上。13-15 个核苷酸的寡核苷酸足以在酵母 DNA 的限制性消化液中鉴定出一个独特的基因(Szostak 等人,1979 年)。
The relationships derived for long hybridization probes have been modified for estimating the melting points of oligonucleotides paired with their complementary DNA sequences. The relationships have been empirically derived and should be treated only as a starting point for establishing appropriate hybridization and wash conditions. These relationships and a corresponding set of example calculations are given below. 为估算与其互补 DNA 序列配对的寡核苷酸的熔点,对长杂交探针得出的关系进行了修改。这些关系是根据经验得出的,只能作为确定适当杂交和洗涤条件的起点。下文给出了这些关系和相应的计算示例。
A variation of eq. (5) (Thomas and Dancis, 1973; Lathe, 1985) is used to estimate the dissociation temperature of oligonucleotides between lengths 10 and 50 : 公式 (5) 的变式(Thomas 和 Dancis,1973 年;Lathe,1985 年)用于估算长度在 10 到 50 之间的寡核苷酸的解离温度:
T_(d)=81.5+16.6 log M+0.41[%(G+C)]-820//nT_{\mathrm{d}}=81.5+16.6 \log M+0.41[\%(\mathrm{G}+\mathrm{C})]-820 / n
For 50%(G+C)50 \%(\mathrm{G}+\mathrm{C}) and 0.3 M concentration of monovalent cations this equation is simplified to (Lathe, 1985) 对于 50%(G+C)50 \%(\mathrm{G}+\mathrm{C}) 和 0.3 M 浓度的单价阳离子,该方程简化为(Lathe,1985 年)
T_(d)=94-820//nT_{\mathrm{d}}=94-820 / n
A simplified estimate of oligonucleotide duplex stability sums the contribution of GC and AT pairs (Suggs et al., 1981). For hybridization in 6xx6 \times SSC the T_(d)T_{\mathrm{d}} of the oligonucleotide duplex is estimated as follows. 对寡核苷酸双链稳定性的简化估计是将 GC 和 AT 对的贡献相加(Suggs 等人,1981 年)。对于 6xx6 \times SSC 中的杂交,寡核苷酸双链的 T_(d)T_{\mathrm{d}} 估计如下。
Where N_(G+C)N_{\mathrm{G}+\mathrm{C}} and N_(A+T)N_{\mathrm{A}+\mathrm{T}} are the numbers of G and C and of A and T . 其中 N_(G+C)N_{\mathrm{G}+\mathrm{C}} 和 N_(A+T)N_{\mathrm{A}+\mathrm{T}} 分别是 G 和 C 以及 A 和 T 的编号。
Figure 8.2 displays a comparison of these relationships for washing probes of 50%(G+C)50 \%(\mathrm{G}+\mathrm{C}) content in 6xx6 \times SSC. As is evident in the figure, agreement is reasonably good for probes between 10 and 26 nucleotides in length. 图 8.2 显示了对 6xx6 \times SSC 中 50%(G+C)50 \%(\mathrm{G}+\mathrm{C}) 含量的洗涤探针的这些关系的比较。从图中可以看出,长度在 10 到 26 个核苷酸之间的探针的一致性相当好。
The following examples compare estimations of oligonucleotide melting points (derived from the above relationships) to each other and to empir- 下面的示例将寡核苷酸熔点的估计值(根据上述关系得出)与其他估计值和经验值进行了比较。
Fig. 8.2 Relationship between T_(d)T_{\mathrm{d}} and oligonucleotide probe length. Comparison of two relationships between T_(d)(^(@)C:}T_{\mathrm{d}}\left({ }^{\circ} \mathrm{C}\right. ) and probe length (number of nucleotides). The solid line (Suggs et al., 1981) and broken line (Lathe, 1985) are plotted for 50%50 \% (G+ C) and a salt concentration of 6xx6 \times SSC (1.17M)(1.17 \mathrm{M}). 图 8.2 T_(d)T_{\mathrm{d}} 与寡核苷酸探针长度的关系。 T_(d)(^(@)C:}T_{\mathrm{d}}\left({ }^{\circ} \mathrm{C}\right. ) 和探针长度(核苷酸数)之间的两种关系比较。实线(Suggs 等人,1981 年)和折线(Lathe,1985 年)是针对 50%50 \% (G+ C) 和 6xx6 \times SSC (1.17M)(1.17 \mathrm{M}) 的盐浓度绘制的。
ically determined T_(d)T_{\mathrm{d}} values. The empirically derived values are for DNARNA hybrids (oligonucleotide probes and immobilized rRNA targets). T_(d)T_{\mathrm{d}} 值。根据经验得出的值适用于 DNARNA 杂交(寡核苷酸探针和固定 rRNA 靶标)。
EXAMPLE 2. The T_(d)T_{\mathrm{d}} of the 21-mer 5^(')5^{\prime}-CCGCATCGATGAATCTTTCGT-3’ was empirically determined to be 52^(@)C52^{\circ} \mathrm{C} in 1xx1 \times SSC (Stahl, unpublished). How does this compare to the estimations determined by eqs (6) and (8)? 例 2.根据经验,21 聚体 5^(')5^{\prime} -CCGCATCGATGAATCTTTCGT-3' 的 T_(d)T_{\mathrm{d}} 在 1xx1 \times SSC 中为 52^(@)C52^{\circ} \mathrm{C} (Stahl,未发表)。这与公式(6)和(8)确定的估计值相比如何?
Recall that eq. (8) (Suggs et al., 1981) is only valid for a salt concentration of 6xx6 \times SSC. Therefore, the use of this equation to estimate T_(d)T_{d} in 1xx1 \times SSC must be corrected for the change in monovalent cation concentration: 请注意,公式 (8) (Suggs 等人,1981 年)只适用于盐浓度为 6xx6 \times SSC 的情况。因此,使用该公式估算 1xx1 \times SSC 中的 T_(d)T_{d} 时,必须根据单价阳离子浓度的变化进行修正:
Thus, in this example, both estimates of the T_(d)T_{\mathrm{d}} agree to within about a degree and are within 3^(@)C3^{\circ} \mathrm{C} of the measured T_(d)T_{\mathrm{d}}. 因此,在此示例中, T_(d)T_{\mathrm{d}} 的两个估计值相差约一度,并且与测量值 T_(d)T_{\mathrm{d}} 相差 3^(@)C3^{\circ} \mathrm{C} 。
EXAMPLE 3. Estimate the T_(d)(1xxSSC)T_{\mathrm{d}}(1 \times \mathrm{SSC}) for the 23-mer5^(')23-\mathrm{mer} 5^{\prime}-AGTACCTCCGA AGAGGCCTTTCC-3’. 例 3.估计 23-mer5^(')23-\mathrm{mer} 5^{\prime} -AGTACCTCCGA AGAGGCCTTTCC-3' 的 T_(d)(1xxSSC)T_{\mathrm{d}}(1 \times \mathrm{SSC}) 。
{:[M=0.195],[%(G+C)=56.6],[n=23]:}\begin{aligned}
M & =0.195 \\
\%(\mathrm{G}+\mathrm{C}) & =56.6 \\
n & =23
\end{aligned}
From eq. (6) (Lathe, 1985): 根据公式 (6)(Lathe,1985 年):
The empirical measurement of the T_(d)T_{\mathrm{d}} was 60^(@)C60^{\circ} \mathrm{C}. Both estimations are within 3^(@)C3^{\circ} \mathrm{C} and reasonably approximate the empirical value. T_(d)T_{\mathrm{d}} 的经验测量值为 60^(@)C60^{\circ} \mathrm{C} 。两个估计值都在 3^(@)C3^{\circ} \mathrm{C} 范围内,与经验值比较接近。
The above relationships should only be considered working approximations to be used as the basis for experimental characterization of oligonucleotide probes. In addition, the following are generalizations concerning the use of oligonucleotide probes. These should at least be considered when designing oligonucleotide probes or interpreting the results of oligonucleotide probe hybridization: 上述关系应仅被视为工作近似值,可用作寡核苷酸探针实验表征的基础。此外,以下是有关寡核苷酸探针使用的概括。在设计寡核苷酸探针或解释寡核苷酸探针杂交结果时,至少应考虑这些因素:
1. Position of mismatch 1.不匹配的位置
A mismatch near the end of a short duplex is generally less destabilizing than an internal mismatch. For example, the study of various single mismatches between a 12-mer probe and target sequence suggested that all the nucleotides on the short side of the mismatch were unpaired. Thus, for a short probe the ‘effective probe size’ was suggested to be equal to the number of base pairs before the mismatch minus 1 (the second deduction is to account for the destabilizing effect of the mismatch on the adjacent base pair). However, experimental observation has shown that the composition of a mismatch can in some instances override positional effects (Szostak et al., 1979). Thus, there are no absolute ‘rules’ for predicting the influence of mismatch position on hybrid stability. 与内部错配相比,短双链末端附近的错配通常不那么不稳定。例如,对 12 聚体探针和目标序列之间的各种单一错配进行的研究表明,错配短边的所有核苷酸都是未配对的。因此,对于短探针,"有效探针大小 "被认为等于错配前的碱基对数减去 1(第二个推论是考虑到错配对相邻碱基对的不稳定影响)。然而,实验观察表明,错配的构成在某些情况下可以超越位置效应(Szostak 等人,1979 年)。因此,预测错配位置对杂交稳定性的影响并没有绝对的 "规则"。
2. Mismatch composition 2.构成不匹配
As indicated above, the composition of the mismatch also influences’ the degree of destabilization. Slightly destabilizing base pairs include G-T, GA, (G-G?). Significant destabilization has been observed for: A-A, T-T, C-T, C-A (Ikuta et al., 1987). However, these are relative stabilities and will vary according to sequence context, e.g. stabilization resulting from stacking contributions of adjacent base pairs (nearest-neighbor contributions). 如上所述,错配的组成也会影响脱稳的程度。轻微脱稳的碱基对包括 G-T、GA、(G-G?)已观察到明显不稳定的碱基对有A-A、T-T、C-T、C-A(Ikuta 等人,1987 年)。不过,这些都是相对稳定性,会因序列背景而异,例如,相邻碱基对的堆叠作用(最近邻作用)会导致稳定。
3. Degenerate probe positions 3.退化探头位置
For certain applications it may be desirable to suppress the contribution of a specific mismatch to duplex destabilization. Such a situation could be a degenerate target position at a homologous position within a gene family or multiple copies of the same functional gene (e.g. between rRNA gene operons). For hybridization to such a target, the use of the G analog inosine (I) has been suggested (Takahashi et al., 1985; Corfield et al., 1987). Inosine pairs with C, T and A without significant destabilization. 在某些应用中,可能需要抑制特定错配对双链失稳的影响。这种情况可能是一个基因家族或同一功能基因的多个拷贝(如 rRNA 基因操作子之间)同源位置上的退化靶位。有人建议使用 G 类似物肌苷(I)与这种目标杂交(Takahashi 等人,1985 年;Corfield 等人,1987 年)。肌苷可与 C、T 和 A 配对,不会产生明显的不稳定性。
In practice, the contribution of these ‘additional’ factors to duplex stability are of greater importance for oligonucleotide probes. With increasing probe length, the influence of individual mismatch composition and position become less significant. 实际上,对于寡核苷酸探针来说,这些 "附加 "因素对双链稳定性的影响更为重要。随着探针长度的增加,单个错配成分和位置的影响就变得不那么重要了。
4. General considerations 4.一般考虑因素
Mismatch discrimination (stringency) is adjusted at one (or both) of two points; during hybridization (generally by adjusting temperature or concentration of formamide) or at a post-hybridization wash step (by adjusting both salt concentration and temperature). Again, the same general considerations discussed above apply. It is emphasized that, as yet, the rules for predicting stability of secondary structure (or higher structure) in nucleic acid are only approximations. The rules have been derived from a relatively limited collection of model structures. This is particularly true for predictions of oligonucleotide duplex stability, where stability is greatly influenced by both sequence and composition. 错配识别(严格程度)在两点中的一点(或两点)进行调整:杂交过程中(一般通过调整温度或甲酰胺浓度)或杂交后的清洗步骤(通过调整盐浓度和温度)。同样,上文讨论的一般注意事项也适用。需要强调的是,目前预测核酸二级结构(或高级结构)稳定性的规则还只是近似值。这些规则是从相对有限的模型结构集合中推导出来的。对于寡核苷酸双链体稳定性的预测来说尤其如此,因为稳定性受序列和组成的影响很大。
5. RNA-DNA versus DNA-DNA duplexes 5.RNA-DNA 与 DNA-DNA 双链体
The above relationships are derived from DNA-DNA duplexes. However, ribonucleotide and deoxyribonucleotide duplex structure differ in relative stability for the same sequence, in the following order: RNA-RNA > RNA-DNA > DNA-DNA (Saenger, 1984). This chapter will not attempt to address this contribution to duplex stability other than in passing. For short RNA-DNA duplex structures the increase in stability appears negligible (as illustrated by the above examples). However, for the use of long RNA transcripts as probes (riboprobes), this effect could markedly alter hybridization results. As a practical example, it may be possible to detect low-homology targets by taking advantage of the greater stability of RNA-DNA duplexes. 上述关系源自 DNA-DNA 双链。然而,对于相同序列,核糖核苷酸和脱氧核苷酸双链结构的相对稳定性不同,顺序如下:RNA-RNA > RNA-DNA > DNA-DNA(Saenger,1984):RNA-RNA>RNA-DNA>DNA-DNA(Saenger,1984 年)。本章将不试图讨论这种对双链稳定性的贡献,只是顺带一提。对于短 RNA-DNA 双链结构,稳定性的增加似乎可以忽略不计(如上述例子所示)。然而,对于使用长 RNA 转录本作为探针(核糖探针)来说,这种效应可能会明显改变杂交结果。举个实际例子,利用 RNA-DNA 双链体更高的稳定性,也许可以检测到低同源性目标。
C. PROBE DESIGN C.探头设计
(i) Empirical probe design (i) 经验性探针设计
Examples of the determinative use of nucleic acid probes are largely restricted to the clinical arena. Also, in large part, these probes have been empirically derived. There are two general approaches to design. The first 核酸探针的确定性应用实例主要局限于临床领域。而且,这些探针在很大程度上是根据经验推导出来的。设计方法一般有两种。第一种
requires the generation of a genomic recombinant library (using either phage or plasmid-based vectors) of the target microorganism followed by randomly screening selected clones (potential probes) for appropriate specificity. The alternative approach uses total genomic DNA (radioactively or non-radioactively labeled) derived from the target organism as a hybridization probe for identifying identical or closely related organisms. The reader is referred to standard references for preparation of DNA, cloning and screening of recombinant clones (Maniatis et al., 1982; Davis et al., 1986; Ausubel et al., 1987). Recent representative examples of both strategies are listed in Table 8.1. 这种方法需要生成目标微生物的基因组重组文库(使用噬菌体或质粒载体),然后随机筛选出具有适当特异性的克隆(潜在探针)。另一种方法是使用来自目标生物的总基因组 DNA(放射性或非放射性标记)作为杂交探针,用于鉴定相同或近缘生物。读者可参阅有关 DNA 制备、克隆和重组克隆筛选的标准参考文献(Maniatis 等人,1982 年;Davis 等人,1986 年;Ausubel 等人,1987 年)。表 8.1 列出了这两种策略的最新代表性实例。
TABLE 8.1 Examples of empirical probe design 表 8.1 经验探针设计示例
Organism or group identified 已确定的生物体或群体
Reference 参考资料
A. Whole-cell DNA probes A.全细胞 DNA 探针
Bacteroides intermedius 中间菌
Moncla et al. (1988) 蒙克拉等人(1988 年)
Bacteroides strains 菌株
Morotomi et al. (1988) Morotomi 等人(1988 年)
Campylobacter strains 弯曲杆菌菌株
Ng et al. (1987) Ng 等人(1987 年)
Cheverier et al. (1989) Cheverier 等人(1989 年)
B. Cloned genomic DNA probes B.克隆基因组 DNA 探针
Bacteroides fragilis 脆弱拟杆菌
Groves and Clark (1987) 格罗夫斯和克拉克(1987 年)
Bacteroides ruminocola 反刍小球杆菌
Attwood et al. (1988) 阿特伍德等人(1988 年)
Campylobacter jejuni 空肠弯曲杆菌
Picken et al. (1987) 皮肯等人(1987 年)
Mycobacterium leprae 麻风分枝杆菌
Clark-Curtiss and Docherty (1989) 克拉克-柯蒂斯和多切蒂(1989 年)
Mycobacterium tuberculosis 结核分枝杆菌
Roberts et al. (1987) 罗伯茨等人(1987 年)
立 :
Pao et al. (1988) Pao 等人(1988 年)
Mycoplasma gallisepticum 胆囊支原体
Santha et al. (1987) 桑塔等人(1987 年)
Mycoplasma pneumoniae, M. genitalum 肺炎支原体、生殖器支原体
Hyman et al. (1987) 海曼等人(1987 年)
Organism or group identified Reference
A. Whole-cell DNA probes
Bacteroides intermedius Moncla et al. (1988)
Bacteroides strains Morotomi et al. (1988)
Campylobacter strains Ng et al. (1987)
Cheverier et al. (1989)
B. Cloned genomic DNA probes
Bacteroides fragilis Groves and Clark (1987)
Bacteroides ruminocola Attwood et al. (1988)
Campylobacter jejuni Picken et al. (1987)
Mycobacterium leprae Clark-Curtiss and Docherty (1989)
Mycobacterium tuberculosis Roberts et al. (1987)
立 : Pao et al. (1988)
Mycoplasma gallisepticum Santha et al. (1987)
Mycoplasma pneumoniae, M. genitalum Hyman et al. (1987)| Organism or group identified | Reference |
| :---: | :---: |
| A. Whole-cell DNA probes | |
| Bacteroides intermedius | Moncla et al. (1988) |
| Bacteroides strains | Morotomi et al. (1988) |
| Campylobacter strains | Ng et al. (1987) |
| | Cheverier et al. (1989) |
| B. Cloned genomic DNA probes | |
| Bacteroides fragilis | Groves and Clark (1987) |
| Bacteroides ruminocola | Attwood et al. (1988) |
| Campylobacter jejuni | Picken et al. (1987) |
| Mycobacterium leprae | Clark-Curtiss and Docherty (1989) |
| Mycobacterium tuberculosis | Roberts et al. (1987) |
| 立 : | Pao et al. (1988) |
| Mycoplasma gallisepticum | Santha et al. (1987) |
| Mycoplasma pneumoniae, M. genitalum | Hyman et al. (1987) |
(ii) Rational or directed probe design (ii) 合理或定向探针设计
The use of directed probes (genes specifying surface epitopes, toxins, plasmid-encoded functions and conserved gene families) offers a rational and more readily interpretable basis for determinative hybridization. A partial listing of recently described determinative probes designed within a rational or comparative framework is given in Table 8.2. Perhaps the most powerful approach, as now developed, is the application of the large data collection of 16 S16 S rRNA sequences to the design of determinative hybridization probes. Several examples of the use of the ribosomal RNAs in such a directed fashion are given in Table 8.2. A more in-depth discussion is reserved for the section on the use of the 165 rRNA and a comparative framework for determinative hybridization. 定向探针(指定表面表位、毒素、质粒编码功能和保守基因家族的基因)的使用为确定性杂交提供了合理和更易于解释的基础。表 8.2 列出了最近在合理或比较框架内设计的确定性探针的部分清单。目前开发的最有力的方法也许是将大量的 16 S16 S rRNA 序列数据应用于确定性杂交探针的设计。表 8.2 给出了以这种定向方式使用核糖体 RNA 的几个例子。关于 165 rRNA 的使用和确定性杂交的比较框架的章节将保留更深入的讨论。
TABLE 8.2 Examples of rational probe design 表 8.2 合理探针设计示例
A. Targeting genes specifying surface epitopes Campylobacter jejuni antigenic whole-cell and membrane genes A. 指定表面表位的靶向基因 空肠弯曲杆菌抗原全细胞和膜基因
B. Targeting genes specifying virulence factors Listeria monocytogenes beta\beta-hemolysin gene B. 指定毒力因子的靶向基因 单核细胞增多性李斯特菌 beta\beta -溶血素基因
Enteric toxin and invasion genes 肠毒素和入侵基因
Escherichia coli enterotoxin genes 大肠杆菌肠毒素基因
C. Targeting conserved genes C. 锁定保守基因
Subclones of Micrococcus luteus 23S rRNA gene 黄体微球菌 23S rRNA 基因子克隆
rRNA coding and spacer regions, actin and discoidin gene families cloned from Dictyostelium discoideum 从盘状竹荪中克隆的 rRNA 编码区和间隔区、肌动蛋白和盘状蛋白基因家族
Oligonucleotide probe complementary to variable regions of Mycoplasma spp. rRNA 与支原体 rRNA 可变区互补的寡核苷酸探针
D. Targeting viral genomes D. 针对病毒基因组
Oligonucleotide probes complementary to Papilloma virus genomic DNA 与乳头状瘤病毒基因组 DNA 互补的寡核苷酸探针
Cubie and Norval (1988) 库比和诺瓦尔(1988 年)
D. LABELING TECHNIQUES D.标记技术
One of the most rapidly changing areas of molecular biology is in the development of alternative systems for detecting target-probe :hybridization. In large part, development is moving away from systems requiring the use of radioisotopes. The issues here are safety and shelf-life, balanced against the general requirement for high sensitivity. For research applications, the use of radioisotopes remains the preferred detection system for most applications requiring high sensitivity. However, more recent nonradioactive systems approach (or possibly surpass in certain applications) radioactive probes. Complete description of experimental or commercial non-radioactive labeling and detection systems is beyond the scope of this chapter and detailed labeling protocols are given only for the more commonly used radioactive systems (Tables 8.5-8.10). These are ‘tried and true’ techniques and are well suited to most research laboratories. It should be kept in mind that there are many variations of these basic protocols, many provided by the manufacturers of kits, vectors and reagents. In part the detailed listing of protocols in this chapter serves to remove some of the mystery from a variety of fairly straightforward techniques of molecular biology. 分子生物学中变化最快的领域之一是开发用于检测目标-探针杂交的替代系统。在很大程度上,开发工作正在摆脱需要使用放射性同位素的系统。这里的问题是安全性和保存期限,以及对高灵敏度的一般要求。在研究应用中,使用放射性同位素仍然是大多数要求高灵敏度的应用的首选检测系统。不过,最新的非放射性系统已接近(或在某些应用中可能超过)放射性探针。对实验性或商业性非放射性标记和检测系统的完整描述超出了本章的范围,本章仅针对较常 用的放射性系统(表 8.5-8.10)给出了详细的标记方案。这些都是 "屡试不爽 "的技术,非常适合大多数研究实验室。需要注意的是,这些基本方案有许多变体,其中许多由试剂盒、载体和试剂制造商提供。本章中详细列出的操作步骤,在一定程度上是为了揭开分子生物学中各种相当直 接的技术的神秘面纱。
There are two basic approaches for the detection of a specific hybrid. One can either directly label the probe (Table 8.3) or attach a reporter group 检测特定杂交种有两种基本方法。一种是直接标记探针(表 8.3),另一种是附加报告基团
TABLE 8.3 Direct labels for radioactive and non-radioactive hybridization probes 表 8.3 用于放射性和非放射性杂交探针的直接标签
Diagrammatic representation of direct labeling and detection of probes hybridized with target sequence. 直接标记和检测与目标序列杂交的探针的示意图。
Label Reference 标签参考
Horseradish peroxidase 辣根过氧化物酶
Horseradish peroxidase detected by chemiluminescence 化学发光检测辣根过氧化物酶
Microperoxidase detected by 微过氧化物酶检测
chemiluminescence 化学发光
Gillespie and Spiegelman (1965); Southern (1975); Collins and Hunsaker (1985); Giovannoni et al. (1988) Gillespie 和 Spiegelman (1965);Southern (1975);Collins 和 Hunsaker (1985);Giovannoni 等人 (1988)
Gillespie and Spiegelman (1965); Cannon et al. (1985); ThomasCavallin and Ait-Ahmed (1988) Gillespie 和 Spiegelman (1965);Cannon 等人 (1985);ThomasCavallin 和 Ait-Ahmed (1988)
Collins and Hunsaker (1985); Collins 和 Hunsaker(1985 年);
Giovannoni et al. (1988) 乔凡诺尼等人(1988 年)
Edelstein (1986); Lewis et al. (1986); Allen et al. (1987) Edelstein (1986);Lewis 等人 (1986);Allen 等人 (1987)
Bauman et al. (1981a, b); DeLong et al. (1989), Amann et al. (1990) Bauman 等人(1981a, b);DeLong 等人(1989),Amann 等人(1990)
Draper (1984) 德雷珀(1984 年)
Renz and Kurz (1984); Jablonski et al. (1986); Li et al. (1987); Edman et al. (1988) Renz 和 Kurz(1984 年);Jablonski 等人(1986 年);Li 等人(1987 年);Edman 等人(1988 年)
Urdea et al. (1987) 乌尔迪亚等人(1987 年)
Amersham Corporation (1989); Thorpe et al. (1985) Amersham 公司(1989 年);Thorpe 等人(1985 年)
Heller and Shneider (1983) 海勒和施奈德(1983 年)
Diagramatic representation of indirect detection of labeled probes hybridized with target sequence. 标记探针与目标序列杂交的间接检测示意图。 (" Reporter group ")/(" Biotin ")quad\frac{\text { Reporter group }}{\text { Biotin }} \quad Leference (1985); Matthews et al (1985); McInnes et al (1987); Kumar et al. (1988); Guitteny et al. (1988); Denman and Miller (1989) (" Reporter group ")/(" Biotin ")quad\frac{\text { Reporter group }}{\text { Biotin }} \quad Leference (1985);Matthews et al (1985);McInnes et al (1987);Kumar et al. (1988);Guitteny et al. (1988);Denman and Miller (1989)
Digoxigenin 地高辛
2,4-Dinitrophenyl 2,4-二硝基苯基
N -2-Acetylaminofluorene N -2-乙酰氨基芴
Sulfone 砜
Boehringer Mannheim Biochemicals (1988); Heiles et al. (1988) 勃林格曼海姆生化公司(1988 年);海尔斯等人(1988 年)
Arnold (1984); Keller et al. (1988,1989)(1988,1989) Arnold (1984); Keller et al.
Tchen et al. (1984);Syvaenen et al. (1986); Chevrier et al. (1989); Landegent et al. (1984) Tchen 等人(1984 年);Syvaenen 等人(1986 年);Chevrier 等人(1989 年);Landegent 等人(1984 年)
Verdlov et al. (1974); Orgenics Ltd. (1987); Syvaenen et al. (1986) Verdlov 等人(1974 年);Orgenics 有限公司(1987 年);Syvaenen 等人(1986 年)。(1987); Syvaenen 等人 (1986)
to the probe and detect this reporter with a labeled binding protein. In Table 8.4 we concentrate on commercially available reporter molecules. For a comprehensive listing of the tremendous variety of secondary labeling systems, the reader is referred to a review article by Matthews and Kricka (1988). 在表 8.4 中,我们集中介绍了市场上可买到的报告分子。在表 8.4 中,我们主要介绍了市售的报告分子。如需全面了解各种二次标记系统,请参阅 Matthews 和 Kricka(1988 年)的综述文章。
(i) Methods for labeling probes with radioisotopes (i) 用放射性同位素标记探针的方法
Nick translation (Table 8.5) 尼克翻译(表 8.5)
A combination of DNase I and Escherichia coli polymerase I serves to incorporate radioactive nucleotides in double-stranded DNA. The small amount of DNase I included in the reaction introduces a controlled number DNase I 和大肠杆菌聚合酶 I 的组合可将放射性核苷酸结合到双链 DNA 中。反应中含有的少量 DNase I 会引入数量可控的放射性核苷酸。
TABLE 8.5 Nick translation (Rigby et al., 1977) 表 8.5 尼克翻译(里格比等人,1977 年)
A typical reaction mix contains: 典型的反应混合物包括 1mug1 \mu \mathrm{~g} double-stranded DNA 1mug1 \mu \mathrm{~g} 双链 DNA 5mu5 \mu I nucleotide mix (dGTP, dCTP, dTTP) 5mu5 \mu I 核苷酸混合物(dGTP、dCTP、dTTP) 5mu1[alpha^(32)P]dATP5 \mu 1\left[\alpha{ }^{32} \mathrm{P}\right] \mathrm{dATP} (NEN, Boston Mass., USA) with a volume specific activity of 10 muCi//mul10 \mu \mathrm{Ci} / \mu \mathrm{l} (specific activity 3000Ci//mM3000 \mathrm{Ci} / \mathrm{mM} ) 5mu1[alpha^(32)P]dATP5 \mu 1\left[\alpha{ }^{32} \mathrm{P}\right] \mathrm{dATP} (美国波士顿马萨诸塞州 NEN 公司),体积比活性为 10 muCi//mul10 \mu \mathrm{Ci} / \mu \mathrm{l} (比活性 3000Ci//mM3000 \mathrm{Ci} / \mathrm{mM} ) 5mul5 \mu \mathrm{l} DNase I-Escherichia coli Polymerase I mixture 5mul5 \mu \mathrm{l} DNase I-大肠杆菌聚合酶 I 混合物 50 mul50 \mu \mathrm{l} final volume with reaction buffer 50 mul50 \mu \mathrm{l} 含有反应缓冲液的最终体积
Mix and incubate at 15^(@)C15^{\circ} \mathrm{C} for 1 hour. 混合并在 15^(@)C15^{\circ} \mathrm{C} 温度下培养 1 小时。
Remove unincorporated nucleotides by standard methods (see Table 8.11). 用标准方法去除未结合的核苷酸(见表 8.11)。
of nicks (each containing a free 3^(')OH3^{\prime} \mathrm{OH} group) into the DNA duplex. Polymerase I initiates a replacement strand synthesis at these nicks by removing preceding nucleotides (using an intrinsic 5^(')-3^(')5^{\prime}-3^{\prime} exonuclease activity) and simultaneously synthesizing DNA. A variety of radioactive and non-radioactive nucleotides are incorporated by polymerase I. Nick translation kits (e.g. Bethesda Research Laboratories) are now commonly used and avoid the sometimes laborious optimization of reaction conditions. 聚合酶 I 在 DNA 双链上形成一个个缺口(每个缺口都含有一个游离的 3^(')OH3^{\prime} \mathrm{OH} 基团)。聚合酶 I 通过去除前面的核苷酸(利用固有的 5^(')-3^(')5^{\prime}-3^{\prime} 外切酶活性)并同时合成 DNA,从而在这些缺口处启动替换链合成。聚合酶 I 可合成各种放射性和非放射性核苷酸。目前,人们普遍使用尼克翻译试剂盒(如 Bethesda 研究实验室),它可避免有时费力的反应条件优化工作。
2. Random primer labeling (Table 8.6) 2.随机引物标记(表 8.6)
An increasingly popular alternative to nick translation is random primer labeling. Denatured DNA is primed with a random mixture of hexamers and incubated with the large fragment of Escherichia coli DNA polymerase I (Klenow fragment) in the presence of the four dNTPs (generally three unlabeled and one labeled). The label is uniformly incorporated in both strands, yielding a probe with high specific activity. Probes of even higher specific activity can be synthesized by substitution of two or more labeled dNTPs in the reaction mix. 随机引物标记是缺口翻译的一种日益流行的替代方法。变性 DNA 以随机六聚体混合物为引物,与大肠杆菌 DNA 聚合酶 I 的大片段(Klenow 片段)在四种 dNTPs(通常是三种未标记和一种已标记)的存在下孵育。标签会均匀地结合到两条链中,从而产生具有高特异性的探针。通过在反应混合物中替换两种或更多标记的 dNTP,可以合成比活性更高的探针。
3. Riboprobes 3.核糖体
Transcription labeling generally requires initial cloning of the DNA probe fragment to serve as template for synthesis of labeled RNA transcripts (riboprobes) (Rotbart et al., 1988). Several plasmid- and phage-based vectors are commercially available for cloning and transcription (Bethesda Research Laboratories, Gaithersberg, Md., USA; Pharmacia LKB Biotech., Sweden). An alternative to cloning is complete chemical synthesis of complementary oligonucleotides containing a phage promoter sequence and flanking probe sequence (Emson et al., 1988). Table 8.7 details the 转录标记通常需要先克隆 DNA 探针片段,作为合成标记 RNA 转录本(核糖体)的模板(Rotbart 等人,1988 年)。市场上有几种基于质粒和噬菌体的载体可用于克隆和转录(美国马里兰州盖瑟斯堡贝塞斯达研究实验室;瑞典 Pharmacia LKB 生物技术公司)。克隆的另一种方法是用化学方法合成含有噬菌体启动子序列和侧翼探针序列的互补寡核苷酸(Emson 等人,1988 年)。表 8.7 详细介绍了
TABLE 8.6 Random primer labeling (Feinberg and Vogelstein, 1983) 表 8.6 随机引物标记(Feinberg 和 Vogelstein,1983 年)
Oligonucleotide solution 寡核苷酸溶液 1mug//mul1 \mu \mathrm{~g} / \mu \mathrm{l} random hexanucleotides (Pharmacia LKB Biotech., Piscataway, N.J., USA) 1mug//mul1 \mu \mathrm{~g} / \mu \mathrm{l} 随机六核苷酸(Pharmacia LKB Biotech.)
2. Denature 100 ng of linear DNA in 10 muI10 \mu \mathrm{I} TE-Buffer ( 10 mM Tris-HCl, pH 7.2; 1 mM EDTA) by boiling ( 100^(@)C100^{\circ} \mathrm{C} ) for 3 minutes 2.在 10 muI10 \mu \mathrm{I} TE 缓冲液(10 mM Tris-HCl,pH 7.2;1 mM EDTA)中煮沸( 100^(@)C100^{\circ} \mathrm{C} )3 分钟,使 100 纳克线性 DNA 变性。
3. Quench on ice 3.冰上淬火
4. Add in the following order 4.按以下顺序添加 10 mu10 \mu linearized, denatured DNA ( 100 ng ) 10 mu10 \mu 线性化变性 DNA ( 100 纳克 ) 4mul4 \mu \mathrm{l} oligonucleotide solution 4mul4 \mu \mathrm{l} 寡核苷酸溶液 2.5 muI2.5 \mu \mathrm{I} 3dNTP mix 2.5 muI2.5 \mu \mathrm{I} 3dNTP混合物 2.5 mu2.5 \mu Klenow fragment buffer 2.5 mu2.5 \mu 克勒诺片段缓冲区 5mu1[gamma^(-32)P]dATP(50 muCi5 \mu 1\left[\gamma^{-32} \mathrm{P}\right] \mathrm{dATP}(50 \mu \mathrm{Ci}; Table 8.5) 5mu1[gamma^(-32)P]dATP(50 muCi5 \mu 1\left[\gamma^{-32} \mathrm{P}\right] \mathrm{dATP}(50 \mu \mathrm{Ci} ;表 8.5) 1mu11 \mu 1 Klenow fragment (3-8 units) 1mu11 \mu 1 克勒诺片段(3-8 个单位)
5. Incubate 2 hours at room temperature 5.室温下孵育 2 小时
6. Remove unincorporated nucleotides by standard methods (see Table 8.11) 6.用标准方法去除未结合的核苷酸(见表 8.11) 7.
synthesis of an RNA probe using a plasmid-based T7 promotor/transcription system. 使用基于质粒的 T7 启动子/转录系统合成 RNA 探针。
4. Polymerase chain reaction 4.聚合酶链反应
The PCR method can produce unlimited amounts of double-stranded DNA starting from very small amounts of DNA or RNA (if cDNA is synthesized with reverse transcriptase prior to the chain reaction). Label can be incorporated during the PCR reaction as outlined in Table 8.8 or following amplification by using standard techniques (see Tables 8.5 and 8.6). PCR 方法可以从极少量的 DNA 或 RNA(如果在链反应前用逆转录酶合成了 cDNA)开始产生无限量的双链 DNA。标签可在 PCR 反应过程中加入,如表 8.8 所示,或在扩增后使用标准技术加入(见表 8.5 和 8.6)。
5. 5^(')5^{\prime}-End labeling with ^(32)P{ }^{32} P (Table 8.9) 5. 5^(')5^{\prime} -以 ^(32)P{ }^{32} P 结束标记(表 8.9)
T4 polynucleotide kinase catalyzes the transfer of the terminal phosphate group from ATP to the 5^(')5^{\prime}-hydroxyl group of RNA or DNA. Thiolated ( ^(35){ }^{35} S) T4 多核苷酸激酶催化 ATP 的末端磷酸基转移到 RNA 或 DNA 的 5^(')5^{\prime} - 羟基上。硫醇化( ^(35){ }^{35} S)
400 mM Tris-HCl pH 7.5 60mMMgCl_(2)60 \mathrm{mM} \mathrm{MgCl}_{2}
100 mM DTT
40 mM spermidine 40 mM 亚精胺 1mg//ml1 \mathrm{mg} / \mathrm{ml} BSA
4 mM GTP
4 mM ATP
4 mM TTP
0.8 mMCTP
2. Add in the following order ( 20 mul20 \mu \mathrm{l} final volume) 2.按以下顺序添加( 20 mul20 \mu \mathrm{l} 最后一卷) 10 mu10 \mu DNA template (0.6 mug)(0.6 \mu \mathrm{~g}) 10 mu10 \mu DNA 模板 (0.6 mug)(0.6 \mu \mathrm{~g}) 2mu110 xx2 \mu 110 \times T7-RNA-polymerase buffer 2mu110 xx2 \mu 110 \times T7-RNA 聚合酶缓冲液 7mu][alpha^(32)P]7 \mu]\left[\alpha{ }^{32} \mathrm{P}\right] CTP (ICN Radiochemicals, Irvine, Calif., USA) with a volume specific activity of 10 muCi//mu110 \mu \mathrm{Ci} / \mu 1 (specific activity 3000Ci//mM3000 \mathrm{Ci} / \mathrm{mM} ) 7mu][alpha^(32)P]7 \mu]\left[\alpha{ }^{32} \mathrm{P}\right] CTP(ICN Radiochemicals,Irvine,Calif, USA),体积比活度为 10 muCi//mu110 \mu \mathrm{Ci} / \mu 1 (比活度 3000Ci//mM3000 \mathrm{Ci} / \mathrm{mM} ) 1muIT71 \mu \mathrm{IT7}-RNA-polymerase (Boehringer Mannheim Biochemicals, Indianapolis, Ind., USA), 20 units/ mu\mu I 1muIT71 \mu \mathrm{IT7} -RNA-聚合酶(Boehringer Mannheim Biochemicals,Indianapolis,Ind.
3. Mix and incubate at 37^(@)C37^{\circ} \mathrm{C} for 1 hour 3.混合并在 37^(@)C37^{\circ} \mathrm{C} 温度下培养 1 小时
4. Remove unincorporated nucleotides (see Table 8.11) 4.去除未结合的核苷酸(见表 8.11)
ATP analogs also serve as substrate for this enzyme. This method can therefore be used to label virtually any polynucleotide (e.g. fragmented rRNA, DNA restriction fragments and oligonucleotides) with either ^(32)P{ }^{32} \mathrm{P} or ^(35)S{ }^{35} \mathrm{~S}. The primary limitation of this labeling strategy is incorporation of a single radioactive group per strand of RNA or DNA. As a consequence, the specific activity (and therefore the associated sensitivity of target detection) may not be as high as that realized by other labeling techniques. ATP 类似物也可作为这种酶的底物。因此,这种方法几乎可以用来用 ^(32)P{ }^{32} \mathrm{P} 或 ^(35)S{ }^{35} \mathrm{~S} 标记任何多核苷酸(如片段 rRNA、DNA 限制片段和寡核苷酸)。这种标记策略的主要局限性在于每条 RNA 或 DNA 链只有一个放射性基团。因此,特异性活性(以及相关的目标检测灵敏度)可能不如其他标记技术高。
Terminal transferase catalyzes the addition of deoxyribonucleotides to the 3^(')3^{\prime} termini (containing available 3^(')OH3^{\prime} \mathrm{OH} groups) of double- or singlestranded DNA in a template-independent reaction. Since multiple labels can be added to an oligonucleotide, yery high specific activities can be achieved. The tail length is controlled by adjusting the relative molar concentrations of 3^(')3^{\prime} ends and dNTPs. By using [ {: alpha^(-32)P]ddNTP\left.\alpha{ }^{-32} \mathrm{P}\right] \mathrm{ddNTP} (or [ {: alpha-^(32)P]\left.\alpha-{ }^{32} \mathrm{P}\right] cordycepin), incorporation can be limited to a single nucleotide. 末端转移酶在一个与模板无关的反应中,催化脱氧核苷酸添加到双链或单链 DNA 的 3^(')3^{\prime} 端部(含有可用的 3^(')OH3^{\prime} \mathrm{OH} 基团)。由于可以在一个寡核苷酸上添加多个标签,因此可以实现极高的比活度。通过调整 3^(')3^{\prime} 末端和 dNTPs 的相对摩尔浓度,可以控制尾部长度。通过使用[ {: alpha^(-32)P]ddNTP\left.\alpha{ }^{-32} \mathrm{P}\right] \mathrm{ddNTP} (或[ {: alpha-^(32)P]\left.\alpha-{ }^{32} \mathrm{P}\right] 虫草素)],可将掺入限制在单个核苷酸内。
7. Purification of labeled products 7.标记产品的纯化
There is a variety of methods for purification of labeled nucleic acid probes. Most simply, this involves only the removal of unincorporated 纯化标记核酸探针的方法多种多样。最简单的方法是只去除未结合的核酸探针。
TABLE 8.8 Probe labeling using the polymerase chain reaction (Saiki et al., 1986; Schowalter and Sommer, 1989) 表 8.8 利用聚合酶链反应进行探针标记(Saiki 等人,1986 年;Schowalter 和 Sommer,1989 年)
Prepare solutions 准备解决方案 20 xx20 \times minus dCTP buffer 20 xx20 \times 减去 dCTP 缓冲液
1 M KCl
0.2 M Tris-HCl, pH 8.3 0.2 M Tris-HCl,pH 8.3 30mMMgCl_(2)30 \mathrm{mM} \mathrm{MgCl}{ }_{2} 0.2%0.2 \% (w/v) gelatin 0.2%0.2 \% (重量/体积)明胶
4 mM dATP
4 mMdTTP
4 mMdGTP
Primer mix: 20 muM20 \mu \mathrm{M} of both primers (for a 15-base oligonucleotide, c, 100ng//mul100 \mathrm{ng} / \mu \mathrm{l} ) 引物混合物:两种引物的 20 muM20 \mu \mathrm{M} (对于 15 个碱基的寡核苷酸,c, 100ng//mul100 \mathrm{ng} / \mu \mathrm{l} )
Add reagents to a 500 mul500 \mu \mathrm{l} Eppendorf tube in the following order: 按以下顺序将试剂加入 500 mul500 \mu \mathrm{l} Eppendorf 试管中: 1muI1 \mu \mathrm{I} template DNA ( 100pg//muI100 \mathrm{pg} / \mu \mathrm{I} ) 1muI1 \mu \mathrm{I} 模板 DNA ( 100pg//muI100 \mathrm{pg} / \mu \mathrm{I} ) 2mu12 \mu 1 primer mix 2mu12 \mu 1 混合底漆 15 mul[alpha-^(32)P]dCTP15 \mu \mathrm{l}\left[\alpha-{ }^{32} \mathrm{P}\right] \mathrm{dCTP} (ICN Radiochemicals, Irvine, Calif., USA) with a volume specific activity of 10 muCi//mul10 \mu \mathrm{Ci} / \mu \mathrm{l} (specific activity 3000Ci//mM3000 \mathrm{Ci} / \mathrm{mM} ) 1mul20 xx1 \mu \mathrm{l} 20 \times minus dCTP buffer 15 mul[alpha-^(32)P]dCTP15 \mu \mathrm{l}\left[\alpha-{ }^{32} \mathrm{P}\right] \mathrm{dCTP} (ICN Radiochemicals,Irvine,Calif,USA),体积比活度为 10 muCi//mul10 \mu \mathrm{Ci} / \mu \mathrm{l} (比活度 3000Ci//mM3000 \mathrm{Ci} / \mathrm{mM} ) 1mul20 xx1 \mu \mathrm{l} 20 \times 减去 dCTP 缓冲液 1muH_(2)O1 \mu \mathrm{H}_{2} \mathrm{O}
Overlay with 20 mu120 \mu 1 mineral oil and heat to 94^(@)C94^{\circ} \mathrm{C} for 10 minutes 覆上 20 mu120 \mu 1 矿物油,加热至 94^(@)C94^{\circ} \mathrm{C} 10 分钟
Add 1 unit of Taq-Polymerase (Perkin-Elmer/Cetus, Emeryville, Calif., USA) 加入 1 个单位的 Taq-聚合酶(Perkin-Elmer/Cetus,美国加利福尼亚州埃默里维尔市)
Run 30 cycles (DNA thermal cycler) of denaturation, primer annealing and transcription (e.g. 1 minute 94^(@)C94^{\circ} \mathrm{C} denaturation, 2 minutes 50^(@)C50^{\circ} \mathrm{C} annealing, and 3 minutes 72^(@)C72^{\circ} \mathrm{C} elongation). Annealing temperature will vary with primer composition. 运行 30 个变性、引物退火和转录循环(DNA 热循环仪)(例如,1 分钟 94^(@)C94^{\circ} \mathrm{C} 变性,2 分钟 50^(@)C50^{\circ} \mathrm{C} 退火,3 分钟 72^(@)C72^{\circ} \mathrm{C} 延伸)。退火温度因引物成分而异。
Remove unincorporated label by using standard techniques (see Table 8.11) 使用标准技术清除未加入的标签(见表 8.11)
radiolabel. However, separation of intact probe from probe degradation products or unwanted side-reaction products may also be necessary. A common approach to simultaneous isolation of intact probe and removal of unincorporated label (commonly used for the purification of oligonucleotides) is fractionation of reaction products on a polyacrylamide gel. The region of the gel containing the labeled probe is excised and the oligonucleotide is eluted from the gel matrix (Miyada and Wallace, 1987). The approach detailed here (Table 8.11) is designed only to remove unincorporated label. However, it is suitable for purification of all of the probes described in this chapter. This technique takes advantage of a Dupont Corporation product (Nensorb ^(TM)20{ }^{\mathrm{TM}} 20 nucleic acid purification cartridge). The Nensorb cartridge can remove protein, salt and unincorporated label from either DNA or RNA. The recovery of nucleic acid from the cartridge is excellent ( 1 ng to 20 mug20 \mu \mathrm{~g} ) and it is suitable for purification of oligonucleotides as well as longer nucleic acid fragments. 放射性标记。不过,也有必要将完整探针与探针降解产物或不需要的副反应产物分离开来。同时分离完整探针和去除未结合标记(常用于纯化寡核苷酸)的常用方法是在聚丙烯酰胺凝胶上对反应产物进行分馏。含有标记探针的凝胶区域被切除,寡核苷酸从凝胶基质中洗脱出来(Miyada 和 Wallace,1987 年)。此处详述的方法(表 8.11)仅用于去除未结合的标记。不过,它也适用于本章所述所有探针的纯化。这项技术利用了杜邦公司的产品(Nensorb0 ^(TM)20{ }^{\mathrm{TM}} 20 核酸纯化盒)。Nensorb 盒可以去除 DNA 或 RNA 中的蛋白质、盐和未结合的标签。该试剂盒的核酸回收率极高(1 ng 至 20 mug20 \mu \mathrm{~g} ),适用于纯化寡核苷酸和较长的核酸片段。
TABLE 8.9 5’-End labeling using T4 polynucleotide kinase (Maxam and Gilbert, 1980) 表 8.9 使用 T4 多核苷酸激酶进行 5'-末端标记(马克萨姆和吉尔伯特,1980 年)
Prepare reaction buffer 准备反应缓冲液 10 xx10 \times kinase buffer 10 xx10 \times 激酶缓冲液
0.5 M Tris-HCl, pH 7.6 0.5 M Tris-HCl,pH 7.6 0.1MMgCl_(2)0.1 \mathrm{M} \mathrm{MgCl}_{2}
50 mMDTT
1 mM spermidine 1 mM 亚精胺
1 mM EDTA
Add in the following order: 按以下顺序添加 1-501-50 pmol dephosphorylated DNA 5^(')5^{\prime} ends 1-501-50 毫摩尔去磷酸化 DNA 5^(')5^{\prime} 末端 10 mul10 xx10 \mu \mathrm{l} 10 \times kinase buffer 10 mul10 xx10 \mu \mathrm{l} 10 \times 激酶缓冲液 1mu1[gamma^(32)P]1 \mu 1\left[\gamma{ }^{32} \mathrm{P}\right] ATP (ICN Radiochemicals, Irvine, Calif., USA) with a volume specific activity of 160mCi//ml( >= 7000Ci//mM)160 \mathrm{mCi} / \mathrm{ml}(\geqslant 7000 \mathrm{Ci} / \mathrm{mM}) 1mu1[gamma^(32)P]1 \mu 1\left[\gamma{ }^{32} \mathrm{P}\right] ATP(ICN Radiochemicals,Irvine,Calif, USA)的体积比活性为 160mCi//ml( >= 7000Ci//mM)160 \mathrm{mCi} / \mathrm{ml}(\geqslant 7000 \mathrm{Ci} / \mathrm{mM}) 。
Adjust volume to 100 mul100 \mu \mathrm{l} with water 用水将体积调整到 100 mul100 \mu \mathrm{l}
Incubate at 37^(@)C37^{\circ} \mathrm{C} for 30 minutes 在 37^(@)C37^{\circ} \mathrm{C} 条件下孵育 30 分钟
Remove unincorporated nucleotides by standard techniques (see Table 8.11) 用标准技术去除未结合的核苷酸(见表 8.11)
TABLE 8.10 quad3^(')\quad 3^{\prime}-End labeling of oligonucleotides using terminal deoxynucleotidyl transferase (Ratcliff, 1981) 表 8.10 quad3^(')\quad 3^{\prime} 使用末端脱氧核苷酸转移酶对寡核苷酸进行末端标记(Ratcliff,1981 年)
500 mM potassium cacodylate 500 mM 水合碘酸钾
10 mM Tris pH 7.2
2. Add in the following order: 2.按以下顺序添加 10 mul10 \mu \mathrm{l} potassium cacodylate 10 mul10 \mu \mathrm{l} 卡可地酸钾 5mu10mM5 \mu 10 \mathrm{mM} cobalt chloride 5mu10mM5 \mu 10 \mathrm{mM} 氯化钴
10 ng oligonucleotide 10 纳克寡核苷酸 10 mul[alpha^(-32):}10 \mu \mathrm{l}\left[\alpha^{-32}\right. P]dATP (NEN, Boston, Mass., USA) with a volume specific activity of 10 muCi//mu10 \mu \mathrm{Ci} / \mu (specific activity 3000Ci//mM3000 \mathrm{Ci} / \mathrm{mM} ) 10 mul[alpha^(-32):}10 \mu \mathrm{l}\left[\alpha^{-32}\right. P]dATP(NEN,波士顿,马萨诸塞州,美国),体积比活性为 10 muCi//mu10 \mu \mathrm{Ci} / \mu (比活性 3000Ci//mM3000 \mathrm{Ci} / \mathrm{mM} )
3. Adjust to a final volume of 50 mul50 \mu \mathrm{l} with water 3.用水调节到 50 mul50 \mu \mathrm{l} 的最终体积
4. Add 0.5 mul0.5 \mu \mathrm{l} terminal deoxynucleotidyl transferase ( 55U//mul55 \mathrm{U} / \mu \mathrm{l}, Boehringer Mannheim Biochemicals, Indianapolis, Ind., USA) 4.加入 0.5 mul0.5 \mu \mathrm{l} 末端脱氧核苷酸转移酶( 55U//mul55 \mathrm{U} / \mu \mathrm{l} ,Boehringer Mannheim Biochemicals,Indianapolis,Ind.)
5. Incubate at 37^(@)C37^{\circ} \mathrm{C} for 1 hour 5.在 37^(@)C37^{\circ} \mathrm{C} 下培养 1 小时
6. Remove unincorporated nucleotides using standard techniques (see Table 8.11) 6.使用标准技术去除未结合的核苷酸(见表 8.11) 7.
TABLE 8.11 Purification of labeled hybridization probes using the Nensorb ^(TM){ }^{\mathrm{TM}} 20 cartridge (Dupont Corporation; Johnson et al., 1986) 表 8.11 使用 Nensorb ^(TM){ }^{\mathrm{TM}} 20 滤芯(杜邦公司;Johnson 等人,1986 年)纯化标记杂交探针
Prepare solutions 准备解决方案
Reagent AA 试剂 AA
0.1 M Tris-HCl pH 7.7
10.0 mM triethylamine 10.0 mM 三乙胺
1.0 mMEDTA
Reagent B 试剂 B
50% methanol 50% 甲醇
2. Prerinse the cartridge with 2ml100%2 \mathrm{ml} 100 \% methanol 2.用 2ml100%2 \mathrm{ml} 100 \% 甲醇预洗滤芯
3. Equilibrate with 2 ml of reagent AA 3.用 2 毫升试剂 AA 平衡
4. Load the sample in a volume greater than 400 mul400 \mu \mathrm{l}, wash with several ml reagent A and elute in a small volume of 50%50 \% methanol (reagent B) 4.以大于 400 mul400 \mu \mathrm{l} 的体积装载样品,用几毫升试剂 A 冲洗,然后用小体积的 50%50 \% 甲醇(试剂 B)洗脱。
E. IMMOBILIZATION OF NUCLEIC ACID ON MEMBRANE SUPPORTS E.将核酸固定在膜支持物上
Detailed discussion of hybrid detection will be restricted to systems having the target nucleic acid immobilized on nylon or nitrocellulose membranes prior to hybridization with probe. Although solution hybridization coupled with capture/detection or detection techniques is an alternative approach, it is not a widely used research tool for determinative studies. Detailed discussion of solution hybridization is addressed elsewhere (see Chapters 2 and 3 ) and is beyond the scope of this chapter. 关于杂交检测的详细讨论将仅限于在与探针杂交前将目标核酸固定在尼龙膜或硝酸纤维膜上的系统。虽然溶液杂交与捕获/检测或检测技术相结合是一种替代方法,但它并不是确定性研究中广泛使用的研究工具。有关溶液杂交的详细讨论将在其他章节中进行(见第 2 章和第 3 章),不在本章讨论范围之内。
Immobilization of DNA on nitrocellulose was first described by Nygaard and Hall (1963, 1964). Two years later, Gillespie and Spiegelman (1965) described the detection of membrane-bound nucleic acid by hybridization with a radioactive probe. Denhardt (1966) further developed the method for application to multiple samples, making it a standard tool in molecular biology. Publication of the paper ‘Detection of specific sequences among DNA fragments separated by gel electrophoresis’ was another landmark in molecular biology (Southern, 1975). Since this time, a variety of alternative supports has been fabricated (e.g. activated nitrocellulose, nylon membranes) and many modifications of transfer and hybridization protocols have been described. Again for a complete listing the reader is referred to a recent review (Meinkoth and Wahl, 1984). Tables 8.12 and 8.13 detail two protocols using the much easier to handle nylon support membranes. These are derivatives of the basic techniques originally described by Denhardt and Southern. Nygaard 和 Hall(1963 年、1964 年)首次描述了将 DNA 固定在硝酸纤维素上的方法。两年后,Gillespie 和 Spiegelman(1965 年)描述了通过与放射性探针杂交来检测膜结合核酸的方法。Denhardt(1966 年)进一步发展了这一方法,将其应用于多个样本,使其成为分子生物学的标准工具。通过凝胶电泳分离的 DNA 片段中特定序列的检测》一文的发表是分子生物学的另一个里程碑(Southern,1975 年)。从那时起,人们开始制造各种替代支撑物(如活化硝酸纤维素、尼龙膜),并对转移和杂交方案进行了许多修改。如需完整清单,读者可参阅最近的一篇综述(Meinkoth 和 Wahl,1984 年)。表 8.12 和 8.13 详细介绍了使用更容易处理的尼龙支撑膜的两种方案。这些都是 Denhardt 和 Southern 最初描述的基本技术的衍生物。
In response to the increasing use of nylon membranes for nucleic acid hybridization, the traditional Southern transfer has been slightly modified 由于越来越多地使用尼龙膜进行核酸杂交,传统的 Southern 转印也略有改动
TABLE 8.12 Immobilization of RNA and DNA on nylon membranes using a dot blot apparatus 表 8.12 使用点印迹仪将 RNA 和 DNA 固定在尼龙膜上
Denaturation of RNA RNA 的变性
Add three volumes 2%2 \% glutaraldehyde in 50 mM sodium phosphate ( pH 7.0 ) to the RNA solution (c. 100 mug//ml100 \mu \mathrm{~g} / \mathrm{ml} ) 在 RNA 溶液中加入三体积 2%2 \% 戊二醛(50 mM 磷酸钠,pH 7.0) (c. 100 mug//ml100 \mu \mathrm{~g} / \mathrm{ml} )
Dilute the denatured RNA in dye/poly A(1mug//ml\mathrm{A}(1 \mu \mathrm{~g} / \mathrm{ml} polyadenylic acid, 0.0002%0.0002 \% bromophenol blue) 将变性 RNA 稀释在染料/聚 A(1mug//ml\mathrm{A}(1 \mu \mathrm{~g} / \mathrm{ml} 多腺苷酸、 0.0002%0.0002 \% 溴酚蓝中)。
Denaturation of DNA (Kafatos et al., 1979; Chen and Seeburg, 1985) DNA 的变性(Kafatos 等人,1979 年;Chen 和 Seeburg,1985 年)
Add one volume denaturation buffer ( 0.4MNaOH,4mM0.4 \mathrm{M} \mathrm{NaOH}, 4 \mathrm{mM} EDTA) to the DNA and incubate at room temperature for 10 minutes 向 DNA 中加入一体积变性缓冲液( 0.4MNaOH,4mM0.4 \mathrm{M} \mathrm{NaOH}, 4 \mathrm{mM} EDTA),室温下孵育 10 分钟
Neutralize by adding 1//101 / 10 volume 2 M ammonium acetate, place on ice and apply immediately to the membrane 加入 1//101 / 10 体积为 2 M 的醋酸铵进行中和,放在冰上并立即涂抹到膜上。
Application of denatured nucleic acid to the nylon membrane 变性核酸在尼龙膜上的应用
Pre-wet the nylon membrane in water and place in the dot blot apparatus (Schleicher & Schuell, FRG) 用水预湿尼龙膜,并将其放入点印迹仪(德国施莱歇尔和舒尔公司)
Apply between 0.5 ng and 2mug2 \mu \mathrm{~g} of denatured nucleic acid in a volume of about 100 mu1100 \mu 1 to the membrane by using a slight vacuum 使用轻微真空将 0.5 毫微克至 2mug2 \mu \mathrm{~g} 的变性核酸以约 100 mu1100 \mu 1 的体积涂抹到膜上
Remove filter, rinse in 2xx2 \times SSC, and air dry (no additional fixation steps are required for nylon membranes 取出过滤器,在 2xx2 \times SSC 中冲洗,然后晾干(尼龙膜不需要额外的固定步骤
TABLE 8.13 Southern transfer using nylon membranes 表 8.13 使用尼龙膜进行南方转印
Place the agarose gel of electrophoretically separated DNA fragments in a tray containing 0.2 M HCl in a volume sufficient to cover the gel (depurination step) 将电泳分离出 DNA 片段的琼脂糖凝胶放入装有 0.2 M HCl 的托盘中,HCl 的体积应足以覆盖凝胶(去质化步骤)。
Incubate with slight shaking for 15 minutes 轻微振荡孵育 15 分钟
Exchange the acid solution and incubate an additional 15 minutes 交换酸溶液并再培养 15 分钟
Pour the acid away and wash several times with water 倒掉酸液,用清水清洗几遍
Wash 15 minutes each in two exchanges of 0.4 M NaOH 在两次 0.4 M NaOH 交换中各清洗 15 分钟
Wet the nylon membrane in water followed by immersing in 0.4 M NaOH 用水浸湿尼龙膜,然后将其浸入 0.4 M NaOH 溶液中。
Assemble the blotting pyramid; a common arrangement for a one-sided transfer includes (assembled from bottom to top): 组装印迹金字塔;单面转移的常见排列方式包括(从下到上组装):
Wick in tray with 0.4 M NaOH 将蜡烛芯放入装有 0.4 M NaOH 的托盘中
Pretreated gel 预处理凝胶
NyIon membrane 离子膜
10 sheets of Whatman 3 MM paper 10 张 3 毫米 Whatman 纸
Paper towels 纸巾
Weight 重量
8. Transfer is completed in 12-24 hours at room temperature 8.室温下 12-24 小时内完成转移
9. Disassemble the blotting pyramid and rinse the filter in 2xx2 \times SSC 9.拆卸印迹金字塔,在 2xx2 \times SSC 中冲洗过滤器
(Reed and Mann, 1985). The method differs from the standard protocol (Maniatis et al., 1982) by performance of the transfer under denaturing conditions (Table 8.13). (里德和曼恩,1985 年)。该方法与标准方案(Maniatis 等人,1982 年)的不同之处在于变性条件下的转移性能(表 8.13)。
(i) RFLP analysis (i) RFLP 分析
An approach of increasing interest for determinative microbiology takes advantage of restriction fragment length polymorphism among conserved genes or conserved gene families. Total DNA from those organisms to be compared is digested with a variety of restriction enzymes, transferred to nylon (or nitrocellulose) membranes (Table 8.13) and hybridized with a suitable probe. The most common application of this technique to microbial systematics again takes advantage of the highly conserved rRNA genes (Grimont and Grimont, 1986; Stull et al., 1988; Yogev et al., 1988). A probe commonly used is base-hydrolyzed and 5 -end-labeled rRNA. Since cross-kingdom hybridization of these probes has been demonstrated, selection of the organism used as source of the rRNA probe is not critical. Organisms are identified or distinguished by characteristic banding patterns. One shortcoming of this approach is the lack of a quantitative relationship between banding pattern and the similarity of organisms compared. 限制性片段长度多态性是确定性微生物学中越来越受关注的一种方法,它利用了保守 基因或保守基因家族之间的限制性片段长度多态性。用各种限制性酶消化要比较的生物的总 DNA,将其转移到尼龙(或硝酸纤维素)膜上(表 8.13),然后与合适的探针杂交。这种技术在微生物系统学中最常见的应用还是利用高度保守的 rRNA 基因(Grimont 和 Grimont,1986 年;Stull 等人,1988 年;Yogev 等人,1988 年)。常用的探针是碱基水解和 5 端标记的 rRNA。由于这些探针的跨领域杂交已经得到证实,因此选择何种生物作为 rRNA 探针的来源并不重要。生物体可通过特征条带模式来识别或区分。这种方法的一个缺点是缺乏条带模式与所比较生物相似性之间的定量关系。
(ii) The 16S rRNA and a comparative framework for determinative hybridization (ii) 16S rRNA 和确定性杂交的比较框架
As stated earlier, there is as yet no preferred strategy for probe design. However, directed (rational) probe design will probably prevail; only within a well-defined comparative framework can probe hybridization be properly interpreted. Microbiology has strong traditions of determinative classifications. Molecular comparisons have fostered a rebirth of naturally based classifications. It is within the determinative tradition that the empirically designed probes fall. Although experimental validation is a necessary ingredient of probe characterization, it is not a satisfying foundation for probe design. As traditional determinative classification schemes are increasingly merged with molecularly-based phylogenetic classifications, the design of determinative probes will increasingly reflect the natural classification. Comparative molecular analyses, most importantly comparative sequencing, are dramatically changing the character of microbiology and will increasingly influence the determinative arena. The most powerful determinative framework is provided now by the 16 S rRNA sequence collection. 如前所述,探针设计还没有首选策略。不过,定向(合理)探针设计可能会占上风;只有在一个明确的比较框架内,才能正确解释探针杂交。微生物学有很强的确定性分类传统。分子比较促进了自然分类法的重生。经验设计的探针正是属于确定性传统。虽然实验验证是探针特征描述的必要组成部分,但它并不是探针设计的令人满意的基础。随着传统的确定性分类方案越来越多地与基于分子的系统发育分类合并,确定性探针的设计将越来越多地反映自然分类。比较分子分析,最重要的是比较测序,正在极大地改变微生物学的特征,并将对确定性领域产生越来越大的影响。目前,16 S rRNA 序列收集提供了最强大的确定性框架。
The application of the 16 S16 S rRNA data collection to determinative studies in microbiology is presented in the form of a story. This investigative story should serve to illustrate the preceding theoretical, anecdotal and rote methodological listings. The intent is not to specify a preferred method- 16 S16 S rRNA 数据收集在微生物学确定性研究中的应用以故事的形式呈现。这个调查故事应有助于说明前面列出的理论、轶事和生搬硬套的方法。其目的并不是指定一种首选方法
ology, but to offer, by example, one approach to unifying molecular and determinative systematics using the described techniques of nucleic acid hybridization. The foundation of probe design, and experimental interpretation, is comparative sequencing of the ribosomal RNAs. The use of the 16 S rRNA sequence collection for the study of the natural microbial community of the bovine rumen will be described in detail. This includes the more recent development of fluorescent probes for microscopic identification of single cells in pure or mixed culture and in the environment. 在此,我只想举例说明一种利用所述核酸杂交技术统一分子系统学和确定系统学的方法。探针设计和实验解释的基础是核糖体 RNA 的比较测序。将详细介绍如何利用 16 S rRNA 序列集研究牛瘤胃的天然微生物群落。这包括最近开发的荧光探针,用于在显微镜下鉴定纯培养物或混合培养物以及环境中的单细胞。
An ongoing study in our laboratory concerns the study of ruminal microbial ecology. Most effort to this time has been directed to the study of the principal fiber-digesting microbiota. Among ruminal prokaryotes, three genera have been implicated as the principal fiber-digesting organisms. These include species of Butyrivibrio, Ruminococcus and Fibrobacter (formerly Bacteroides succinogenes). The molecular classification of these organisms and design of nucleic acid probes for their identification (within the pure culture collection and the environment) provides a useful framework for discussion of the development and application of probes for genus, species and subspecies identification. For purposes of instruction, details of probe design and application are restricted to discussion of the genus Fibrobacter. However, the development and application of probes for their determinative and environmental characterization could be applied to any microorganism. 我们实验室正在进行的一项研究涉及瘤胃微生物生态学研究。迄今为止,我们的大部分精力都放在了对主要纤维消化微生物群的研究上。在瘤胃原核生物中,有三个属被认为是主要的纤维消化生物。其中包括布氏嗜血杆菌属、反刍球菌属和纤维细菌属(原琥珀酸芽孢杆菌属)。这些生物的分子分类和用于鉴定它们的核酸探针的设计(在纯培养物和环境中)为讨论属、种和亚种鉴定探针的开发和应用提供了一个有用的框架。为了便于教学,探针设计和应用的细节仅限于讨论纤维细菌属。不过,探针的开发和应用可用于任何微生物的确定性和环境特征鉴定。
(iii) The design of phylogenetically based nucleic acid hybridization probes (iii) 设计基于系统发育的核酸杂交探针
The use of ribosomal RNAs for studies of microbial phylogeny and evolution is well developed, as indicated by their prominent representation elsewhere (see Chapters 3, 5, 6 and 7). Although these studies are central to design of the determinative probes described here, background discussion will necessarily be limited. The reader is referred to reviews for detailed discussion of approach and analysis (Olsen et al., 1986; Stahl, 1988; Stahl et al., 1988) 核糖体 RNA 在微生物系统发育和进化研究中的应用已十分成熟,这一点在其他章节(见第 3 章、第 5 章、第 6 章和第 7 章)中也有突出表现(见第 3 章、第 5 章、第 6 章和第 7 章)。尽管这些研究是本文所述决定性探针设计的核心,但背景讨论必然有限。读者可参阅有关方法和分析的详细讨论(Olsen 等人,1986 年;Stahl,1988 年;Stahl 等人,1988 年)。
The ribosomal RNAs have for several reasons proved exceptionally well suited as targets for determinative probes. Although they are highly conserved biopolymers, they exhibit great variation in regional sequence conservation. Some nucleotide positions and locales have remained virtually unchanged since the divergence of all existing life (universal sequences), whereas other regions vary so quickly that they can be used to differentiate among species or subspecies of bacteria. In addition, their high copy number per cell lends greater sensitivity to direct determinative tests. A diagrammatic representation of relative positional conservation is shown in Fig. 8.3. For determinative studies, probes of 15-2515-25 nucleotides in length are commonly used for hybridization to whole cells or total 由于多种原因,核糖体 RNA 被证明非常适合作为确定性探针的目标。尽管核糖体 RNA 是高度保守的生物聚合物,但它们在区域序列保守性方面却表现出很大的差异。有些核苷酸的位置和位置自所有现存生命分化以来几乎保持不变(通用序列),而其他区域则变化极快,可用于区分细菌的物种或亚种。此外,它们在每个细胞中的拷贝数很高,因此对直接确定性测试的灵敏度更高。图 8.3 是相对位置保持的示意图。在确定性研究中,长度为 15-2515-25 核苷酸的探针通常用于与全细胞或总细胞杂交。
Fig. 8.3 Positional conservation representation of the 16S rRNA secondary structure derived from the comparison of 27 diverse eubacterial species. Shading intensity varies according to the relative conservation of each homologous nucleotide. Invariant positions are black. (Reproduced with permission, Nature Publishing Co.) 图 8.3 通过比较 27 个不同的真细菌物种得出的 16S rRNA 二级结构的位置保护表示法。阴影强度根据每个同源核苷酸的相对保守性而变化。不变位置为黑色。(经授权转载,自然出版公司)。
nucleic acid extracted from cultures or the environment. The assemblage of organisms addressed by a single hybridization probe varies according to the region of the molecule selected as the hybridization target. Subspeciesspecific probes target the most variable regions. More general probes (those identifying species or larger, phylogenetically coherent, assemblages) target more conserved regions of the molecule (Fig. 8.3). Examples of the design of kingdom-specific (eubacteria, archaebacteria; Woese and Fox, 1977) and species-specific (Fibrobacter succinogenes, F. intestinalis) oligonucleotide probes are shown in Fig. 8.4. 从培养物或环境中提取的核酸。单个杂交探针所针对的生物群体因分子中被选为杂交目标的区域而异。亚种特异性探针针对的是变化最大的区域。更通用的探针(识别物种或更大的、系统发育一致的集合体的探针)针对分子中更保守的区域(图 8.3)。图 8.4 显示了王国特异性(真细菌、古细菌;Woese 和 Fox,1977 年)和物种特异性(琥珀酸纤毛杆菌、肠道纤毛杆菌)寡核苷酸探针的设计实例。
Fig. 8.4 Target sequences for kingdom- and species-specific oligonucleotide probes. Sequence alignment of 16 S rRNAs in the ukaryotes). Left: target region for the eubacterial prope Eub338 the three primary kingdoms (eubacteria, archaebacteria and 图 8.4 王国和物种特异性寡核苷酸探针的目标序列。图 8.4 王国和物种特异性寡核苷酸探针的目标序列(图 8.4ukaryotes 中 16 S rRNA 的序列比对)。图 8.4 王国和物种特异性寡核苷酸探针的目标序列。
Nucleic Acid Techniques in Bacterial Systematics. Edited by E. Stackebrandt and M. Goodfellow 《细菌系统学中的核酸技术》。E. Stackebrandt 和 M. Goodfellow 编辑