生物技术专业英语翻译

Lesson 1 DNA CLONING: AN OVERVIEW

第一课克隆：概要

DNA cloning：DNA doning facilitates the isolation and manipulation of fragments of an organism's genome by replicating them independently as part of an autonomous vector.

DNA克隆：DNA克隆通过独立复制可以用分离和操作方法使生物体基因组片段成为自发性载体的一部分。

Hosts and vectors: Most of the routine manipulations involved in gene cloning use Escherichia coli as the host organism. Plasmids and bacteriophages may be used as cloning vectors in E. coli. Vectors based on plasmids, viruses and whole chromosomes have been used to carry foreign genes into other prokaryotic and eukaryotic organisms.

宿主和载体：大多数的基因克隆使用的常规操作，以大肠杆菌为宿主生物体。质粒和噬菌体可作为大肠杆菌的克隆载体。以质粒、病毒和整条染色体为载体，将外源基因导入其他原核和真核生物。

Subcloning: Subcloning is the simple transfer of a cloned fragment of DNA from one vector to another; it serves to illustrate many of the routine techniques involved in gene cloning.

亚克隆：克隆是克隆的DNA片段从一个载体到另一个载体的简单传递；它可以用来说明基因克隆中的许多常规技术。

DNA libraries : DNA libraries, consisting of sets of random cloned fragments of either genomic or cDNA, each in a separate vector molecule, are used in the isolation of unknown genes.

基因库：基因库，由两组随机克隆片段组成，无论是基因组还是基因，每一个在一个单独的载体分子中，都被用来分离未知的基因。

Screening libraries: Libraries are screened for the presence of a gene sequence by hybridization with a sequence derived from its protein product or a related gene, or through the screening of the protein products of the cloned fragments.

筛选库：通过与来自其蛋白产物或相关基因的序列杂交，或者通过克隆片段的蛋白产物的筛选，筛选出一个基因序列的存在。

Analysis of a clone: Once identified, a cloned gene may be analyzed by restriction mapping, and ultimately by DNA sequencing, before being used in any of the diverse applications of DNA cloning.

克隆分析:在用于基因克隆的各种应用中之前，一旦发现某个克隆基因可以通过限制性图谱进行分析，则用这方法进行DNA序列测定。

DNA cloning: detailed molecular analysis of proteins or other constituents of most organisms was rendered difficult or impossible by their scarcity and the consequent difficulty of their purification in large quantities. One approach is to isolate the gene(s)

responsible for the expression of a protein or the formation of a product. However, every organism's genome is large and complex (see Section D), and any sequence of interest usually occurs only once or twice per cell. Hence, standard chemical or biochemical methods cannot be used to isolate a specific region of the genome for study, particularly as the required sequence of DNA is chemically identical to all the others. The solution to this dilemmas is to place a relatively short fragment of a genome, which might contain the gene or other sequence of interest, in an autonomously replicating piece of DNA, known as a vector, forming recombinant DNA, which can be replicated independently of the original genome, and normally in another host species altogether. Propagation of the host organism containing the recombinant DNA forms a set of genetically identical organisms, or a clone. This process is hence known as DNA cloning.

DNA克隆：一般上，因为大多数生物体的蛋白质或其他成分的稀缺性及其在大量的净化过程中的困难，导致它们的详细分子分析变得困难甚至不可能。一种方法是分离负责蛋白质表达的基因或者其产品的构成。然而每个生物体的基因组都是大而复杂的（见第4节），每个细胞中任何目标序列通常只发生一次或两次。因此，标准的化学或生化方法不能用于分离特定区域的基因组进行研究，特别是所需的DNA序列与所有其他的序列的化学性质相同。这种难点的解决方法是放置一个可能包含目标基因或序列相对短的基因组片段在一个自主复制片段的基因，称为载体，在另一个宿主物种中它可以复制独立的原始基因组，形成重组DNA。含有重组DNA的宿主生物体的繁殖形成一组基因相同的生物体，或克隆。这个过程就是DNA克隆。

Amongst the exploding numbers of applications of DNA cloning, often collected together under the term genetic engineering, are the following:

在长期的基因工程中，克隆的应用程序的数量激增，这是以下几项：

(1)DNA sequencing, and hence the derivation of protein sequence (see Topic J2).

DNA序列测定，也即蛋白质序列的推导（见主题J2）。

(2)Isolation and analysis of gene promoters and other control sequences (see Topic

J4).

基因启动子与其它控制序列的分离与分析（见主题J4）。

(3)Investigation of protein/enzyme/RNA function by large-scale production of normal

and altered forms (see Topic JS).

蛋白质/酶/ RNA功能的正常大规模生产和改变的研究（见主题J5）。

(4)Identification of mutations, for example gene defects leading to disease (see Topic

J6).

突变的识别，例如基因缺陷导致的疾病（见主题J6）。

(5)Biotechnology; the large-scale commercial production of proteins and other

molecules of biological importance, for example human insulin and growth

hormone (see Topic J6).

生物技术；蛋白质和其他分子生物学意义的大规模商业化生产，例如人胰岛素和生长激素（见主题J6）。

(6)Engineering animals and plants, and gene therapy (see Topic J6).

转基因动物和植物，和基因治疗（见主题J6）。

(7)Engineering proteins to alter their properties (see Topic J6).

通过蛋白质工程来改变它们的性质（见主题J6）

Hosts and vectors: The initial isolation and analysis of DNA fragments is almost always carried out using the bacterium E. coli as the host organism, although the yeast Saccharomyces cerevisiae is being used to manipulate very large fragments of the human genome (see Topic H3). A wide variety of natural replicons have the properties required to allow them to act as cloning vectors. Vectors must normally be capable of being replicated and isolated independently of the host's genome, although some are designed to incorporate DNA into the host genome for longer term expression of cloned genes. Vectors also incorporate a selectable marker, a gene which allows host cells containing the vector to be selected from amongst those which do not, usually by conferring resistance to a toxin(see Topic G2), or enabling their survival under certain growth conditions (see Topic H3).

宿主和载体DNA片段的初步分离和分析几乎总是使用大肠杆菌作为宿主生物体，虽然酿酒酵母被用来操纵人类基因组大片段（见主题H3）。各种各样的自然的复制，要求他们拥有作为克隆载体的性质。载体通常是能够被复制和独立的主机的基因组中分离，虽然一些被设计成将核酸到宿主基因组中的长期表达的克隆基因。载体也将选择标记允许宿主细胞中没有相同序列载体的基因，通常用抗毒素（见主题G2），或使他们在一定的生长条件生存（见主题H3）。

The first E. coli vectors were extrachromosomal (separate from the chromosome) circular plasmids (see Topic G2), and a number of bacteriophages(viruses infecting bacteria; see Topic R2) have also been used in E. coli. Phage k can be used to clone fragments larger than plasmid vectors, and phage M13 allows cloned DNA to be isolated in single-stranded form (see Topic H2). More specialist vectors have been engineered to use aspects of plasmids and bacteriophages, such as the

plasmid-bacteriophage k hybrids known as cosmids(see Topic H3). Very large genomic fragments from humans and other species have been cloned in S. cerevisiae as yeast artificial chromosomes (YACs; seeTopic H3). Plasmid and phage vectors have been used to express genes in a range of bacteria other than E. coli, and .some phages may be used to incorporate DNA into the host genome, for example phage k (see Topic H2). Plasmid vectors have been developed for use in yeast (yeast episomal plasmids), while in plants; a bacterial plasmid (Agrobacterium tumefaciens Ti plasmid) can be used to integrate DNA into the genome. In other eukaryotic cells in culture, vectors have often

been based on viruses which naturally infect the required species, either by maintaining their DNA extrachromosomally or by integration into the host genome (examples include SV40, baculovirus, retroviruses; see Topic H4).

第一个大肠杆菌质粒染色体外（从染色体分离）的环状质粒（见主题G2），和一些噬菌体（感染细菌的病毒；见主题R2）也被用于大肠杆菌。噬菌体K可用于克隆片段大于质粒，噬菌体M13和允许克隆的DNA被孤立单链形式（见主题H2）。更专业的载体已被设计为使用质粒和噬菌体方面，如质粒，噬菌体K杂交称为粘粒（见主题H3）。非常大的基因组片段从人类和其他物种已被克隆的酿酒酵母的酵母人工染色体（YAC克隆；seetopic H3）。质粒和噬菌体载体已被用于在一个范围内的其他比大肠杆菌，细菌和噬菌体的基因表达。一些可能被用来将DNA插入宿主基因组，例如噬菌体K（见主题H2）。质粒载体已经开发使用的酵母（酵母着丝粒质粒），而植物；细菌质粒（根癌农杆菌Ti质粒）可以用来整合进基因组DNA。在文化的其他真核细胞中，载体通常是根据其自然感染病毒所需的物种，或者通过保持他们的DNA染色体外或整合到宿主基因组（包括SV40，杆状病毒，逆转录病毒；见主题H4）。

Subcloning: The simplest kind of cloning experiment, which exemplifies many of the basic techniques of DNA cloning, is the transfer of a fragment of cloned DNA from one vector to another, a process known as subcloning. This might be used to investigate a short region of a large cloned fragment in more detail, or to transfer a gene to a vector designed to express it in a particular species, for example. In the case of plasmid vectors in E. coli, the most common situation, the processmay be divided into the following steps, which are considered in greater detail in Topics G2-G5:

亚克隆：最简单的克隆实验，这体现了许多DNA克隆的基本技术，是克隆的片段从一个载体到另一个载体的转移，这一过程称为亚克隆。这可更详细的用来研究一个大克隆片段的短区域，比如说将基因放入载体使其转移到一个特定物种中进行表达。通常情况下，在质粒载体转移到大肠杆菌的案例中，该方法可分为以下几个步骤，其中在主题g2-g5的说明更详细:

(1)Isolation of plasmid DNA containing the cloned sequence of interest (seeTopic G2).

质粒分离包含目标克隆序列（见主题G2）。

(2)Digestion (cutting) of the plasmid into discrete fragments with restriction

endonucleases (see Topic G3).

用限制性内切酶切（割）离散片段的质粒（见主题G3）。

(3)Separation of the fragments by agarose gel electrophoresis (see Topic G3).

通过琼脂糖凝胶电泳分离片段（见主题G3）。

(4)Purification of the desired target fragment(see topic G3)

纯化所需的目标片段（见主题G3）。

(5)Ligation (joining) of the fragment into a new plasmid vector, to form a new

recombinant molecule (see Topic G4).

将片段插入新的质粒载体中，以形成新的重组分子（见主题G4）。

(6) Transfer of the ligated plasmid into an E. Coil strain (transformation) (see topic G4).

把连接质粒转移到大肠杆菌菌株（变换）（见主题G4）。

(7) selection of transformed bacteria (see topic G4).

转化菌的筛选（见主题G4）。

(8) Analysis of plasmids (see Topic G4).

质粒分析（见主题G4）。

DNA libraries:

克隆库:

There are two main sources from which DNA is derived for cloning experiments designed to identify an unknown gene - bulk genomic DNA from the species of interest and, in the case of eukaryotes, bulk mRNA from a cell or tissue where the gene is known to be expressed. They are used in the formation of genomic libraries and cDNA libraries respectively (see Topic I2).

有2个主要来源，一是基因的克隆实验，目的是鉴定未知的基因——来自目标物种的大部分基因组DNA。二是就真核生物来说，大多数mRNA来自已知其基因表达的细胞或组织。它们分别用于基因组文库和基因库的形成（见主题I2）。

DNA libraries are sets of DNA clones (a clone is a genetically distinct individual or set of identical individuals), each of which has been derived from the insertion of a different fragment into a vector followed by propagation in the host. Genomic libraries are prepared from random fragments of genomic DNA. However, genomic libraries may be an inefficient method of finding a gene, particularly in large eukaryotic genomes, where much of the DNA is noncoding (see Topic D4). The alternative is to use as the source of the library the mRNA from a cell or tissue which is known to express the gene. DNA copies (cDNA) are synthesized from the mRNA by reverse transcription and are then inserted into a vector to form a cDNA library, cDNA libraries are efficient for cloning a gene sequence, but yield only the coding region, and not the surrounding genomic sequences.

克隆库是一组基因的克隆（克隆是一个基因不同的个体或相同的个体），每一个都来自于一个不同的片段插入到载体中，然后在宿主中传播。基因组文库是从基因组的随机片段制备的。然而，基因组文库中可能是一个低效率的寻找基因的方法，特别是在大型的真核生物的基因组，其中大部分是非编码DNA（见主题D4）。另一种方法是利用细胞或组织中的细胞或组织来表达基因的来源。通过反转录合成基因的复制品，并将其插入

到载体中，形成一个文库，文库是有效的，用于克隆一个基因序列，但只产生编码区，而不是周围的基因组序列。

Screening libraries:

筛选库:

Since it is not apparent which clone in a library contains the gene of interest, a method for screening for its presence is required. This is often based on the use of a radioactively labeled DNA probe which is complementary or partially complementary to a region of the gene sequence, and which can be used to detect it by hybridization (see Topic C3). The probe sequence might be an oligonucleotide derived from the sequence of the protein product of the gene, if it is available, or from a related gene from another species (see Topic I3). An increasingly important method for the generation of probes is the polymerase chain reaction (PCR;.see Topic J3). Other screening methods rely on the expression of the coding sequences of the clones in the library, and identification of the protein product from its activity, or with a spedfic antibody, for example (see Topic I3).

因为在克隆库中目标基因是不明显的，所以其筛选的方法的存在是必需的。这通常基于应用的放射性标记DNA探针是互补的或部分互补的一个区域的基因序列，可以通过杂交来检测它（见主题C3）。探针序列可能是来自于基因蛋白质产物序列的一种寡核苷酸，也可能来自另一种相关基因（见主题I3）。聚合酶链反应是一种日益重要的探针生成方法（PCR；。见主题J3）。其他筛选方法依赖于在克隆库中的编码序列的表达，比如说用特异抗体进行蛋白质产物的活性鉴定（见主题I3）。

Analysis of a clone:

克隆分析:

Once a clone containing a target gene is identified, the structure of the cloned fragment may be investigated further using restriction mapping, the analysisof the fragmentation of the DNA with restriction enzymes (see Topic J1), or ultimately by the sequencing of the entire fragment (see Topic J2). The sequence can then be analyzed by comparison with other known sequences from data-bases, and the complete sequence of the protein product determined (see TopicJ2). The sequence is then available for manipulation in any of the applicationsof cloning described above.

一旦一个克隆含有目标基因，克隆的片段的结构可以用限制性内切酶图谱进一步调查，分析酶切的DNA碎片（见主题J1），或通过整个片段的顺序进行分析（见主题J2）。那么序列可通过与其他已知序列数据库进行对比分析，进而确定蛋白质产物的完整序列（见主题j2）。因而序列可用于操纵上述的任何克隆应用。

Many enzymes are used in vitro in DNA cloning and analysis. The properties of the common enzymes are given in Table 1, along with the section wheretheir use is discussed in more detail.

许多酶应用于基因克隆与分析中。常见酶的性质见表1，根据他们的使用部分进行更详细的讨论。

Lesson3 RESTRICTION ENZYMES AND ELECTROPHORESIS

第三课限制酶和电泳

Restriction endonucleases: Restriction endonucleases are bacterial enzymes which cut (hydrolyze) DNA into defined and reproducible fragments. In bacteria, they form part of the restriction-modification defense mechanism against foreign DNA. They are the basic tools of gene cloning.

限制性内切酶：限制性内切酶是把DNA切割或者水解成明确的且可重复片段的细菌酶。在细菌中，它们构成部分限制性修饰对抗外源基因的防御机制。它们是基因克隆的基本工具。

Recognition sequence: Restriction enzymes cleave DNA symmetrically in both strands at short palindromic (symmetrical) recognition sequences to leave a 5'-phosphate and a 3'-OH. They leave blunt ends, or protruding 5'- or 3'-termini.

识别序列：限制内切酶在短的对称识别序列的位点上对称性地切割双链DNA，而留下一个5端的磷酸和一个3端的羟基。它们留下迟钝的末端或者突出5'末端或3’末端。Cohesive ends:Restriction enzyme products with single-stranded termini are said to have cohesive or 'sticky' ends, since they can anneal by base pairing to any other fragment with a complementary terminus.

粘性末端：由限制酶切割产生的单链终点处据说是拥有有粘着力的的末端，后来它们能够退火，通过其他片段与末端进行碱基互补配对。

Restriction digests: Commercially supplied enzymes are used to digest plasmid DNA before analysis or purification of the fragments by agarose gel electrophoresis.

限制性消化：在分析或者通过琼脂糖凝胶电泳进行片段的提纯和分析之前，质粒DNA 要用市场上能买到的酶消化分解。

Agarose gel electrophoresis: Agarose gels separate linear DNA on the basis of size, by the migration of DNA through a matrix under the influence of an electric

field.Electrophoresis may be used to determine the gross organization of plasmid molecules.

琼脂糖凝胶电泳：琼脂糖凝胶根据大小分离线状DNA，在电场的作用下将DNA迁移到基质中。电泳可以用于确定质粒分子总的组织。

Isolation of fragment: Specific DNA fragments may be cut out of agarose gels and purified for use in subsequent cloning experiments.

分离片段：特殊DNA片段可以被琼脂糖凝胶切割开来以及纯化，用在随后的克隆实验。Restriction endonucleases:

限制性内切酶：

To incorporate fragments of foreign DNA into a plasmid vector, methods for the cutting and rejoining of dsDNA are required. The identification and manipulation of restriction endonucleases in the 1960s and early 1970s was the key discovery which

allowed the cloning of DNA to become a reality. Restriction-modification systems occur in many bacterial species, and constitute a defense mechanism against the introduction of foreign DNA into the cell. They consist of two components; the first is a restriction endonuclease, which recognizes a short, symmetrical DNA sequence (Fig.

1), and cuts (hydrolyzes) the DNA backbone in each strand at a specific site within that sequence. Foreign DNA will hence be degraded to relatively short fragments. The second component of the system is a methylase, which adds a methyl group to a C or A base within the same recognition sequences in the cellular DNA (see Topic Cf). This modification renders the host DNA resistant to degradation by the endonuclease.

将外来DNA片段整合到质粒载体中需要双链DNA切割和再结合的方法。在上世纪60年代和上世纪70年代早期，限制性内切酶的识别和操纵是使DNA克隆成为事实的重要发现。限制修饰系统在许多细菌物种中都有，并且构成了一个抵制外来DNA进入细胞中。它们由两个要素构成；第一个是限制性内切酶。限制性内切酶识别短的对称DNA 序列，并在每条链的那段序列的特殊位点上切割。因此外来DNA将被退化到相对短的片段。系统的第二个重要要素是甲基化酶。在细胞DNA中，甲基化酶在相同的识别序列中对C或者A添加一个甲基基团。这种改变使宿主DNA能抵制由于内切酶而导致的降解。

Recognition sequences: The action of restriction endonudeases (restriction enzymes for short) is illustrated in Fig. la including the archetypal enzyme EcoRI as an example. This enzyme, which acts as a dimer, will only recognize a 6 bp palindromic sequence (the sequence is the same, reading 5'→3', on each strand). The product of the cutting reaction at this site on a linear DNA is two double-Stranded fragments (restriction fragments), each with an identical protruding single-stranded 5'-end with a phosphate group attached. The 3'-ends have free hydroxyl groups.

识别序列：在图1a阐明了限制性内切酶的行动特征还以原型的EcoRI酶作为例子。这种酶是一个二聚物，只能识别6个碱基对的复发序列（这类序列都是一样的，在每条单链上都是从5'→3'阅读）。在每个线状DNA上的特殊位点切割得到的产物是两个双链片段，每个片段带有相同的突出的单链带有磷酸基团的5’末端。3’端具有自由的羟基基团。

A 6 bp recognition sequence will occur on average every 46 = 4096 bp in random sequence DNA; hence, a very large DNA molecule will be cut into specific fragments averaging 4 kb by such an enzyme. Hundreds of restriction enzymes are now known, and a large number are commercially available. They recognize sites ranging in size from 4 to 8 bp or more, and may give products with protruding 5'- or 3'-tails or blunt ends. The newly formed 5'-ends always retain the phosphate groups. Two further examples are illustrated in Fig. 1. The extremely high specificity of restriction enzymes for their sites of action allows large DNA molecules and vectors to be cut reproducibly into defined fragments.

在随机的DNA序列里平均每46 = 4096 bp就会出现一个6bp的识别序列；因此，一个人非常大的DNA分子会被这样的酶切割成明确的平均具有4kb的片段。现在许许多多的限制性内切酶已经被知道，并且大多数用在市场上。它们识别在4到8bp的大小范围或者更多的位点，并且能给出带有突出5’尾巴或者3’尾巴或者是迟钝末端的产物。新形成的5’末端总是保留磷酸基团。在图1中阐明了两个更深一层的例子。限制性内切酶对于它们位置处理的高度特异性允许DNA分子和载体可再生地被切割成为明确的片段。

Cohesive ends: Those products of restriction enzyme digestion with protruding ends have a further property; these ends are known as cohesive, or 'sticky' ends, since they can bind to any other end with the same overhanging sequence, by base pairing (annealing) of the single-stranded tails. Hence, for example, any fragment formed by an EcoRI cut can anneal to any other fragment formed in the same way (Fig. lb), and may subsequently be joined covalently by ligation (see Topic E4). In fact, in some cases, DNA ends formed by enzymes with different recognition sequences may be compatible, provided the single-stranded tails can base-pair together.

粘性末端：那些由限制性酶消化得到的带有突出末端的产物具有深一步的特性；这些末端是粘性末端，因此它们能和相同的悬垂序列通过单链尾部的碱基配对与其他末端连接在一起。因此，例如任何由EcoRI切割得到的片段能通过相同的方法退火成任何其他片段，并且随后可以被加到共价连接。事实上，在某些情况上由酶形成的DNA末端与不同识别序列能够兼容，使单链的尾部能与碱基互补。

Restriction digests: Digestion of plasmid or genomic DNA (see Topic I1) is carried out with enzymes for analytical or preparative purposes, using commercial enzymes and buffer solutions. All restriction enzymes require Mg2+, usually at a concentration of up to 10 mM, but different enzymes require different pHs, NaC1 concentrations or other solution constituents for optimum activity. The buffer solution required for a particular enzyme is supplied with it as a concentrate. The digestion of a sample plasmid with two different restriction enzymes, BamHI and EcoRI, is illustrated in Fig. 2.

限制性消化：质粒或者基因组DNA的消化是通过酶进行的，为了分析或制备的目的，利用市场上的酶和缓冲液。所有限制性内切酶都需要Mg2+,时常达到10mM的浓度，但为了达到最适活力，不同的酶需要不同的pHs ，NaCl浓度或其他溶液成分。需要特殊酶的缓冲液作为一种浓缩液供应给它。在图2阐明了一个样本质粒通过两种限制性内切酶BamHI 和EcoRI的消化。

The digestion of a few hundred nanograms(<1μg) of plasmid DNA is sufficient for analysis by agarose gel electrophoresis; preparative purposes may require a few micrograms. The former amount corresponds to a few percent of a miniprep sample, as described in Topic G2. The DNA is incubated with the enzyme and the appropriate buffer at the optimum temperature (usually 37℃),

通过琼脂糖凝胶电泳，几百微毫克的质粒的消化足以让分析；制备目的可能需要几微克。如在Topic G2所描述，前面的数量对应于小量制备样本的很少的百分比。DNA是在最适温度（一般是37℃）由酶和适当缓冲液培养的。

In a valume of perhaps 20μl. A dye mixture is then added to the solution ,and the sample is loaded on an agarose gel.

然后将染色的混合物加到溶液中，接着样本装到琼脂糖凝胶上。

Agarose gel electrophoresis: Agarose is a polysaccharide derived from seaweed, which forms a solid gel when dissolved in aqueous solution at concentrations between 0.5 and 2%(w/V). Agarose used for electrophoresis is a more purified form of the agar used to make bacterial culture plates.

琼脂糖凝胶电泳：琼脂糖是一种来自于海草的多聚糖并且当以0.5到2%的浓度溶解在水溶液的时候它能形成固体凝胶。用于电泳的琼脂比用于制作细菌培养基的是更加纯化的形式。

When an electric field is applied to an agarose gel in the presence of a buffer solution which will conduct electricity, DNA fragments move through the gel towards the positive electrode (DNA is highly negatively charged; see Topic C1) at a rate which is dependent on its size and shape (Fig. 3). Small linear fragments move more quickly than large ones, which are retarded by entanglement with the network of agarose fibers forming the gel. Hence, this process of electrophoresis may be used to separate mixtures of DNA fragments on the basis of size. Different concentrations of gel [1%, 1.5% (w/v), etc.] will allow the optimal resolution of fragments in different size ranges. The DNA samples are placed in wells in the gel surface (Fig. 3), the power supply is switched on and the DNA is allowed to migrate through the gel in separate lanes or tracks.

当一个电场是应用于有缓冲溶液存在的琼脂糖凝胶时其会导电，DNA片段以根据自身大小和形状形成的速率从琼脂糖向正极移动（DNA是高度带负电的，看Topic C1）小的线状片段比大的移动得更快，因为的大的会被琼脂糖纤维形成的网所阻碍。因此，电泳的过程可以根据大小被用于分里DNA片段的混合物。不同浓度的凝胶允许片段在不同大小范围里有最佳的分辨率。DNA 样本被放在凝胶表面的井里，打开电源后DNA被允许在分隔开的分道或轨道上迁移通过凝胶。

The added dye also migrates, and is used to follow the progress of electrophoresis. The DNA is stained by the inclusion of ethidium bromide (see Topic C4) in the gel, or by soaking the gel in a solution of ethidium bromide after electrophoresis. The DNA shows up as an orange band on illumination by UV light.

加进染料也迁移，接着背用于紧随电泳过程。在凝胶中，DNA被内含物溴化乙锭染色或者在电泳后在溴化乙锭里浸染凝胶。在紫外灯的照射下DNA以橙黄色的带出现。

Figure 4a illustrates the result of gel electrophoresis of the fragments formed by the digestions in Fig. 2. The plasmid has been run on the gel without digestion (track U), and after digestion with BamHI (track B) and EcoRI (track E). A set of linear marker DNA fragments of known sizes (tracks M) have been run alongside the samples at two different concentrations; the sizes are marked on the figure. A number of points may be noted.

图4a阐明片段的凝胶电泳的结果，而通过消化形成的片段在图二中。没有消化的质粒已经在凝胶上面跑起来（轨道U），之后被BamHI（轨道B）和EcoRI（轨道E）消化。一整套已知大小的线状标记的DNA片段和两个不同浓度的样本已经跑起来；在图中大小有标记。点的数量可以记下来。

Undigested plasmid DNA (track U) run on an agarose gel commonly consists of two bands. The lower, more mobile, band consists of negatively supercoiled plasmid DNA isolated intact from the cell. This has a high mobility, because of its compact conformation (see Topic C4). The upper band is open-circular, or nicked DNA, formed from supercoiled DNA by breakage of one strand; this has an Opened-out circular conformation and lower mobility. The lanes containing the digested DNA dearly reveal a single fragment (track B) and five fragments (track E), whose sizes can be estimated by comparison with the marker tracks (M). The intensities of the bands in track E are proportional to the sizes of the fragments, since a small fragment has less mass of DNA at a given molar concentration. This is also true of the markers, since in this case they are formed by digestion of the 48.5 kb linear DNA of bacteriophage k (see Topic

H2). The amount of DNA present in tracks U, B and E is not equal; the quantifies have been optimized to show all the fragments clearly.

在琼脂糖凝胶上运行的未消化的质粒DNA通常由两条带组成。较低移动更快的带由从细胞中完好无缺分离出来的负螺旋质粒DNA组成。由于它自身高凑的体型结构，这样具有高度的移动性。（见主题4）上一条带是开环或者是微缺的DNA，形成于一套破损超螺旋的DNA带；这种带有带开了的循环构象和较低的迁移。带有小胡DNA非得轨道深深地揭露单一的片段和5个片段，片段的大小能通过跟标记轨道M比较被估计得到。在轨道E中，带的强度跟片段的大小成比例，在给定的摩尔浓度里因为每小片段拥有小量DNA。这对标记是真实的，由于这样的情况他们通过噬菌体K的48.5kb线状DNA 消化而形成。在轨道U，B和E上DNA的数量分布是不均匀的；确定的数量最佳化清晰地展现所有片段。

A more accurate determination of the sizes of the linear fragments can be made by plotting a calibration curve of the log of the size of the known fragments in track M against the distance migrated by each fragment. This plot(Fig. 4b) is a fairly straight line, often with a deviation at large fragment sizes. This may be used to derive the size of an unknown linear fragment on the same gel from its mobility, by reading off the log(size) as shown. It is not possible to derive the sizes of undigested circular plasmids by the same method, since the relative mobility of circular and linear DNA on a gel depends on the conditions (temperature, electric field, etc.).

更多线状片段的大小的精确测定能通过在轨道M上依靠每一个片段迁移距离测绘成已知片段的校准曲线的记录制作。这种情况是一条相当直的线，经常在庞大的片段大小时有背离。这可以在相同的凝胶上根据片段的迁移被用于导出未知大小的线状片段，根据出来的结果读取数据。通过相同的方法导出未消化的环状质粒是不可能的，因为在凝胶上的环状和线状DNA的相关迁移性是需要条件的。

Isolation of fragments:

Agarose gels may also be used preparatively to isolate specific fragments for use in subsequent ligation and other cloning experiments. Fragments are excised from the gel, and treated by orie of a number of procedures to purify the DNA away from the contaminating agarose and ethidium bromide stain. If we assume that the EcoRI fragment containing the gene X (Fig. 2) is the target DNA for a subcloning experiment (see Topic Gl), then the third largest fragment in track E of Fig. 4a could be purified from the gel ready for ligation into a new vector (see Topic G4).

分离片段：琼脂糖凝胶也能被用于分离特殊片段，从而用在随后的连接和其他克隆实验。从凝胶上切除的片段，接着通过大量步骤从污染的琼脂糖中纯化出DNA，再用溴化乙锭染色。如果我们假定含有基因X的EcoRI片段是亚克隆实验的目的DNA，图4a的在轨道上的第三大的片段能从凝胶中被纯化出来，为连接进一个新的载体做准备（看主题G4）

Lesson 4 LIGATION, TRANSFORMATION AND ANALYSIS OF RECOMBINANTS

第4课结扎，重组转型分析

DNA ligation: T4 DNA ligase repairs breaks in a dsDNA backbone and can covalently rejoin annealed cohesive ends in the reverse of a restriction enzyme reaction, to create new DNA molecules.

DNA 连接：T4 DNA连接酶在双链DNA的分水岭处修复破损的地方，然后在反向限制酶反应中，能共价地再加入退火的粘性末端，从而形成新的DNA分子。Recombinant DNA molecules: The use of a restriction enzyme, followed by DNA ligase, can create recombinant plasmids, with a target DNA fragment inserted into a vector plasmid.

重组DNA分子：限制性内切酶的应用，紧随DNA连接酶，能通过将目标片段插入一个人载体质粒形成重组质粒。

Alkaline phosphatase: Treatment of the linear vector molecule with alkaline phosphatase will remove the 5'-phosphates and render the vector unable to llgate into a circle without an inserted target, so reducing the proportion of recreated vector in the mixture.

碱性磷酸酶：碱性磷酸酶对线状载体分子的处理是移除5’的磷酸键和会使没带出入目标基因的载体连接成一个环，从而混合物中减少重新形成的载体的比率。Transformation: Transformation is the process of take-up of foreign DNA, normally plasmids, by bacteria. Plasmids are cloned by transfer into strains of E. coli with defined genetic properties. The E. coli cells can be made competent to take up plasmid DNA by treatment with Ca2+. The cells are plated out on agar and grown to yield single colonies, or clones.

转换：转换是提取外来DNA的过程，一般是在细菌里的质粒。带有明确遗传特性的质粒是通过转移到大肠杆菌菌株才被克隆的。大肠杆菌细胞能通过Ca2+ 的处理提取质粒DNA。细胞被平铺在琼脂上，然后逐渐产出单一的菌落或是克隆体。

Selection: Bacteria which have taken up a plasmid are selected by growth on a plate containing an antibiotic to which the plasmid vector encodes resistance.

筛选：已经提取了质粒的细菌是通过含有看抗生素的生长培养基选出来的，而这种抗生素是阻碍质粒编码的。

Transformation efficiency: The efficiency of the transformation step is given by the number of anti-biotic-resistant colonies per microgram of input plasmid DNA.

转换效率：转换步骤的效率是通过输出质粒DNA每微克抗生素抗性菌落的数量所定的。

Screening transformants: In many cases, such as when using DNA libraries, plasmid and other vectors have been designed to facilitate the screening of transformants for recombinant plasmids. In the case of a simple subcloning experiment, transformants are screened most easily by digesting the DNA from minipreparations of the transformants, followed by analysis on an agarose gel.

筛选转化株：在许多情况里，如利用DNA文库、质粒和其他载体是已经设计好促进筛选重组质粒的转换株的。在亚克隆实验情况里，转换株通过消化来自转换株的小量准备的DNA最容易被筛选出来，它是紧随琼脂糖凝胶分析的。

Growth and storage of transformants: Single colonies from a transformation plate are grown in liquid medium, maintaining the antibiotic selection for the plasmid, and a portion of the culture is stored for later use as a frozen glycerol stock.

转换株的生长和储存：来自转换平板的单一菌落是在一体培养基中生长的，继续抗生素对质粒的选择，也是被储存到冰冻的丙三醇库存储存起来为以后使用的培养菌的一部分。

Gel analysis: Recombinant plasmids can be distinguished from vectors by size on an agarose gel and by excising the inserted fragment with the same restriction enzyme(s) used to insert it.

凝胶分析：重组质粒子能在琼脂糖凝胶上通过大小来区别于载体和通过相同的用来插入到载体的限制酶切除插入的片段。

Fragment orientation: The orientation of the insert in the vector may be determined using an agarose gel by digestion of the plasmid with a restriction enzyme known to cut asymmetrically within the insert sequence.

片段定位：载体的插入物的定位通过在插入序列里不对称地切割质粒的消化利用琼脂糖凝胶可以准确测定。

D NA ligation:

DNA连接：

To insert a target DNA fragment into a vector, a method for the covalent joining of DNA molecules is essential. DNA ligase enzymes perform just this function; they will repair (ligate) a break in one strand of a dsDNA molecule (see Topic E2), provided the 5'-end has a phosphate group. They require an adenylating agent to activate the phosphate group for attack by the 3'-OH; the E. coli enzyme uses NAD+, and the more commonly used enzyme from bacteriophage T4 uses ATP. Ligases are efficient at sealing the broken phosphodiester bonds in an annealed pair of cohesive ends (see Topic G2), essentially the reverse of a restriction enzyme reaction, and T4 ligase can even ligate one blunt end to another, albeit with rather lower efficiency.

将目标DNA片段插入到载体中必须需要DNA分子的共价连接方法。只在这种作用下DNA连接酶才表现；它们会在一条双链DNA分子的一条带修复破损的位置，给予带有磷酸基团的5’末端。为了来自于3'-OH的制止，它们需要一种腺苷化剂去激活磷酸基团；大肠杆菌酶利用NAD+ ，而通常大多数来自于噬菌体T4的适应酶利用ATP。在一个粘性末端的退火碱基处，连接酶对确定破损的磷酸二酯键是有效的，基本上是限制内切酶的反转防御，以及T4连接酶甚至能连接一个迟钝的末端到另个上面唔；即使是更低的效率。

Recombinant DNA molecules:

重组DNA分子：

We can now envisage an experiment in which a DNA fragment containing a gene (X) of interest (the target DNA) is inserted into a plasmid vector (Fig. 1). The target DNA may bea single fragment isolated from an agarose gel (see Topic G3), or a mixture of many fragments from, for example, genomic DNA (see Topic Il). If the target has been prepared by digestion with EcoRI, then the fragment can be ligated with vector DNA cut with the same enzyme (Fig. 1) In practice, the vector should have only one site for cleavage with the relevant enzyme, since otherwise,, the correct product could only be formed by the ligation of three or more fragments, which would be very inefficient. There are many possible products from this ligation reaction, and the outcome will depend on the relative concentrations of the fragments as well as the conditions, but the products of interest will be circular molecules with the target fragment inserted at the EcoRI site of the vector molecule (with either orientation), to form a recombinant molecule (Fig. 1). The recreation of the original vector plasmid, by circularization of the linear vector alone, is a competing side reaction which can make the identification of recombinant products problematic. One solution is to prepare both the target and the vector using a pair of distinct restriction enzymes, such that they have noncompatible cohesive ends at either end. The likelihood of ligating the vector into a circle is then much reduced.

现在我们可以设想一个这样的实验，含有目标基因的片段被插入到一个智力载体当中（图1）。目标DNA可能是来自于琼脂糖凝胶分离出来的单一片段，或者是如来自于基因组许多片段的混合物。如果目标基因已经由EcoRI消化准备好，然后通过切割DNA 相同的酶的作用连被连接。事实上，载体应该只有一个与相关酶的切割位点，否则，正确的产物只能够形成于3个或更多片段的连接，这样的话将非常没效率。来自于连接反应有许多可能的产物，结果依靠不同相对浓度的片段和条件，但将于插入在载体分子的EcoRI位点目标产物的目标片段形成环状的分子。通过线状的单一载体的通函询证，原始在体质粒的反应是一个竞争的副反应，这个反应可以识别不确定的重组的产物。一个方法是利用一对不同的限制性内切酶制备目标和载体，如他们在另外的末端有不兼容的粘性末端。连接在环里的载体有很大可能被分解掉。

Alkaline phosphatase:

碱性磷酸酶：

If it is inconvenient to use two restriction enzymes, then the linear vector fragment may be treated with the enzyme alkaline phosphatase after restriction enzyme digestion. Alkaline phosphatase removes phosphate groups from the 5'-ends of DNA molecules. Thelinear vector will hence be unable to ligate into a circle, since no phosphates are available for the ligation reaction (Fig. 2). A ligation with a target DNA insert can still proceed, since one phosphate is present to ligate one strand at each cut site (Fig. 2). The remaining nicks in the other strands will be repaired by cellular mechanisms after transformation (see following).

如果不能使用两种限制性内切酶，可以在限制性内切酶消化后用碱性磷酸酶处理线性载体片段。碱性磷酸酶移走位于DNA分子5'端的磷酸基团。因此线性载体将不能连接在环里，因为没有磷酸基团不能进行连接反应。一个连接目标DNA插入物仍然可以前进，因为每个磷酸基团在每个位点用来连接每条带的。在转换之后，剩余的在其他带的割伤能通过细胞机理被修补好。

Transformation:

改造：

The components of the mixture of recombinant and other plasmid molecules formed by ligation (Fig. I) must now be isolated from one another and replicated (doned) by transfer into a host organism, By far the most common hosts for simple cloning experiments are strains of E. coli which have specific genetic properties. One obvious requirement, for example, is that they must not express a restriction-modification system(see Topic G3).

通过连接形成的重组或其他质粒分子混合物的成分现在必须从另外一个中被分离出来，然后转移到宿主有机体中进行复制。到目前为止用于简单的克隆实验的大多数普通的宿主是大肠杆菌菌株，因为其有特殊的遗传特性。例如，一个明显的必要条件是它们必须不能表达限制修饰系统。

It was discovered that E. coli cells treated With solutions containing Ca2+ ions were rendered susceptible to take up exogenous DNA, a process known as transformation. Cells pre-treated With Ca2+ (and sometimes more exotic metal ions such as Rb+ and Mn2+), in order to render them able to take up DNA, are known as competent cells.

被含有Ca2+ 的溶液处理的大肠杆菌细胞易被影响利用用外生的DNA，这个过程叫转换。利用Ca2+ 处理细胞是为了能让它们能够利用好DNA。

In transformation of E. coli, a solution of a plasmid molecule, or a mixture of molecules formed in a ligation reaction, is combined with a suspension of competent cells for a period, to allow the DNA to be taken up. The precise mechanism of the transfer of DNA into the cells is obscure. The mixture is then heat-shocked at 42℃for 1-2 min. This induces enzymes involved in the repair of DNA and other cellular

co.mponents, which allow the cells to recover from the unusual conditions of the transformation process, and increases the efficiency. The cells are then incubated in a growth medium and finally spread on an agar plate and incubated until single colonies of bacteria grow (Fig. 3). All the cells within a colony originate from division of a single

individual. Thus, all the cells will have the same genotype, barring spontaneous