生物技术专业英语翻译——《限制性内切酶与电泳技术》.doc

G3 RESTRICTION ENZYMES AND ELECTROPHORESISG3 限制性内切酶与电泳技术Restriction endonucleases: 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.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 53, 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.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.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. The digestion of a few hundred nanograms(1g) 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), in a valume of perhaps 20l. A dye mixture is then added to the solution ,and the sample is loaded on an agarose gel. 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. 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.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. 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. 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.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.).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).中文译文：限制性内切酶：限制性内切酶是将外源DNA整合到质粒载体时将双链DNA切断与使其重新连接到DNA上所必需的酶。限制性内切酶的识别和操纵是20世纪60年代和70年代初的关键发现，也促使了DNA克隆技术成为了现实。限制修饰系统发生在很多种类的细菌当中，并构成了对引入的外源DNA进入细胞的防御机制。限制修饰系统由两部分构成：首先是限制性内切酶，它能识别一个简短且对称的DNA序列（图1），并能消减（水解）在该序列中某一特定位点的DNA骨干。因此，外源DNA会被降解为相对较短的片段。这个系统的第二部分是一个甲基化酶，当C或A在同一识别序列的细胞DNA（主题比照）的基础上，能增加碱基的甲基对。这一修改可以使抗宿主DNA内切酶降解。序列的识别：限制性内切酶（限制酵素含量较低）的作用机理为插图1(a) 中的过程，其中包括了原型酵素EcoRI。这一个酵素作为一个二聚体，只能识别一个6 bp长度的回文序列（回文序列是指在每一股DNA序列上，从53还是从35开始读，DNA序列上的碱基顺序都是一样的）。在一个线性DNA的某个位点上进行切割反应所得到的产物是两个双链片段（限制性片段），每一个相同凸出的单链5黏性末端都附着这一个磷酸基团。3末端有一个游离的羟基基团。一个6 bp的碱基识别序列会发生在平均每46 = 4096 bp的随机DNA序列；因此，一个非常大的DNA分子将会被一种类似限制酵素的酶切割成平均4 kb的特异片段。现在已经有数百种限制性酶被发现了，而且有大多数的酶类能在市面上买到。它们能识别位点大小不一，由4 bp 至8 bp或更多的位点，并可能产生以凸出的5或3为尾巴或者尾部钝化的产物。新形成的5末端始终保留着磷酸基团。另外两个例子如图1所示。极高的特异性限制性内切酶能使它们的识别位点能将大地DNA分子和载体可被再次被切割成更小定义片段。黏性末端：限制性内切酶消化得到的产物具有更进一步的功能，这些产物具有已知的粘着力，或者是“粘性”末端，因为他们可以通过碱基配对（退火）结合与它具有相同碱基对得单链尾巴。因此，例如，通过形成一个EcoRI位切割任何片段可以通过相同的方法（图. lb）退火到任何的其他片段，以及随后得到的片段可以通过共价键相互进行碱基配对。事实上，在某些情况下，DNA末端酶在对不同的序列识别时可能会发生兼容，也就促使了单链尾巴可以进行碱基配对。限制性酶切：对质粒或基因组DNA（见主题I1）进行分析或制备时所需要的酶一般为商业酶和缓冲溶液。所有的限制性内切酶，通常都需要Mg2+ 的催化，且Mg2+ 的溶度需要高达10 mM，但是不同的酶需要不同的pH值、NAC1浓度或者其他最佳的环境条件。这种缓冲液能使一个特定的酶集中在一起。样本质粒在两个不同的限制性内切酶BamHI和EcoRI中的酶切作用，如图2所示。我们可以使用琼脂糖凝胶电泳分析方法来酶切几百毫微克（1g）的质粒DNA；如果用于制备时，可能需要几微克了。前者数额相当于一个小量样品的百分之几，见于主题G2。DNA酶切需要相应的酶、适合的缓冲溶液还需要一个最适宜的温度（通常为37）。我们还可以通过加入一些染料混合物，使DNA片段在琼脂糖凝胶电泳显示出来，称为显带。琼脂糖凝胶电泳：琼脂糖是从海藻中提取的一种多糖，当琼脂溶解在水溶液中的浓度在0.5和2（W / V）之间时，琼脂糖会凝固成固体凝胶。琼脂糖凝脂电泳是用于将细菌培养板中的琼脂纯化的方法。 当缓冲溶液接上电流后，在电力的作用下，琼脂糖凝胶上的DNA片段会朝向正极移动（DNA是高度带有负电荷；主题见C1），DNA片段在凝胶上的移动速率主要受它的大小和形状所控制（如图3）。小的线性片段移动的速度比大的要快，这是由于琼脂糖凝胶纤维形成了网络状的缠绕系统，让大的片段不易通过。因此，电泳技术用于分离不同程度的DNA片段混合物。不同浓度的凝胶1，1.5（W / V）等，分别为不同尺寸的DNA碎片的最佳分辨凝胶固体。将DNA样本放置在装有凝胶的井表面（图3），接通电源后，DNA会通过单独的涌道或者轨道进行移动。如果在凝胶中加入染料，染料也会随着电泳而进行移动，根据这个原理，