Experimental Techniques

1、Appendix D:Experimental Techniques                附录D:实 验 技 术        Elucidating the molecular basis of life is no easy task.  Our current understanding of complex processes like replication, transcription or translation has requi

2、red thousands of scientists laboring for decades.  Yet, considering the tiny scale on which molecular biologists work, the amount known is admirable.  This understanding is due to a powerful set of techniques, tools that allow our immense hands to cut and past molecules, and our

3、blurry eyes to peer inside cells.   In this chapter we review the most important of these techniques used today.                要弄清楚生命的分子基础并不是一件容易的事。我们现在了解的复杂过程，如复制、转录或转译，是几千位科学家通过几十年的辛勤研究得来的。不过，考虑到分子生物学家工作的对象是如此微小，已获得的知识还是值得称道的。这样的理解得益于一套有效的技术，这是一些使

4、我们的大手能够去切割并粘贴分子和使我们模糊的眼睛能够窥探细胞内部的工具。本章我们回顾现今使用的一些最重要的技术。        D.1  DNA Manipulation         Techniques                D.1  DNA操作技术        D.1.1  Gel Electrophoresis and

5、         Southern Blotting                D.1.1  凝胶电泳与        Southern印迹法        Gel electrophoresis is a technique used to separate a mixture of DNA molecules according to length (Figure D.

6、1). DNA molecules are pulled through a gel by a voltage that attracts the negative charges on phosphodiester bonds.  The gel is a tangle of polymers that is quite dense, but contains holes through which DNA molecules can pass.  Shorter molecules fit more easily through these hole

7、s than larger molecules; as a result, the speed at which a DNA molecule moves down the gel is directly related to its size.                凝胶电泳是用来将DNA分子混合物根据它们的长度分开的技术（图D.1）。DNA分子的磷酸二酯键上带有负电荷，在电场作用下会在凝胶中发生移动。凝胶是一团密度较大但有孔的聚合物，DNA分子可以从孔中穿过。较小的分子比大分子更容易进入这些孔；结果，一

8、个DNA分子在凝胶中的移动速度直接与它的大小有关。        After the gel has been run for some time, DNA molecules of different lengths will each occupy different places on the gel, appearing as individual bands.  Very fine gels can be made that can allow DNA molecules to be resolved even if the

9、y differ in length by only one base pair.  Note that gel electrophoresis does not differentiate molecules according to sequence.  Two molecules with different sequences but with the same number of base pairs will appear as one band on a gel.          

10、0;     在凝胶被跑了一段时间以后，每种不同长度的DNA分子将在凝胶上占据不同的位置，呈现出不同的条带。甚至可以制备出很精细的凝胶用于分开长度只差一个碱基对的DNA分子。注意，凝胶电泳不能根据序列区分分子。具有相同长度但序列不同的两个分子将出现在凝胶的同一个位置。        Figure D.1  DNA gel electrophoresis. (a) DNA molecules migrate towards anode. The smaller molecules move faster than t

11、he larger ones. (b) DNA bands on the gel after electrophoresis and staining.                图D.1  DNA凝胶电泳。（a）DNA分子向正极迁移。较小的分子比大分子移动更快。（b）电泳后凝胶经过染色显示的DNA条带。        Gel electrophoresis is often used to determine whether a specific D

12、NA molecule is present in a mixture.  Size is often enough to identify the specific molecule on a gel.  The gel is stained with a chemical that colors DNA, so that all bands become apparent.  Meanwhile, in a parallel lane of the gel, a set of DNA molecules of known size

13、 is run.  By comparing the bands of experimental DNA to the DNA bands of known size, a good estimate of the size of the experimental DNA molecules can be assessed.                凝胶电泳也常用于确定某种特殊的DNA分子是否出现在某个混合物中。通常根据分子的大小就足以鉴定在凝胶上是否有这一特殊分子。凝胶用一种化学物质将D

14、NA染上颜色，使所有条带都清楚易见。同时，在凝胶的平行泳道上跑一套已知大小的DNA分子。通过比较实验DNA的条带与已知大小DNA的条带，就可以很好地估计实验DNA分子的大小。        Figure D.2  Southern blotting. The probe binds to a band of complementary sequence and makes it visible.                图D.2  

15、;Southern印迹法。探针与互补序列条带结合而让它能被看到。        Sometimes it is necessary to check for the presence of a specific DNA molecule on a gel even though its size is not known.  In this case, a technique called Southern blotting is used (Figure D.2).  Short pieces of DNA ca

16、lled probes are made that are complementary to the DNA molecule of interest, and therefore bind only to that molecule.  The probes are made to be radioactive or fluorescent.  They are then applied to the gel (or an imprint of the gel).  If they bind to a DNA molecule in

17、 the gel, the band where that DNA is present will become visible by the radioactivity or the fluorescence of the probe.  Southern blotting is also used when there are so many genes on the gel that size alone cannot be used to determine the presence of a specific gene, as other genes may ha

18、ve the same size.                  有时甚至在不知道实验DNA分子大小的情况下也需要检查某种特殊的DNA分子是否在凝胶上。这时，需要采用称为Southern印迹法的技术（图D.2）。先制备与感兴趣的DNA分子互补的称为探针的DNA小片段，探针只能与那个分子结合。之后使探针带上放射性或荧光标记。然后将它们与凝胶（或凝胶的印迹）混合。如果它们能与凝胶上的某种DNA分子结合，那么这一DNA出现的地方会由于探针上的放射性或荧光而被看到。Southern印迹法也用在许多基

19、因出现在凝胶中仅靠分子大小不能确定某种特殊基因是否存在的情况下，因为其它基因可能具有相同的大小。        D.1.2  Polymerase Chain Reaction         (PCR)                D.1.2  聚合酶链式反应        （PCR）        Figure

20、D.3  The polymerase chain reaction                图D.3  聚合酶链式反应        Polymerase chain reaction is a technique used to copy specific regions of a DNA molecule (Figure D.3).  As with normal replication in cells

21、, PCR depends on DNA polymerase and deoxyribonucleotides in synthesis.  However, most other enzymes used in normal replication are replaced during PCR by machine functions and artificial molecules.                聚合酶链式反应是用来拷贝DNA分子特殊区域的技术（图D.3）。与细胞中的正

22、常复制一样，PCR在合成中需要依靠DNA聚合酶和脱氧核糖核苷酸。但是，在正常复制中用到的大多数其它酶被PCR仪器的功能和其它人造分子取代。        A mixture of components, including DNA molecules, is placed into a machine that controls temperature.  The machine initially raises the temperature of the mixture, denaturing the double-strande

23、d DNA into single-strands.  This essentially mimics the function of helicase.  DNA polymerase can use these single strands as template for synthesis.  However, before it can do so, primers must present on the DNA.  In PCR, primers are artificially-made DNA oli

24、gomers that are added to the reaction mixture.  They are not RNA, and they are not made by primase.  The machine lowers the reaction temperature to allow the DNA primers to hydridize or anneal to the DNA template.  Following this, DNA polymerase is able to copy each str

25、and, beginning at the primer.                将各种成分的混合物（包括DNA分子）放进能控制温度的仪器里。PCR仪先升高混合物的温度，将双链DNA变性成为单链。这实际上模仿了解旋酶的功能。DNA聚合酶能够使用这些单链为模板进行合成。但在它开始合成前，必须有引物出现在DNA上。在PCR中，引物是人工制备的DNA寡聚物，它们也被加到了反应混合物中。它们不是RNA，也不是由引发酶产生的。PCR仪将温度降下来以便DNA引物与DNA模板杂交或退火。接着，DNA聚合酶便能够从引物开始

26、拷贝每一条链了。        After one round of synthesis, two DNA molecules have been made for each initial template molecule present.  Now another cycle begins.  The machine heats the reaction once again, denaturing the DNA molecules including the newly made ones then co

27、ols to allow annealing of primers and synthesis.  From the two molecules made in the previous round of PCR, four new molecules can be made.  In the next round, these four molecules will be used to make 8 new molecules.  Thus, with every round of PCR, the number of DNA m

28、olecules increases exponentially.  After several dozen rounds, billions of copies can be made.                在完成一轮合成后，从最初的一个模板分子得到了两个DNA分子。现在另一个循环开始了。PCR仪又一次加热使DNA分子变性包括新合成的分子然后降温使引物退火并进行DNA合成。从前一轮PCR得到的两个分子出发可以产生四个新的分子。下一轮合成中，这四个分子将被用来产生8个新的分子。这样，随

29、着每一轮PCR的进行，DNA分子的数量呈指数式增长。经过几十轮后，可以产生几十亿个拷贝。        A powerful feature of PCR is that specific regions of the template molecule can be copied, rather than the whole template molecule (Figure D.4).  The region that will be replicated is determined by which sites on the t

30、emplate the artificial primers are designed to anneal to.  One primer determines one end of the region to be copied, and a second primer determines the other end.  Each primer always binds to one site and only binds to one strand of the DNA.   Using PCR, many copies can

31、 be made of any gene in a cells genome.                PCR的一个有用之处是它可以用来拷贝模板分子的特殊区域，而不是整个模板分子（图D.4）。将要被复制的区域由根据模板上的退火位点设计的人工引物决定。一个引物决定了需要拷贝区域的一个末端，另一个引物决定了另一个末端。每个引物总是与一个位点结合并且只与模板DNA的一条链结合。应用PCR可以产生细胞基因组中任何基因的许多拷贝。        PCR reactions oft

32、en produce a variety of undesirable side-products.  Gel electrophoresis is quite useful for separating the desired product from the side-products and purifying it.                PCR反应经常会产生一些不希望出现的副产物。凝胶电泳在将需要的产物从副产物中分离出来方面相当有用。      &

33、#160; Figure D.4  PCR is used to copy specific region of DNA.                图D.4  PCR用来拷贝DNA的特殊区域。        D.1.3  Recombining DNA                D.1.3  重组DNA

34、60;       In addition to being copied and separated, DNA molecules can also be rearranged in the laboratory.  Whole regions of DNA can be moved around, deleted, and reattached, for example, using common techniques.                除了能被拷

35、贝和分离外，在实验室里也能对DNA分子进行重排。例如，可以使用常规技术对DNA的整个区域进行移动、删除和重新连接。        Recombinant DNA technology is made possible by restriction endonucleases, which are proteins that cut DNA molecules at precise sequences (Figure D.5). Usually, the target sequences are palindromes, meaning they read

36、 the same backward and forward.  The enzyme makes two single-stranded cuts several bases apart on separate strands.  This produces two ends, each with one single-strand slightly longer than the other.  There are hundreds of different restriction enzymes, each recognizin

37、g different sites.  However, every time a particular restriction enzyme cuts a DNA molecule, it produces the same exact ends.                限制性内切核酸酶使重组DNA技术成为可能，这是一些能在准确的位置切割DNA分子的蛋白质（图D.5）。通常，它们的目标序列具有回文结构，即从反向读和从正向读它们都是一样的。这种酶在两条链上相距几个碱基各产生一个单链切口。

38、这样就得到了两个末端，每个末端各有一条单链比另一条略长。共有几百种不同的限制酶，每一种识别不同的位点。不过，每次一种特殊的限制酶切割DNA分子时，它产生的都是完全相同的末端。        Figure D.5  EcoR I, a restriction endo- nuclease, recognizes a palindromic sequence and cleaves it to yield sticky ends.               

39、 图D.5  限制性核酸内切酶EcoR I识别回文序列并将其切割产生粘性末端。        The staggered ends produced by restriction enzymes are often called sticky ends.  This is because one end can bind to a complementary end, sticking two molecules together.  In fact, after a DNA molecule

40、is cut by a restriction enzyme, the two complementary ends produced often continue to stick together.  But they dont have to.  If another DNA molecule is cut with the same enzyme, the same sticky ends will be produced.  These ends are also complementary to those in the

41、first molecule, and can bind to them (Figure D.6).                限制酶产生的交错切口常被称为粘性末端。这是因为一个末端可以与另一个互补的末端结合，把两个分子粘合在一起。事实上，在DNA分子被限制酶切开后，两个互补的末端常常继续粘合在一起。但它们并不是非这样不可。如果另外一个DNA分子也用相同的酶切割，就会得到相同的粘性末端。这些末端与从第一个分子得到的粘性末端是一样的，也能结合在一起（图D.6）。       

42、; Figure D.6  Recombinant DNA molecule created with restriction enzyme and ligase.                图D.6  由限制酶和连接酶产生的重组DNA分子。        This is a very useful property, because it allows two different molecules to be glued to

43、each other in a specific way.  If different molecules are cut with the same restriction enzymes, the ends produced at each one have the capacity to bind to each other, joining together the two different molecules.  To create covalent bonds where the sticky ends have hybridized, a

44、n enzyme called ligase is required.                这是一种很有用的性质，因为它可以让两个不同的分子以特殊的方式结合在一起。如果不同的分子用相同的限制酶切割，从每个分子产生的末端都能互相结合，从而将两种不同的分子连接在一起。要在粘性末端已经发生杂交的地方形成共价键，还需要一种称为连接酶的酶。        D.1.4  DNA Sequencing        &#

45、160;       D.1.4  DNA序列测定        Understanding, manipulating, and identifying a DNA molecule often requires knowing the exact sequence of bases it contains.  A base sequence would be important, for example, in making probes for Southern blotting

46、, or in determining which restriction enzymes can cut a gene.  One of the most convenient aspects about working with DNA in the laboratory is the ease with which its sequence can be determined.  There are various ways of determining a DNA sequence, and new techniques are being ra

47、pidly developed.  The first very successful method, which is still quite popular, is called the dideoxy method.                了解、操作和鉴定DNA分子常常需要知道它含有哪些确切的碱基序列。例如，在为Southern印迹法制备探针或确定哪种限制酶能够切割某一基因的时候，清楚地知道它的碱基序列就很重要。在实验室中对DNA开展工作最方便的一个方面就是可以很容易地测得它的序列

48、。有几种测定DNA序列的方法，新技术也在快速发展。第一个最成功的、目前仍然相当通行的方法称为双脱氧法。        In this technique a DNA molecule is copied in a reaction similar to PCR.  However, small amounts of nucleotides called dideoxyribonucleotides are present in the reaction mixture (Figure D.7).  These sp

49、ecial nucleotides do not contain the 3-OH group present on normal nucleotides.  As a result, once they are incorporated into a growing chain, no further nucleotides can be attached to the DNA molecule, and synthesis is terminated.                在这一技

50、术中，DNA分子在一个 PCR类似的反应中被拷贝。但是，反应混合物中含有少量的双脱氧核糖核苷酸（图D.7）。这些特殊的核苷酸没有正常核苷酸中所具有的3-OH。结果，一旦它们被整合到生长链中，将不再有核苷酸能够连接到DNA分子上，合成就终止了。        Figure D.7  Structure of a dideoxycytocine- 5-triphosphate                图D.7  双脱氧胞嘧啶核苷-5-

51、三磷酸的结构        To sequence DNA, four separate reactions are prepared (Figure D.8).  Each one receives a small amount of a different dideoxy - G, A, T, or C, as well as larger quantities of all four normal nucleotides.  Let us see what happens in the mixture tha

52、t receives dideoxy-C (Figure D.8a).  Many DNA synthesis reactions are occurring in the mixture at the same time.  Each time that DNA polymerase must add a C to the growing DNA molecule, there is a chance that the dideoxy-C will be incorporated.  Sometimes this happens e

53、arly, so synthesis stops early and a small DNA molecule is produced.  Sometimes it happens late, and long molecule is produced before synthesis stops.  In short, adding dideoxy-C causes a variety of DNA molecules with different lengths to be synthesized, each one ending in C.

54、0;               为了DNA进行测序，需要分开准备四组反应（图D.8）。每组含有少量不同的双脱氧-G、A、T或C以及大量的所有四种正常的核苷酸。让我们来看一看含有双脱氧-C的混合物中会发生什么情况（图D.8a）。在这一混合物中同一时间发生着许多DNA合成反应。每次DNA聚合酶向生长中的DNA分子添加C的时候，双脱氧-C都会有机会被整合进去。有时这样的事发生得较早，这样合成终止得也早，得到的是一个较小的DNA分子。有时它发生得较晚，则在合成终止前得到了较长的分子。简言之，添加上双脱氧-C导致合成一系列不同长度的

55、DNA分子，每个分子的结尾处都是C。        Next, the length of each of these molecules is determined by gel electrophoresis.  The length of a DNA molecule is directly related to the number of bases.  We also know that all DNA molecules from the dideoxy-C reaction end in C.

56、0; By determining the lengths of these various molecules, each position of the DNA that contains C can be determined.  For example, if the molecules are 24, 27, and 32 base pairs in length, this means that C is present at positions 4, 7, and 12 in the normal molecule (after subtractio

57、n of 20, length of the primer).  The same process is repeated using the other three dideoxy nucleotides, and this way, the entire sequence of the DNA molecule can be determined (Figure D.8 b d).                之后，用凝胶电泳来测定这些分子的长度。DNA分子的长度与它具有的碱基数直接相关联

58、。我们还知道，来自于双脱氧-C反应的所有DNA分子都以C结尾。通过测定这些不同分子的长度，DNA中每个含有C的位置能够被确定下来。例如，如果得到的分子长度是24、27和32个碱基对长，这就意味着在正常的分子中，C出现在第4、第7和第12的位置（在减去引物长度20后得出）。再用其它三种双脱氧核苷酸重复同样的过程，这样，DNA分子的全部序列就能被测出（图D.8 b d）。        Figure D.8  (a)(d): Products of sequencing reactions with ddCTP, ddTTP, ddATP

59、 and ddGTP, respectively. (e) Electrophoresis of the products.                图D.8  （a）（d）：分别使用ddCTP、ddTTP、ddATP和ddGTP进行的测序反应产物。（e）反应产物的电泳图。        D.1.5  Molecular Cloning             

60、   D.1.5  分子克隆        The first step in studying a gene is to isolate it, and to have access to many identical copies of it.  This is called cloning the gene.  The process by which a gene is cloned is called molecular cloning (Figure D.9). This

61、technique draws on the more basic techniques discussed above.                研究基因的第一步是分离它并获得许多与它完全相同的拷贝。这称为克隆基因。一个基因被克隆的过程称为分子克隆（图D.9）。这一技术牵涉到上面讨论过的更基本的技术。        Cloning begins with a PCR reaction in which the many copies are made of the g

62、ene of interest.  It might seem as though the process of molecular cloning could end with PCR.  The gene has been isolated, and many copies are available.  However, the PCR reaction often makes mistakes, and the product contains a mixture of accurate copies and inaccura

63、te copies.  Whether the gene will be sequenced, recombined, or expressed, such a hodgepodge is not desirable.                克隆开始于PCR反应，先从中获得许多目的基因拷贝。看起来好像分子克隆的过程可以在PCR这儿结束了，因为基因已经被分离了，也获得了很多拷贝。然而，PCR反应常常会出错，得到的产物是准确拷贝和不准确拷贝的混合物。不管是要对它进行测序、重组或表达，这样一堆

64、乱七八糟的东西总是不行的。        Figure D.9  A general process of molecular cloning (plasmid size not drawn to scale)                图D.9  分子克隆的一般过程（质粒大小未按比例画）        In order to have accurate copies of the origi

65、nal gene, it needs to be placed into a living cell, usually E. coli, which has the ability to copy genes with very few errors.  DNA copied by PCR is linear, and will be degraded if placed in an E. coli cell.  Therefore, the PCR product must first be inserted into plasmids, circul

66、ar pieces of DNA which act as artificial chromosomes.                为了获得原始基因的准确拷贝，需要将它放入活细胞（通常是大肠杆菌）中，活细胞具有准确地拷贝基因的能力（很少出错）。PCR拷贝得到的DNA是线状的，如果放入大肠杆菌细胞的话会被降解掉。因此，PCR产物必须先插入到质粒中，质粒是环状DN*段，能起到人工染色体的作用。        Placing a linear piece of DNA int

67、o a circular plasmid requires recombinant DNA techniques discussed above.  Both the PCR product and the plasmid are cut with the same restriction enzymes, in two places.  This produces sticky ends on either side of the gene fragment, and also creates an opening in the plasmid whe

68、re the fragment can enter.  Because the gene and the plasmid have complementary sticky ends after cutting, they can be joined together. Afterwards they are covalently linked by the protein ligase.                将线状DN*段放入环状质粒需要采用上面讨论过的DNA重组技术。PCR产物和质

69、粒均用相同的限制酶在两个位置进行切割，在基因片段的两端产生粘性末端，同时在质粒上打开一个缺口以便基因片段进入。由于酶切后基因和质粒具有相同的粘性末端，所以它们能结合在一起。之后由连接酶将它们共价连接起来。        Once the gene fragments made by PCR have been inserted in plasmids, the plasmids must be placed inside E. coli cells.  In this case, the insertion of DNA into a

70、 cell is called transformation.  Usually, the plasmid is mixed with a population of E. coli cells, and then a shock is applied, such as heat or electricity, that temporarily makes the cells permeable to large molecules like DNA.  In order to distinguish cells that have taken the plasmid from those that have not, plasmids are usually designed to contain a gene for resistance to an antibiotic.  After transformation, the cells are placed on medium containing antibiotic.  Only those cells that have taken up the plasmid are able to grow on t