Experimental Techniques

1、Appendix D:Experimental Techniques                附錄D:實(shí) 驗(yàn) 技 術(shù)        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礎(chǔ)并不是一件容易的事。我們現(xiàn)在了解的復(fù)雜過(guò)程，如復(fù)制、轉(zhuǎn)錄或轉(zhuǎn)譯，是幾千位科學(xué)家通過(guò)幾十年的辛勤研究得來(lái)的。不過(guò)，考慮到分子生物學(xué)家工作的對(duì)象是如此微小，已獲得的知識(shí)還是值得稱道的。這樣的理解得益于一套有效的技術(shù)，這是一些使

4、我們的大手能夠去切割并粘貼分子和使我們模糊的眼睛能夠窺探細(xì)胞內(nèi)部的工具。本章我們回顧現(xiàn)今使用的一些最重要的技術(shù)。        D.1  DNA Manipulation         Techniques                D.1  DNA操作技術(shù)        D.1.1  Gel Electrophoresis and

5、         Southern Blotting                D.1.1  凝膠電泳與        Southern印跡法        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.                凝膠電泳是用來(lái)將DNA分子混合物根據(jù)它們的長(zhǎng)度分開(kāi)的技術(shù)（圖D.1）。DNA分子的磷酸二酯鍵上帶有負(fù)電荷，在電場(chǎng)作用下會(huì)在凝膠中發(fā)生移動(dòng)。凝膠是一團(tuán)密度較大但有孔的聚合物，DNA分子可以從孔中穿過(guò)。較小的分子比大分子更容易進(jìn)入這些孔；結(jié)果，一

8、個(gè)DNA分子在凝膠中的移動(dòng)速度直接與它的大小有關(guān)。        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;     在凝膠被跑了一段時(shí)間以后，每種不同長(zhǎng)度的DNA分子將在凝膠上占據(jù)不同的位置，呈現(xiàn)出不同的條帶。甚至可以制備出很精細(xì)的凝膠用于分開(kāi)長(zhǎng)度只差一個(gè)堿基對(duì)的DNA分子。注意，凝膠電泳不能根據(jù)序列區(qū)分分子。具有相同長(zhǎng)度但序列不同的兩個(gè)分子將出現(xiàn)在凝膠的同一個(gè)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.                圖D.1  DNA凝膠電泳。（a）DNA分子向正極遷移。較小的分子比大分子移動(dòng)更快。（b）電泳后凝膠經(jīng)過(guò)染色顯示的DNA條帶。        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.                凝膠電泳也常用于確定某種特殊的DNA分子是否出現(xiàn)在某個(gè)混合物中。通常根據(jù)分子的大小就足以鑒定在凝膠上是否有這一特殊分子。凝膠用一種化學(xué)物質(zhì)將D

14、NA染上顏色，使所有條帶都清楚易見(jiàn)。同時(shí)，在凝膠的平行泳道上跑一套已知大小的DNA分子。通過(guò)比較實(shí)驗(yàn)DNA的條帶與已知大小DNA的條帶，就可以很好地估計(jì)實(shí)驗(yàn)DNA分子的大小。        Figure D.2  Southern blotting. The probe binds to a band of complementary sequence and makes it visible.                圖D.2  

15、;Southern印跡法。探針與互補(bǔ)序列條帶結(jié)合而讓它能被看到。        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.                  有時(shí)甚至在不知道實(shí)驗(yàn)DNA分子大小的情況下也需要檢查某種特殊的DNA分子是否在凝膠上。這時(shí)，需要采用稱為Southern印跡法的技術(shù)（圖D.2）。先制備與感興趣的DNA分子互補(bǔ)的稱為探針的DNA小片段，探針只能與那個(gè)分子結(jié)合。之后使探針帶上放射性或熒光標(biāo)記。然后將它們與凝膠（或凝膠的印跡）混合。如果它們能與凝膠上的某種DNA分子結(jié)合，那么這一DNA出現(xiàn)的地方會(huì)由于探針上的放射性或熒光而被看到。Southern印跡法也用在許多基

19、因出現(xiàn)在凝膠中僅靠分子大小不能確定某種特殊基因是否存在的情況下，因?yàn)槠渌蚩赡芫哂邢嗤拇笮　?#160;       D.1.2  Polymerase Chain Reaction         (PCR)                D.1.2  聚合酶鏈?zhǔn)椒磻?yīng)        （PCR）        Figure

20、D.3  The polymerase chain reaction                圖D.3  聚合酶鏈?zhǔn)椒磻?yīng)        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.                聚合酶鏈?zhǔn)椒磻?yīng)是用來(lái)拷貝DNA分子特殊區(qū)域的技術(shù)（圖D.3）。與細(xì)胞中的正

22、常復(fù)制一樣，PCR在合成中需要依靠DNA聚合酶和脫氧核糖核苷酸。但是，在正常復(fù)制中用到的大多數(shù)其它酶被PCR儀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.                將各種成分的混合物（包括DNA分子）放進(jìn)能控制溫度的儀器里。PCR儀先升高混合物的溫度，將雙鏈DNA變性成為單鏈。這實(shí)際上模仿了解旋酶的功能。DNA聚合酶能夠使用這些單鏈為模板進(jìn)行合成。但在它開(kāi)始合成前，必須有引物出現(xiàn)在DNA上。在PCR中，引物是人工制備的DNA寡聚物，它們也被加到了反應(yīng)混合物中。它們不是RNA，也不是由引發(fā)酶產(chǎn)生的。PCR儀將溫度降下來(lái)以便DNA引物與DNA模板雜交或退火。接著，DNA聚合酶便能夠從引物開(kāi)始

26、拷貝每一條鏈了。        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.                在完成一輪合成后，從最初的一個(gè)模板分子得到了兩個(gè)DNA分子。現(xiàn)在另一個(gè)循環(huán)開(kāi)始了。PCR儀又一次加熱使DNA分子變性包括新合成的分子然后降溫使引物退火并進(jìn)行DNA合成。從前一輪PCR得到的兩個(gè)分子出發(fā)可以產(chǎn)生四個(gè)新的分子。下一輪合成中，這四個(gè)分子將被用來(lái)產(chǎn)生8個(gè)新的分子。這樣，隨

29、著每一輪PCR的進(jìn)行，DNA分子的數(shù)量呈指數(shù)式增長(zhǎng)。經(jīng)過(guò)幾十輪后，可以產(chǎn)生幾十億個(gè)拷貝。        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的一個(gè)有用之處是它可以用來(lái)拷貝模板分子的特殊區(qū)域，而不是整個(gè)模板分子（圖D.4）。將要被復(fù)制的區(qū)域由根據(jù)模板上的退火位點(diǎn)設(shè)計(jì)的人工引物決定。一個(gè)引物決定了需要拷貝區(qū)域的一個(gè)末端，另一個(gè)引物決定了另一個(gè)末端。每個(gè)引物總是與一個(gè)位點(diǎn)結(jié)合并且只與模板DNA的一條鏈結(jié)合。應(yīng)用PCR可以產(chǎn)生細(xì)胞基因組中任何基因的許多拷貝。        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反應(yīng)經(jīng)常會(huì)產(chǎn)生一些不希望出現(xiàn)的副產(chǎn)物。凝膠電泳在將需要的產(chǎn)物從副產(chǎn)物中分離出來(lái)方面相當(dāng)有用。      &

33、#160; Figure D.4  PCR is used to copy specific region of DNA.                圖D.4  PCR用來(lái)拷貝DNA的特殊區(qū)域。        D.1.3  Recombining DNA                D.1.3  重組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、貝和分離外，在實(shí)驗(yàn)室里也能對(duì)DNA分子進(jìn)行重排。例如，可以使用常規(guī)技術(shù)對(duì)DNA的整個(gè)區(qū)域進(jìn)行移動(dòng)、刪除和重新連接。        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.                限制性內(nèi)切核酸酶使重組DNA技術(shù)成為可能，這是一些能在準(zhǔn)確的位置切割DNA分子的蛋白質(zhì)（圖D.5）。通常，它們的目標(biāo)序列具有回文結(jié)構(gòu)，即從反向讀和從正向讀它們都是一樣的。這種酶在兩條鏈上相距幾個(gè)堿基各產(chǎn)生一個(gè)單鏈切口。

38、這樣就得到了兩個(gè)末端，每個(gè)末端各有一條單鏈比另一條略長(zhǎng)。共有幾百種不同的限制酶，每一種識(shí)別不同的位點(diǎn)。不過(guò)，每次一種特殊的限制酶切割DNA分子時(shí)，它產(chǎn)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、 圖D.5  限制性核酸內(nèi)切酶EcoR I識(shí)別回文序列并將其切割產(chǎn)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).                限制酶產(chǎn)生的交錯(cuò)切口常被稱為粘性末端。這是因?yàn)橐粋€(gè)末端可以與另一個(gè)互補(bǔ)的末端結(jié)合，把兩個(gè)分子粘合在一起。事實(shí)上，在DNA分子被限制酶切開(kāi)后，兩個(gè)互補(bǔ)的末端常常繼續(xù)粘合在一起。但它們并不是非這樣不可。如果另外一個(gè)DNA分子也用相同的酶切割，就會(huì)得到相同的粘性末端。這些末端與從第一個(gè)分子得到的粘性末端是一樣的，也能結(jié)合在一起（圖D.6）。       

42、; Figure D.6  Recombinant DNA molecule created with restriction enzyme and ligase.                圖D.6  由限制酶和連接酶產(chǎn)生的重組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.                這是一種很有用的性質(zhì)，因?yàn)樗梢宰寖蓚€(gè)不同的分子以特殊的方式結(jié)合在一起。如果不同的分子用相同的限制酶切割，從每個(gè)分子產(chǎn)生的末端都能互相結(jié)合，從而將兩種不同的分子連接在一起。要在粘性末端已經(jīng)發(fā)生雜交的地方形成共價(jià)鍵，還需要一種稱為連接酶的酶。        D.1.4  DNA Sequencing        &#

45、160;       D.1.4  DNA序列測(cè)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、操作和鑒定DNA分子常常需要知道它含有哪些確切的堿基序列。例如，在為Southern印跡法制備探針或確定哪種限制酶能夠切割某一基因的時(shí)候，清楚地知道它的堿基序列就很重要。在實(shí)驗(yàn)室中對(duì)DNA開(kāi)展工作最方便的一個(gè)方面就是可以很容易地測(cè)得它的序列

48、。有幾種測(cè)定DNA序列的方法，新技術(shù)也在快速發(fā)展。第一個(gè)最成功的、目前仍然相當(dāng)通行的方法稱為雙脫氧法。        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.                在這一技

50、術(shù)中，DNA分子在一個(gè) PCR類(lèi)似的反應(yīng)中被拷貝。但是，反應(yīng)混合物中含有少量的雙脫氧核糖核苷酸（圖D.7）。這些特殊的核苷酸沒(méi)有正常核苷酸中所具有的3-OH。結(jié)果，一旦它們被整合到生長(zhǎng)鏈中，將不再有核苷酸能夠連接到DNA分子上，合成就終止了。        Figure D.7  Structure of a dideoxycytocine- 5-triphosphate                圖D.7  雙脫氧胞嘧啶核苷-5-

51、三磷酸的結(jié)構(gòu)        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;               為了DNA進(jìn)行測(cè)序，需要分開(kāi)準(zhǔn)備四組反應(yīng)（圖D.8）。每組含有少量不同的雙脫氧-G、A、T或C以及大量的所有四種正常的核苷酸。讓我們來(lái)看一看含有雙脫氧-C的混合物中會(huì)發(fā)生什么情況（圖D.8a）。在這一混合物中同一時(shí)間發(fā)生著許多DNA合成反應(yīng)。每次DNA聚合酶向生長(zhǎng)中的DNA分子添加C的時(shí)候，雙脫氧-C都會(huì)有機(jī)會(huì)被整合進(jìn)去。有時(shí)這樣的事發(fā)生得較早，這樣合成終止得也早，得到的是一個(gè)較小的DNA分子。有時(shí)它發(fā)生得較晚，則在合成終止前得到了較長(zhǎng)的分子。簡(jiǎn)言之，添加上雙脫氧-C導(dǎo)致合成一系列不同長(zhǎng)度的

55、DNA分子，每個(gè)分子的結(jié)尾處都是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，用凝膠電泳來(lái)測(cè)定這些分子的長(zhǎng)度。DNA分子的長(zhǎng)度與它具有的堿基數(shù)直接相關(guān)聯(lián)

58、。我們還知道，來(lái)自于雙脫氧-C反應(yīng)的所有DNA分子都以C結(jié)尾。通過(guò)測(cè)定這些不同分子的長(zhǎng)度，DNA中每個(gè)含有C的位置能夠被確定下來(lái)。例如，如果得到的分子長(zhǎng)度是24、27和32個(gè)堿基對(duì)長(zhǎng)，這就意味著在正常的分子中，C出現(xiàn)在第4、第7和第12的位置（在減去引物長(zhǎng)度20后得出）。再用其它三種雙脫氧核苷酸重復(fù)同樣的過(guò)程，這樣，DNA分子的全部序列就能被測(cè)出（圖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.                圖D.8  （a）（d）：分別使用ddCTP、ddTTP、ddATP和ddGTP進(jìn)行的測(cè)序反應(yīng)產(chǎn)物。（e）反應(yīng)產(chǎn)物的電泳圖。        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離它并獲得許多與它完全相同的拷貝。這稱為克隆基因。一個(gè)基因被克隆的過(guò)程稱為分子克?。▓DD.9）。這一技術(shù)牽涉到上面討論過(guò)的更基本的技術(shù)。        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.                克隆開(kāi)始于PCR反應(yīng)，先從中獲得許多目的基因拷貝?？雌饋?lái)好像分子克隆的過(guò)程可以在PCR這兒結(jié)束了，因?yàn)榛蛞呀?jīng)被分離了，也獲得了很多拷貝。然而，PCR反應(yīng)常常會(huì)出錯(cuò)，得到的產(chǎn)物是準(zhǔn)確拷貝和不準(zhǔn)確拷貝的混合物。不管是要對(duì)它進(jìn)行測(cè)序、重組或表達(dá)，這樣一堆

64、亂七八糟的東西總是不行的。        Figure D.9  A general process of molecular cloning (plasmid size not drawn to scale)                圖D.9  分子克隆的一般過(guò)程（質(zhì)粒大小未按比例畫(huà)）        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.                為了獲得原始基因的準(zhǔn)確拷貝，需要將它放入活細(xì)胞（通常是大腸桿菌）中，活細(xì)胞具有準(zhǔn)確地拷貝基因的能力（很少出錯(cuò)）。PCR拷貝得到的DNA是線狀的，如果放入大腸桿菌細(xì)胞的話會(huì)被降解掉。因此，PCR產(chǎn)物必須先插入到質(zhì)粒中，質(zhì)粒是環(huán)狀DN*段，能起到人工染色體的作用。        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.                將線狀DN*段放入環(huán)狀質(zhì)粒需要采用上面討論過(guò)的DNA重組技術(shù)。PCR產(chǎn)物和質(zhì)

69、粒均用相同的限制酶在兩個(gè)位置進(jìn)行切割，在基因片段的兩端產(chǎn)生粘性末端，同時(shí)在質(zhì)粒上打開(kāi)一個(gè)缺口以便基因片段進(jìn)入。由于酶切后基因和質(zhì)粒具有相同的粘性末端，所以它們能結(jié)合在一起。之后由連接酶將它們共價(jià)連接起來(lái)。        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