Unit3 Biochemistry and Molecular Biology
Keyto the Exercises
1.Preparation
Watcha short video clip and fill in each of the blanks in the following paragraph.
1 ) infants 2) molecules 3) intestine
4) enzyme 5) site 6) enzyme
7) enzyme 8) capillaries 9) redistributed
Afterwatching, can you tell what plays an important part in helping young mammals to
absorbnutrition from milk?
Theenzyme lactase.
Script
Like all young mammals, human infantsdepend on milk for nutrition. Milk is rich
in lactose9 a disaccharide made from twosugar molecules. Lactose cannot be directly
absorbed in the small intestine. To digestlactose, mammals produce the enzyme lactase.
the lactose binds to the active site of thelactase enzyme. The enzyme hydrolyzes lactose
and produces two simple sugars, glucose andgalactose. These sugars are then absorbed
into the capillaries of the small intestineand redistributed to the rest of the body.
II.Readlnq
TextA
ComprehensionCheck
Pairwork Discuss and answer the following questions. Try not to look back at thetext.
1) Because knowledge of factors thatcontrol the rates of enzyme-catalyzed reactions is
essential to an understanding of themolecular basis of disease. (Para. 1)
2)The author draws an analogy theunidirectional flow of metabolites through a
pathway with a large overall negativechange in free energy is analogous to the flow of
water through a pipe in which one end islower than the other. (Para. 3)
3) proteins synthesized proproteins includethe hormone insulin ( Proprotein-
proinsulin). The digestive enzymes pepsin,trypsin, and chymotrypsin ( Proproteins-
pepsinogen : trypsinogenr andchymotrypsinogen9 respectively), several factors of the
blood clotting and blood clot dissolutioncascades, and the connective tissue protein
collagen ( proprotein-procollagen). ( Para.4)
4) The synthesis and secretion of proteasesas catalytically inactive proenzymes protects
the tissue of origin ( e. g., the pancreas)from autodigestion，such as can occur in
pancreatitis. In addition, enzymes neededintermittently but rapidly often are secreted
as inactive proenzymes since the secretionprocess or new synthesis of the required
proteins might be insufficiently rapid forresponse to a pressing pathophysiologic
demand such as the loss of blood. ( Para.5)
5) Activation of prochymotrypsin requiresselective proteolysis. Selective proteolysis involves
highly specific proteolytic clips that mayor may not be accompanied by
separation of the resulting peptides. Mostimportantly, selective proteolysis often results in
conformational changes that "create"the catalytic site of an enzyme. The conformational
changes that accompany selectiveproteolysis of prochymotrypsin (chymotrypsinogen) align
the three residues of the charge-relaynetwork, forming the catalytic site. (Para. 6)
LanguagePractice
1.Read the text carefully and then fill out the missing information in thefollowing short
passage.The beginning letter of each missing word has been given.
1 ) Enzymes 2) catalytic 3) active 4)proteins 5) catalysis
6) reactions 7) substrates 8) bonds 9)catalyze
2.paraphrase the following sentences. Pay special attention to the underlinedparts.
1) Claude Bernard, a physiologist who livedin the 19'h century, expressed clearly and
exactly the conceptual basis for metabolicregulation.
2) Later, Water Cannon invented the term "homeostasis"to describe the ability of
animals to maintain a constant environmentwithin cells in spite of changes in their
external environment.
3) Curves or twists in the pipe imitate thefeatures of single enzyme-catalyzed steps :
with a small negative or positive change infree energy.
4) Proenzymes make the mobilization of anactivity happen rapidly to satisfy a
physiologic requirement.
3.Identify the expression from each of the following sentences which correspondsin
meaningto one of the Chinese expressions given below. (These sentences are wellwritten,
Itis suggested that you read them aloud and learn them by heart. )
1)respond to对······做出反应应对
2)(be)characterized by以······为特征
3)contribute to对······有影响促进
4) (be) responsible for 负责 · · · · · 。
5)knowledge of知道······，了解·····。
(be)essential to对······至关重要
6)in a...Manner以······方式
7) (be) accompanied by 由 · · · · · · 伴随
8)(be) analogous to类似于与······相似
9)correspond to与······相应；与······一致
4.Translate the following sentences into English with some expressions you haveidentified
inexercise 3.
Cells could respond tosignaling molecules even if those molecules do not pass into
the cell.
2) The most complex genomes arecharacterized by the presence of many extra genes
compared with animal and yeastmitochondrial genomes.
3) High serum levels of cholesterol causedisease and death by contributing to the
formation of atherosclerotic plaguesthroughout the body.
4) The properties of a protein, which are responsible for its biologicalfunction, are
determined by its three-dimensionalstructure.
5) Knowledge of the functional andstructural properties of proteins is essential to
understanding the significance of the humangenome sequence.
6) Each different DNA fragment is placed ata unique position on the microarray, and
whole genomes can thereby be representedin an orderly manner.
7) Cell division is not accompanied bynuclear division until about 6000 nuclei have
formed and migrated outward to the plasmamembrane.
8) The folded domains of RNA molecules notonly are structurally analogous to the a helices
and P strands found in proteins, but insome cases also have catalytic capacities.
9)These hypersensitive sites correspondto regions that have few nucleosomes or have
nucleosomes in an altered conformationalstate.
NoteThe underlining points out the expressions identified in exercise 3.
TextB
ComprehensionCheck
Answerthe following questions. Try not to look back at the text.
1) Isolation and manipulation of DNA.(Para. 1 )
2) Because their presence in a givenbacterium restricted the growth of certain bacterial
viruses called bacteriophages. (Para, 2)
3) Because in such cells, the site-specificDNA methylase methylates the host DNA and
turns it into an unsuitable substrate fordigestion by the restriction enzyme. (para. 2)
4) Chimeric DNA molecules are constructedin cloning vectors, and then the vectors
replicate in a host cell under their owncontrol systems. In this way, the chimeric DNA
is amplified. (Para. 4)
5) Cloning vectors mentioned here areplasmids, phages, cosmids, and the BAC, YAC,
and PAC vectors. (Para 4 & 9)
6) The BAC, YAC, and Pl vectors arepreferred since they can accept very large
fragments of DNA and thus offer a betterchance of isolating an intact gene on a single
DNA fragment. (Para. 9)
7) Expression vectors are now commonly usedto detect specific eDNA molecules in
libraries and to produce proteins bygenetic engineering techniques. (para. 1o)
LanguagePractice
1.Translate the following sentences into Chinese. pay special attention to theunderlined parts.
1)有时，人工合成的带有一个适宜的限制性核酸内切酶识别序列的平头末端双链寡核
苷酸接头会被连接到平头末端的DNA分子上。
2)一个克隆就是源自一个共同祖先的一大群相同的分子、细菌或细胞。
3)细菌质粒是小型环状双链DN八，它们的天然功能是把抗生素抗性赋予宿主细胞。
4)黏粒是含有被称作黏性位点的DNA序列的质粒，黏性位点是将lambdaDNA包装入
噬菌体颗粒所必需的。
5)更大长度的DNA片段能被整合到细菌人工染色体、酵母人工染色体、或者是以P1菌
噬菌体为基础的人工染色体(PAC)载体中。
6)因此，可以很容易地将对四环素和氨苄西林都具有抗性的亲代质粒从嵌合质粒(只有
四环素抗性)中分离出来。
7)一些载体&至还包含编码蛋白酶抑制剂的基因，从而导致产物终产量的增加。
8)探针通常是用含放射性磷(P)的核苷酸或者荧光标记的核苷酸(目前后者应用更为普
遍)标记的DNA或RNA片段。
2.Paraphrase the following sentences. Pay special attention to the underlinedparts.
1 ) These vectors will accept and multiplyDNA inserts of several hundred kilobases or
more in length.
2) The idea is to produce rather largefragments so that most genes will keep complete
and undamaged.
III.Video Watching and Speaking
ClipOne
Watchthe video clip and do the exercises given below.
1.Decide whether the following statements are true or false. Write T for true andF for false.
1) F 2) T 3) F
2. Takenotes while watching and then answer the following questions.
1 ) The active site reorients thesubstrates and hence reduces the amount of energy
needed to activate the reaction.
2) ATP contains three phosphate groups heldtogether by high energy bounds. The
phosphate bounds can be broken apart veryeasily. When they are broken, they
release large amounts of energy.
3) ADP makes ATP and vice versa. When ATPreleases energy9 the end phosphate
group of ATP breaks off, leaving ADP and aphosphate molecule an ADP molecule
captures the energy and uses it to bondwith a phosphate group, making a new
molecule of ATP.
3. omitted.
script
Catalysts coordinate the flow of energybetween exergonic and endergonic reactions.
The catalysts in cells are proteins calledenzymes. Molecules involved in the reactions are
substrates. Enzymes catalyze exergonic andendergonic reactions. During a catalytic
reaction, a substrate fits into a groove onthe surface of an enzyme called an active site.
The active site reorients the substrates.Reorienting the molecules reduces the amount of
energy needed to activate the reaction.Each cell contains thousands of different
enzymes, and each one has a specificsubstrate that it can catalyze. A catalytic process
does not destroy or use up enzymes. Afterthe reaction is completed, enzymes are reused
with new substrates. The amount of energyan organism needs depends on its level of
activity. Running requires more energy thanwalking. Cells have a way of transferring
energy and releasing it when it is needed.Energy is transferred in a molecule adenosine
triphosphate or ATP. ATP contains threephosphate groups held together by high
energy bounds. The phosphate bounds can bebroken apart very easily. When they are
broken, they release large amounts ofenergy. When enzymes catalyze an endergonic
reaction, simple molecules are chemicallyjoined to create a more complex molecule.
ATP supplies the energy required to drivethe reaction. ATP releases energy when the
high energy bounds between the phosphategroups break down. The end phosphate
group of ATP breaks off, releasing a largeamount of energy for the endergonic reaction.
This leaves adenosine diphosphate or ADPand a phosphate molecule. An exergonic
reaction breaks the chemical bonds ofsubstrate which releases energy. An ADP
molecule captures the energy and uses it tobond with a phosphate group, making a new
molecule of ATP. Cells always have a supplyof ATP, ADP and phosphate groups for
energy reactions. This is how cellstransfer and release energy that organisms need to
live. Thanks to ATP and chemical reactions,we have enough energy to get through
the day.
ClipTwo
Witchthe video clip and do the exercise below.
Answerthe following questions.
1 ) the enzyme and the substrate in thelock and key model are compared to a space
station and a space ship, respectively.
2) The enzyme changes shape to accommodatethe substrate.
3) Without enzymes, these reactions wouldhappen too slowly to sustain life, or would
require extreme conditions of pH ortemperature.
4) Competitive inhibition means when asimilar shaped molecule competes for the active
site it reduces substrate activity.
script
Enzymes are proteins which catalyzemetabolic reactions involving the conversion of a
substrate into a product. Enzymes bind withsubstrates due to their complementary shape This
is called the lock and key model. Ratherlike a space ship docking into a space station. In the
induced fit model, the enzyme changes shapeto accommodate the substrate. Enzymes enable
metabolic processes to proceed rapidly atroom temperature by providing an alternative reaction
pathway of lower activation energy. Withoutenzymes, these reactions would happen too slowly
to sustain life, or would require extremeconditions of pH or temperature. Carbonic anhydrase
catalyzes the following reaction betweencarbon dioxide and water. ?his reaction proceeds 10
million times faster with the enzymecatalyst than without. A water molecule is coordinated to
its zinc atom in the protein. A proton isremoved, leaving a hydroxyl ion. Carbon dioxide binds
the hydroxyl ion on the zinc and a productis released. When a similar shaped molecule
competes for the active site, it reducessubstrate activity--this is called competitive inhibition.
Adding more substrate increases activity.In non-competitive (allosteric) inhibition, a molecule
binds to a remote part of the protein,preventing substrate bin · ding byaltering the shape of the
protein.
ClipThree
Watchthe video clip and do the exercises below.
Fillin the blanks in the following paragraph.
1 ) biotechnology 2) plasmid 3) interest
4) resistant 5) isolated 6) restriction
7) sequences 8) base 9) manageable
10) fragments 1 1) recombined 12)complementary
13) sticky 14) transformation 15) cloned
Script
The tools of biotechnology start with theconstruction of a recombinant DNA molecule, an
insertion of DNA from one species into aplasmid vector. The first step is to identify a gene of
interest, in this case, a pest-resistantgene from the BT bacterium. In creating recombinant
DNA for plants, the most commonly usedvector is a plasmid isolated from Agrobaczerium
tumefaciens. Next, restriction enzymeswhich recognize specific DNA sequences cut both the
plasmid and the DNA of interest betweencorresponding base pairs.
"SO the restriction enzymes are a wayof getting down to smaller pieces, more
manageable pieces of DNA and even singlegenes."
Any two fragments of DNA cut by the samerestriction enzyme can then be
recombined because they have complementarysequences on their dangling or sticky ends
that pair back up.
During transformation, the recombinantplasmid with the gene of interest can be
reintroduced into Agrobacterium and clonedas the bacterium multiplies naturally. The
bacteria can be used as vectors to infectplant cells growing in culture.
IV.Writing
Untanglethe noun clusters in the following sentences by adding the appropriate preposition
orprepositions, and other words as needed. Start at the end of the cluster andwork your
wayto the beginning. The underlining may serve you as a clue.
1 ) The specificity of binding depends onthe precisely defined arrangement of atoms in an
active site.
2 ) Studies of the thermal stability ofsynthetic polypeptides have been especially
informative.
3) Enzymes serve as catalysts by decreasingthe free energy of activation of chemical reactions.
4) Enzymes accelerate reactions byfacilitating the formation of the transition state.
5) the binding energy is the free energyreleased in the formation of a large number of
weak interactions between the enzyme andthe substrate.
6) Enzymes, the catalysts of biologicalsystems, are remarkable molecular devices that
determine the patterns of chemicaltransformations.
7) In general, synthesizing compounds thatmore closely resemble the transition state
than the substrate itself can producehighly potent and specific inhibitors of enzymes.
8) PKC-activation-induced translocation ofRACK1 is specific....
V.Autonomous Learning
VocabularyDevelopment
1. Fill in each of thefollowing blanks with an appropriate word.
1) Nucleus 2) enzyme 3) glucose
4) growth 5) formation 6) sugar
7) network/reticulum 8) proteins 9) discharge
2.Match each of the following terms with its meaning and write the appropriateletter to the
leftof each number.
Group 1 1) e) 2) a) 3) 4) c) 5) b) D
Group2 6) D 7) P 8) D 9) g) 10) h)
3.Give meaning for each of the following terms.
1) an enzyme that digests proteins
2) poor nourishment of tissue
3) formation of fibrous tissue
4) producing cells
5) a short nucleic acid polymer
SupplementaryListening
Script
The first step in cloning a gene is toisolate the DNA from the organism that
contains the desired gene. The isolated DNAis purified and then fragmented with a
restriction enzyme. Restriction enzymesused in cloning produce staggered cuts in
specific sequences in the DNA, generatingfragments with cohesive ends. Each fragment
has a single-stranded sequence ofnucleotides on its ends that is capable of hybridizing
with DNA that has been fragmented with thesame restriction enzyme. The DNA
fragments are then incorporated intoplasmids. The type of plasmid used for cloning has
a single restriction site, and when cleavedby the restriction enzyme, generates the same
cohesive ends that are in the fragments ofthe DNA to be cloned. The cohesive ends of
the plasmid and DNA fragments now line upand the enzyme, DNA ligase, is used to
form phosphodiester bonds. The next step isto incorporate the plasmids into bacterial
host cells by transformation. Each cellcontains a different segment of DNA from the
original organism. Taken together, thesecells represent a DNA library. The cells can
now be plated out on an agar medium. Thecolony of cells containing the desired cloned
gene can then be identified and isolated.
Translationof the Texts
TextA
酶：生命活动的调节者
分子生物学重要性
十九世纪的生理学家ClaudeBernard阐述了代谢调节的基础概念。他观察到生物以在数量上和时间上适宜的方式做出应答以战胜因内外环境改变而引发的多种挑战，从而得以生存。随后，WalterCannon提出了“内环境稳态”的概念，来描述即使在外环境变化的情况下，动物仍能保持一个持续稳定的内环境的能力。我们现在知道，生物通过平衡协调地改变特异的代谢反应率来应对内外环境的改变。许多人类疾病，比如癌症、糖尿病、囊性纤维化及阿尔茨海默病(老年痴呆症），均以病原体或基因突变诱发的调节性功能异常为特征。例如，许多致癌病毒可产生蛋白酪氨酸激酶，这种激酶能够改变控制基因表达模式的调节事件，从而促进了癌症的发生和进展。在小肠上皮细胞中，霍乱弧菌毒素，即霍乱的致病毒素，通过对连接细胞表面受体和腺苷酸环化酶的GTP（鸟苷三磷酸）结合蛋白（G-蛋白）进行ADP（腺苷二磷酸）-核糖基化，从而使细胞的感受器-应答途径失活。由此导致的腺苷酸环化酶的激活引起水流入肠道，造成严重腹泻和脱水。鼠疫杆菌，即鼠疫病原体，可产生一种蛋白酪氨酸磷酸酶，该磷酸酶能水解细胞关键骨架蛋白上的磷酰基。通常认为，负责降解缺陷或异常蛋白的系统的功能障碍与神经退行性疾病（如阿尔茨海默病和帕金森病）相关。因此，了解控制酶催化反应速率的因素对于理解疾病的分子基础至关重要。
代谢物流动的主动调节与被动调节
酶的催化速率在达到最大值后便不会再随底物浓度增加而改变，仅在底物浓度骤降时发生改变。因此，对于大多数的酶，细胞内的平均底物浓度常常接近Km值，所以底物浓度的变化导致了相应的代谢流量改变（图1）。对反应底物水平变化的应答是一个重要但被动的手段，用于在静息细胞中协调代谢物流动和维持稳态。然而，它们对外环境变化的应对是有限的。下面将讨论在应对内外信号时对酶活性进行积极调节的机制。
图1 当底物浓度等于Km或远高于Km时，相同的底物浓度变化（△S）对于酶促反应速率的影响不同（△VA或△VB）。
代谢物的流动往往是单向的
尽管代谢物浓度和酶的水平存在短期振荡变化，但活细胞处于一个动态的稳态中，代谢中间产物的平均浓度长期保持相对稳定（图2）。同时，虽然所有的化学反应在一定程度上是可逆的，但在活细胞内，反应产物成为其它酶催化反应的底物并被去除。因此许多名义上的可逆反应实际上是单向的。伴随这一连串的相互偶联的代谢反应，会发生自由能的总体改变，这种改变有利于代谢物单向流动。代谢物通过一条途径单向流动，并伴随整体自由能的巨大负性变化，类似于水流经一根一端较低的管道。管道的弯曲或扭结模拟了个别伴有较小负（正）自由能改变的酶催化步骤。尽管存在弯曲或扭结，由于管道高度的整体改变，水流经过管道时仍然是单向的。这与一条途径的自由能的总变化是相应的（图3）。
图2 处于稳态的理想化细胞。注意：代谢物的流动是单向的
图3 具有限速步骤（A）和接近0的△G数值步骤（B）的代谢途径的流体静力学模型
蛋白酶可以无催化活性的酶原形式被分泌
某些蛋白质以无活性的前体蛋白形式被合成和分泌，称为蛋白原。酶的蛋白原被称为酶原。选择性蛋白水解通过一个或多个连续的蛋白分解“修剪”将蛋白原转化为展现出成熟蛋白特性（例如酶活性）的形式。作为蛋白原被合成的蛋白质包括胰岛素（其蛋白原为胰岛素原），属于消化酶的胃蛋白酶、胰蛋白酶和糜蛋白酶（其蛋白原分别为胃蛋白酶原、胰蛋白酶原、糜蛋白酶原），凝血和血块溶解级联反应中的几个因子，以及结缔组织蛋白胶原（其蛋白原为前胶原）。
酶原形式有利于快速活化以响应生理需求
蛋白酶以无催化活性的酶原形式被合成和分泌，以保护来源组织（例如胰腺）不被自身消化（自身消化有可能在胰腺炎中发生）。某些生理过程（例如消化）是间歇性的，但相当有规律并且可预见。另一些生理过程（例如血液凝块的形成、血块溶解和组织修复）只有在应对紧迫的生理或病理生理需要时才被启动。血块的形成和溶解显然必须在时间上相协调以实现内稳态。被生物体间歇性但迅速需要的酶通常以非活性初始形式分泌。这是由于如果面对紧迫的病理生理需要（如失血）时才分泌或重新合成所需蛋白质，便难以实现迅速应答。
激活糜蛋白酶原需要选择性蛋白水解
选择性的蛋白水解涉及一次或多次高度特异的蛋白分解剪切，可能伴有（或不伴有）产物肽的分离。最重要的是，选择性的蛋白水解常常导致构象的变化，“创造”出酶的催化部位。注意，第57位的组氨酸和第102位的门冬氨酸位于α-糜蛋白酶的B肽链上，而第195位的丝氨酸位于C肽链上（图4）。伴随糜蛋白酶原的选择性水解而产生了构象改变，从而形成了三个氨基酸残基的电荷传递网络，产生了催化位点。还要注意，接触和催化残基可以位于不同的肽链，但仍在结合底物的键形成距离内。
图4 选择性蛋白水解和相关构象变化形成糜蛋白酶的活性位点，这一位点包含由Asp102（102位门冬氨酸）、His57（57位组氨酸）和Ser195（195位丝氨酸）三个氨基酸残基构成的催化三联体。连续的蛋白水解依次形成糜蛋白酶原（pro-CT）、π-糜蛋白酶（π-CT），并最终形成α-糜蛋白酶（α-CT）。α-CT是一种活性蛋白酶，其三条肽链通过链间二硫键共价结合。
总结
内环境稳态（内稳态）指在广泛波动的外环境条件下，通过适当改变由生理需要引发的生化反应的速率，保持细胞和组织内环境的相对恒定。对于大多数酶而言，底物通常以接近Km值的浓度存在。这有利于被动控制产物形成率以应对代谢中间产物的水平变化。主动控制代谢物流量涉及催化一个特定的限速反应的酶的浓度或/和催化活性的改变。选择性水解无催化活性的酶原启动构象变化形成活性位点。无催化活性的酶原的分泌有利于酶原活性的迅速动员以应对损伤或生理需要并可以保护酶原的来源组织（例如避免蛋白酶对来源组织的自身消化）。
TextB
重组DNA技术指分离和操作DNA以获得嵌合分子
对DNA分子的分离和操作是重组DNA研究的本质，包括将不同来源的DNA序列首尾相接以获得嵌合分子（例如由人类和细菌DNA序列以序列非依赖性方式连接到一起的嵌合分子）。这一研究涉及一些独特的技术和试剂。
限制性核酸内切酶在特定位点切割DNA链
内切酶——在DNA分子内的特定序列切割DNA的酶（与从DNA分子末端切割DNA的核酸外切酶相反）——是重组DNA研究中的关键工具。这些酶被称做限制性内切酶，因为它们在特定细菌中的存在限制了被称作噬菌体的细菌病毒的生长。与大多数其它随机切断DNA的酶学的、化学的、物理的方法相反，限制性内切酶可将任何来源的DNA分子以序列特异的方式切割成短片段。这种防御性的酶（数百种已被发现）保护了宿主DNA免受外来生物（主要是感染性噬菌体）DNA的干扰。然而，它们仅存在于含有与之并存的能甲基化宿主DNA的酶的细胞中，使宿主DNA无法成为限制性内切酶的适合底物。因此，位点特异性的DNA甲基化酶和限制性内切酶总是成对地存在于细菌中。
限制性酶和DNA连接酶被用来制备嵌合DNA分子
黏性末端连接从技术层面来看很简单，但常常需要一些特殊的技术来克服这一途径固有的问题。载体的黏性末端可以自己重新连接而不会导致DNA分子的净增加。片段的黏性末端也可以退火，从而形成串联的异质性插入序列。此外，也可能存在黏性末端不可用或者处于不合适的位置的情况。为了避免这些问题，可以使用一种可以产生平头末端的酶，同时可以用末端转移酶添加新的末端。如果多聚d(G)添加到载体的3′末端，而多聚d(C) 添加到外源DNA的3′末端，这两种分子便只能相互退火，这样就可以避免前述的问题。这个步骤被称为同聚物加尾法。有时，人工合成的带有一个适宜的限制性核酸内切酶识别序列的平头末端双链寡核苷酸接头被连接到平头末端的DNA分子上。直接的平头末端连接由噬菌体T4 DNA连接酶催化完成。虽然这种技术没有黏性末端连接那么高效，但是它具有可以连接任何一对末端的优点。它的缺点是不能控制插入序列的方向和退火的分子的数目，并且也没有简便的方法来重新获得插入序列。
克隆技术扩增DNA
一个克隆就是源自一个共同祖先的一大群相同的分子、细菌或细胞。分子克隆可以产生数量巨大的相同的DNA分子，它们可以被赋予某种特性或用于其他目的。这项技术基于一个事实，那就是嵌合DNA分子和杂交DNA分子可以在克隆载体——通常是细菌质粒、噬菌体、或者是黏粒——中构建，并在宿主细胞中在自身的控制系统的控制下继续复制。通过这种方式，嵌合DNA分子得到扩增。大体过程如图1所示。
图1 用限制性核酸酶构建新的重组（或嵌合）DNA分子。重组质粒重新被摄入细菌细胞后（这一过程称为转化，通常一个细胞只接纳一个质粒），质粒DNA在自我复制的同时对与之相连的新插入的DNA序列进行复制。如图所示，限制性核酸酶能识别特定的DNA序列，则粘性末端重新结合后仍能被最初切割形成该粘性末端的限制性酶所识别和切割，因此，我们便能用这一限制性内切酶将克隆的DNA片段从重组环状质粒中完整地切除。如果用单一的限制性核酸酶切割人类总DNA，并将所得DNA片段的混合物用作人类DNA的来源，就能获得约100万种不同的重组DNA分子，每一个细菌克隆仅含单一的DNA分子的拷贝（经授权，修改再版自Cohen SN：The manipulation ofgenes. Sci Am [July] 1975; 233: 25. © The Estate of Bunji Tagawa）。
细菌质粒是小型环状双链DNA，他们的天然功能是把抗生素抗性赋予宿主细胞。质粒的若干特性使之成为极其有用的克隆载体。它们在细菌中以单拷贝或多拷贝形式存在，并独立于细菌DNA单独复制。许多质粒的完整的DNA序列已经明确。因此，确定用于插入外源DNA的限制性酶切位点的精确位置便成为可能。质粒比宿主染色体小，因此也很容易从后者分离，并且插入质粒的目的DNA片段可以通过用（用于原始DNA片段的插入的）位点特异的限制性酶切质粒的方法来移除。
噬菌体通常含有线性的DNA分子，外源的DNA分子由数个限制性酶切位点插入该DNA分子。嵌合DNA分子可以在噬菌体完成裂解周期并且产生成熟的、感染性噬菌体颗粒后加以收集。噬菌体载体最主要的优势在于：质粒只能接受6-10 kb长度的DNA片段，而噬菌体能接受10-20 kb长度的DNA片段。这一极限是由噬菌体头部所能最大限度容纳的DNA量决定的。
更大的DNA片段可以在黏粒中被克隆，黏粒结合了质粒和噬菌体的最佳优点。黏粒是含有被称作黏性位点的DNA序列的质粒，黏性位点是将lambda DNA包装入噬菌体颗粒所必需的。这种载体在细菌中以质粒的形式生长，但是由于很多不必要的lambda DNA都已经被移除，更多的嵌合DNA分子可以被包装入颗粒头部。黏粒携带长达35-50 kb的嵌合DNA分子的情况并不少见。更大长度的DNA片段能被整合到细菌人工染色体、酵母人工染色体、或者是以P1菌噬菌体为基础的人工染色体（PAC）载体。这些载体可接受并且扩增数百kb（乃至更大）的DNA插入序列，因此在某些基因克隆和基因作图应用方面，在很大程度上取代了质粒、噬菌体和黏粒载体。关于这些载体的比较见表1。
表1 常用克隆载体的克隆能力比较
由于DNA片段插入载体的功能区域将会影响这一区域的功能，必须小心，以免妨碍载体的基本功能。然而，这一认识可以进一步发展以提供一种选择技术。例如，常用的质粒载体pBR322同时含有四环素和氨苄青霉素抗性基因。一个单独的PstI限制性酶切位点位于氨苄青霉素抗性基因内，常常被用作一个外源DNA片段的插入位点。在此处插入的DNA片段打断了氨苄青霉素抗性基因，使携带该质粒的细菌成为氨苄青霉素敏感型（图2）。因此，可以很容易地将对四环素和氨苄青霉素都具有抗性的亲代质粒与嵌合质粒（只有四环素抗性）区分开来。YACs在细菌和酵母细胞中都具有选择、复制和分离功能，因此可以在二者中任何一种体内繁殖。
图2 筛选含有DNA插入片段的重组质粒的方法。将一个DNA片段插入质粒pBR322的Pstl位点。这种插入打断了编码氨苄青霉素抗性蛋白的基因，嵌合质粒将无法在含有氨苄青霉素的培养基中生存。因此，质粒对四环素和氨苄青霉素的敏感性的差异可用于筛选含有插入片段的阳性质粒克隆。而另一种筛选方法则是依赖于一个新的DNA插入片段在编码框内的融合，从而产生肽段，补救处于无活性、不完整形式的β-半乳糖苷酶，使其具有功能。细菌因而在加入了染色剂（能被β-半乳糖苷酶水解）的琼脂板上形成蓝-白菌落。β-半乳糖苷酶阳性菌落呈蓝色，这些菌落含有成功插入了DNA片段的质粒。
文库就是重组克隆的集合
限制性核酸内切酶和多种多样的克隆载体的组合使得一个生物体的整个基因组得以被装入一个载体。不同的重组克隆的集合就成为一个文库。基因组文库是基于一个细胞株或是一个组织的总DNA构建而成的。一个cDNA文库是由一个组织中的总mRNA的互补DNA组成的。基因组DNA文库通常是通过对总DNA进行部分（酶切）消化而构建的，使用的限制性核酸内切酶频繁地切割DNA（例如4碱基内切酶TaqI）。人们试图设法产生更大的酶切片段，从而使得绝大多数基因将在酶切后完好地保留。BAC、YAC和 Pl载体受到亲睐，因为他们能接受很大的DNA片段并因此为在单一的DNA片段上分离一个完整的基因提供了很大的可能性。
以重组DNA技术导入的基因所编码蛋白质的载体被称为表达载体。目前这种载体被普遍用于检测文库中的特定cDNA分子和通过基因工程技术生产蛋白质。这种载体是专门构建的，含有非常活跃的诱导型启动子、恰当的同相的翻译起始密码子、转录及翻译终止信号和必要的蛋白质加工信号。一些载体甚至还包含编码蛋白酶抑制剂的基因，从而导致产物终产量的增加。
用探针在文库中寻找特定基因或cDNA分子
多种多样的分子都可以被用于“探查”文库，寻找特定的基因或cDNA分子或者对用不同的凝胶电泳分离的DNA或RNA进行定性、定量。探针通常是用含放射性磷(P)的核苷酸或者荧光标记的核苷酸（目前后者应用更为普遍）标记的DNA或RNA片段。重要的是，任一种修饰（放射性P标记或荧光标记）都不会影响被标记的核酸探针的杂交特性。探针必须识别互补序列从而得以发挥作用。从特定的mRNA合成的cDNA可以用于筛查一个cDNA文库以获得一个更长的cDNA分子，或者用于筛查一个基因组文库以获得一个位于基因编码区的互补序列。一种普遍采用的寻找特定基因的技术是：借助短的氨基酸序列推导出该物种编码相应氨基酸序列的密码子序列，进而合成相应的寡核苷酸探针，用于在基因组文库中探测相应的DNA片段。如果序列完美匹配，15-20个核苷酸长的探针便可杂交。cDNA探针用于在Southern印记杂交转印膜上探测DNA片段以及在Northern印记杂交转印膜上探测RNA并定量。特异的抗体也可被用作探针，但前提是所使用的载体能合成可以被该抗体识别的蛋白质分子。
