02第二讲 目的基因的制备(一)

• The digested DNA molecules are run on agarose electrophoresis for which a suitable range of lengths of DNA pieces are isolated and ligated to vector plasmids. Statistically, 99% of the genes will be incorporated into the plasmids. The plasmids then can be taken up by suitable hosts.
的磁珠加入到总
RNA中，再用磁 铁收集磁珠，最 后洗出mRNA。
（2）合成第一链cDNA oligo(dT)与mRNA的poly(A)结合可以作为合成第一
链cDNA的引物.在逆转录酶的作用下，以mRNA为模板，
以oligo(dT)为引物模板合成cDNA，形成RNA－DNA杂交 分子。这种合成第一链cDNA的方法又称为利用 oligo(dT)引导的cDNA合成法。
• The hosts are kept in liquid media and can be frozen at -80°C for a long period of time. Usually the hosts are bacteria that do not contain any plasmids so as to be sensitive to 是在一系列酶的作用下，使总
poly(A) RNA制剂转变成双链cDNA群体，并插入到适当
的载体中，然后再转化大肠杆菌细胞。基本手段。
（1）分离细胞总RNA，然后从中纯化出poly(A) RNA
原理
• 在真核细胞中，大部分mRNA的3’末端都具有 poly(A)尾。
5’ cap
(AAAAAAAAAA)n
• oligo (dT) can be bound to the poly(A) tail and used to recover the mRNA.
• 通过寡聚（dT）
纤维素柱 • 将结合着（dT）
当目的基因在某一细胞类是小麦中的一种重要的营养蛋白，在发育 的小麦种子中，编码醇溶蛋白的基因高水平表达。这
些细胞总mRNA的30%是麦醇溶蛋白的mRNA。显然，如
果我们从小麦种子中克隆mRNA，将会得到大量特异表
达的麦醇溶蛋白基因的克隆。2.2 cDNA(cDNA library)的构建
cDNA(cDNA library)，将纯化的某种生物的 特定发育阶段或组织的mRNA，经反转录酶作用合 成双链的DNA群体，同适当的载体分子重组之后转 定的发 育阶段特定组织或器官中表达随机地克隆着某种生物、组织、器官或细胞类型 的所有的DNA片段而构成的克隆集合体。在理想的
情况下，它应包含有该生ry）
• The DNA molecules of an organism in interest are isolated. The DNA molecules are then partially digested by an endonuclease restriction enzyme. Sometimes, the DNA molecules are digested to different lengths of time in order to ensure that all the genes have been digested to manageable sizes.
• The central dogma of molecular biology outlines that in synthesizing proteins, DNA is transcribed into mRNA, which is translated into protein. One difference between eukaryotic and prokaryotic genes is that eukaryotic genes can contain introns (intervening sequences), which are not coding sequences, and must be spliced out of the RNA primary transcript before it becomes mRNA and can be translated into protein. Prokaryotic genes have no introns, so their RNA is not subject to splicing.
• 用质粒（pBR322）克隆目的基因（1.5kb）需要 有9×106克隆才能汇集人基因组全部DNA序列;若 用λ噬菌体载体可少到9×105, 而要用柯斯质粒将40kb目的 基因片断所需要重组体克隆数可降到3.5×105左 右,均可满足建库要求。
对细菌、酵母和真菌来讲，一个完整的基因组文
库所需的克隆数还是在可操作的范围内的定出目的克隆，工作量是相当大
的。这时可library
2.1 cDNA • In genetics, complementary DNA (cDNA) is DNA synthesized from a mature mRNA template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to clone eukaryotic genes in proka片段平均长
实际克隆数
1975年L.C
N=ln( 1出现概率 f：插入片段大小/全基因组大小的比值
• Often it is desirable to express eukaryotic genes in prokaryotic cells. A simplified method of doing so would include the addition of eukaryotic DNA to a prokaryotic host, which would transcribe the DNA to mRNA and then translate it to protein. However, as eukaryotic DNA has introns, and since prokaryotes lack the machinery to splice them, the splicing of eukaryotic DNA must be done prior to adding the eukaryotic DNA into the host.
三、基因的化学合成一、从基因中获得目的基因1、基因（ Gene library ） 1.1 定义
A gene library, also called genomic library, is a population of host bacteria, each of which carries a DNA molecule that was inserted into a cloning vector, such that the collection of cloned DNA molecules represents the entire genome of the source organism. A gene library is also called gene bank.
⑶ DNA片段与载体的连接包装
• 常用载体：粘性质粒，λ噬菌体，酵母人工染色体
• ①基因组DNA +粘性质粒——重组DNA——转化细菌
• ②基因组DNA + λ噬菌体 ——重组DNA—包装成噬 菌体——感染细菌 1个克隆含有基因组的某个片段，整个克隆群体就包 含基因组的.5 ×103＝2×106
In（1－0.99）
实际克隆数 ：
N = ——————————————— = 9.2×106 In （1－1.5×103／3×109）
生物体是由很多不同类型的细胞组成的，如神经细 胞、血细胞、肝细胞等。每种细胞都含有相同数目基 因，但不同类型的细胞所表达的基因是有差别的。 因为在任何一种细胞中都只有部分基因得到表达，
我们可以利用这一事实来制备cDNA：制作的起始材料不再是DNA而是信使RNA（mRNA）。由于只有 那些被表达的基因会被转录成mRNA，因此以mRNA为材 料所得到的克隆将只包含细胞所有基因的一部分。
• The process of subdividing genomic DNA into clonable element and inserting them into host is called creating a library, a clone bank or a gene bank. A complete library of host cell will contain all of the genomic DNA of the source organism.
基因工程的操作包含以下步骤： 获得目的基因 构造重组 DNA 分子 转化或转染 表达 蛋白质产物的分离纯化
第二讲 目的基因的制备
需要克隆的DNA片 段
编码某种蛋白质
ห้องสมุดไป่ตู้研究某基因与 疾病的关系
研究某基因结构 和功能的关基因
• This DNA which was made as a complementary to the RNA is called complementary DNA (cDNA). To obtain expression of the protein encoded by the eukaryotic cDNA, prokaryotic regulatory sequences would also be required (e.g. a pro取哺乳动物细胞染色体DNA（酚抽提法） ⑵制备全部基因组的DNA片断 ①机械断裂法 用超声波或高速搅拌DNA溶液，断 裂片段两端没有与克隆载体匹配的粘末端, 因此插 入载体之前还需要进行修饰加工，少用 ②限制性内切酶降解（鸟枪法） 过去用EcoRⅠ,现 在用Sau3A，断裂片段两端有与克隆载体匹配的粘 末端，可以直接与处理过的载体连接。