1. Gene libraries and screening Molecular Biology Course （基因文库与筛选）

2. I1 Genomic libraries （基因组文库） I2 cDNA libraries （ cDNA 文库） I3 Screening procedures （筛选流程） Gene libraries and screening

3. I1 Genomic libraries I1-1 Representative gene libraries I1-2 Size of library I1-3 Genomic DNA I1-4 Vectors Gene libraries and screening

4. Gene libraries ： 来自某种生物的不同 DNA 序列 的总集，这些 DNA 序列已经被克隆在载体上，以便于 纯化、贮存和分析。 Genomic libraries cDNA libraries Gene libraries (made from genomic DNA) (made from cDNA- copy of mRNA) I1 Genomic libraries （基因组文库） （ cDNA 文库） ( 根据来源不同 )

5. I1-1 Representative gene libraries --- Contain all the original sequences 1. Certain sequences have not been cloned. 2. Library does not contain sufficient clones. Missing original sequence I1 Genomic libraries

6. I1-2 Size of library (ensure enough clones) The formula to calculate the number of recombinants: N = ln (1-P) ln (1-f) P: desired probability f : the fraction of the genome in one insert I1 Genomic libraries

7. For example :for a probability of 0.99 with insert sizes of 20 kb these values for the Ecoli (4.6×10 6 bp) and human (3×10 9 bp) genomes are : N Ecoli = = 1.1 ×10 3 ln( 1-0.99) ln[1-(2×10 4 /4.6×10 6 )] N human = = 6.9 ×10 5 ln(1-0.99) ln[1-(2 ×10 4 /3 ×10 9 )] These values explain why it is possible to make good genomic libraries from prokaryotes in plasmids where the insert size is 5-10kb,as only a few thousand recombinants will be needed. I1 Genomic libraries

8. Break DNA into fragments randomly I1-3 Genomic DNA libraries Eukaryotes Prokaryotes Clone these fragments into vectors （将片段克隆入载体） I1 Genomic libraries 真核生物 原核生物 ( 纯化基因组 DNA) ( 随机切割 DNA) Purify genomic DNA

9. Purification of genomic DNA : Prokaryotes extracted DNA directly from cells remove protein, lipids and other unwanted macro- molecules by protease digestion and phase extraction. Eukaryotes I1 Genomic libraries prepare cell nuclei 真核细胞采用细 胞分级分离法

10. Break DNA into fragments randomly: Physical shearing （物理剪切） pipeting, mixing or sonication （吹打、振荡、超声波） Restriction enzyme digestion （限制性酶解） partial digestion is preferred to get a greater lengths of DNA fragments. I1 Genomic libraries

11. Selection of restriction enzyme 1. 酶切产生的末端是否能和载体直接相连？ 2. 酶的作用是否被 DNA 碱基修饰作用所抑制？ 3. 酶解的时间影响最后产生的目的片段的大小？ I1 Genomic libraries

12. I1-4 Vectors 根据基因组的大小来选择合适的克隆载体 Vectors Plasmid phageλ cosmid YAC insert (kb) 5 23 45 1000 常用的基因组克隆载体 λ relacement vectors （替换型） I1 Genomic libraries

13. I 2 cDNA libraries 1.No cDNA library was made from prokaryotic mRNA. 原核的 mRNA 不稳定 原核的基因组文库更容易制备，并且可以包含 所有的基因组序列 I 2 cDNA libraries

14. 2.cDNA libraries are very useful for eukaryotic gene analysis cDNA 从 mRNA 逆转录而来，代表了基因中的可转录部 分（去除了非转录序列） cDNAs 不含有内含子 （ intron sequences ），可以 直接在大肠杆菌中表达 可以用于鉴定新基因 不同类型的细胞和组织表达特定的基因（奢侈基因） cDNA libraries I 2 cDNA libraries

15. cDNA libraries mRNA isolation 、 purification Check the RNA integrity Fractionate and enrich mRNA Synthesis of cDNA Treatment of cDNA ends Ligation to vector Gene libraries and screening mRNA 的提取、纯化 mRNA 的分级分离、富集 合成 cDNA 与载体连接 cDNA 的末端处理 检测 mRNA 的完整性

16. I2-1 mRNA isolation Most eukaryotic mRNAs are polyadenylated at their 3’ ends oligo (dT) can be bound to the poly(A) tail and used to recover the mRNA. 3’ AAAAAAAAAAn 5’ cap I 2 cDNA libraries

17. 1. 传统的提取方法是把总 RNA 通过寡聚 (dT)- 纤维素 柱。 2. 直接将结合了寡聚 (dT) 的磁珠加入到细胞裂解液中， 利用磁性将吸附了 mRNA 的磁珠分离出来后，再用溶 剂把 mRNA 从磁珠上洗脱。 3. 利用蔗糖梯度，从细胞裂解液中制备 mRNA －核糖 体复合物，再提取 mRNA 。 Three methods to isolate mRNA I2 cDNA libraries

19. Make sure that the mRNA is not degraded. （确定 mRNA 没有被降解） Methods: Translating the mRNA （翻译 mRNA ） Analysis the mRNAs by gel elctrophoresis （ 琼脂糖凝胶电泳或聚丙烯酰胺凝胶电泳） I2-2 Check the mRNA integrity I2 cDNA libraries

20. I2-3 Cloning the particular mRNAs 为了克隆某一特定基因，而不是构建完整的 cDNA 文库，需要对 mRNA 进行分级分离或者富集。 Fractionate on the gel （利用 mRNA 的大小不同，从凝胶中回收） Subtracted cDNA library ( 扣除 cDNA 文库 ) I2 cDNA libraries

21. I2-4 Synthesis of cDNA First stand synthesis reverse transcriptase( 反转录酶 ) 、 primer- oligo(dT) （寡聚 dT 引物） 、 dNTPs （ 4 种） Second strand synthesis 对第一链的 3’ 端 “ 加尾 ” ， 再用与之互补的引物合成第 二链，保证获得全长 cDNA 。 ( Fig 1.1) I2 cDNA libraries

22. 5’ mRNA AAAAA -3’ HO- TTTTT P -5’ 5’ Reverse transcriptase 4 dNTPs AAAAA -3’ TTTTT P -5’ mRNA c DNA Duplex c DNA AAAAACC -3’ TTTTT P -5’ 3’ 3’-CCCCCCC Terminal transferase （末端转移酶） dCTP Alkali (hydrolyaes RNA) Purify DNA oligo(dG) Klenow polymerase or reverse Transcriptase 、 4dNTPs 5’-pGGGG-OH 5’ 3’-CCCCCCC 5’-pGGGG 3’-CCCCCCC TTTTT P -5’ -3’ Fig 1.1 The first and second strands synthesis I2 cDNA libraries primer

23. I2-5 Treatment of cDNA ends 大片段的平末端连接效率很低，所以必需对双链 cDNA 的末 端进行加工，可以通过添加接头（ linkers ）进行改造。 The process : 切除突出的 3’-ends ( 单链特异性核酸酶 ) 补平 3’-ends (DNA 聚合酶的 klenow 片段和 dNTPs) 连接双链 cDNA 的平末端和接头 (T4 DNA ligase) 酶切产生粘性末端 (EcoR I ) 接头中可以嵌入酶切位点 (EcoR I ) I2 cDNA libraries

24. 5’-pGGGG 3’-CCCCCCC HO-CCG/AATTCGGGGGG 3’-GGCTTAA/GCCCCCC 5’-pAATTCGGGGGG TTTTTGGCTTAA/GCC-OH CCG/AATTCGG-3’ 3’-CCCC 3’-GCCCCCC 3’-CCC 5’-pGGGG TTTTTp-5’ -3’ TTTTTp-5’ -3’ TTTTTGGCTTAAp-5’ HO-CCG/AATTCGG-3’ 3’-GGCTTAA/GCC-OH CCG-3’ Duplex cDNA 单链特异性核酸酶 Klenow 聚合酶、 dNTP 用 EcoR I 甲基化酶处理 加入嵌入酶切位点的 接头进行连接 EcoR I 消化 Ligate to vector and transform Fig2.1 end preparation and linker addition to duplex cDNA 接头

25. I2-6 Ligation to vector （载体） Any vectors with an EcoR I site would suitable for cloning the cDNA. The process : 载体去磷酸化（避免自连） 连接载体和 cDNA 片段（ T4 DNA ligase ） (plasmid or λ phage vector) I2 cDNA libraries

26. I3 Screening procedures I3-1 Screening I3-2 Colony and plaque hybridization I3-3 Expression screening I3-4 Hybrid arrest and release I3-5 Chromosome walking (repeat screening) Gene libraries and screening （筛选） （菌落及噬菌斑杂交） （表达筛选） （杂交扣留与释放） （染色体步移）

27. I3-1 Screening Screening ：从基因文库的大量克隆中鉴定出某个含 有目的基因的特定克隆的过程。 1.Using nucleic acid probe （核酸探针） to screen the library based on hybridization （杂交） with nucleic acids. 2.Analyze the protein product. I3 Screening procedures

28. Screening libraries Hybridization to identify the interested DNA or its RNA product 1.Radiolabeled probes （放射性标记的探针） 2.Blotting the DNA or RNA on a membrane （将目的 DNA 或 RNA 吸附在膜上） 3.Hybridize the labeled probe with DNA membrane (Southern) or RNA (Northern) membrane （加入探针，在膜上进行杂交） Searching the genes of interest in a DNA library I3 Screening procedures

29. I3-2 Colony and plaque hybridization Transfer the DNA in the plaque or colony to a Nylon or nitrocellulose membrane Phage DNA bind to the membrane directly Bacterial colonies must be lysed to release DNA on the membrane surface. Hybridization (in a solution Containing Nucleic acid probe) Wash to remove unhybri- dization probe and visualize X-ray film(radio- actively labeled ) antibody or enzyme ( 抗体或酶） Line up the hybridizated region or repeated hybridization ( 菌落，碱裂解 ) I3 Screening procedures （噬菌斑） （与探针杂交） ( 标出被杂交区域，重复杂交 )

30. Transfer to nitrocellulose or nylon membrane Denature DNA(NaOH) Bake onto membrane Probe with 32 p-labled DNA complementary to gene of interest Expose to film Select positive from master plate Keep master plate Screening by plaque hybridization I3 Screening procedures

31. Identify the protein product of an interested gene 1.Protein activity 2.Western blotting using a specific antibody I3 Screening procedures I3-3 Expression screening （表达筛选）

32. Protein activity If the inserts are cloned into an expression sites, it may be expressed. Therefore, we can screen for the expressed proteins. （ However, this screening may miss the right clone. ） Example: the EcoR I site of lgt11 vector. The inserted genes have one in six change (1/6) to be in both the correct orientation (2 possibilities;  ) and reading frame (three possibilities; three nucleotide code XXX). I3 Screening procedures

33. ‘Plaque lift’ ( taken by placing a membrane on the dish of plaque ) Immersed in a solution of the antibody Detected by other antibodies Repeat cycles of screening to isolate pure plaques Antibodies （抗体） can be used to screen the expression library. The procedure has similarities to the plaque hybridization （噬菌斑杂交） protocol. I3 Screening procedures 噬菌斑复制 抗体结合 抗体识别 重复循环 筛选

34. I3-4 Hybrid arrest and release Individual cDNA clones or pools of clones can be used to hybridize to mRNA preparation. 单个 cDNA 克隆或克隆群都可以用来与 mRNA 样品进行杂交。 I3 Screening procedures ( 杂交扣留与释放）

35. Hybrid arrest :translate the mRNA population directly, and the inhibition of translation of some products detected. Hybrid release translation : purify the hybrids and the hybridized mRNAs released from them and translated, it identifies the protein encoded by the cDNA clone

36. I3-5 Chromosome walking （染色体步移） Definition: To clone the desired gene by repeated isolating adjacent genomic clones from the library. to obtain overlapping genomic clones that represent progressively longer parts of a particular chromosome. I3 Screening procedures

37. Process: 1. Prepare a probe from the end insert. 2.The probe are used to re-screen the library by colony or plaque hybridization 3.Analyzed the new isolate clones and posited them relative to the starting clone. some will be overlapping. 4. Repeated the whole process using a probe from the distal end of the second clone. I3 Screening procedures

38. }} }} Vector arm Genomic clone insert Vector arm Prepare probe from ends of insert Re-screen genomic library Restriction Restriction map new genomic clones Prepare new probes from distal ends of least overlapping insert. Re-screen genomic library. Restriction map new genomic clones Chromosome walking

39. over !