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Chapter 6 Cloning strategies

Chapter 6 Cloning strategies. Objectives and Requirements. Should master the strategy to express a gene and the related methods. Need to grasp the basic procedure of DNA cloning and the techniques involved in DNA cloning. Class-periods 9h. Main contents. 6.1. Introduction.

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Chapter 6 Cloning strategies

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  1. Chapter 6 Cloning strategies

  2. Objectives and Requirements Should master the strategy to express a gene and the related methods. Need to grasp the basic procedure of DNA cloning and the techniques involved in DNA cloning Class-periods 9h

  3. Main contents 6.1. Introduction 6.2. Cloning genomic DNA 6.3. cDNA cloning 6.4. Screening strategies

  4. 6.1 Introduction Cloning strategy somewhat like going abroad h

  5. 6.1.1 Four essential parts of a cloning procedure (i) A method for generating the DNA fragment for cloning 基因片断获得 (ii) A reaction that inserts that fragment into the chosen cloning vector; 重组质粒获得 (iii) A means for introducing that recombinant vector into a host cell where it is replicated;受体转化 (iv) A method for selecting recipient cells that have acquired the recombinant 转化子筛选

  6. 同聚物 Taq酶在扩增产物的3末端加A,insert into T vector Generalized overview of cloning strategies Principles of Gene Manipulation by liuzengran Hebei University of Economics and Business

  7. Hebei University of Economics and Business

  8. 涉及外源基因表达的四个词：转化、转染、转导、感染涉及外源基因表达的四个词：转化、转染、转导、感染 Hebei University of Economics and Business

  9. 可以简单理解如下 Hebei University of Economics and Business

  10. 6.1.2 DNA Screening strategy Question 1）What should be done，when the DNA donor source is very complex? • 2）How • to rapidly sift through large numbers of unwanted sequences? • to identify the particular target? two major strategies for isolating sequence

  11. ① The first strategy—build library • to divide the source DNA into manageable fragments and clone everyone. All the clones is known as alibrary • To screen the library to identify the interest clone. ② The second strategy---by PCR • to selectively amplify the target sequence directly from the source DNA by PCR. • clone this individual fragment.

  12. ③ Comparision of the two strategies The library approach • the entire DNA are generated • clone all indiscriminately • need screening The PCR approach • the specific DNA are generated, • need no screening • only clone the selected fragments

  13. 6.2. Cloning genomic DNA 6.2.1 Constructing Genomic DNA libraries By λ cloning vectors ② By high-capacity vectors 6.2.2 By PCR as an alternative ① Long PCR ② Fragment libraries

  14. 6.2.1 Constructing genomic DNA libraries ①Constructing libraries in λ cloning vectors ? How to clone a single-copy gene from the human genome? methods • digest total human DNA with a restriction endonuclease • insert all the fragments into a suitable phage-λ vector • isolate the desired clone. Principles of Gene Manipulation by liuzengran Hebei University of Economics and Business

  15. Facing 2 questions Question1：需要筛选多少个重组子？ How many recombinants have to be screened ? Assuming Human haploid(单倍体）genome (2.8×106 kb) EcoRI Fragments of about 4 kb over 7×105 independent recombinants must be prepared and screened. Question 2 如何获得合适大小的片段 How can appropriate fragment size be produced?

  16. Question 2 如何获得合适大小的片段 How can appropriate fragment size be produced? • Random breakage by mechanical shearing机械剪切 • the average fragment size can be controlled, • insertion reaction requires additional steps (to produce the stick end) 片段大小可控、需要加粘性末端 • restriction endonucleases 基因内部切断，片段大超过载体承载能力 There are two problems • the gene may be cut internally. • the obtained gene may be larger than the vector can accept.

  17. How to solve these problem? by cloning randomDNA fragments of a large size (for λ replacement vectors, ～ 20 kb) Only fewer clones are required for a complete or nearly complete library.

  18. 卡隆载体 random Maniatis’ strategy for producing a representative gene library. 获得代表性基因库的策略 Principles of Gene Manipulation by liuzengran Hebei University of Economics and Business

  19. Creation of a genomic DNA library using phage -λ vector EMBL3A.

  20. 串联体

  21. ② Genomic libraries in high-capacity vectors BACs, PACs，YACs Advantages • the number of recombinants needed to be screened is lower, • large genes are more likely to be contained within a single clone • 需要筛选的重组子少 • 单克隆可能容纳多基因

  22. 6.2.2. PCR as an alternative to genomic DNA cloning Limitation of traditional PCR Standard PCR conditions are suitable only for the amplification of short DNAs. 传统PCR,仅适于小片段 • The maximum product size is about 5 kb, • the typical size is 1–2 kb. ①Long PCR ②Fragment libraries by PCR

  23. ①Long PCR－改变PCR的性能（反应条件/双聚合酶） • Modify the reaction conditions • lower the reaction • temperature • increase the pH Prevent the template from damage improve polymerase processivity 持续合成能力 • 降低反应温度， • 提高pH • Proofreading enzyme • polymerase. • two DNA polymerases 校正酶 the performance of long templates PCR improved

  24. ② Fragment libraries by PCR How to construct a genomic librarybyPCR ? 2 methods: • specific type of primer • 采用特定引物 • random primers • 任意引物 • digest genomic DNA with restriction enzymes, • ligate linkers（接头） carrying annealing sites for the specific primer Select for suitable products by size

  25. 6.3 cDNA cloning 6.3.1 Preparation of cDNA by conventional cloning strategies ①The cDNA synthesis reaction (RT-PCR) involves three major steps: (to synthesis a double-stranded cDNA) • first-strand DNA synthesis on the mRNA template (with a reverse transcriptase) ; 合成第一个DNA链 RNA模板降解 • removal of the RNA template; • Second strand DNA synthesis using the first DNA strand as a template, (with a DNA-dependent DNA polymerase). 合成第二链DNA

  26. ② Development of cDNA cloning strategies cDNA克隆策略的发展 • early cDNA cloning strategy--the hairpin method • improved method • adding a linker to the double-stranded cDNA before the hairpin loop is cleaved • using an oligo-dT primer containing a linker sequence • using primers that are already linked to a plasmid • the Gubler–Hoffman method---widely used

  27. 1 ) An early cDNA cloning strategy the first cDNA strand has the tendency to transiently fold back on itself. a hairpin loop 末端转移酶

  28. A serious disadvantage: 5′序列可能丢失 Cleavaging with S1 nuclease results in the loss of a certain amount of sequence at the 5′ end of the clone.

  29. 3) adding a linker to the double-stranded cDNA before the hairpin loop is cleaved to realize direction cloning Principles of Gene Manipulation by liuzengran Hebei University of Economics and Business

  30. 2) Improved method (adding oligo-dC at the 5' end of first strand cDNA) 合成第一链 +polyC 降解mRNA 纯化 a full-length cDNA 合成第二链 Principles of Gene Manipulation by liuzengran Hebei University of Economics and Business

  31. 4) Using oligo-dT primer containing a linker sequence Principles of Gene Manipulation by liuzengran Hebei University of Economics and Business

  32. 5) using primers that are already linked to a plasmid 退火合成第一链cDNA 引物携带质粒 加聚C HindIII酶切 3' mRNA cDNA 与HindIII+聚G粘末端片段连接 mRNA降解，并合成第二链cDNA 特点：PolyT引物连在载体上。 合成第一链// +polyC// 酶切// +polyG与HindIII片段// 合成第二链

  33. Hebei University of Economics and Business

  34. Summary cDNA cloning Extraction total RNA Template //primer //reverse transcriptase First chain cDNA synthesis Template//primer // RNase // DNA polymerase // DNA ligase Second chain cDNA synthesis Note • Add EDTA to stop the reaction. • The products need to be purified • Primer design with certain technique for facilitating further operation

  35. 6) The Gubler–Hoffman method---widely used a simple and generalmethod for non-directional cDNA cloning. 非定向克隆的一般方法 It combines classical first-strand synthesis with the novel RNase H-DNA polymerase I-mediated second-strand synthesis DNA ligase 取代合成 Gene. 1983,25:263-269 Neither the elaborate vector-primer system nor the classical hairpin loop cleavage by S 1 nuclease are used.

  36. 6.3.2 Full-length cDNA cloning ①two major drawbacks of conventional approaches to the production of cDNA libraries 传统cDNA库克隆策略的缺陷 oligo-dT primers are used to initiate first-strand synthesis, • Trigger a 3′- end bias 引起偏差 • Difficult to gain full-length clones • (when the size of a cDNA increases) 难获得完整的长片段 • Deficiencies in the reverse-transcriptase enzymes used for first-strand cDNA synthesis. 反转录酶有缺陷 酶持续合成能力差、固有的RNA酶活性 • Native enzymes have poor processivity and intrinsic RNase activity.

  37. ②Strategy for the selection of 5 ′mRNA ends To generate full-length clones corresponding to large mRNAs 获得长片段克隆的策略 5'cap Hebei University of Economics and Business

  38. To capture full-length cDNA an oligo-dT primer hybrid molecules P1 digests single-stranded RNA P2 protected digested away eukaryotic initiation factor 翻译起始因子 using the eIF-4E to select mRNAs with caps Principles of Gene Manipulation by liuzengran Hebei University of Economics and Business

  39. oligo-capping(寡聚加帽法） Steps BAP treatment to removes phosphate 脱磷 alkaline phosphatase 碱性磷酸酶 去除5'磷酸基团 TAP treatment to remove the cap 脱帽 acid pyrophosphatase 酸性焦磷酸酶 Primer annealing site 加帽 using the oligo-dT primer and a primer annealing to the oligonucleotide cap. RT- 合成第一链 PCR Principles of Gene Manipulation by liuzengran Hebei University of Economics and Business

  40. Strategy adopted all eukaryotic mRNAs have a 5′ end cap 5′帽子 Combining cap selection and nuclease treatment 筛选完整第一链cDNA to pick out full-length first-strand cDNAs to generate libraries (highly enriched in full-length clones). 富含完整链的库

  41. 6.3.3 PCR as an alternative to cDNA cloning PCR方法克隆cDNA ① RT-PCR RT-PCR can be used to provide the cDNA for library construction, when the source is unsuitable for conventional approaches. RT-PCR可用于建cDNA库(起始材料稀有） universal primers 通用引物 In cDNA form amplification of all mRNAs mRNAs扩增 subcloned into suitable vectors 插入合适载体 cDNA library cDNA 库

  42. ②Rapid amplification of cDNA ends (RACE) 通过PCR进行cDNA末端快速扩增技术  is a technique used to obtain the full sequence of an RNA transcript found within a cell.  Based on incomplete clone from a cDNA library if limited knowledge of the mRNA sequence is required 从一段RNA转录子，获得全长cDNA序列 通过RT-PCR技术、借助cDNA的一段已知序列, 通过向两端延伸，快速扩增得到完整的cDNA 的5‘ 和3’末端。

  43. Can we get the cDNA of the following whole mRNA? 5'RACE 上游未知序列 3'RACE 上游未知序列 已知序列 未知区域 Hebei University of Economics and Business

  44. Isolation of Total RNA Strategy First strand cDNA synthesis from total RNA 5'RACE 3'RACE Amplification of a Target cDNA Clone and Sequence RACE Fragments Full-length cDNA

  45. Anneal oligo(dT)-containing Adapter Primer (AP) to mRNA 3’ end RACE Extend AP using SuperScript™ II RT Degrade RNA template using RNase H PCR amplify cDNA user-designed Primer Universal Amplification Primer Reamplify primary PCR product Hebei University of Economics and Business

  46. TdT酶法 5′ end RACE

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