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CHAPTER 7 Sequencing and mutagenesis

CHAPTER 7 Sequencing and mutagenesis. Objective & Requirements. Know Principle of Cassette mutagenesis Know Basic DNA sequencing method. Master the methods used to make a site-directed mutagnesis. Class period 3h. Main content. 7.1 Introduction 7.2 Basic DNA sequencing

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CHAPTER 7 Sequencing and mutagenesis

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  1. CHAPTER 7 Sequencing and mutagenesis

  2. Objective & Requirements Know Principle of Cassette mutagenesis KnowBasic DNA sequencing method Master the methods used to make a site-directed mutagnesis Class period 3h

  3. Main content 7.1 Introduction 7.2 Basic DNA sequencing 7.3 Changing gene: site-directed mutagenesis

  4. 7.1 Introduction Why sequence and make mutant? 为何测序、制造突变子 • Sequence information is a prerequisite for planning any substantial manipulation of the DNA. 序列信息是设计基因操作的先决条件 突变子是遗传研究的先决条件 • Mutants are an prerequisite for genetic study Esp: gene structure and function relationships . 尤其是基因结构与功能之间的关系

  5. Example of mutant usages Used in protein engineering 突变子在蛋白工程中的应用 Main aim: to generate a protein with an improvement in some operating parameter 获得某些操作参数优化的蛋白 What factors should be considered to embark on protein mutagenesis? 要考虑的因素 • Construction of the mutant DNA • The expression vector, • The expression system • Strategies for purification • Assay methods of proteins activity. Gene expression

  6. Via changing Gene sequence composition Site-directed mutagnesis Mutagenesis Classical mutagenesis • Have to carry out following steps: • mutagenize cells or organisms with mutagens (chemical or physical agents), • analyze many offspring, • isolate a desired mutant.

  7. Disadvantages of classical mutagenesis 1) Any gene has low chance to be mutated 2) Relation of phenotype andmutant geneis unsure mutants with the desired phenotype ≠ mutation occurred in the interest gene 3) know nothing of the mutagenizing principles • where the mutation occurred • whether it is arisen by • a single base change • an insertion of DNA • a deletion. site-directed mutagenesis.

  8. 7.2 Changing genes: Site-directed mutagenesis Methods used 7.3.1 Cassette mutagenesis 盒式诱变 引物延伸 基于PCR 7.3.2 Primer extension: single-primer method 7.3.3 Based on PCR methods

  9. 7.3.1 Cassette mutagenesis Principle A synthetic DNA fragment containing the desired mutant sequence is used to replace the corresponding sequence in the wild-type gene. • The disadvantages • the requirement for unique restriction sites flanking the interest region. • the limitation on the realistic number of different oligonucleotide replacements that can be synthesized.

  10. 7.3.2 Primer extension: single-primer method Two requirements of the method 二个前提条件 • Primer carries a base mismatch with the complementary sequence. • the DNA to be mutated is available in single-stranded form. • 引物与互补链间有一个碱基错配 • 欲突变的DNA可以获得单链形式

  11. 异源双链质粒 E. coli

  12. heteroduplex 异性 c T Homoduplex 同型 to pick out mutants, the clones can be screened by hybridization under stringency condition.

  13. Oligonucleotide-directed mutagenesis used for multiple point mutation,insertion mutagenesis and deletion mutagenesis. 携带有要插入序列的突变寡聚核苷酸，夹在两个区中间，这两个区的序列与模板中目标位点二侧的位点互补。

  14. 7.3.3 PCR methods of site-directed mutagenesis PCR-based mutagenesis Two methods • Four primers and three PCRs method 四个引物、三次PCR • The megaprimer method 大引物：三个引物，2次PCR

  15. Primer B/C bearing the same mutation in the overlap region to amplify the overlapping segments 配对组合 Denature & anneal • Results: • to fuse the two segments • Knock out a fragment in the middle four primers and three PCRs

  16. The megaprimer method of mutagenesis. P1 • utilizes three primers • perform two rounds of PCR. P2 megaprimer P3 The mutant molecule, produced in the early rounds of PCR, acts as a primer (‘megaprimer’) for later rounds of PCR.

  17. 获得待突变的基因 第一步：设计诱变引物 二步：首次PCR 三步：纯化大引物 四步：第二次PCR 五步：纯化诱变的基因 六步：亚克隆基因

  18. 7.2 Basic DNA sequencing  双脱氧法 7.2.1 The chain-termination method dideoxy procedure • Key point: • use 2′,3′dideoxynucleoside triphosphate as a chain terminator • use 2′ deoxynucleotides as substrates 双脱氧核苷酸 链终止剂 脱氧核苷 chain terminators

  19. DNA sequencing----by synthesizingDNA by PCR Steps in manual sequencing

  20. Four PCR reaction Every mixture contains a low concentration of one dideoxynucleoside triphosphate analogues Four group PCR products • there is a population of partially synthesized radioactive DNA molecules, • each having a common 5′end, but each varying in length and having a base-specific 3′end

  21. manual sequencing anneal PCR mixture Extension products

  22. high-resolution gel CGTAAGGCCACGTATG

  23. EG autoradiograph of a DNA sequencing method

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