1. How to obtain a clone of a specific gene 1. The problem of selection 2. Two basic strategies for obtaining the clone you want 2. Two basic strategies for obtaining the clone you want 3. Methods for clone identification 3. Methods for clone identification

2. The problem of selection

3. How to obtain a clone of a specific gene 1. The problem of selection 2. Two basic strategies for obtaining the clone you want 2. Two basic strategies for obtaining the clone you want 3. Methods for clone identification 3. Methods for clone identification

4. Two basic strategies for obtaining the clone you want Two basic strategies for obtaining the clone you want 1. Direct selection 2. Library screening

5. The simplest example 1. Direct selection

6. Marker Rescue Mutant strain No tryptophan auxotrophic

7. 2. Gene Library 2. Gene Library Genomic library construction Or transformation and culture collection Fraction12 3 4 5 6

8. How many clones are needed for a genomic library?

9. Or we can use another type of library! Mammoth task! → YAC, BAC or PAC cDNA library

10. Not all genes are expressed at the same time Complementary DNA, cDNA Gliadin( 醇溶蛋白） high level in developing wheat seeds 30% mRNA for it Reverse transcriptase

11. ⑤ second strand DNA synthesis ⑥ ⑦ ⑧ collection ① Total RNA extraction mRNA 1~5% mRNA 1~5% ② mRNA isolation ③ ④ The steps of cDNA library construciton:

12. Now we can use reverse transcript –PCR to obtain double strand cDNA

13. How to obtain a clone of a specific gene 1. The problem of selection 2. Direct selection 2. Direct selection 3. Identification of a clone from gene library 3. Identification of a clone from gene library 4. Methods for clone identification 4. Methods for clone identification

14. 8.4 Methods for clone identification 8.4 Methods for clone identification 8.4.1 Complementary nucleic acid strands hybridize to each other to each other 8.4.2 Colony and plaque hybridization probing 8.4.3 Examples of the practical use hybridization probing probing 8.4.4 Identification methods based on detection of the translation product of the cloned gene the translation product of the cloned gene

15. 1. Complementary nucleic acid strands hybridize 1. Complementary nucleic acid strands hybridize

16. 2. Colony and plaque hybridization probing 2. Colony and plaque hybridization probing 硝酸纤维素膜或尼龙膜

17. The probe must now be labeled with a radioactive or other type of marker, denatured by heating, and applied to the membrane in a solution of chemicals that promote nucleic acid hybridization

18. Probe ( Molecular probe, Hybridization probe ) Probe ( Molecular probe, Hybridization probe ) The labeled nucleic acid molecules that can be used to identify complementary or homologous molecules through the formation of stable base-paired hybrids The labeled nucleic acid molecules that can be used to identify complementary or homologous molecules through the formation of stable base-paired hybrids. ● Radioactive labeling 32 P, 35 S, etc. ● Non-radioactive labeling Labeling methods: ☆ Nick translation ☆ End filling ☆ Random priming Avidin ( 抗生物素蛋白） Horseradish peroxidase （辣根过氧化物酶）

19. Random priming results in a probe with higher activity The gentlest method

20. ● Non-radioactive labeling Avidin 抗生 物素蛋白 Biotin ( 生物素）

21. Horseradish peroxidase 辣根过氧化物酶戊二醛 氨基苯二酰肼氨基苯二酰肼氨基苯二酰肼氨基苯二酰肼

22. 3. Examples of the practical use hybridization probing 3. Examples of the practical use hybridization probing If the gene itself is not available, what can be used as the probe? (a) Abundancy probing to analyse a cDNA library

23. (b) Oligonucleotide probes for genes whose translation products have been characterized translation products have been characterized

24. Trp-Asp-Glu-Asn-Asn-Met 1 2 2 2 2 1 1 2 2 2 2 1 (16 species) (16 species) Ser-Glu-Try-Leu-Thr-Asn 6 2 2 6 4 2 6 2 2 6 4 2 (1152 species) (1152 species) 编码 20 种氨基酸的密码子 AlaAGCU, GCC, GCA, GCGLeuLUUA, UUG, CUU, CUC, CUA, CUG ArgRCGU, CGC, CGA, CGG, AGA, AGG LysKAAA, AAG AsnNAAU, AACMetMAUG AspDGAU, GACPheFUUU, UUC CysCUGU, UGCProPCCU, CCC, CCA, CCG GlnQCAA, CAGSerSUCU, UCC, UCA, UCG, AGU,AGC GluEGAA, GAGThrTACU, ACC, ACA, ACG GlyGGGU, GGC, GGA, GGGTrpWUGG HisHCAU, CACTyrYUAU, UAC IleIAUU, AUC, AUAValVGUU, GUC, GUA, GUG 起始 AUG, GUG 终止 UAG, UGA, UAA

25. Oligonucleotide probe synthesis

26. DMT, 二甲氧三苯 甲基（在 DNA 合 成中用作羟基保护 剂） 二甲稀丙基焦磷酸

27. Use an end-labeled oligonucleotide to identify a clone

29. (c) Heterologous probing allows related genes to be identified

31. Heterologous probing allows related genes to be identified

32. Southern hybridization enables a specific restriction fragment containing a gene to be identified

35. transfer of RNA molecules (“northern” transfer) or proteins (“western” transfer)

36. 8.4.4 Identification methods based on detection of the translation product of the cloned gene the translation product of the cloned gene Immunological screening Antibody 抗体 （ Ab 抗体） Antigen 抗原 （ Ag 抗原）

37. Using a purified antibody to detect protein in recombinant colonies Need a expression library Or expression vector primary antibody secondary antibody

38. Identificaiton of the desired gene Direct selection: antibiotic resistant gene marker rescue marker rescue Hybridization selection: ● the method of selection: based on the abundance of the gene based on the abundance of the gene oligonucleotide probe oligonucleotide probe heterologous probe heterologous probe protein products probe (Western) protein products probe (Western) ● the method of Probe synthesis: nick translation end tilling end tilling random primer random primer ● Types of probe: radioactive biotin biotin

39. The end of Chapter 8