基因工程與原理

1. 基因工程與原理 GENE CLONING & DNA ANALYSIS T. A. BROWN Chapter 8 How to obtain a clone of specific gene?

3. 8 How to Obtain a Clone of a Specific Gene 8.1 The problem of selection 8.1.1 There are two basic strategies for obtaining the clone you want 8.2 Direct selection 8.2.1 Marker rescue extends the scope of direct selection 8.2.2 The scope and limitations of marker rescue 8.3 Identification of a clone from a gene library 8.3.1 Gene libraries 8.3.2 Not all genes are expressed at the same time 8.3.3 mRNA can be cloned as complementary DNA 8.4 Methods for clone identification 8.4.1 Complementary nucleic acid strands hybridize to each other 8.4.2 Colony and plaque hybridization probing Labeling with a radioactive marker Non-radioactive labeling 8.4.3 Examples of the practical use of hybridization probing Abundancy probing to analyse a cDNA library Oligonucleotide probes for genes whose translation products have been characterized Heterologous probing allows related genes to be identified Southern hybridization enables a specific restriction fragment containing a gene to be identified 8.4.4 Identification methods based on detection of the translation product of the cloned gene Antibodies are required for immunological detection methods Using a purified antibody to detect protein in recombinant colonies The problem of gene expression

4. 8.1 The problem of selection Figure 8.1 The problem of selection.

5. 8.2 Direct selection Figure 8.2 The basic strategies that can be used to obtain a particular clone: (a) direct selection; (b) identification of the desired recombinant from a clone library.

6. Figure 8.3 Direct selection for the cloned R6-5 kanamycin resistance (kanR) gene. Chloramphenicol, Strptomycin, Suphonamide.

7. Tryptophan synthase Auxotroph營養變異菌 Figure 8.4 Direct selection for the trpA gene cloned in a trpA− strain of E. coli.

8. 8.3 Identification of a clone from a gene library Partial restriction digestion Cosmid, replacement vector, YAC, yeast artificial chromosome, BAC, bacterial artificial chromosome, P1 vector. Figure 8.5 Preparation of a gene library in a cosmid vector.

9. 8.3.2 Not all genes are expressed at the same time Figure 8.6 Different genes are expressed in different types of cell.

10. 8.3.3 mRNA can be cloned as complementary DNA Figure 8.7 One possible scheme for cDNA cloning. Poly(A) = polyadenosine, oligo(dT) = oligodeoxythymidine.

11. 8.4 Methods for clone identification 8.4.1 Complementary nucleic acid strands hybridize to each other Figure 8.8 Nucleic acid hybridization. (a) An unstable hybrid molecule formed between two non-homologous DNA strands. (b) A stable hybrid formed between two complementary strands. (c) A DNA–RNA hybrid, such as may be formed between a gene and its transcript.

12. Figure 8.9 Colony hybridization probing. In this example, the probe is labelled with a radioactive marker and hybridization detected by autoradiography, but other types of label and detection system can also be used.

13. Figure 8.10 The structure of α-32P-deoxyadenosine triphosphate ([α-32P]dATP).

14. Klenow fragment: enzyme lack the nuclease activity of DNA polymerase I Figure 8.11 Methods for labeling DNA. Q: How to detect mRNA?

15. Figure 8.12 Two methods for the non-radioactive labelling of DNA probes. Biotin生物素 (維生素H) - avidin卵蛋白

16. Detection of gliadin gene from a cDNA library from developing wheat seeds. Figure 8.13 Probing within a library to identify an abundant clone.

17. Trp-Asp-Glu-Asn-Asn-Met TGG-GAT-GAA-AAT-AAT-ATG C G C C Figure 8.14 The amino acid sequence of yeast cytochrome c. The hexapeptide that is highlighted red is the one used to illustrate how a nucleotide sequence can be predicted from an amino acid sequence.

18. Figure 8.15 A simplified scheme for oligonucleotide synthesis. Each nucleotide is modified by attachment of an activating group to the 3′ carbon and a protecting group to the 5′ carbon. The activating group enables the normally inefficient process of nucleotide joining to proceed much more rapidly. The protecting group ensures that individual nucleotides cannot attach to one another, and instead react only with the terminal 5′ group of the growing oligonucleotide, this 5′ group being deprotected by chemical treatment at the appropriate point in each synthesis cycle.

19. Figure 8.16 The use of a synthetic, end-labelled oligonucleotide to identify a clone of the yeast cytochrome c gene.

20. Figure 8.17 Heterologous probing.

21. Figure 8.18 A long cloned DNA fragment may contain several genes in addition to the one in which we are interested. B = BamHI restriction site.

22. Figure 8.19 Southern hybridization. Northern: RNA, Western: protein.

23. Antibodies are required for immunological detection methods Figure 8.20 Antibodies. (a) Antibodies in the bloodstream bind to foreign molecules and help degrade them. (b) Purified antibodies can be obtained from a small volume of blood taken from a rabbit injected with the foreign protein.

24. Using a purified antibody to detect protein in recombinant colonies Immunological screening Q: The problem of gene expression Figure 8.21 Using a purified antibody to detect protein in recombinant colonies. Instead of labelled protein A, the antibody itself can be labelled, or alternatively a second labelled antibody which binds specifically to the primary antibody can be used.

25. 8.4 Methods for clone identification 8.4.1 Complementary nucleic acid strands hybridize to each other 8.4.2 Colony and plaque hybridization probing Labeling with a radioactive marker Non-radioactive labeling 8.4.3 Examples of the practical use of hybridization probing Abundancy probing to analyse a cDNA library Oligonucleotide probes for genes whose translation products have been characterized Heterologous probing allows related genes to be identified Southern hybridization enables a specific restriction fragment containing a gene to be identified 8.4.4 Identification methods based on detection of the translation product of the cloned gene Antibodies are required for immunological detection methods Using a purified antibody to detect protein in recombinant colonies The problem of gene expression