1. Part Two and Three
The Applications of Gene Cloning and DNA Analysis in Research and Biotechnology
Chapter 10. Studying gene location and structure
Chapter 11. Studying gene expression and function
Chapter 12. Studying genomics and post genomics
Chapter 13. Production of protein from cloned genes
Chapter 14 . Gene cloning and DNA analysis in medicines
Chapter 15 . Gene cloning and DNA analysis in agriculture
Chapter 16 . Gene cloning and DNA analysis in forensic science

2. Studying gene expression
Chapter 11
Studying gene expression
And function
Studying the RNA transcript of a gene
Studying the regulation of gene expression
Identifying and studying the translation product of a cloned gene ●

3. The process of gene expression
Figure 11.1

4. exons
Figure 11.1

5. 5’ -UTR
3’ -UTR
Figure 11.1

6. 1. Northern hybridization
11.1 Studying the RNA transcript of a gene
Detecting the presence of transcripts of a gene and determining its nucleotide sequence
1. Northern hybridization

7. Northern hybridization
If RNA from different tissues is run in different lanes of the gel, then the possibility that the gene is differentially expressed can be examined.
R S L
h Darkness
Northern hybridization

8. Comparison between the sequence of the cDNA and the sequence of its gene will reveal the positions of introns and possibly the start and end points of the transcript.
However, The cDNA synthesis might not continue all the way to the 5′end of the transcript, especially if the RNA is more than a few hundred nucleotides in length.

10. 2. Transcript mapping by hybridization between gene and RNA
The size can be measured by gel electrophoresis.

11. Locating a transcription start point by S1 nuclease mapping
Figure 11.5

12. 3. Transcript analysis by primer extension
Figure 11.6

13. 4. Transcript analysis by PCR
Rapid Amplification of cDNA Ends (RACE)

14. 11.1 Studying the transcript of a cloned gene
11.2 Studying the regulation of gene expression
11.3 Identifying and studying the translation
product of a cloned gene
R S L
h Darkness

15. 11.2 Studying the regulation of gene expression
Figure Possible positions for control sequences in the region upstream of a gene.
The gene is switched on or off by the attachment of regulatory proteins to the control sequences
Positive role or negative role
Firstly, how to obtain the upstream of a gene?

16. 11.2.1 The methods of cloning the promoter of a gene
Using PCR
Adapter PCR (接头PCR法)
Reverse PCR (反向PCR法)
Tail PCR (热不对称交错PCR)

17. ◆ 接头PCR法

18. ◆ 反向PCR法 Genomic DNA Known sequence analysis Genomic DNA digestion
Ligation
PCR
Cloned cDNA sequence
Upstream sequence
Downstream sequence
Genomic DNA RE Pu Pd RE Pd Pu Pd RE Pu

19. ◆ Tail-PCR法 (Termal Asymmetric Interlaced PCR)又叫热不对称交错PCR
Genomic walking

20. 11.2.2 To determine the possible positions for control sequences in the upstream of a gene

21. 1. Identifying protein-binding sites on a DNA molecule
(a) Gel retardation of DNA-protein complex
(凝胶阻滞检测）
(b) Footprinting with DNase I
(c) Modification interference assays
2. Identifying control sequences
by deletion analysis
(a) Reporter genes
(b) Carrying out a deletion analysis

22. (a) Gel retardation of DNA-protein complex
(凝胶阻滞检测）
A bound protein decreases the mobility of a DNA fragment during gel electrophoresis.

23. Carrying out a gel retardation experiment
Fig 11.10
Carrying out a gel retardation experiment
① Extraction of gDNA ② ③ ④

24. 3 3 4 4 1 1 5 5 2 2

25. 11.2.2.1 Identifying protein-binding sites on a DNA molecule
(a) Gel retardation of DNA-protein complex
(凝胶阻滞检测）
(b) Footprinting with DNase I
(c) Modification interference assays
Identifying control sequences
by deletion analysis
(a) Reporter genes
(b) Carrying out a deletion analysis

26. (b) Footprinting (足迹法）
Fig A bound protein protects a region of a DNA from degradation by a nuclease such as DNase I

27. Footprinting
(足迹法） ① ② ③ ④

28. ④

29. 11.2.2.1 Identifying protein-binding sites on a DNA molecule
(a) Gel retardation of DNA-protein complex
(凝胶阻滞检测）
(b) Footprinting with DNase I
(c) Modification interference assays
Identifying control sequences
by deletion analysis
(a) Reporter genes
(b) Carrying out a deletion analysis

30. (c) Modification interference assays

31. 11.2.2.1 Identifying protein-binding sites on a DNA molecule
(a) Gel retardation of DNA-protein complex
(凝胶阻滞检测）
(b) Footprinting with DNase I
(c) Modification interference assays
Identifying control sequences
by deletion analysis
(a) Reporter genes
(b) Carrying out a deletion analysis

32. Reporter genes
半乳糖苷酶，新霉素磷酸转移酶，氯霉素乙酰转移酶，二氢叶酸还原酶，潮霉素磷酸转移酶，荧光素酶，葡糖醛酸酶

34. Deletion analysis by restriction enzyme
Fig Deletion analysis. A reporter gene has been attached to the upstream region of a seed-specific gene from a plant.

35. Deletion analysis by PCR
-232 ~ -1
-617 ~ -1
-1078 ~ -1
GUS
GhNP1
GhNP3
GhNP2
GhNP1
GhNP2

36. 11.1 Studying the transcript of a cloned gene
11.2 Studying the regulation of gene expression
11.3 Identifying and studying the translation
product of a cloned gene
HRT (Hybrid-release translation) and
HART (hybrid-arrest translation) can
identify the translation product of a
cloned gene
Analysis of proteins by in vitro
mutagenesis

37. Cell-free translationsystems
are cell extracts, usually prepared from germinating wheat seeds or from rabbit reticulocyte cells and containing ribosomes, tRNAs, and all the other molecules needed for protein synthesis. AA
Fig 11.18

38. ① Hybrid-release translation
Fig 11.19

39. ② Hybrid-arrest translation
Fig 11.20

40. ③ We can also use the pGEM3Z vector to synthesize RNA in vitro
Use these proteins, we can do some experiments, such as pull down, co-immunoprecipitation, etc, to uncover the mechanism of a gene product.

41. 11.1 Studying the transcript of a cloned gene
11.2 Studying the regulation of gene expression
11.3 Identifying and studying the translation
product of a cloned gene
HRT (Hybrid-release translation) and HART
(hybrid-arrest translation) can identify the
translation product of a cloned gene
Analysis of proteins by in vitro mutagenesis

42. Using mutation in the coding region to determine protein function
Fig 11.21

43. Different types of in vitro mutagenesis techniques
Fig 11.22

44. Arabidopsis T-DNA insertional mutants
Different types of in vitro mutagenesis techniques
Fig 11.24
Arabidopsis T-DNA insertional mutants

45. Oligonucleotide-directed point mutagenesis
Fig 11.23
Protein function research
important amino acid

46. Creating a point mutation in a cloned gene
Fig 11.25
One method of using PCR to create a directed mutation

47. The end of chapter 11