1. The Outline of Molecular Biotechnology
Molecular biology（Chapter 10）-2
The Outline of Molecular Biotechnology
李 芬 年11月

2. Main Content Main Content Main Content 1 2 3 4 5 6
Genetic engineering and molecular cloning 1
DNA amplification 2
Molecular hybridization 3
DNA sequencing 4 5
Gene function identification techniques 6
Interactions between biological macromolecules

3. DNA sequencing Dideoxy chain-termination method 12
Maxam-Gilbert Sequencing 3
Automatic DNA sequencing

4. DNA sequencing Dideoxy chain-termination method
Concept：Dideoxy chain-termination method is a method of DNA sequencing based on the selective incorporation of chain-terminating dideoxynucleotides
by DNA polymerase during in vitro DNA replication.

6. DNA sequencing Maxam–Gilbert sequencing
This method is based on nucleobase-specific partial chemical modification of DNA and subsequent cleavage of the DNA backbone at sites adjacent to the modified nucleotides.

8. DNA sequencing Automatic DNA sequencing
Introduction：Automatic DNA sequencing take some of the drudgery out of DNA sequencing and speed up data production , The instrument is a microchemical robot that can carry out the fragmentations of 16 dna solutions-that is it can test for the four bases(A,G,C and T) in each of four different dna solutions. it performs the fragmentation in four to five hours, following the Maxam-Gilbert protocol.

10. Gene function identification techniques
Transgenic technology I
Gene knockout II
Gene Silencing
III

11. Topic I. Transgenic technology1
Introduction 2
Transgenic plants 3
Transgenic animals 4
Applications 5
Benefits and Disadvantage

12. Topic1. Transgenic technology
Introduction
Concept： Transgenesis is the process that introducing an exogenous gene — called a transgene — into a living organism so that the organism will exhibit a new property and transmit that property to its offspring.

13. Topic1. Transgenic technology
Introduction

15. Transgenic plants Topic1. Transgenic technology
What are transgenic plants?
Methods for producing transgenic plants.
The flow chart of producing transgenic plants.

16. What are transgenic plants?
Transgenic plants are plants that have been genetically engineered, a breeding approach that uses recombinant DNA techniques to create plants with new characteristics.

17. Methods for producing transgenic plantsVS

18. 1. Transgenic technology
Methods for producing transgenic plants

24. Transgenic plants
The flow chart of producing transgenic plants

25. Transgenic animals Topic I. Transgenic technology
What are transgenic animals?
Methods for producing transgenic animals.
The flow chart of producing transgenic mice.

26. What are transgenic animals?
Transgenic animals are genetically modified organisms which are animals. They have had their DNA altered specifically by having the DNA of another animal inserted into their own code.
They have in some way had their genetic material changed, for any number of reasons. In some cases these animals may be designed simply to be visually interesting, to study, to yield more meat, or to perform a specific task better.
Transgenic animals are used as experimental models to perform phenotypic and for testing in biomedical research.
The simplest type of transgenic animals are those which have genetic material inserted into their own code for research purposes. One notable example of this is the injection of material from a certain species of jellyfish into other creatures. This material is responsible for a fluorescent protein, GFP, which then allows researchers to track proteins tagged with GFP in the animal it has been inserted into.
There are valuable uses of transgenic animals in medicine, as well, with many animals altered in order to make them produce something needed by human beings. One of the first uses of transgenesis, for example, was to make the E. Coli bacteria produce human insulin, which could then be gathered cheaply, rather than having to be harvested from more expensive animals like pigs. A more contemporary example can be seen in the use of transgenic goats to product an anticoagulant in their milk. The milk can then be harvested from the animals, and the anticoagulant, ATryn, can be extracted and used in situations such as surgeries where the blood cannot be allowed to clot.
In research, transgenic animals may also allow for specific research possibilities. Modified mice, for example, are often used in laboratory testing. They can be modified so that researchers can observe specific responses their tissue has to diseases. This can lead to the development of drugs and treatments for humans suffering from those same diseases.
For the most part, transgenic animals have not become widely commercially available. This is in part because the public is still wary about eating them, and there is some concern over what would happen if certain super-species were to escape into the wild and overrun native populations. Many fish farms, for example, have developed transgenic versions of popular food fish, which can grow many times the size of their un-modified counterparts. Eventually this may allow for a much cheaper rearing of food fish, driving down the price on these fish for consumers.
One of the most iconic transgenic animals is the brand GloFish®. These are zebrafish that have been modified to include genes that make them glow fluorescent colors. The fish were created in 1999 with the goal of helping to detect pollutants, but it quickly became apparent that they had huge potential as a novelty item. They come in three colors, with green GloFish® derived from the GFP protein from jellyfish, a red GloFish® derived from a type of sea coral, and a yellow GloFish® derived from a variant of the jellyfish protein.

27. Methods for producing transgenic animals
Transgenic plants
Methods for producing transgenic animals
Microinjection （显微注射法）
Retroviral infection embryo （逆转录病毒感染胚胎法）
Embryonic stem cell method （胚胎干细胞法）
Sperm carrier method （精子载体法）
Nuclear transplantation method（细胞核移植法）
Germ cell transfection method （生殖细胞转染法）

28. Transgenic mice expressed GFP

30. A 抗生素筛选抗性愈伤组织 B 抗性愈伤组织分化培养 C 遗传转化筛选后得到的试管苗
Topic1. Transgenic technology
Applications
Food
转基因抗性植株筛选
A 抗生素筛选抗性愈伤组织 B 抗性愈伤组织分化培养 C 遗传转化筛选后得到的试管苗

35. Benefits and Disadvantage
Topic1. Transgenic technology
Benefits and Disadvantage
1. Reduce the production cost.
2. Improve the output.
Benefits
3. To reduce the harmful material.
4. Add some beneficial to human body.

36. Disadvantage

38. Topic II. Gene knockout 1
Concept 2
Method 3
Use

39. Gene knockout
Concept
A gene knockout is a genetic technique in which one of an organism‘s genes are made inoperative.
. they are used in learning about a gene that has been sequenced, but which has an unknown or incompletely known function.

40. Gene knockout
Method
基因同源重组法敲除靶基因的基本步骤

41. Knockout mice / 基因敲除小鼠
For small organisms, such as C. elegans, siRNAs can be used to prevent expression of a protein in all cells. However, for larger model organisms like mice, it is so far impossible to introduce the siRNAs into all of the body’s cells. Instead, a technique called gene knockout is used (Figure D.27). Knockout causes the removal of a gene from the genome of an organism.对小的生物如线虫而言，siRNA可以用来防止某种蛋白在所有细胞中的表达。然而，对更大的模式动物如小鼠而言，目前还不可能通过将siRNA引入所有的体细胞。此时可以采用另一种技术，即基因敲除（图D.27）。敲除使某个基因从一种生物的基因组中去除。

42. Knockout mice / 基因敲除小鼠
For small organisms, such as C. elegans, siRNAs can be used to prevent expression of a protein in all cells. However, for larger model organisms like mice, it is so far impossible to introduce the siRNAs into all of the body’s cells. Instead, a technique called gene knockout is used (Figure D.27). Knockout causes the removal of a gene from the genome of an organism.对小的生物如线虫而言，siRNA可以用来防止某种蛋白在所有细胞中的表达。然而，对更大的模式动物如小鼠而言，目前还不可能通过将siRNA引入所有的体细胞。此时可以采用另一种技术，即基因敲除（图D.27）。敲除使某个基因从一种生物的基因组中去除。

43. Knockout mice / 基因敲除小鼠
A fragment of DNA is made that has sequences homologous to both sides of the gene to be removed. However, the fragment does not contain a functional copy of the gene. The fragment is introduced into a plate containing millions of embryonic cells. Some cells take up the fragment. In a very small percentage of the cells, recombination occurs between the gene in the chromosome and the artificial fragment. This replaces the normal gene with the nonfunctional version present in the fragment. The rare cells in which this occurs are identified by various selective properties present on the artificial fragment, such as resistance to drugs that normally kill the cells. These recombinant cells are now heterozygous for the targeted gene. The copy on one chromosome is normal, but the other copy on the other chromosome is disrupted.制备一个DNA片段，它具有与需要去除的基因的两边同源的序列。然而，该片段并不含有目标基因的有功能的拷贝。将此片段引入到含有几百万个胚细胞的平板中。有些细胞能吸收这一片段。在很小比例的细胞中染色体和该人工构建的片段之间会发生重组。这样的重组会导致正常的基因被片段中的无功能版本取代。发生了这种情况的少数细胞可以通过人工构建片段上的不同选择特性而进行鉴定，比如对某些药剂的抗性，这些药剂会杀死普通的细胞。对目标基因来说这些重组细胞现在是杂合的。该基因在一条染色体上的拷贝是正常的，但在另一条染色体上的拷贝是被破坏了的。

44. Gene knockout
USE
Understand the role of a gene or DNA region by comparing the knockout organism to a wildtype with a similar geneticbackground.
Development of drugs, to target specific biological processes or deficiencies .
understand the mechanism of action of a drug by using a library of knockout organisms spanning the entire genome, such as in Saccharomyces cerevisiae.
Knockouts are primarily used to understand the role of a specific gene or DNA region by comparing the knockout organism to a wildtype with a similar geneticbackground.
Knockouts organisms are also used as screening tools in the development of drugs, to target specific biological processes or deficiencies by using a specific knockout, or to understand the mechanism of action of a drug by using a library of knockout organisms spanning the entire genome, such as in Saccharomyces cerevisiae.

45. Topic III. Gene Silencing1
Concept 2
Method 3
Use

46. Gene Silencing Concept
Gene silencing is a general term used to describe the epigenetic regulation of gene expression. In particular, this term refers to the ability of a cell to prevent the expression of a certain gene.

47. Gene Silencing Method 3、RNA interference
1、Antisense oligonucleotides 2、Ribozymes
General mechanism utilized by ribozymes to cleave RNA molecules
General mechanism utilized by ribozymes to cleave RNA molecules
3、RNA interference

48. RNA interference
Left:Overview of RNA interference.

49. The gene knockdown / 基因敲低

50. Gene Silencing
Use
What happens if it is removed or altered? 如果它被移去或改变会怎样？

51. Interactions between biological macromolecules
Interaction between DNA and Protein 1 2
Interaction between Protein and Protein

52. Interaction between DNA and Protein1
Gel Shift assay 2
DNase I footprinting 3
chromatin immunoprecipitation 4
ChIP-chip/ChIP-seq 5
yeast one-hybrid screening system.

53. Interaction between DNA and Protein Gel Shift assay
Concept：Gel Shift assay (Electrophoretic Mobility Shift Assay )is capable of binding to a given DNA or RNA sequence, and can sometimes indicate if more than one protein molecule is involved in the binding complex.

55. Interaction between DNA and Protein DNase I footprinting
Concept：A DNase footprinting assay is a DNA footprinting technique from molecular biology/ biochemistry that detects DNA-protein interaction using the fact that a protein bound to DNA will often protect that DNA from enzymatic cleavage.

58. Interaction between DNA and Protein Chromatin immunoprecipitation
Concept：Chromatin Immunoprecipitation (ChIP) is a type of immunoprecipitation experimental technique used to investigate the interaction between proteins and DNA in the cell.

60. Interaction between DNA and Protein ChIP-chip/ChIP-seq
Concept ：ChIP-seq is a method used to analyze protein interactions with DNA.

62. Interaction between DNA and Protein Yeast one-hybrid screening system
Concept ：The yeast one-hybrid screening system is a versatile and efficient method to identify transcription factors that can bind and regulate a given gene-of-interest.

64. Interaction between Protein and Protein1
Yeast two-hybrid assay 2
Co- Immunoprecipitation 3
GST Pull-Down 4
FRET 5
Phage display 6
BiFC 7
protein chip

65. Interaction between Protein and Protein Yeast two-hybrid assay
Concept ：Yeast two-hybrid system is a molecular biology technique used to discover protein–protein interactions and protein–DNA interactions by testing for physical interactions (such as binding) between two proteins or a single protein and a DNA molecule, respectively.

68. Overview of two-hybrid assay, checking for interactions between two proteins, called here Bait and Prey.
A. Gal4 transcription factor gene produces two domain protein (BD and AD), which is essential for transcription of the reporter gene (LacZ).
B,C. Two fusion proteins are prepared: Gal4BD+Bait and Gal4AD+Prey. None of them is usually sufficient to initiate the transcription (of the reporter gene) alone.
D. When both fusion proteins are produced and Bait part of the first interact with Prey part of the second, transcription of the reporter gene occurs.

69. Interaction between Protein and Protein Co- Immunoprecipitation
Principle：Co-IP works by selecting an antibody that targets a known protein that is believed to be a member of a larger complex of proteins. By targeting this known member with an antibody it may become possible to pull the entire protein complex out of solution and thereby identify unknown members of the complex.

71. Interaction between Protein and Protein GST Pull-Down
Concept：GST can be added to a protein of interest to purify it from solution in a process known as a pull-down assay.
Principle： This is accomplished by inserting the GST DNA coding sequence next to that which codes for the protein of interest.

73. Interaction between Protein and Protein FRET
Fluorescence resonance energy transfer (FRET) is a mechanism describing energy transfer between two light-sensitive molecules A donor chromophore, initially in its electronic excited state, may transfer energy to an acceptor chromophore through nonradiative dipole–dipole coupling. The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor, making FRET extremely sensitive to small changes in distance.

77. Interaction between Protein and Protein Phage display
Concept ：Phage display is a laboratory technique for the study of protein–protein, protein–peptide, and protein–DNA interactions that uses bacteriophages to connect proteins with the genetic information that encodes them.

78. The sequence of events that are followed in phage display screening to identify polypeptides that bind with high affinity to desired target protein or DNA sequence.

79. Interaction between Protein and Protein BiFC
Concept ：Bimolecular fluorescence complementation (BiFC) is based on the association of fluorescent protein fragments that are attached to components of the same macromolecular complex. Proteins that are postulated to interact are fused to unfolded complementary fragments of a fluorescent reporter protein and expressed in live cells.

80. Protein complex formation using BiFC
Protein complex formation using BiFC. Interaction between protein A and protein B occurs first, followed by the re-formation and fluorescence of fluorescent reporter protein

81. BiFC
N fragment
C fragment

82. BiFC
N fragment
C fragment
linker
Protein A
Protein B
A&B interaction

83. Interaction between Protein and Protein Protein chip
Concept：A protein chip is a high-throughput method used to track the interactions and activities of proteins, and to determine their function, and determining function on a large scale.

85. Gene chip
Introduction：A DNA chip is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome. Each DNA spot contains picomoles (10−12 moles) of a specific DNA sequence, known as probes.

86. Gene chip
Principle：The core principle behind microarrays is hybridization between two DNA strands, the property of complementary nucleic acid sequences to specifically pair with each other by forming hydrogen bonds between complementary nucleotide base pairs.

87. Hybridization of the target to the probe