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Molecular biology ( Chapter 10 ) -1 李 芬 2014 年 11 月 The Outline of Molecular Biotechnology.

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Presentation on theme: "Molecular biology ( Chapter 10 ) -1 李 芬 2014 年 11 月 The Outline of Molecular Biotechnology."— Presentation transcript:

1 Molecular biology ( Chapter 10 ) -1 李 芬 2014 年 11 月 The Outline of Molecular Biotechnology

2 Question 1 Question 2 Question 3 How to clone a gene ? How to study the function of a gene ? How to determine whether there is interaction between two molecules ?

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

4 Genetic engineering and molecular cloning Enzymes for gene cloning 1 Molecular Cloning 2

5 DNA Ligase DNA polymerase Reverse Transcriptase Kinase Methyltransferases Restriction enzyme Enzymes 1.Enzymes for gene cloning

6 Concept : A restriction enzyme is an enzyme that cuts DNA at or near specific recognition nucleotide sequences known as restriction sites Enzymes for gene cloning Restriction enzyme

7 该酶的作用是一个 DNA 片段的 5’- P 与另一 DNA 片段的 3’- OH 连接成磷酸二酯键。 Enzymes for gene cloning DNA Ligase DNA ligase is a specific type of enzyme, a ligase, that facilitates the joining of DNA strands together by catalyzing the formation of a phosphodiester bond.

8 Enzymes for gene cloning DNA Ligase

9 Concept : The DNA polymerases are enzymes that create DNA molecules by assembling nucleotides, the building blocks of DNA. Enzymes for gene cloning DNA polymerase

10 Concept : A Reverse transcriptase (RT) is an enzyme used to generate complementary DNA (cDNA) from an RNA template, a process termed reverse transcription. Enzymes for gene cloning Reverse Transcriptase

11 Enzymes for gene cloning Methyltransferases Concept : The general mechanism for methyl transfer is a SN2-like nucleophilic attack where the methionine sulfur serves as the nucleophile that transfers the methyl group to the enzyme substrate.

12 Enzymes for gene cloning Kinase Concept : A kinase is a type of enzyme that transfers phosphate groups from high-energy donor molecules, such as ATP, to specific substrates, a process referred to as phosphorylation. General reaction that is catalyzed by kinases.

13 2 . Molecular Cloning A.History of molecular cloning( 略 )History of molecular cloning B.OverviewOverview C.Steps in molecular cloningSteps in molecular cloning D.Applications of molecular cloningApplications of molecular cloning

14 Concept : Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. Overview Molecular Cloning Molecular cloning and PCR Simliar: replication of DNA sequence. Difference: PCR replicates DNA in an in vitro solution, while molecular cloning involves replication of the DNA in a living microorganism.in vitro

15 Molecular Cloning Steps in molecular cloning ① Choice of host organism and cloning vector, ② Preparation of vector DNA, ③ Preparation of DNA to be cloned, ④ Creation of recombinant DNA, ⑤ Introduction of recombinant DNA into host organism, ⑥ Selection of organisms containing recombinant DNA, ⑦ Screening for clones with desired DNA inserts and biological properties.

16 (1)An origin of DNA replication. (2)One or more unique restriction endonuclease recognition sites. (3)A selectable genetic marker gene. (4)An additional gene. Four important DNA segments in vector: Choice of host organism and cloning vector Host organisms: bacterium E. coli (Escherichia coli)Escherichia coli Vector: E. coli cloning vector, bacterial or yeast artificial chromosomebacterialyeast artificial chromosome

17 Preparation of DNA to be cloned

18 Creation of recombinant DNA

19 The methods used to get DNA into cells are varied, and the name applied to this step in the molecular cloning process will often depend upon the experimental method that is chosen. e.g. : Transformation: microorganismsTransformation Transfection: mammalian cell cultureTransfection Transduction Transduction: uses high voltage electrical pulses Electroporation: using these virus-like particlesElectroporation Introduction of recombinant DNA into host organism

20 导入大肠杆菌 导入大肠杆菌(转化:热激或电激法; 噬菌体载体:侵染 ) 导入真核细胞 导入真核细胞( 酵母、植物细胞、动物细胞、原生质体等) ( 1 )化学方法:磷酸钙法、脂质体转染法(动物细胞) ( 2 )物理方法:电穿孔法、显微注射、基因枪法 ( 3 )生物方法:农杆菌介导的转化法(植物细胞) Methods used to get DNA into cells

21 Selection of organisms containing recombinant DNA ( 1 ) Feature of Vector ( 依载体的特征进行筛选 ) 抗性标记、 β- 半乳糖苷酶显色 (蓝白斑筛选) ( 2 ) The sequence of interest gene 菌落 PCR 、酶切鉴定、筛选基因文库 ( 3 ) The products of interest gene 如导入的重组 DNA 可在受体细胞表达,可用免疫筛选法 The selection of organisms containg recobinant DNA usually based on the features of recobinant DNA.

22  载体的选择和制备 —— 分;  制备目的基因片段 —— 切;  DNA 片段的重组连接 —— 接;  重组 DNA 导入受体细胞 —— 转;  转化子的筛选 —— 筛;  重组子的筛选 —— 筛;  重组子的鉴定 —— 鉴;  克隆扩增或表达 —— 扩 / 表达; Molecular Cloning

23 Genome organization and gene expression Production of recombinant proteins Transgenic organisms Gene therapy Molecular Cloning Applications of molecular cloning

24 DNA amplification Polymerase Chain Reaction PCR 1 Several modified PCR 2

25 1. PCR Introduction : The polymerase chain reaction (PCR) is a biomedical technology in molecular biology used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.

26 PCR

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28 DNA template: This is the DNA from which you amplify your fragment of interest. Primers: A pair of short single-stranded oligonucleotides that are identical to the 5’-end of the sense and anti-sense strands that will be amplified. Taq DNA polymerase: was isolated from the bacterium Thermus aquaticus, is stable at high temperatures, which means it is stable under the extreme temperature condition of PCR. dNTP: Just like in all other DNA sequencing reactions, the nucleotide building blocks must be present. PCR Components of PCR

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34 PCR PCR efficiency Amplification decrease with increase in length Too long primer decrease efficiency. Annealing temp, play imp (higher than ideal reduces effi). Primer sequence: if primers have complementary regions, dimers will be formed and effi. Will go down. Sequence of target: GC rich will form 2ndary str. And hair-pin loop will be formed. Addtion of BSA enhance eff. By protecting DNA polymerase and by binding to PCR inhibitor. Certain inhibitors also affects the efficiency

35 Diagnodsis of genetic disorders. The genetic identification of samples such as hair, sperm, blood stain involved in criminal cases, maternal/paternal disputes, rape cases etc. Analysis of homologous genes in evolutionary biology. DNA polymorphism. Gene transfer detection. Location of gene. Genmerate ssDNA for sequencing. Formation of cDNA from mNRA. PCR General Application

36 PCR Advantages of PCR Large amount of amplification from small amount of DNA is possible. Nanogram (ng) amounts of DNA can be generated from a single template molecule. Technique is quick and simple. Technique is very sensitive. The biggest advantage is that the DNA need not be in pure form. Cheap. Versatile. Large number of modifications possible. Large number of modifications possible.

37 PCR Limitation of PCR Nucleotide sequence of boundary region must be known. Error rate has to be checked by proper conditions.. As PCR is very sensitive technique, it may generate false signals due to previous contamination. Sequence upto 3kb is possible...Ideal for < 1kb. Amplicon size is very important. There are several differences between PCR and gene cloning. Amplification is upto 30 cycles..If you increase cycle, errors in annealing and amplification.

38 2. Several modified PCR Real-time PCR Multiplex PCR Touchdown PCR Nested PCR Anchored PCR Inverse PCR Hot start PCR Asymmetric PCR Assembly PCR Reverse transcription PCR RACE- PCR …………………

39 PCR Real-time PCR Concept : A real-time polymerase chain reaction is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR), which is used to amplify and simultaneously detect or quantify a targeted DNA molecule.

40 Real time quantitative PCR uses fluorophores in order to detect levels of gene expression

41 PCR Inverse PCR Concept : Inverse polymerase chain reaction (Inverse PCR) is a variant of the polymerase chain reaction that is used to amplify DNA with only one known sequence.

42 Limitation : One limitation of conventional PCR is that it requires primers complementary to both termini of the target DNA, but this method allows PCR to be carried out even if only one sequence is available from which primers may be designed. PCR Inverse PCR

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44 PCR Concept : Multiplex polymerase chain reaction (Multiplex PCR) is a modification of polymerase chain reaction in order to rapidly detect deletions or duplications in a large gene. Multiplex PCR Multiplex-PCR consists of multiple primer sets within a single PCR mixture to produce amplicons of varying sizes that are specific to different DNA sequences.amplicons

45 1. Pathogen Identification 2. High Throughput SNP Genotyping 3. Mutation Analysis 4. Gene Deletion Analysis 5. Template Quantitation 6. Linkage Analysis 7. RNA Detection 8. Forensic Studies 9. Diet Analysis PCR Applications : Multiplex PCR

46 PCR Reverse transcription PCR Concept : Reverse transcription PCR is used to clone expressed genes by reverse transcribing the RNA of interest into its complementary DNA (cDNA) through the use of reverse transcriptase.

47 Protocol of Reverse Transcription- Polymerase Chain Reaction (RT-PCR) In the one-step protocol, the components of RT and PCR are mixed in a single tube at the same time. The one-step protocol generally works well for amplifying targets that are reasonably abundant. Alternatively, RT-PCR can be done in two steps, first with the reverse transcription and then the PCR. The two-step protocol is usually more sensitive than the one-step method; yields of rare targets may be improved by using the two-step procedure. We recommend the two-step protocol for this class.

48 TD-PCR employs an initial annealing temperature above the projected melting temperature (T(m)) of the primers being used, then progressively transitions to a lower, more permissive annealing temperature over the course of successive cycles. Any difference in T(m) between correct and incorrect annealing will produce an exponential advantage of twofold per cycle. PCR Touchdown PCR

49 Concept : The hot start PCR is a modified form of Polymerase chain reaction (PCR) which avoids a non-specific amplification of DNA by inactivating the taq polymerase at lower temperature. PCR Hot start PCR

50 Principle : In this dsDNA is denatured by heating the sample at its denaturing temperature and then the temperature is suddenly reduced to 55 ℃ at which primer and Taq-polymerase is added, but here the difference arises i.e. specific antibodies are used to block this Taq-polymerase at annealing temperature. PCR Hot start PCR

51 Concept : Nested polymerase chain reaction (Nested PCR) is a modification of polymerase chain reaction intended to reduce non-specific binding in products due to the amplification of unexpected primer binding sites. PCR Nested PCR

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53 Preferentially amplifies one DNA strand in a double-stranded DNA template. It is used in sequencing and hybridization probing where amplification of only one of the two complementary strands is required. PCR Asymmetric PCR

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55 Concept : Assembly PCR is a method for the assembly of large DNA oligonucleotides from shorter fragments. PCR Assembly PCR

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57 Concept : Anchored PCR is a technique used in molecular biology to obtain the full length sequence of an RNA transcript found within a cell. PCR Anchored PCR

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59 3’ RACE-PCR 5’ RACE-PCR

60 ……………...

61 Molecular hybridization Southern Blotting Northern Blotting Western blotting in situ hybridization fluorescence in situ hybridization

62 Molecular hybridization Southern Blotting Southern blot is a method used in molecular biology for detection of a specific DNA sequence in DNA samples. First described by E.M. Sorthern in 1975.

63 Molecular hybridization The flow chart of Southern Blotting

64 RFLP’s, VNTR’s and DNA fingerprting. Checking of the gene knockout mice ……. Molecular hybridization Applications of Southern Blotting

65 Molecular hybridization Detection of RFLP by Southern Blotting

66 Molecular hybridization Detection of the sickle-cell globin gene by Southern Blotting

67 Molecular hybridization Checking of the gene knockout mice by Southern Blotting

68 Technique for detecting specific RNAs separrated by electrophoresis by hybridization to a labeled DNA probe. Used in molecular biology research to study gene expression by detection of RNA (or isolated mRNA) in a sample. Molecular hybridization Northern Blotting

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70 Molecular hybridization The flow chart of Northern Blotting

71 Molecular hybridization Western blotting Technique for detecting specific proteins separated by electrophoresis by use of labeled antibodies. widely used analytical technique used to detect specific proteins in a sample of tissue homogenate or extract.

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73 Molecular hybridization The flow chart of Western Blotting

74 Molecular hybridization Example of Western blotting

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77 Concept : In situ hybridization (ISH) is a type of hybridization that uses a labeled complementary DNA or RNA strand to localize a specific DNA or RNA sequence in a portion or section of tissue. Molecular hybridization In situ hybridization

78 Localization and level of RNALocalization of DNA

79 In situ hybridization of wild type Drosophila embryos at different developmental stages for the RNA from a gene called hunchback.

80 FISH is a powerful technique that uses non- toxic fluorescent DNA probes to target any given sequence within a nucleus, resulting in colored signals that are detected with a fluorescence microscope. Molecular hybridization Fluorescence in situ hybridization

81 Scheme of the principle of the FISH Experiment to localize a gene in the nucleus.


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