1 / 70

Section G – Gene manipulation

Section G – Gene manipulation. The Beadle-Tatum experiment for the isolation of genetic mutants in Neurospora ( 脉胞菌 ) showed that each mutant had a gene defect that resulted in an enzyme deficiency that prevented the cells from catalyzing a particular metabolic reaction.

saima
Download Presentation

Section G – Gene manipulation

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Section G – Gene manipulation

  2. The Beadle-Tatum experiment for the isolation of genetic mutants in Neurospora(脉胞菌) showed that each mutant had a gene defect that resulted in an enzyme deficiency that prevented the cells from catalyzing a particular metabolic reaction. “One gene-one enzyme” hypothesis “One gene-one polypetide” hypothesis

  3. An overview of the flow of information through the cell Segments of DNA are transcribed Pre-mRNAs mRNAs mRNAs are translated Proteins

  4. Contents G1 DNA cloning: an overview DNA cloning, Hosts and vectors, Subcloning, DNA libraries, Screening libraries, Analysis of a clone G2 Preparation of plasmid DNA Plasmids as vectors, Plasmid minipreparation, Alkaline lysis, Phenol extraction, Ethanol precipitation, Cesium chloride gradient G3 Restriction enzymes and electrophoresis Restriction endonucleases, Recognition sequences, Cohesive ends, Restriction digesis, Agarose gel electrophoresis, Isolation of fragment G4 Ligation, transformation and analysis of recombinants DNA ligation, Recombinant DNA molecules, Alkaline phosphatase, Transformation, Selection, Transformation efficiency, Screening transformants, Growth and storage of transfoemants, Gel analysis, Fragment orientation

  5. G1 DNA cloning: an overview — DNA cloning • DNA cloning facilitates the isolation and manipulation of fragments of an organism’s genome by replicating them independently as part of an autonomous vector.

  6. Question: Why do we have to carry out DNA cloning?

  7. Applications of DNA cloning • Sequencing, hence to derive protein sequence; • Isolation and analysis of gene promoter etc; • Investigation of protein/enzyme/RNA function in various forms; • Identification of mutations; • Biotechnology; • Transgenic plants and animals； • Gene therapy.

  8. G1 DNA cloning: an overview — Hosts and vectors • Hosts: Escherichia coli Saccharomyces cerivisiae e.g. E. coli DH5α: Host for Blue/White screening utilizing the activity of β-galactosidase (α-complementation) in combination use of pUC vectors. As this strain does not carry lac l, basically IPTG is not needed. Therefore, DH5α allows easy selection of recombinant DNA with X-Gal when constructing gene library or subcloning recombinant plasmid. • Host organism/cell: where the plasmids get multiplied and propagated faithfully

  9. Vectors: Plasmid, Bacteriophages,Viruses BACs, YACs, cosmid, Ti plasmid • A wide variety of natural replicons have the properties of cloning vector. • General features of a vector for longer term expression of cloned genes in the host (1) Autonomously replicating DNA independent of host’s genome; (2) Easily to be isolated from the host cell; (3) Contains at least one selective marker, which allows host cells containing the vector to be selected among those which do not; (4) Contains a multiple cloning site (MCS).

  10. Types of vectors (1) Cloning vectors (2) Expression vectors (3) Integration vectors (4) Viral vectors

  11. (1) Cloning vectors Allowing the exogenous DNA to be inserted, stored, and manipulated at the DNA level. E. coli cloning vector: plasmids, bacteriophages (l and M13), plasmid-bacteriophage l hybrids (cosmids). Yeast cloning vector: yeast artificial chromosomes (YACs)

  12. Earlier plasmid Rop:一种调节蛋白

  13. Versatile cloning plasmid Phagemid (噬菌粒）

  14. (2) Expression vectors • Allowing the exogenous DNA to be inserted and expressed. Promoter and terminator for RNA transcription are required. • bacterialexpression vectors • yeast expression vectors • mammalian expression vectors

  15. (3) Integration vectors • Allowing the exogenous DNA to be inserted and integrated into a chromosomal DNA after a transformation. The integration is a random insertion by homologous recombinationbetween the homologous sequence shared by the plasmid and the genome of the recipient cells. • Bacterialintegration vectors (Agrobacterium tumefaciens Ti plasmid is used to integrate DNA into plant genome) • Yeastintegration vectors • Mammalian integration vectors

  16. (4) Viral vectors （1）Bacterial phage: Lambda, M13 （2）Insect:baculoviruses （3）Mammalian viruses: SV40 Pox virus Adenovirus Retroviruses （4）Plant viruses: TMV

  17. G1 DNA cloning: an overview — Subcloning • Subcloning is a technique used to move a particular gene of interest from a parent vector to a destination vector in order to further study its functionality.

  18. Transfer of a fragment of cloned DNA from one vector to another. • Enables us to investigate a short region of a large cloned fragment in more detail. • To transfer a gene from one plasmid to a vector designed to express it in a particular species.

  19. DNA Cloning: a simplified flow chart Genomic fragment (restriction, PCR), cDNA (insert) Plasmid preparation (vector) Restriction digestion (trimming the DNA ends) Ligation (join the insert and the vector) Transformation (introduce the plasmids into host cells) Analysis of the recombinants Electrophoresis (check your DNA)

  20. G1 DNA cloning: an overview — DNA libraries

  21. Genomic libraries Prepared from random fragments of genomic DNA, which may be inefficient to find a gene because of the huge abundance of the non-coding DNA cDNA libraries DNA copies (cDNA) synthesized from the mRNA by reverse transcription are inserted into a vector to form a cDNA library. Much more efficient in identifying a gene, but yield only the coding region, and not surrounding genomic sequence.

  22. G1 DNA cloning: an overview — Screening libraries • Searching the interested genes in a DNA library (1) Colony or plaque hybridization Radiolabeled probes of the interested gene Probes: *An oligonucleotide derived from the sequence of a protein product of the gene * A DNA fragment/oligo from a related gene of another species * PCR product (2) Identify the protein product of an interested gene (1)Protein activity (2)Western blotting（ Western ） using a specific antibody (3)In vivo expression and functional assay

  23. Plating the cells carrying the library. Colony or plaque lift on membrane and then hybridize with the labeled probe

  24. G1 DNA cloning: an overview — Analysis of a clone (1) Restrictionmapping: digestion of the plasmids with restriction enzymes. (2) Sequencing the cloned DNA

  25. You may have to fully understand the function and application of all the listedenzymes if you want to manipulate genes Enzymes commonly used in DNA cloning (1) Alkaline phosphotase

  26. (2) DNA ligase (连接dsDNA,T4)

  27. (3) DNA pol I 、 Klenow fragment 、Taq

  28. (4) Exunuclease III

  29. (5) Mung bean nuclease and S1 nuclease

  30. (6) Polynucleotide kinase

  31. (7) Restrictionenzymes: e.g. EcoRI, HindIII

  32. (8) Reverse transcriptase

  33. (9) RNase A、 RNase H (10) T7, T3 and SP6 RNA polymerases

  34. 5’ AAAAA-3’ TTTTTP-5’ 3’ Terminal transferase dCTP AAAAACCC-3’ 5’ 3’-CCCCCCC TTTTTP-5’ (11) Terminal transferase

  35. G2 Preparation of plasmid DNA — Plasmids as vectors • Plasmids: small, extrachromosomal circular DNA molecules, from 2 to ~200 kb in size, which exist in multiple copies within the host cells. (1) Contain an origin of replication and replicate independently (2) Usually carry a few genes, one of which may confer resistance to antibacterial substance.

  36. G2 Preparation of plasmid DNA — Plasmid minipreparation • A plasmid may be obtained on a small scale for analysis by isolation from a few milliliters of culture, a process known as a minipreparation or miniprep.

  37. G2 Preparation of plasmid DNA — Alkaline lysis • An alkaline solution of SDS lyses E. coli cells and denatures protein and DNA. • Neutralization precipitates the chromosomal DNA and most of the protein, leaving plasmid DNA and RNA in solution.

  38. G2 Preparation of plasmid DNA — Phenol extraction • Extraction with phenol or a phenol-chloroform mixture removes any remaining protein from an alkaline lysate.

  39. G2 Preparation of plasmid DNA — Ethanol precipitation • Nucleic acid may be precipitated from solution by the addition of sodium acetate and ethanol, followed by centrifugation. • The method is used to concentrate the sample.

  40. G2 Preparation of plasmid DNA —Cesium chloride gradient • A CsCl gradient can be used as part of a large-scale plasmid preparation to purify supercoiled plasmid DNA away from protein, RNA and linear or nicked DNA.

  41. 1.Growth of the cells containing plasmids; 2.Collect the cells by centrifugation; 3. Alkaline lysis Resuspend the cells in a buffer solution Cell lysis in lysis buffer containing SDS ，disrupts cell membrane and denatures proteins and NaOH (denatures DNA); Neutralization buffer containing KOAc renaturation of plasmid DNA (supercoiled) and precipitation of denatured proteins and chromosomal DNA. Centrifugation ：plasmid in supernatant (lysate) 4.Phenol extraction to get rid of the protein contaminants 5. Ethanol precipitation to concentrate the nucleic acids remained (0.3M NaAc, 2-3 vol ethanol). 6. Resuspend in TE buffer

  42. G3 Restriction enzymes and electrophoresis —Restriction endonucleases

  43. G3 Restriction enzymes and electrophoresis —Recognition sequences Recognize 4-8 bp palindromic sequences. Most commonly used enzymes recognize 6 bp which occurs at a rate of 46=4096 bp. (44=256 bp; 48=65536 bp) 5’ GAATTC 3’ 3’ CTTAAG 5’ e.g. EcoRI site:

  44. SmaI p -GGG-3’ OH-CCC-5’ 5’-CCCGGG-3’ 3’-GGGCCC-5’ 5’-CCC-OH 3’-GGG- p G3 Restriction enzymes and electrophoresis —Cohesive ends 5’ protruding ends 3’ protruding ends Sticky ends + blunt ends

  45. G3 Restriction enzymes and electrophoresis —Restriction digesis • (1) Commercially available; • (2) Require Mg2+ for enzymatic activity,10M; • (3) Different enzymes, different pHs, NaCl, other solution constituents; • (4) A few hundred nanograms for analysis by electrophoresis, preparative purposes, a few micrograms; • (5) 37℃ , 20ul.

More Related