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Recombinant DNA technology allows for the isolation, cloning, and manipulation of genes. Two key advances enabled this field: genetic engineering using restriction enzymes to isolate and modify genes in vitro, and DNA sequencing to determine the order of nucleotides. Recombinant DNA is generated by joining DNA from different sources, and molecular cloning produces large quantities of a particular DNA fragment through construction of a recombinant vector, introduction into a host cell, selective propagation of cells containing the vector, and extraction of the cloned DNA.

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Recombinant DNA technology and Cloning Mai Masri Dep. of Zoology University of Khartoum
Recombinant DNA technology Two primary advances helped in the new era of molecular genetics in the late 1970s to the early 1980s: (1) The use of recombinant DNA technology (genetic engineering) used to isolate and manipulate genes in vitro in order to endow cells with new synthetic capabilities (restriction enzymes) (2) The ability to synthesize and determine the linear order of nucleotides of DNA molecules (DNA sequencing).
Advances of DNA technology Recombinant DNA technology has made it possible to: Clone (isolate and make copies of individual genes). Transfer genes between bacterial species and strains or from eukaryotes into bacteria (or vice versa). Causing the engineered cells to produce, sometimes in relatively large quantities, proteins of great economic importance such as enzymes (e. g. , amylases, proteases), hormones (e. g. , insulin, growth hormone).
Recombinant DNA Recombinant DNA is generated by covalently joining DNA molecules from different sources. The technology associated with the construction application of recombinant DNA is referred to as genetic engineering.
cloning, sudan 2016.pdf
Molecular coloning Molecular cloning is an in vivo technique for the production of large quantities of a particular DNA fragment. It contains four major steps: 1. Construction of recombinant vector. 2. Induction of the recombinant vector into suitable host cell. 3. Selective propagation of cells containing the vector(cloning). 4. Extraction and purification of the cloned DNA
Construction of recombinant vector, which involve cutting, modifying and joining donor and vector DNA in vitro. Induction of the recombinant vector into suitable host cell. Extraction and purification of the cloned DNA Selective propagation of cells containing the vector(cloning).
Restriction Endonucleases Using restriction enzymes II to create recombinant DNA Cutting DNA by using restriction enzymes is one of the most common Molecular Biology techniques. The cut ends can be joined using DNA ligase. The availability of pure restriction enzymes was one of the first major advances in the new science of Molecular Biology.
Restriction Endonucleases restriction enzymes These enzymes occur naturally in bacteria. Enzymes that cut DNA in a sequence-specific manner. recognition sequence Serve as a natural defense mechanism for bacteria against viral infection (bacteriophage). Bacteria protect their DNA from cutting by their own enzymes through methylation.
The simplest way of cloning is to cut the donor and the vector DNA with the same enzyme and then join them with ligase.
Examples Enzyme Recognition sequence EcoRI GAATTC HindIII AAGCTT BamHI GGATCC EcoRV GATATC Recognition sequences are usually 4-8 base pairs in length and are usually palindromic
Restriction Map
Cloning vectors DNA fragment does not contain origin of replication (or a replicon). It should be joined to a replicon (vector). Those include : Plasmids, Viruses and chromosomes.
An ideal cloning vector should be: 1. Episomal (do not integrate to the host genome, can be separated easily). 2. Replicate autonomously giving high copy number. 3. Allow the identification of DNA-carrying vector those lacking them. 4. Allow the identification of cells (host) crying DNA-carrying vector. 5. Should maintain characters that enable them to be used in applications after cloning.
Plasmids and bacteriophages are considered as naturally poor vectors. So they are manipulated by entering restriction sites without affecting the sequence of the plasmid or bacteriophages. 1. Plasmids Naturally occurring in bacteria. Used to copy already present copy of a gene in the genomic library (sub-cloning). Can be used in post transcriptional activities (small and easy to deal with).
2- Bacteriophage 了: Used in DNA libraries. Can contain larger inserts Can be stored for long periods. 3- Cosmids Vectors constructed from both plasmids and bacteriophages Can contain large inserts thats why used in genomic libraries. 4-Phagemids: Plasmids that contain the OriC of the M13 Bacteriophage. Thus can produce single stranded DNA ( can be used for sequencing, probe synthesis and mutagenesis) 5-phasmids: Used in post-transcriptional applications. 6-Artificail chromosomes Can carry large DNA sequences YACs BACs
Types of Cloning Vector Types size of cloned DNA (kb) Plasmid 20 Lambda Phage 25 Cosmid 45 P1 phage 100 BAC 300 YAC 1000
cloning, sudan 2016.pdf
ex. pBR322 Is a plasmid and was the first widely-used E. coli cloning vectors. Created in 1977 in the laboratory of Herbert Boyer (University of California San Francisco). It was named after the Mexican postdoctoral researchers who constructed it (p stands for "plasmid," and BR for "Bolivar" and "Rodriguez. pBR322 is 4361 base pairs in length and contains the replicon of plasmid pMB1.
cloning, sudan 2016.pdf
cloning, sudan 2016.pdf
DNA ligases DNA ligases catalyze formation of a phosphodiester bond between nucleotides. This enzyme is used to covalently link or ligate fragments of DNA together. Most commonly, the reaction involves ligating a fragment of DNA into a plasmid vector, which is a fundamental technique in recombinant DNA work. One of the most used enzymes to ligate DNA fragments is T4 DNA ligase, which originates from the T4 bacteriophage
DNA transfer to the cloning host Once recombinant vector has been constructed in vitro it should be introduce into a host cell. E.coli is the major host cells in general, however it can not introduce the DNA naturally into the cell. To over come that problem: 1- Electroporation (electron transformation through high voltatge). 2-Heat shock 3-Treatment with calcium ions Ca++.
cloning, sudan 2016.pdf
Vector and recombinant selection Construction of recombinant DNA and transfection are not always 100% successful. Vector selection (test transfection): Markers: Antibiotic resistance marker. Only the colonies that containing the plasmid can grow in a medium containing that antibiotic. Selection of recombinant DNA(test recombination): Blue- white selection: Insert disrupts a marker (gene) that turns the cell into blue if not distrusted hence has white color if disrupted.
Selection of colonies
Copyright 息 2010 Academic Press Inc. 27 Figure 22.16 Copyright 息 2010 Academic Press Inc.
Recovery of cloned DNA The cells are cultured in solid media. The colonies that contain the insert are selected by the previous methods. Transferred into a liquid media to generate large quantities from it. Collection of host cells to re-extract the plasmids: Using cloning we can generate libraries for genes or parts of genes that contain thousands of copies
DNA Library 1. Genomic libraries: Collection of DNA sequences from a living organism that has been copied into a vector so as to be used and stored and analyzed. 2. Complementary DNA (cDNA) libraries. Only coding parts of the genome
How to construct a genomic library The construction of a genomic library begins with cleaving the genome into small pieces by a restriction endonuclease. These genomic fragments are then either cloned into vectors & introduced into a microbe or packed into phage particles that are used to infect the host. At the end many thousands of different clones each with a different genomic DNA insert are created. Therefore each clone will act as a book in this library of DNA fragments. If the genomic library has been inserted into a microbe that expresses the foreign gene, it may be possible to assay each clone for a specific protein or phenotype
Digestion of the chromosomal DNA with restriction endonuclease Production of cDNA (complimentary DNA) Genomic Library Insertion of each DNA fragment into vector (recombinant DNA) Transformation of Bacteria using the recombinant vectors Cloning of the bacterial cells Each clone produced is a book in the Library of DNA fragments cDNA Library Extraction of mRNA Extraction of chromosomal DNA Insertion of each cDNA into vector (recombinant DNA) Transformation of bacteria Cloning of each recombinant Production of Bacterial cell (clones) containing the gene of interest
cloning, sudan 2016.pdf
cloning, sudan 2016.pdf
cloning, sudan 2016.pdf
cloning, sudan 2016.pdf
cloning, sudan 2016.pdf

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cloning, sudan 2016.pdf

  • 1. Recombinant DNA technology and Cloning Mai Masri Dep. of Zoology University of Khartoum
  • 2. Recombinant DNA technology Two primary advances helped in the new era of molecular genetics in the late 1970s to the early 1980s: (1) The use of recombinant DNA technology (genetic engineering) used to isolate and manipulate genes in vitro in order to endow cells with new synthetic capabilities (restriction enzymes) (2) The ability to synthesize and determine the linear order of nucleotides of DNA molecules (DNA sequencing).
  • 3. Advances of DNA technology Recombinant DNA technology has made it possible to: Clone (isolate and make copies of individual genes). Transfer genes between bacterial species and strains or from eukaryotes into bacteria (or vice versa). Causing the engineered cells to produce, sometimes in relatively large quantities, proteins of great economic importance such as enzymes (e. g. , amylases, proteases), hormones (e. g. , insulin, growth hormone).
  • 4. Recombinant DNA Recombinant DNA is generated by covalently joining DNA molecules from different sources. The technology associated with the construction application of recombinant DNA is referred to as genetic engineering.
  • 6. Molecular coloning Molecular cloning is an in vivo technique for the production of large quantities of a particular DNA fragment. It contains four major steps: 1. Construction of recombinant vector. 2. Induction of the recombinant vector into suitable host cell. 3. Selective propagation of cells containing the vector(cloning). 4. Extraction and purification of the cloned DNA
  • 7. Construction of recombinant vector, which involve cutting, modifying and joining donor and vector DNA in vitro. Induction of the recombinant vector into suitable host cell. Extraction and purification of the cloned DNA Selective propagation of cells containing the vector(cloning).
  • 8. Restriction Endonucleases Using restriction enzymes II to create recombinant DNA Cutting DNA by using restriction enzymes is one of the most common Molecular Biology techniques. The cut ends can be joined using DNA ligase. The availability of pure restriction enzymes was one of the first major advances in the new science of Molecular Biology.
  • 9. Restriction Endonucleases restriction enzymes These enzymes occur naturally in bacteria. Enzymes that cut DNA in a sequence-specific manner. recognition sequence Serve as a natural defense mechanism for bacteria against viral infection (bacteriophage). Bacteria protect their DNA from cutting by their own enzymes through methylation.
  • 10. The simplest way of cloning is to cut the donor and the vector DNA with the same enzyme and then join them with ligase.
  • 11. Examples Enzyme Recognition sequence EcoRI GAATTC HindIII AAGCTT BamHI GGATCC EcoRV GATATC Recognition sequences are usually 4-8 base pairs in length and are usually palindromic
  • 13. Cloning vectors DNA fragment does not contain origin of replication (or a replicon). It should be joined to a replicon (vector). Those include : Plasmids, Viruses and chromosomes.
  • 14. An ideal cloning vector should be: 1. Episomal (do not integrate to the host genome, can be separated easily). 2. Replicate autonomously giving high copy number. 3. Allow the identification of DNA-carrying vector those lacking them. 4. Allow the identification of cells (host) crying DNA-carrying vector. 5. Should maintain characters that enable them to be used in applications after cloning.
  • 15. Plasmids and bacteriophages are considered as naturally poor vectors. So they are manipulated by entering restriction sites without affecting the sequence of the plasmid or bacteriophages. 1. Plasmids Naturally occurring in bacteria. Used to copy already present copy of a gene in the genomic library (sub-cloning). Can be used in post transcriptional activities (small and easy to deal with).
  • 16. 2- Bacteriophage 了: Used in DNA libraries. Can contain larger inserts Can be stored for long periods. 3- Cosmids Vectors constructed from both plasmids and bacteriophages Can contain large inserts thats why used in genomic libraries. 4-Phagemids: Plasmids that contain the OriC of the M13 Bacteriophage. Thus can produce single stranded DNA ( can be used for sequencing, probe synthesis and mutagenesis) 5-phasmids: Used in post-transcriptional applications. 6-Artificail chromosomes Can carry large DNA sequences YACs BACs
  • 17. Types of Cloning Vector Types size of cloned DNA (kb) Plasmid 20 Lambda Phage 25 Cosmid 45 P1 phage 100 BAC 300 YAC 1000
  • 19. ex. pBR322 Is a plasmid and was the first widely-used E. coli cloning vectors. Created in 1977 in the laboratory of Herbert Boyer (University of California San Francisco). It was named after the Mexican postdoctoral researchers who constructed it (p stands for "plasmid," and BR for "Bolivar" and "Rodriguez. pBR322 is 4361 base pairs in length and contains the replicon of plasmid pMB1.
  • 22. DNA ligases DNA ligases catalyze formation of a phosphodiester bond between nucleotides. This enzyme is used to covalently link or ligate fragments of DNA together. Most commonly, the reaction involves ligating a fragment of DNA into a plasmid vector, which is a fundamental technique in recombinant DNA work. One of the most used enzymes to ligate DNA fragments is T4 DNA ligase, which originates from the T4 bacteriophage
  • 23. DNA transfer to the cloning host Once recombinant vector has been constructed in vitro it should be introduce into a host cell. E.coli is the major host cells in general, however it can not introduce the DNA naturally into the cell. To over come that problem: 1- Electroporation (electron transformation through high voltatge). 2-Heat shock 3-Treatment with calcium ions Ca++.
  • 25. Vector and recombinant selection Construction of recombinant DNA and transfection are not always 100% successful. Vector selection (test transfection): Markers: Antibiotic resistance marker. Only the colonies that containing the plasmid can grow in a medium containing that antibiotic. Selection of recombinant DNA(test recombination): Blue- white selection: Insert disrupts a marker (gene) that turns the cell into blue if not distrusted hence has white color if disrupted.
  • 27. Copyright 息 2010 Academic Press Inc. 27 Figure 22.16 Copyright 息 2010 Academic Press Inc.
  • 28. Recovery of cloned DNA The cells are cultured in solid media. The colonies that contain the insert are selected by the previous methods. Transferred into a liquid media to generate large quantities from it. Collection of host cells to re-extract the plasmids: Using cloning we can generate libraries for genes or parts of genes that contain thousands of copies
  • 29. DNA Library 1. Genomic libraries: Collection of DNA sequences from a living organism that has been copied into a vector so as to be used and stored and analyzed. 2. Complementary DNA (cDNA) libraries. Only coding parts of the genome
  • 30. How to construct a genomic library The construction of a genomic library begins with cleaving the genome into small pieces by a restriction endonuclease. These genomic fragments are then either cloned into vectors & introduced into a microbe or packed into phage particles that are used to infect the host. At the end many thousands of different clones each with a different genomic DNA insert are created. Therefore each clone will act as a book in this library of DNA fragments. If the genomic library has been inserted into a microbe that expresses the foreign gene, it may be possible to assay each clone for a specific protein or phenotype
  • 31. Digestion of the chromosomal DNA with restriction endonuclease Production of cDNA (complimentary DNA) Genomic Library Insertion of each DNA fragment into vector (recombinant DNA) Transformation of Bacteria using the recombinant vectors Cloning of the bacterial cells Each clone produced is a book in the Library of DNA fragments cDNA Library Extraction of mRNA Extraction of chromosomal DNA Insertion of each cDNA into vector (recombinant DNA) Transformation of bacteria Cloning of each recombinant Production of Bacterial cell (clones) containing the gene of interest