cloning

• Cloning


  The process of creating an identical copy of something
• Cloning (in biology)


  Processes used to create copies of DNA fragments, cells, or organisms
• What cloning covers in biology

  • Processes used to create copies of DNA fragments, cells, or organisms
  • Asexual reproduction in organisms like bacteria, insects, or plants
• Cloning vector


  A small piece of DNA taken from a virus, a plasmid, or the cell of a higher organism, that can be stably maintained in an organism and into which a foreign DNA fragment can be inserted for cloning purposes
• Most cloning vectors are genetically engineered
• How cloning vector is chosen

  According to the size and type of DNA to be cloned
• Cloning vector


  • Features that allow for the convenient insertion or removal of DNA fragment in or out of the vector, for example by treating the vector and the foreign DNA with a restriction enzyme and then ligating the fragments together
• What can be done after a DNA fragment has been cloned into a cloning vector

  It may be further subcloned into another vector designed for more specific use
• Vector (in molecular cloning)

  A DNA molecule used to carry foreign genetic material into another cell
• Purpose of a vector in molecular cloning

  To replicate and/or express foreign genetic material in another cell
• Recombinant DNA


  A vector containing foreign DNA
• Purpose of a cloning vector

  To artificially carry foreign genetic material into another cell for replication and expression
• How a cloning vector is used to amplify DNA

  It is used to amplify a single molecule of DNA into many copies
• What cloning vectors are used to transport

  Cloned sequences
• Significance of cloning vectors in molecular gene cloning

  It makes molecular gene cloning possible
• Herbert Boyer, Keiichi Itakura, and Arthur Riggs recognized a general cloning vector with unique restriction sites for cloning in foreign DNA and the expression of antibiotic resistance genes
• The vector 'pBR 322' was constructed

  1977
• Vector 'pBR 322'


  • Small, ~4 kb in size, and had two antibiotic resistance genes for selection
• Origin of replication (ori) in a cloning vector

  It allows autonomous replication in the vector
• Cloning site in a cloning vector

  It is a place where the vector DNA can be digested and desired DNA can be inserted by the same restriction enzyme
• Multiple cloning site (MCS) in recombinant plasmids


  It contains many (up to ~20) restriction sites
• Selectable marker in cloning


  A gene that confers resistance to particular antibiotics or selective agents
• Purpose of a selectable marker in a cloning vector

  To confer on the host cell an ability to survive and proliferate in a selective growth medium containing specific antibiotics
• Reporter genes in cloning vectors

  To facilitate the screening of successful clones by using features that allow successful clones to be easily identified
• Blue-white selection in cloning vectors

  To use a feature present in cloning vectors for screening successful clones
• Desirable properties of a plasmid cloning vector

  • Replicate autonomously
  • Have restriction sites
  • Self-replicate in multiple copies
  • Have a replication origin site
  • Be small in size
  • Have low molecular weight
  • Be non-pathogenic
  • Be easily isolated and purified
  • Be easily isolated into host cell
  • Have control elements like promoter, operator, and ribosome binding site
• Importance of restriction sites in a plasmid cloning vector


  To allow for the insertion of foreign DNA fragments
• Significance of a replication origin site in a plasmid cloning vector

  It is the site where DNA replication begins
• Why it is preferable for a plasmid cloning vector to be small in size

  Larger plasmids are more difficult to characterize restriction mapping and replicate to lower copy numbers
• Purpose of control elements in a plasmid cloning vector

  To regulate gene expression
• What determines the choice of vector in cloning

  Insert size, vector size, restriction sites, copy number, cloning efficiency, and ability to screen for inserts
• Insert size for different vectors

  • Plasmid vectors: Less than 10 kb
  • Bacteriophage vectors: 9 - 15 kb
  • Cosmid vectors: 23 - 45 kb
  • BACs (Bacterial Artificial Chromosomes): Less than or equal to 300 kb
  • PACs (P1-derived Artificial Chromosomes): 100 - 300 kb
  • YACs (Yeast Artificial Chromosomes): 100 - 3000 kb
• What are cloning vectors used for

  To carry foreign DNA into host cells for replication and expression
• Purpose of expression vectors

  To produce a specific protein in large quantities
• Difference between cloning and expression vectors

  Cloning vectors are used to replicate DNA, while expression vectors are used to produce proteins
• Insert capacity of different vectors

  • Plasmid vector: 0.1 - 10 kb
  • Ti & Ri plasmid vector: Varies
  • Bacteriophages lambda vector: Bacteria
  • Bacteriophages P1 vector: 80 - 100 kb
  • Cosmid/Fosmid vector: 35 - 45 kb
  • Bacterial Artificial Chromosome (BAC) vector: 50 - 300 kb
  • Yeast Artificial Chromosome (YAC) vector: 100 - 2000 kb
  • Human Artificial Chromosome (HAC) vector: More than 2000 kb
  • Retroviral Vectors: Varies
• Targeted hosts for different vectors

  • Plasmid vector: Bacteria, Streptomyces
  • Ti & Ri plasmid vector: Higher plants
  • Bacteriophages lambda vector: Bacteria
  • Cosmid/Fosmid vector: Bacteria/E. coli
  • Bacterial Artificial Chromosome (BAC) vector: Bacteria
  • Yeast Artificial Chromosome (YAC) vector: Yeast
  • Human Artificial Chromosome (HAC) vector: Human cultured cells
  • Retroviral Vectors: Human and Animal Cells
• Viral vectors


  Viral vectors are tools used by molecular biologists to deliver genetic material into cells
• Main function of viral vectors

  To transfer genetic material into a target cell's genome
• Examples of viral vectors

  • Adenovirus, Lentivirus, Retrovirus, Adeno-associated virus (AAV)