Genetic vectors

• Genes


  The fundamental physical, biological and functional unit of heredity
• Genetics is a discipline that studies heredity in general and genes in particular
• Genetics as a branch of science began with the study of how the characteristics of organisms are passed on from parents to offspring
• Until the middle of the twentieth century, no one knew for sure what the hereditary material was
• Requirements for hereditary material


  • It had to replicate so that copies could be transmitted from parents to offspring
  • It had to encode information to guide the development, functioning, and behavior of cells and the organisms to which they belong
  • It had to change (mutate), even if only once in a great while, to account for the differences that exist among individuals
• Gregor Mendel


  A Moravian monk who lived in the nineteenth century and worked with Pea plants, crossing them to produce progenies and making observations that led him to postulate the existence of hereditary factors responsible for the traits he studied
• Mendel's discoveries were published in 1866 but not much noticed, and the science of genetics was born in 1900 when the paper finally came to light
• In the middle of the twentieth century, the question "What is a gene?" was finally answered by the breakthrough work and discoveries of James Watson and Francis Crick, who showed that genes consist of complex molecules called nucleic acids

  
• Genes


  Predominantly composed of long strands of the molecule DNA, located in single file within the chromosomes. The genetic material of some viruses such as HIV and Ebola is RNA

  
• Nucleotides


  The elementary building blocks of nucleic acids (genes)
• A gene is an ordered sequence of nucleotides of nucleic acids located in a particular position on a particular chromosome that encodes a specific functional product (i.e., a protein or RNA molecule)

  
• Components of a nucleotide

  • A sugar molecule
  • A phosphate molecule, which has acidic chemical properties
  • A nitrogen-containing molecule, which has slightly basic chemical properties
• Bases in RNA and DNA

  • In RNA: Adenine (A), Guanine (G), Cytosine (C), Uracil (U)
  • In DNA: Adenine (A), Guanine (G), Cytosine (C), Thymine (T)
• Watson and Crick found that DNA molecules consist of two chains of nucleotides held together by hydrogen bonds between particular pairs of bases: A pairs with T, and G pairs with C
• DNA duplex


  A double-stranded DNA molecule in a helical configuration, with the sugar-phosphate backbones on the outside and the bases on the inside
• The "program" or genetic code encoded by DNA resides in the order of base pairs

  
• The decoding of DNA takes place in two separate steps: transcription and translation
• RNA, like DNA, consists of nucleotides linked in a chain, but RNA molecules are usually single-stranded
• There are approximately three billion pairs of nucleotides in the chromosomes of a human cell, and every person has a unique sequence of nucleotides

  
• Scientists believe that every human has about 30,000 genes per cell, and a mutation or imperfection in any one of these genes can result in a disease, physical disability or shortened life span
• A complete set of genes (genetic material i.e., the DNA) in a cell is called a genome

  
• Genetic vectors

  Vehicles for delivering foreign DNA from a donor into a recipient host (microbial, plant, animal) cell
• Genetic vectors

  • They can replicate autonomously and typically include features to facilitate the manipulation of DNA as well as a genetic marker for their selective recognition
  • They act as vehicles to transfer genetic material from one cell to the other for different purposes like multiplying, expressing, or isolation
  • They are used as a tool in molecular cloning procedures to introduce the desired DNA insert into a host cell

  
• Recombinant DNA


  The DNA insert that is transmitted by a vector

  
• Components of a genetic vector

  • An insert, also known as a transgene, that carries the recombinant DNA
  • A larger sequence called the backbone of the vector responsible for the structure of the vector

  
• Even though vectors are usually DNA sequences, viruses and other particles can also function as vectors in processes like transduction
• Essential general characteristics/features and properties of vectors

  • They should be capable of replicating autonomously
  • The size of an ideal vector should be small enough for it to be incorporated into the host genome
  • They should be easy to isolate and purify
  • They should have certain components that facilitate the process of determining whether the host cell has received the vector (e.g. a marker gene like an antibiotic resistance gene)
  • They should have unique restriction endonuclease enzyme recognition sites (multiple cloning sites or polylinker) to enable the insertion of foreign DNA
  • The introduction of vectors into the host cell should be easy
  • They should be capable of integrating themselves or the recombinant DNA into the genome of the host cell
  • The introduction of recombinant DNA into the vector shouldn't affect the replication cycle of the vector
  • They should have a relaxed control of replication to obtain multiple copies

  
• Types of vectors


  • Cloning vectors
  • Expression vectors
  • Shuttle vectors
  • Viral vectors
  • Cosmid vectors
  • Artificial chromosome vectors

  
• Cloning vectors


  Vectors that are capable of replicating autonomously and are used for the replication of recombinant DNA within the host cell

  
• Characteristics of cloning vectors


  • They must be easily introduced into the host bacterium
  • They must be able to replicate in the host bacterium, preferably with a high copy number
  • They should contain unique sites for the action of restriction endonucleases
  • They should encode a means for selecting or screening host cells that contain a copy of the vector

  
• Plasmid-cloning vectors


  Plasmids that are widely used as vectors in bacteria and yeasts, derived from naturally occurring extra-chromosomal circular DNA that can replicate autonomously within the host cell

  
• Important features of plasmid vectors

  • Their small size facilitates separation from host genomic DNA and enables them to carry rDNA up to 20 kb
  • They can replicate by inserting themselves into the bacterial chromosomes (episomes)
  • They can be classified as conjugative (self-transmissible) or non-conjugative
  • They can have low copy number (<10) or high copy number (>20)

  
• Main types of naturally occurring plasmids


  • Fertility/F plasmids
  • Resistant or 'R' plasmids
  • Col plasmids
  • Degradative plasmids
  • Virulence plasmids

  
• High copy number


  Preferred in bacterial system for higher production
• Low copy plasmids


  Automatic choice in transformation experiments

  
• Main types of naturally occurring plasmids

  • Fertility/F plasmids
  • Resistant or 'R' plasmids
  • Col plasmids
  • Degradative plasmids
  • Virulence plasmids

  
• Fertility/F plasmids


  • Carry tra genes which has the ability to promote conjugal transfer of plasmid

  
• Resistant or 'R' plasmids

  • Carry genes conferring on the host bacterium resistance to one or more antibacterial agents, such as chloramphenicol, ampicillin and mercury

  
• Col plasmids

  • Code for colicins-proteins that kills other bacteria; e.g. ColE1 of E.Coli

  
• Degradative plasmids


  • Allow the host bacterium to metabolize unusual molecules such as toluene and salicylic acid