Bacteriophage Vectors
- Bacteriophages are simply phages are viruses that attack bacterial cells by injecting their DNA into these cells.
- The injected DNA is selectively rep licated and expressed in the host bacterial cell resulting in a number of phages which burst out of the cell (lytic pathway) and reinfect neighbouring cells.
- This ability to transfer DNA from the phage genome to specific bacterial hosts during the process of bacterial infection gave scientists the idea that specially designed phage vectors would be useful tools for gene cloning experiments . Several bacteriophages are being used as cloning vectors but most commonly used are lambda phage and M13 phage.
- It has a double - stranded, linear DNA genome of 48, 502bp, in which the 12 bases at each end are unpaired but complementary.
- These ends are, therefore, sticky or cohesive and are referred to as the cos sites (cohesive end sites).
- These sites are important for packaging DNA into phage heads.
- The lambda genome remains linear in the phage head, but within E.coli cells the two cohesive ends join to form a circular molecule necessary for replication.
- These vectors allow cloning of DNA fragments upto 23 Kb length (=Kilobase is a unit designating the length of a nucleic acid sequence , e.g. , 1 Kb = 1000 nucleotide long base sequence).
- It is filamentous phage which infects E.coli having F - pili.
- Its genome is a single stranded,circular DNA of 6407 bp.
- Foreign DNA can be inserted into it without disrupting any of the essential genes.
- After the M13 phage DNA enters the bacterial cell, it is converted to a double-stranded molecule known as the replicative form or RF, which replicates until there are 100 copies in the cell and single- stranded copies of the genome are produced and extruded from the cell as M13 particles.
- The major advantages of developing vectors based on M13 are that its genome is less than 10Kb length.
- The RF can be purified and manipulated exactly like a plasmid.
- In addition, genes cloned in M13 based vectors can be obtained in the form of single - stranded DNA
- Bac teriophage vectors can be used for large DNA fragments and.
- These can easily be detected at the time of cloning experiments.
Some other Cloning Vectors
- Cosmid (= cos + plasmid) Vectors
- The term cosmid is a combination of two words.
- COS + MID. COS is taken from COS site of Lambda phage and MID is taken from plasmid DNA.
- Cosmid was developed for the first time by Collins and Hons (1978).
- Cosmids can be used to clone DNA fragments upto 45 kb in length.
- Bacterial Artificial Chromosome (BAC) Vectors
- These are vectors based on natural , extra chromosomal plasmid of E. coli
- These vectors can accommodate upto 300-350 kb of foreign DNA and are also being used in genome sequencing project.
- Yeast Artificial Chromosome (YAC) Vectors
- These are used to clone DNA fragments of more than 1 Mb - Megabase pairs (100 bp) in size, therefore, they have been exploited extensively in mapping the large genomes, e.g. , in the Human Genome Project.
- These vectors contain the telomeric sequence, the centromere and the autonomously replicating sequence from yeast chromosomes.
- They also contain restric tion enzyme sites and genes which act as selectable markers in yeast.
- Phagemid Vectors
- Phagemid is a composite structure made of bacteriophage and plasmid . They are used for carrying larger DNA sequences.
- Animal and Plant Viral Vectors
- A vector based on Simian Virus 40 (SV40) was used in the first cloning experiment involving mammalian cells in 1979.
- Since 1979, a number of vectors based on other types of viruses like Adenovirus and Papillomavirus have been used to clone genes in mammals.
- At present, retroviral vectors are the most commonly used vectors for cloning genes in mammalian cells.
- In case of plants, plant viruses like Cauliflower Mosaic Viruses, Tobacco Mosaic Viruses and Gemini Viruses were used but with limited success.
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