Cosmids vector
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1. A cosmid is a type of plasmid vector that contains sequences from bacteriophage lambda, specifically the cos sites. This allows DNA fragments up to 45kb to be packaged into phage particles and transduced into bacteria. 2. Cosmids are developed by combining features of plasmid and phage vectors. They can accept large DNA inserts through packaging and transduction while also replicating stably inside bacteria like plasmids. 3. Cloning with cosmids involves inserting DNA fragments into the cosmid, ligating to form concatemers, in vitro packaging into phage particles, and transducing the particles into bacteria where the cosmids replicate as plasmids.
Cosmids vector
- 1. Dr. Manikandan Kathirvel M.Sc., Ph.D., (NET) Assistant Professor, Department of Life Sciences, Kristu Jayanti College (Autonomous), Reaccredited with "A++" Grade by NAAC K. Narayanapura, Kothanur (PO) Bengaluru Cosmids
- 2. Cosmid Definition A cosmid is a plasmid that contain phage sequence that allows the vector to be packaged and transmitted to bacteria like phage vector. Or • A cosmid is a type of hybrid plasmid that contains a Lambda phage cos sequence. Cosmids (cos sites + plasmid = cosmids) DNA sequences are originally from the lambda phage.
- 3. Cosmids 1. Cosmids are plasmids, medium sized cloning vectors. 2. Cosmid vector are developed by combining the features of plasmid vector and bacteriophage vector. 3. The first cosmid vector was described by Collins in 1978. 4. They are capable of incorporating the bacteriophage λ DNA segment. This DNA segment contains cohesive terminal sites (cos sites). 5. Cos sites are necessary for efficient packaging of DNA into λ phage particles. 6. Large DNA fragments of size varying from 35 to 45 kb can be cloned. 7. They are also packaged into λ. This permits the foreign DNA fragment or genes to be introduced into the host organism by the mechanism of transduction. Advantages of using cosmids as vectors: i. They have high transformation efficiency and are capable of producing a large number of clones from a small quantity of DNA. ii. Also, they can carry up to 45 kb of insert compared to 25 kb carried by plasmids and λ. Disadvantages of using cosmids as vectors: i. Cosmids cannot accept more than 50 kb of the insert.
- 4. Properties of cosmid vector Contains Features of plasmid • Origin of replication • Multiple cloning site • Selectable marker Contains Features of lambda phage • Only cohesive site or cos site region. • A linear DNA molecule, has a 12 base long , single stranded complementary overhang at both ends. Which emerge during the packaging process into phage particle through splitting of cos site. •• A cosmid vector may have one or two cos site. Uses: •• Cosmid vector are used in construction of genomic libraries. •• Cosmid vector have cloning capacity up to 45 kbp.
- 5. Cloning of foreign DNA in cosmid vector involves the following steps: 1. The cosmid is opened at its unique restriction site and new DNA fragments inserted -Ligation of foreign DNA. 2. Making a concatemeric DNA. 3. In vitro packaging to introduce the DNA into phage head to form the matured phage particle. 4. Introduction of the cloned DNA into E. coli by transduction. 5. After their entry into host cell the cosmid are maintained as plasmid. A concatemer is a long continuous DNA molecule that contains multiple copies of the same DNA sequence linked in series. Examples of Cosmid Vector
- 6. Cosmid pJB8 Cloning of foreign DNA in cosmid vector involves the following steps: 1. The cosmid is opened at its unique restriction site and new DNA fragments inserted -Ligation of foreign DNA. 2. Making a concatemeric DNA. 3. In vitro packaging to introduce the DNA into phage head to form the matured phage particle. 4. Introduction of the cloned DNA into E. coli by transduction. 5. After their entry into host cell the cosmid are maintained as plasmid.
- 7. A cloning experiment with a cosmid is carried out as follows. • The cosmid is opened at its unique restriction site and new DNA fragments inserted. • These fragments are usually produced by partial digestion with a restriction endonuclease, as total digestion almost invariably results in fragments that are too small to be cloned with a cosmid. • Ligation is carried out so that catenanes are formed. Providing the inserted DNA is the right size, in vitro packaging cleaves the cossites and places the recombinant cosmids in mature phage particles. • These lambda phage are then used to infect an E. coli culture, though of course plaques are not formed. • Instead, infected cells are plated onto a selective medium and antibiotic-resistant colonies are grown. All colonies are recombinants, as non-recombinant linear cosmids are too small to be packaged into lambda heads.