Cosmid Vectors, YAC and BAC Expression Vectors
- 1. COSMID VECTORS, YAC’S AND BAC’S EXPRESSION VECTORS By:CharthaGaglani
- 3. INTRODUCTION • Cosmid vectors are hybrid vectors derived from plasmids which contain cos site of phage lambda. • Vector- In molecular cloning, a vector is a DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and/or expressed. • Cos site- "Cos" is the abbreviation of "cohesive end site". This is a speciality of the lambda phage which has to be linearized to fit into the phages head, but circularizes in the host cell. • Cosmid= cos site + plasmid. • They are about 45 kb.
- 4. structure • Cosmids are essentially plasmids that contain a minimum of 250bp of ˄ DNA, which includes the following sequences from ˄ genome: 1. The cos site( the sequence yielding cohesive ends ) 2. Sequences needed for binding of and cleavage by terminase so that under appropriate conditions they are packaged in vitro into empty ˄ phage particles. • A typical cosmid has (1) Replication origin (2) Unique restriction sites (3) A selectable markers from a plasmid.
- 5. • The cosmid vectors are opened by the appropriate restriction enzyme at a unique site, are then mixed with DNA inserts prepared by using the same enzyme and annealed. • Among the several types of products, long cancatemers are present, which are the appropriate precursors of packaging in ˄ particles. • This procedure selects the long DNA inserts since for packaging the distance between two cos sites must be between 38 and 52 kb. • The DNA fragments used for cloning are usually produced by partial digestion with a restriction enzyme. • This is because complete digestion almost always produces fragments that are too small for cloning in a cosmid. • Cosmids can accommodate upto 40 kb long DNA inserts.
- 6. • The typical features of cosmids are as follows: 1. They can be used to clone DNA inserts of upto 40 kb. 2. They can be packaged into ˄ particles, which infect host cells; this is many-fold more efficient than plasmid transformation. 3. Selection for recombinant DNA is based on the procedure applicable to the plasmid making up the cosmid. 4. Finally, these vectors are amplified and maintained in the same manner as the contributing plasmid.
- 7. DRAWBACKS OF COSMID • Cosmids are particularly attractive for construction of genomic libraries of eukaryotes since they cam be used for cloning large fragments. • This approach is very sensitive to the exact ratio of DNA insert-to- vector DNA because vector-to-vector ligation can occur. • Recombination cosmids having duplicated vector sequences are unstable during cloning.
- 9. YEAST ARTIFICIAL CHROMOSOME • Yeast artificial chromosome are genetically engineered chromosome derived from DNA of the yeast. • It is human-engineered DNA molecule used to clone DNA sequences in yeast cells. • They are the products of a recombinant DNA cloning methodology to isolate and propagate very large segments of DNA in a yeast host. • The amount of DNA that can be cloned into YAC is on average from 200 to 500 kb. • However as much as 1 MB can be cloned into a YAC.
- 10. STRUCTURE OF YAC • The YAC cloning vectors consist of two copies of a yeast telomeric sequence, a yeast centromere, a yeast ars (an autonomously replicating sequence where DNA replication begins) and appropriate selectable markers.
- 11. WORKING PRINCIPLE • The principle is similar to that chromosome to that for plasmids or cosmids. • The experimenter introduces some typical elements that are necessary for correct replication. • In the case of YAC’s, the replication origins are the centromeres and telomeres of yeast chromosomes, which must be inserted into the DNA being cloned.
- 12. ADVANTAGES OF YAC • YAC provide the largest insert capacity of any cloning system. • Yeast expression vectors, such as YAC’s, YIPs( Yeast Integrating Plasmids), and YEP’s(Yeast Episomal Plasmids), have advantageous over BAC’S. They can be used to express eukaryotic proteins that require post-translational modification. • A major advantage of cloning in yeast, a eukaryote, is that many sequences that are unable, underrepresented or absent when cloned into prokaryotic systems, remain stable and intact in YAC clones.
- 13. LIMITATIONS OF YAC • YAC clones frequently contain deletions, rearrangements or noncontiguous pieces of cloned DNA. As a result, each YAC clone must be carefully analyzed to be sure that no rearrangements of the DNA have occurred. • The efficiency of cloning is low. • YAC have been found to be less stable than BAC.
- 15. BAC • A Bacterial Artificial Chromosome is a DNA construct, based on a functional fertility plasmid, using for transforming and cloning in bacteria, usually E-coli. • They are capable of carrying approximately upto 300 kbp of inserted DNA sequence. • BAC was created because of the problems faced with YAC e.g. recombination between copies of inserts and more particularly, deletions in the DNA inserts. • The first BAC vector was pBAC108L.
- 16. COMMON GENE COMPONENTS IN BAC • RepE- for plasmid replication and regulation of copy number. • ParA and ParB- for partitioning F plasmid DNA to daughter cells during division and ensures stable maintenance of BAC. • Selectable marker- for antibiotic resistance; some BAC’s also have lacZ at the cloning site for blue/white selection. • T7 & Sp6- phage promotors for transcription of inserted genes. • OrlS- origin of replication.
- 17. APPLICATION OF BAC • BAC’s are being greatly used in modelling genetic diseases in order to study their effects in the experimentation on transgenic mice. • BAC have been used to study the neurological diseases such as Alzheimer’s disease or in case of Down’s syndrome. • The genome of several large DNA viruses and RNA viruses have been cloned by BAC’s. These constructs are referred to as ‘infectious clones’.
- 18. DRAWBACKS • The chief disadvantage of BAC vectors is the somewhat laborious construction of BAC libraries as in vitro manipulations, such as, restriction digestion, etc. have to be performed in agarose plugs to avoid shearing of the large DNA molecules.
- 19. REFERENCES • Biotechnology Expanding Horizons by B.D.Singh • www.microbenotes.com