5. ARTIFICIAL CHROMOSOMES
Genetic manipulation is much easier in prokaryotes and lowers eukaryotes but when it comes for higher eukaryotes and multicellular plants and animals, it becomes a daunting task. The basic aim of genetic engineering is to express a gene in plant and animal cells in tissue culture and study is expression or production of useful proteins secondly it deals with the development of transgenic organism by altering the genetic makeup of the organism. But genetic changes or introduction of a new gene must be done at initial stages of development so that recombinant sequence can spread throughout the organism.
The sequencing of the one also requires vectors. But for sequencing, the carrying capacity of the vector should be large. Therefore, vectors were developed for the sequencing purpose. Bacterial artificial chromosome or BAC was used for the human gene sequencing project. This vector is based on F plasmid. F plasmid is larger than the standard plasmid, therefore it can integrate larger fragments. It also contains the origin of replication of bacterial chromosome and has the capacity to carry around 300 kb long DNA fragment.
1. BAC (Bacterial artificial chromosomes )
2. PAC (P1 derived artificial chromosomes)
3. YAC (Yeast artificial chromosomes)
4. MAC (Mammalian artificial chromosomes)
BAC (Bacterial artificial chromosomes)
BACs are F-plasmid based technology that enables integration of insert up to a few hundred kilobases into the host genome. BAC used in human genome project to sequence the genome of the human. A short piece of the organism's DNA is amplified as an insert in BACs and then sequenced.
i. Cloning of large DNA size of 100 - 300 kb in E. coli.
ii. BAC contains ori of F-plasmid (fertility). F-plasmid controls the replication and maintains the low copy number.
iii. Transfer to conjugation prosses between F+ to F–
BAC contain elements
i. RepE for plasmid for replication and regulation of copy number.
ii. Par A and Par B Partition of F-plasmid during cell division for even distribution of cloned gene.
iii. Selectable marker Also has Lac Z the cloning site for blue and white screening and Lac Z gene contains sac-B gene if negative selection marker because the sac-B gene produces levansucrase enzyme convert the sucrose to levan. Levan is toxic for bacteria.
iv. T7 and Sp6 Phage promoter transcription gene
Exp. : pBAC 108L, pBeloBAC11, pECBAC1
PAC (P1 derived artificial chromosomes)
Developed by Sternberg and co-worker in 1990. Drive by phage P1 virus. Phage P1 is a temperate bacteriophage which has been used for genetic analysis of E. coli because it mediates generalized transduction process. It can carry 100-200 kb size of desired DNA. It contains Kanamycin resistant marker and high copy number can be induced by exploitation of P1 lytic replicon. It is used in the sequence of the genome and from the genetic library of the mouse, human and Drosophila
Exp. : pESAC13
YAC (Yeast artificial chromosomes)
YAC is a human-engineered DNA molecule used to clone DNA sequence in yeast cells. It was the first time developed by Murray and Szostak in 1983. It can carry the DNA of size 1-2 Mb. Yeast artificial chromosome plasmid contains both ori of E. coli and ARS of yeast. Thus it is used as a shuttle vector. It is indicated by pYAC.
The element of YAC
i. It contains Yeast origin of replication (ARS)
ii. It also contains the selection marker of both the host.
iii. It also contains Centromere and Telomere region of yeast chromosome
MAC (Mammalian artificial chromosomes)
MAC is similar to YAC because of presence centromeric and telomeric and origin of replication.
They also undergo autonomous replication in segregation in mammalian cells.
In one method, telomere-directed fragmentation of natural chromosomes is used. For example, using human chromosome number 21 for making human artificial chromosome. Another example involves de novo assembly of cloned centromeric and replication origins in vitro.
Mammalian DNA has a higher degree of repetition and larger centromere and telomere regions.
- TOOL AND TECHNOLOGY
- HYBRID PLASMID / PHAGE VECTORS
- ARTIFICIAL CHROMOSOMES
- SHUTTLE VECTORS
- ENZYMES USED FOR RECOMBINANT DNA TECHNOLOGY
- DNA LIBRARY
- FLUROSCENT ACTIVATED CELL SORTER
- DNA MICROARRAY OR GENE CHIP OR BIO CHIP
- ANTIBODY GENERATION
- RADIOIMMUNOASSAY (RIA)
- ELISA OR ENZYME LINKED IMMUNOSORBANT ASSAY
- POLYMERASE CHAIN REACTION
- TYPE OF HYDROLYSIS PROBE
- X-RAY DIFFRACTION
- NMR (NUCLEAR MAGNETIC RESONANCE)
- CIRCULAR DICHROISM
- DNA SEQUENCING
- TRANSGENIC ANIMALS
- CRE–LOX P RECOMBINANT SYSTEM
- GENE THERAPY
- TRANSGENIC PLANTS
- PLANT TISSUE CULTURE (PTC)
- MICRO PROPAGATION
- ARTIFICIAL SEEDS
- PRACTICAL APPLICATIONS OF PLANT TISSUE CULTURE
- ANIMAL CELL CULTURE