Human and other mammalian genomes are very large, therefore, vectors are needed to clone large DNA fragments. Many vectors have been prepared that aid in the characterisation and expression of large genes or gene complexes. These vectors exhibit varied cloning capacity (table).
Table Maximum DNA Insert Possible with Different Cloning Vectors
|λ phage||E. coil||5-15kb|
|λ cosmids||E. coil||35-45kb|
|P1 phage||E. coil||70-100kb|
|BACs||E. coil||≤ 300kb|
Bacterial Artificial Chromosome (BAC) Vectors
Vectors used for DNA cloning in bacterial cells have high to moderate number of replicons. Greater number of DNA clones is formed by these vectors depending on the replication efficiency of its replicon.
Disadvantage : regarding these vectors is structural instability of the inserts, causing deletion or rearrangement of a segment of cloned DNA. This is observed in DNA inserts of eukaryotic origin where repetitive sequences frequently occur. Hence, cloning and maintaining large DNA in bacterial cells is not easy.
This limitation is however overcome by vectors with low copy number of replicons. An example is a vector prepared from the E. coli fertility plasmid or the F-factor. It has two genes (par A and B) to sustain the copy number for F-factor at 1-2 per E. coli cell.
This vector accepts large foreign DNA fragment (>300 kb). Thus, the recombinant vector can be transferred into bacterial cells through electroporation method (in which cells are exposed to high voltage to relax selective permeability of the plasma membrane). Such vectors provide a low yield of recombinant DNA from the host cells (figure).
Bacteriophage P1 and P1-derived Artificial Chromosome (PAC) Vectors
Certain bacteriophages carry large genomes and hold larger DNA fragments. An example is bacteriophage P1 that packages linear DNA (110-115kb) in P1 protein coat. The P1 cloning vectors are prepared from components of P1 phase provided in a circular plasmid.
The plasmid vector when cleaved generates two vector arms that allow in vitro ligation and packaging of ∼100 kb of foreign DNA into a P1 protein coat. This recombinant Pl phage is then adsorbed on a suitable host, and then the recombinant PI DNA is injected into the cell, circularised and amplified (figure). Employing bacteriophage T4 in vitro packaging system with P1 vectors aid in the recovery of inserts (of around 122 kb). The features of P1 and F-factor systems when assembled together develop P1-derived Artificial Chromosome (PAC) cloning system.
Yeast Artificial Chromosome (YAC) Vectors
Yeast artificial chromosomes are advantageous in cloning of bacterial cells as it can clone very large DNA fragments.
A YAC has the following features:
- At each end of YAC a yeast telomere (TEL) is present.
- In order to facilitate regulated segregation during mitosis, a yeast centromere sequence (CEN) is present.
- Each arm has a selectable marker to detect the YAC in yeast (for example, TRPI and URAIII for tryptophan and uracil independence in trpI and uraIII mutant strains respectively).
- To allow the replication of vector in a yeast cell, an origin of replication ARS (Autonomously Replicating Sequence) is present.
- For inserting foreign DNA, restriction sites unique to the YAC are present (figure). To have a successful cloning, a restriction enzyme is used to cut a circular YAC in multiple cloning sites and another restriction enzyme is used to cut between the two TELs; thus, the left and right arms are formed. The DNA with high molecular weight is ligated to the two arms.
The transfection of so produced YAC into yeast cells cannot be achieved directly. So yeast cells are treated appropriately to eradicate the external cell wall. The yeast spheroplasts obtained accept the exogenous fragments, and are yet osmotically unstable and thus embedded in agar.
|Read More Topics|
|Study of cloning vectors|
|Insertion of target DNA into a vector|
|Introduction for recombinant DNA technology|