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1. Plasmids Plasmids are closed circular, double-stranded, extrachromosomal DNA molecules which occur naturally in bacteria, yeast, and some higher eukaryotic cells, and exist in a parasitic or symbiotic relationship with their host cell (Lodish et al. , 2000) The main application of plasmids is as cloning vectors in gene cloning. In gene cloning, a fragment of DNA, containing the gene to be cloned is inserted into a circular molecule called the “vector” to produce recombinant DNA molecule. Plasmids are one of the most commonly used “vectors” for this purpose.
They transport the gene into a host cell, such as a bacterium, which is said to be transformed with the recombinant molecule. Here, these plasmid vectors multiply, producing numerous identical copies of itself and the gene that it carries. Like the host-cell chromosomal DNA, plasmid DNA is duplicated before every cell division; they replicate independent of the chromosomal DNA. During cell division, at least one copy of the plasmid DNA is segregated to each daughter cell, assuring continued propagation of the plasmid through successive generations of the host cell.
Thus after a number of successive cell divisions, various identical host cells are produced, thus, the gene carried by the plasmid is “cloned” (Brown, 2006; Lodish et al. , 2000). Furthermore, the transformed bacterial host cells may have the ability to express the gene, thus producing proteins encoded by the gene included in the recombinant plasmid. This application can be used for producing proteins in large quantities which can be purified further, as is done for the production of recombinant insulin.
Also, plasmids could be used in gene therapy, for delivery of therapeutic gene of interest into human host cells, without causing cell injury, oncogenic mutations and an immune response (Daugherty, 2007; Lipps, 2008). Various features of plasmid that make it a suitable cloning vector are: 1. Plasmids contain an “Origin of replication” sequence (ori), which allows the plasmid to replicate/ multiply inside the cell independent of the bacterial chromosome, and thus maintains the plasmid DNA in the cell population as the host organism grows and divides, allowing a large number of copies of the plasmid to be made (Brown, 2006). . Plasmids have antibiotic resistance genes which allow the plasmid to survive in normally toxic concentrations of antibiotics such as ampicillin, and this feature can be used as a selectable marker to conveniently select for “transformed” bacterial cells containing a particular plasmid from a culture (Brown, 2006). 3. Plasmids have a region that contains several unique restriction endonuclease sites in a short stretch of the DNA, known as a “polylinker” region or Multiple Cloning Site (MCS).
This provides a variety of choices of sites available for insertion of DNA fragments during recombinant DNA production, because it provides a greater choice for restriction enzymes that can be used in cloning (Brown, 2006; Nicholl, 2008). 4. Plasmids have a packaging capacity (that is they can take foreign DNA) upto 10 kb in length which is usually very suitable for cloning. Moreover, a cloning vector should be ideally less than 10kb in size, as large molecules tend to break down during purification.
Plasmids usually range from about 1. 0kb to over 250 kb for the largest plasmids. Thus, plasmids, especially small ones, are very relevant and useful for cloning purposes, as far as size is concerned (Brown, 2006; Lipps, 2008). 5. Plasmids are inherited stably, without integration and they have reduced toxicity, features that make plasmids more advantageous than viral vectors in gene therapy (Stoll & Calos, 2002). They can be transferred into many cell types, thus increasing their usefulness (Brown, 2006). . Also it is easy to find plasmids with a high copy number, which is useful because multiple copies of the cloning vector need to be present in a cell so that large quantities of the recombinant DNA molecule can be obtained. Relaxed plasmids (which replicate without bacterial chromosome replication), have a high copy number (Brown, 2006). 2. Cosmids Cosmid libraries are used for cloning genes with large introns and for sequencing larger chunks of the genome.
Their main application is in the construction of genomic libraries, where the large insert capacity is an advantage in minimising the number of clones required for a complete representative gene library. Apart from this, they also enhance the likelihood that a cloned gene will be present as an intact copy in a cosmid library (because the gene is very long) (Brown, 2006; Lodish et al. , 2000). Cosmid vectors have also been used for rapid genomic walking, gene transfer, restriction mapping and determining the functional and structural organization of complex eukaryotic genomes (Wahl et al. 1987). Features of cosmids: Cosmids are artificially constructed plasmids of about 40-50kb which combine the high efficiency of lambda transfection with the ability to clone large pieces of DNA into plasmid vectors (Brown, 2006; Lodge, Lund & Minchin, 2007). Thus cosmids are essentially plasmids which have had a small piece of bacteriophage lambda DNA including the cos site (that is, containing the single stranded cohesive/sticky endings of ? phage which are complimentary to each other) cloned into them (Lodge, Lund & Minchin, 2007).
Their plasmid features enable the vectors to have an origin of replication, to replicate at a high copy number, a selectable marker such as an antibiotic resistant gene which can be amplified in the presence of antibiotics, and which provides a means of selection for recombinant cells and a restriction site which can be cleaved with restriction endonucleases. Their lambda phage features, that is, the presence of the cos sequences in them allows them to undergo in vitro packaging for efficient recovery of recombinant vectors.
They have an origin of replication and selectable markers which, together with the lambda cos sites, account for a very small size, about 6-12 kb of DNA. Thus these cosmids can accommodate 40-45 kb of exogenous/foreign DNA, (with a lower limit of 32 kb and an upper limit of about 47 kb) before reaching the upper DNA carrying capacity of the phage particle which is about 52 kb, thus allowing them to carry much larger amount of DNA than conventional lambda vectors (Brown, 2006; Primrose & Twyman, 2006).
While the normal lambda infection process is used to introduce the cosmid into an E. coli cell, once inside the cell the cosmid behaves as a very large plasmid. When plated on selective media, colonies of antibiotic resistant, recombinant cosmids are grown, and no plaques are formed (because cosmids lack all the lambda genes that are involved in plaque formation) (Brown, 2006). 3. M13 phage Recombinant filamentous bacteriophages like M13 vectors are used to provide single strand copies of DNA fragments cloned in other vectors, which is used to separate the strands of DNA segments.
The single stranded DNA fragments are used mainly as templates for site-directed mutagenesis, sequencing of DNA fragments by the dideoxy chain termination method, construction of subtractive cDNA libraries, and synthesis of strand-specific probes especially in mRNA selection. Specialised M13 vectors can also be used for display of foreign peptides and proteins on the surface of the bacteriophage particles (Sambrook & Russell, 2001). Features of M13 phage: The features that make it suitable are that firstly, the M13 phage genome is 6. 4kb (less than 10kb) in size, which is suitable for a cloning vector.
However, since it has a smaller size than the phage lambda, it can accommodate fewer genes than lambda vector. In the M13 phage, 0. 5kb of genome is non-essential and thus 1. 5kb of foreign DNA can be inserted. Since its genome is circular, single stranded DNA, one of the most important features of genes cloned with an M13 based vector is that they can be obtained in the form of single stranded DNA easily, which are useful for the purpose of providing single strand copies of DNA fragments (these ssDNA can be used as probes for mRNA selection) and several techniques like DNA sequencing and in vitro mutagenesis mentioned above.
Furthermore, the double stranded replicative form (RF) of the M13 genome behaves like a plasmid, and can be treated as such for experimental purposes, and the copy numbers of the RF (dsDNA) can exceed 100 per cell, which is high enough as a cloning vector. It is also easy to recover the single stranded molecules because the mature M13 phage particles are released into the extracellular medium (the bacterial host cells are not lysed) and then the ssDNA can be recovered from the extracellular medium.
The concentrations of RF DNA and of progeny phage extruded into the culture fluid are very high (usually 1011 ??? 1012 phage particles/ml) so it is relatively easy to isolate large quantities of both dsDNA and ssDNA versions of the phage genome. Moreover, it is easily prepared from a culture of infected E. coli cells and can be reintroduced by transfection. The filamentous phage capsid is little more than a coating of proteins and, in contrast to phage lambda, does not present a barrier to the accommodation of extra DNA.
Last but not the least, it provides an easy way for selecting recombinants from non-recombinants, if the lacZ gene is incorporated in an intergenic region of the phage genome; this provides selection on the basis of formation of blue/white colour plaques (Brown, 2006; Primrose & Twyman, 2006; Nicholl, 2008). 4. Phage Lambda Bacteriophage ? as a cloning vector, is used to clone large DNA fragments (larger than those cloned in plasmids) and it has many uses from subcloning of genomic DNA sequences initially cloned to vectors with larger capacity (eg.
BACs) to construction of complex cDNA or genomic DNA libraries. ? phage is not suitable for cloning small DNA fragments, as the selection of recombinants is usually determined by size (Brown, 2006; Sambrook & Russell, 2001). Features of phage lambda: The linear form of ? phage is a double stranded molecule with two free ends; however, the advantage of using it as a cloning vector is that at each end of the molecule there is a short 12-nucleotide stretch in which the DNA is single-stranded and the two strands are complementary to each other.
These ends, called cohesive ends, or “cos” sites can base pair with each other allowing the linear molecule to circularise, which is first of all, necessary for its insertion into the bacterial genome. In lambda phage genome, related genes are clustered and this is an important feature as a cloning vector. This is because the regions of the ? genome that are ‘non-essential’ for cloning (stuffer fragments) can be deleted from the central region of the genome and then replaced with upto 18-20 kb of foreign DNA (much more than the capacity of plasmid), without affecting the ability of the phage to grow lytically.
Since the stuffer fragment (from positions 20 to 35 on the genome) contains genes required for the lysogenic functions of the ? phage, removing it abolishes it lysogenic function, and retains only the lytic function which is desirable for a vector as it ensures a high copy number and induction is not needed before plaques are formed. Since about 15 kb of DNA can be removed, DNA fragments about 15-20 kb long can be inserted into the genome to reach the upper limit of about 52 kb that is packageable into the phage head.
It is very advantageous to have large pieces of DNA cloned in one vector because some genes are very big and it is obviously better to have them all together in one vector (Brown, 2006; Primrose & Twyman, 2006; Sambrook & Russell, 2001; Sofer, 1991). Moreover, when the prophage excises from the host genome and a number of new ? DNA molecules are produced by the rolling circle mechanism of replication, a catenane is formed consisting of a series of linear ? genomes joined together at cos sites. Now, these sequences of cos sites are recognised by endonucleases, which cleaves the catenane at these sites to produce individual ? enomes, which are then further packaged into phage head structures. However, an advantage of this process important for cloning is that the cleavage and packaging processes recognise only the “cos” sites, and thus, even if new genes are inserted (replaced) in the internal regions of ? genome without altering the length of the phage genome, there is no effect on these events and the overall process still proceeds efficiently carrying the foreign genes while being packaged into progeny phages (Brown, 2006).
A main advantage of bacteriophage lambda is that it can be reconstituted in a test tube by simply mixing phage DNA with a mixture of phage proteins, that is, through the process of “in vitro” packaging. Furthermore, through genetic engineering, now numerous derivatives of lambda have been constructed that contain only one or two sites for a variety of restriction enzymes, which is a very suitable property as a cloning vector (Sofer, 1991). Also, the fact that only a particular size of DNA molecule will be effectively packaged into the phage head provides an easy way of distinguishing recombinant from non-recombinant molecules.
This is because when the stuffer fragment is removed, the spacing between the cos sites becomes too short for successful packaging. Thus, if no replacement for it is made, then it will not produce infective phage particles. In addition, some lambda strains have a stuffer fragment that carries the beta-galactosidase gene. When it is removed or when foreign DNA is cloned within the gene, beta-galactosidase activity may be abolished. The accompanying loss of activity may be used to select recombinant clones (Brown, 2006; Sofer, 1991).
The major advantage of the lambda (? ) phage vector is its high transformation efficiency, which is about 100 times more efficient than the plasmid vector. The ? genome is 48. 5 kb of linear dsDNA and can carry upto 20 kb of foreign DNA, as mentioned earlier. 5. Artificial chromosomes Artificial chromosomes are simple vectors that mimic the natural construction of chromosomal DNA, the telomeres, a centromere, and an origin of replication in addition to features designed for ease of use, such as, selectable markers (Nicholl, 2008).
The main use of artificial chromosomes is that they can be used to clone DNA fragments up to several hundred kb in length and are especially useful in the construction of eukaryotic gene libraries (Brown, 2006). BAC libraries are essential elements in doing large-scale genome research such as genome sequencing, large-scale physical maps, and gene cloning. BAC libraries allow direct genome walking instead of sequencing directly from genomes, which is a very difficult proposition and allows study of gene structure using the entire genome.
BAC libraries can be used for isolation of gene clusters to scan for gene pathways and metabolic pathways in drug discovery. They have been used to prepare FISH probes (Fluorescence In Situ Hybridization) to analyze tissue samples to understand chromosomal rearrangements, amplifications, deletions and aneusomies. YAC libraries are used for cloning very large DNA fragments (of more than 1 Mb), and are useful for cloning large genes (such as the 250 kb cystic fibrosis gene) and for creating libraries of large overlapping clones, such as for individual chromosomes isolated from organisms (chromosomal libraries).
These have been used extensively for mapping genomes of complex organisms and studying gene expression (e. g. Homo sapiens) (Brown, 2006; Lodge, Lund & Minchin, 2007; Nicholl, 2008). Features: Artificial chromosomes contain a centromere, telomeric sequences, origins of replication and selectable markers, along with the gene to be cloned. Thus, they contain all the DNA sequences necessary for them to be replicated as a chromosome.
They rely on the presence of centromeric sequences, sequences that can initiate DNA replication, and telomeric sequences. When it is introduced into the cell, artificial chromosomes behave as normal chromosomes, replicating each time the yeast cell divides. They are very advantageous because they can accommodate a foreign DNA insert of up to 300 kb, which is much larger than even a cosmid. Artificial chromosomes are very stable, especially BACs which are most stable than even YACs.
Furthermore, bacterial clones and libraries (in BACs) grow very fast, are easier to prepare and handle. They also allow for many efficient screening by either hybridization or PCR based screening. An engineered BAC vetor is about 7. 4 kb in size including its origin of replication, cloning sites and selectable markers and can thus accommodate a very large amount of DNA, even though they have a low copy number (Brown, 2006; Lodge, Lund & Minchin, 2007; Nicholl, 2008; Sambrook & Russell, 2001).