Gene cloning is a process in which multiple copies of genetic material, such as genes, are created. To do this, scientists utilize vectors – specific strands of DNA that can be inserted into cells and transport the desired genes with them. Through gene cloning, researchers have been able to gain valuable insight into genetics and further our understanding of biology.
Plasmids
- Plasmid is circular molecules of DNA that independently existence in the bacterial cell
- Plasmids almost always carry one or more genes, and often these genes are responsible for a useful characteristic displayed by the host bacterium such as Antibiotic resistance
- when plasmids used for gene cloning Size and copy number of a plasmid are particularly important as far as cloning is concerned
- Copy number – number of molecules of an individual plasmid in a single bacterial cell.
Characters of the ideal plasmids.
- contains single sites for many restriction enzymes
- Low molecular weight
- Gives higher copy number
- Gives higher copy
- Can accept larger pieces of DNA
- Easier to handle (less susceptible to breakage)
E. coli plasmids – pBR322
Advantages of pBR322
- The plasmid is much smaller than a natural plasmid – More resistant to damage by shearing, and
- Increases the efficiency of uptake by bacteria
- It carries two sets of antibiotic resistance genes – ampicillin or tetracycline useful in identification of transformed cells
- There are single recognition sites for a number of restriction enzymes at various points around the plasmid.
E. coli plasmids – pUC8: A Lac selection plasmid
- There is an antibiotic resistance gene for tetracycline
- Origin of replication for E. coli
- The cluster of cloning sites are contained polylinker
- lacZ′ gene
- This gene may be switched on by adding the inducer IPTG (isopropyl-b-D thiogalactopyranoside)
In presence of IPGT lacZ gene get activated, and produce β-galactosidase enzyme, it can hydrolyse the colourless X-gal into blue colour insoluble materials.
when the lacZ gene is disturbed by the foreign fragment of DNA, X-gal does not hydrolysis therefore recombinant pUC plasmids appears as white colour.
Non-recombinant pUC plasmid will be blue since its gene is not disrupted
pUC9, has the same polylinker as pUC8, but inserted into the lacZ gene in the opposite orientation.
Therefore be used to clone a DNA fragment in both forward and backward directions.
E. coli plasmids – pGEM3Z
it carries the ampR gene and lacZ′gene very similar to a pUC vector
pGEM3Z is a cloning vector that contains two short DNA sequences (promoters), on either side of the Multiple Cloning Site (MCS). These promoters enable efficient transcription of the inserted DNA fragment and can be used for in vitro transcription and expression of cloned genes. This makes pGEM3Z an ideal choice for downstream applications such as gene expression studies.
E. coli plasmids – Phage cloning vectors
Bacteriophages, or viruses that infect bacteria, are used to create phage cloning vectors. (Lambda λ Phage cloning vectors)
- The Lambda DNA vector can be modified by removing certain segments and replacing them with a stuffer fragment containing marker genes. stuffer fragment helps maintain the correct size of the vector and allowing for selection of colonies that contain it.
- When the Lambda DNA is intact, beta-galactosidase reacts with X-gal and the colonies turn blue.
- When inserting foreign DNA into a vector and replacing the stuffer region, the lacZ gene can be disrupted, preventing the formation of beta-galactosidase since there is no colour.
E. coli plasmids – Cosmid cloning vectors
Cosmids are synthetic cloning vectors that are based on the combination of elements from plasmids and bacteriophages.
- Cosmids combine essential elements of a plasmid and Lambda systems (bacteriophages).
- Cosmids are extracted from bacteria and mixed with restriction endonucleases.
- These are mixed with foreign DNA that has been cleaved with the same endonuclease.
- Recombinant cosmid is injected into the bacterial cell.
Many investigative can be carried out to find out about on clone DNA
- Functions of genes
- Analyses of promoters to understand how a gene is regulated
- Comparison of genes and gene expression
- Express a gene under in vitro conditions
Downstream techniques done by using Cloned DNA
- DNA sequencing
- PCR
- Preparation of gene libraries
DNA sequencing – once the sequence of the gene is known, it can be used in sequence similarity search, phylogenetic analysis, predict function, predict the protein structure, predict the protein functions….
Gene libraries
- Genomic library – contains total chromosomal DNA of organism. Used if the aim is control the protein production for particular gene or analysis the gene architecture.
- cDNA library – contains only mRNA from particular cell, tissue at a specific point of time. Used when production of new oe modified proteins.
