Gene cloning is a new breakthrough in getting a gene that is needed for human life. Gene cloning includes a series of processes to isolate specific DNA fragments from an organism’s genome, determine DNA sequences, form Recombinant DNA molecules, and target gene expression in host cells. Genes or molecular clones, meaning groups of copies of identical genes that are replicated from one gene to gene another.
What is DNA or Gene Cloning?
Determination of DNA sequences aims to ensure that the DNA fragments we isolate are the target genes according to our will. The target gene that we get is then we clone in a vector (plasmid, phage or cosmid) through recombinant DNA technology which then forms recombinant DNA molecules. The recombinant DNA produced is then transformed into the host cell (usually bacterial cells, such as the E. coli strain) to be produced more. The target genes in the host cell if expressed will produce the gene product that we want.
Birth of cloned genes
About a century ago, Gregor Mandel formulated rules explaining the inheritance of biological properties. The characteristics of an organism that can be inherited are governed by a factor called a gene, which is a particle that is inside a cell, precisely on a chromosome. Genes form the basis for the development of genetic research including gene mapping, analyzing the position of genes on chromosomes. A study which is a revolution in medern biology is after the emergence of DNA recombination or genetic engineering technology which is the core process of gene cloning, which is a procedure for obtaining replicas that can be the same from a single cell or organism (Anonimus b)
The Purpose of Cloning Genes
a. Determine the sequence of nucleotide bases that make up the gene
b. Analyze or identify the sequence of nucleotide bases controlling the gene
c. Study the function of RNA / protein / enzymes that are encoded by the gene
d. Identify mutations that occur in gene defects that cause disease default
e. Engineering organisms for certain purposes, for example producing insulin, resistance to pests, etc.
DNA source (gene) to be cloned
1) Chromosome DNA
2) cDNA (complementary DNA) which is synthesized using mRNA as a mold (template)
3) DNA produced from propagation using PCR (Polymerase Chain Reaction)
Materials / Tools for cloning
a. Endonuclease enzyme restriction
b. Ligase enzyme
c. Vectors
d. Host
e. The method for inserting DNA into the host cell
Vector (vehicle) for cloning
Vector is a DNA molecule that functions as a vehicle or vehicle that will carry a DNA fragment into the host cell and allow replication and expression of these foreign DNA fragments. Vectors that can be used in prokaryotic host cells, especially E. coli, are plasmids, bacteriophages, cosmids, and fasmids. Meanwhile, YACs and YEps vectors can be used on yeast. Plasmid Ti, baculovirus, SV40, and retroviruses are vectors that can be used in high-level eukaryotic cells.
A vehicle is needed to insert a piece of DNA into the cell so that the DNA can be stored and propagated in the cell
Plasmids
Plasmids are genetic elements outside the chromosome (extrachromosomal) which carry out automatic replication in bacterial cells. Naturally plasm contains genes for antibiotic resistance, disease virulence factors and gene transfer proteins. The DNA is circular and stranded and plasmids support the genes needed for both the replication of plasmides and other functions carried by plasmides. DNA is not chromosome (extrachromosomal DNA) that is naturally possessed by a body. The form of circular double threads (double strands DNA, dsDNA). Artificial plasmids can be made by adding other pieces of DNA. Plasmids can be modified to be able to carry other pieces of DNA into cells if they have:
a. Originator of replication
b. Easy-to-select markers (eg antibiotic resistance genes)
c. Site for cloning (pieces of DNA that have a sequence of nucleotide bases subject to restriction enzymes but not in the replicator area or marker
Vector in the form of Plasmids
1. Having the origin of replication of the intended host, allowing replication
independently of the host genome.
2. Has a selective marker: Facilitates the selection of plasmid carrier cells inserted by foreign DNA
3. Resistance to multiple antibiotics
4. Blue-white screening
5. Has many cloning sites (multiple cloning sites, MCS)
6. Easy to isolate from host cells.
Two of the plasmide vehicles used for molecular cloning are Psc101 and pBR322. Both types are nonconjugal types and each E. coli cell is an example of episodes. Pbr322 in its extraction is derived from pSC101, the plasmid contains resistance genes for tetracyclic antibiotics and ampicillin. One problem with plasmid vectors is that the insertion of DNA that can be contained is small (<5-8 kb). While the other vectors are more than 10-50 kb.
Bacteriophage (l phage)
Bacteriophage is a virus that infects bacteria used if we are referring to different species (eg lambda phages and T4). Phages have an ikosahedral capsid which is roughly spherical and is attached to a short tail and some are long, contractile and non contractile. The genetic material of most phages is double-stranded DNA (dsDNA).
The lambda phage region contains genes that are involved in and are initiated and maintained by lyogenic conditions and can be replaced by insertion of foreign DNA if the size is 20 kb and still allows the phage DNA to be packed in the phage head. Its lytic or lysogenic life cycle can be used as a cloning vector on bacterial host cells. There are several types of bacteriophages commonly used as cloning vectors, namely bacteriophages 1 and bacteriophages M13
Cosmid Vector
Combined plasmid vector properties and useful properties of l cos site (omitted in vector l). Cosmid can be propagated in bacterial cells or purified by packing invitro into phages. Kosmid is a vector constructed by combining the board of DNA l with a plasmid. Its ability to carry DNA fragments from 32 to 47 kb makes the cosmid more beneficial than fag and plasmid.
Fasmid
Besides the cosmids, there are synthetic vector groups which are a combination of plasmids and phages. This vector, called a fasmid, carries a segment of DNA l containing the att place. That place is used by DNA l to integrate with host cell chromosomes in fascisogenic.
Plasmid Ti Agrobacterium tumefaciens
Plant cells do not contain natural plasmids which can be used as cloning vectors. However, there is a bacterium, namely Agrobacterium tumefaciens, which carries a plasmid measuring 200 kb and is called plasmid Ti (tumor inducing or causing tumors). The bacteria A. tumefaciens can infect dicotyledonous plants such as tomatoes and tobacco and monocot plants, especially rice. When an infection takes place a certain part of the plasmid Ti, called T-DNA, will be integrated into the chromosome DNA of the plant, resulting in uncontrolled growth of plant cells. As a result, a tumor or crown gall will form. Recombinant Ti plasmid with a target gene inserted in the T-DNA region can integrate the gene into plant DNA. This target gene will then be expressed using a plant DNA system.
In practice, the size of a large plasmid Ti is very difficult to manipulate. However, it turns out that if the T-DNA part is separated from other parts of Ti plasmid, integration with plant DNA can still occur as long as the T-DNA and other parts are still in one cell of A. tumefaciens. Thus, manipulation or insertion of foreign DNA fragments is only carried out on T-DNA in the same way as is done on E. coli plasmids. Furthermore, the recombinant T-DNA plasmid produced was transformed into A. tumefaciens cells carrying the Ti plasmid without the T-DNA part. The next improvement procedure is the removal of tumor-forming genes found in T-DNA.
Baculovirus is a virus that infects insects. One of the important proteins encoded by the genome of this virus is the polihedrin, which will accumulate in very large quantities in the nuclei of infected insect cells because the gene has a very active promoter. This promoter can be used to spur overexpression of foreign genes cloned into the bacilovirus genome so that very large quantities of protein products will be obtained in the culture of infected insect cells.
Vector Cloning of Mammals
The vector for cloning mammalian cells is also constructed on the basis of the viral genome. One of them that has long been known is SV40, which infects various mammalian species. The SV40 genome is only 5.2 kb in length. This genome has difficulties in packaging so that the use of SV40 to transfer large sized fragments is limited. Retroviruses have a genome in the form of a single-stranded RNA that is transcribed back into a double-stranded DNA after infection. This DNA is then integrated seamlessly into the host mammalian cell genome so that the retrovirus has been used as a vector in gene therapy. Retroviruses have several strong promoters.
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