METHOD IN GENE CLONING

PROPOSAL

H4T30

INTRODUCTION
Gene cloning is one of the derivatives of the genetic engineering. Genetic engineering is the ability to take a specific gene from one cell and place it to another cell where it is expressed. It is also the direct manipulation of genes for practical purposes. New DNA made by combining different DNA pieces is known as the recombinant DNA. A clone is an exact copy of an organism, organ, single cell, organelle or macromolecule. Cell lines for medical research are derived from a single cell allowed to replicate millions of times, producing masses of identical clones.

Gene cloning is the act of making copies of a single gene. Amplified genes are useful in many areas of research and for medical applications such as gene therapy. Selective amplification of genes depends on our ability to perform the following essential procedures.

There are several methods in gene cloning that is isolation, splicing, insertion, transformation and screening. In isolation, two different sources are selected. One from a plasmid DNA bacterial (as a vector) and another one are from DNA that contain gene of interest such as the gene that coding for human insulin. To "clone a gene," a DNA fragment containing the gene of interest is isolated from chromosomal DNA using restriction enzymes and then united with a plasmid that has been cut with the same restriction enzymes. When the fragment of chromosomal DNA is joined with its cloning vector in the lab, it is called a "recombinant DNA molecule." Following introduction into suitable host cells, the recombinant DNA can then be reproduced along with the host cell DNA

Next step is cut or cleave the DNA donor and plasmid DNA bacterial using same restriction enzyme. The restriction enzyme recognizes a specific sequence of DNA bases and cuts the DNA at or close to that site. This specific sequences of bases occurs within the DNA of the bacterium but is protected from the action of the restriction enzyme by modification of one of the bases within the sequence. Foreign DNA is not similarly protected so that when it enters the cell it is rapidly degraded by the restriction enzyme.

The plasmids used in genetic engineering are relatives of the naturally occurring plasmids found in microorganisms. Generally they have been mutated and tailored with enzymes in order to have specific desired properties. Through genetic engineering (as an advantage over conventional genetic techniques) one can transfer a single specific gene between organisms.

The donor DNA carrying the gene of interest is cut with a restriction enzyme to yield fragments of various sizes, one of which bears the desired gene. A suitable plasmid cut with the same enzyme, is mixed with the donor DNA and the two are joined by an enzyme, DNA ligase, to give a series of hybrid plasmids.

The hybrid plasmid DNA is used to transform the host cell and the cells are plated out onto agar. The ratio of the amount of DNA to cells is adjusted in such a way that each cell takes up only one DNA molecule. Thus each colony which grows represents a different piece of the DNA donor carried on a plasmid. One of these carries the gene of interest and can be recognized by the characteristics it confers on the cell. This gene is then said to have been cloned.

This is the basis of genetic engineering. This method can be applied to DNA from any organisms and the host can be a bacterium, yeast, any other fungus or plant or animal cell. Genetic engineering has made significant contributions to industrial microbiology and in understanding the possible mechanisms of adaptability in microorganisms.

In brief, gene cloning involves (i) isolation and fragmentation of the source DNA and incorporation of the fragments obtained into a cloning vector (segment of DNA used for replication of foreign DNA fragments), with the use of restriction endonuclease to cut and ligase to rejoin DNA molecules, (ii) incorporation of the genetically transformed DNA into a recipient organism that can replicate the cloning vector, (iii) detection of the newly transformed cells containing DNA and isolation of a pure culture there from and (iv) growth of culture of cells containing the cloned DNA fragment.

A cloning vector must be able to replicate autonomously in a suitable host. Plasmids are used as carriers of unrelated DNA for genetic engineering, and the enzymes used for splicing foreign DNA into the plasmid carriers are those used in normal recombination and replication of DNA. However, at the same time there are dangers also associated with this technology. There are possibilities of creating new and uncontrollable pathogens from tame microbes like E. coli and yeast.

Picture 1: The methods in gene cloning.

Recombinant DNA technology is important for learning about other related technologies, such as gene therapy, genetic engineering of organisms, and sequencing genomes. Gene therapy can be used to treat certain genetic conditions by introducing virus vectors that carry corrected copies of faulty genes into the cells of a host organism. Genes from different organisms that improve taste and nutritional value or provide resistance to particular types of disease can be used to genetically engineer food crops. Besides that, gene cloning also is important for medical field. Today, production of insulin by using bacteria such as E coli helps in diabetes. This technique also used for the production of transgenic organism to get the desirable characteristics. For example, in transgenic plants, it can resist the herbicides and pests. In transgenic animal, the production of plasminogen activator, that is human growth hormone in cows milk, helps most people who care about their children growth. As we know, the grass in Labuan Matriculation College (KML) is infertile due to the hot climate in Labuan. The grass is not very suitable in such condition. So, what should we do to improve the quality of the grass? This is where we can apply the method in gene cloning. Our researcher had found that the Bermuda grass, which is one of the field grasses, is a suitable grass to combine with KML grass. This grass has an attractive color and also suitable to live in a hot climate. By combining the genes from both grasses, we can produce a gene of more attractive and high quality grass.

OBJECTIVES

There are three objectives to achieve in our project: To study the method in gene cloning. Gene cloning is the act of making exact copies of a single gene. There are 5 methods that involve in gene cloning that is isolation, cutting or cleavage, insertion, transformation, amplification and transformation (ICITAS). To determine the benefits of gene cloning in daily life. Gene cloning also has benefits especially in agriculture, medical field and genetic engineering. For example, the production of genetically modified organism (GMO) in genetic engineering. To find out whether gene cloning can be apply to improve the quality of grass in KML. The grass in KML is considered infertile due to hot climate in Labuan. The application of gene cloning can be applied to the grass by combining its gene with Bermuda grasss gene which is more resistance towards hot climate.

PROBLEM STATEMENT

Gene cloning is one of the most used technology to improve the quality of and organisms. However there are some problems that cause people not confidence with this technology. One of the reasons is, people did not know the correct methods in gene cloning. As we know, to produce an organism with desirable characteristics, we must used the proper methods, so that any unwanted organism that may harmful the others organisms, can be avoided. The next problem is, people are 100% unsure about the benefit of applying this technology in their daily life. Most of students studying this technology only through the books without experience it practically. Therefore the student did not sure whether this technology surely can give benefit or not. Last but not least, if this technology wants to be applied by the student to improve the quality of the grass in KML by combining the Bermuda grass and the grass in KML, is it possible? As we know, a new combining organism cannot directly adapt to the new environment. Therefore if this grass combines with the grass in KML, do the new grass that produced can adapt normally?