PROCEDURES AND MECHANISM CLONING

The etimologis, said the cloning comes from the Greek word "clone" means the discount that is used to propagate plants. While the terminologis, cloning is the process of making a large number of cells or molecules are identical with the whole cell or molecular origin.

In the field of genetic cloning is a segment of DNA replication without going through the process of sexual (Rekombinasi DNA). This process opens new opportunities in breakthrough technology to change the function and behavior of living beings in accordance with the wishes and needs of men.

Theoretically cloning procedures and mechanisms of living beings through the four (4) stage of the DNA fragment isolation, DNA fragment inserts into the vectors, transformation and selection of cloning.

1) Isolation DNA fragments
Isolation of specific DNA fragments can be done with the method of PCR (polymerase chain reaction) technique, namely amplikasi DNA fragments that are specific in vitro. In general, the DNA used for PCR is the total DNA extracted from the genom of the cell and does not require a high level of purity. DNA sequence that specifically amplication determined by the primary-primary composed of nukleotida. (1)

Materials needed for the process of PCR is a DNA sequence that contains a series that will be (DNA duplication), namely primary, and DNA polimerase mixture of four different deoksiribonukleotida-trifosfat (dATP, dCTP, dGTP and dTTP), and MgCl2.

2) Insertion DNA fragments into vectors
The process of interpolation or continuation molecular DNA fragments with the DNA molecule called a vector ligasi. Ligasi usually occurs between the ends of the truss with the truss hidroksil phosphate. Ligasi between DNA fragments that have the end of the sticky (cohesive ends), which is much more complementary efesien compared with the end of the blunt (blunt ends). Ligasi efficiency is also influenced by the single on the tip of the deoksiadenosin. Ligasi efficiency can be improved, if the DNA fragment that has a single deoksiadenosin at the end of the meeting with the vectors that have timidin on the edge.

3) DNA Transformation
Transformation is the process of transfer of donor DNA molecules from the environment outside the cell. Cloning vector which is the bearer of genes akan dikloning ditransformasi to cells in the wet nurse. Transformation can be either natural or artificial. In the process of natural transformation, the DNA strand ganda shaped and has a string of basa specific membrane proteins into the bacteria through bacterial cell membrane terhidrolisis. In the artificial transformation, bacterial cells into cells made competent by force so that the shroud is permeabel bacterial cells and allows DNA to cells with ties and into sitoplasma, and interact with the genom of bacterial cells. (3)

Competent cells are cells that have a wet nurse competency Entry vectors for cloning. Treatment competent to enter the cells can be made using the method of heat shock (heat shock) or free electric shocks (electroporation method). (2)

4) Selection of cloning
Penyeleksian bacterial colony cloning to obtain the desired way with X-gal or cutting with a restriction enzyme. Selection with X-gal can be used to identify plasmid rekombinan with komplementasi. While cutting with restriction enzyme can be used to select plasmid cloning rekombinan results. Results are dielektroforesis cutting the ribbon and insert DNA fragments that separate from the cloning vector band. (4)

In the application level, the process sequence cloning can be done with the follow the following steps:
1. Preparing for the stem cells, the cells will start to grow into different body cells. Cells obtained from this organism that would dikloning.
2. Stem cells taken selnya nucleus containing the genetic information and then separated from the cells.
3. Preparing egg cells, a cell taken from the adult organism and essentially separated.
4. Nucleus cells from stem cells diimplimentasikan to egg cells.
5. Triggered so that the egg cell division and growth occurs. After a split embryo.
6. Embryo cells that continue to split (blastosis) began to separate themselves and ready to be implemented in the womb.
7. Fetus in the womb grow a fetus with the genetic code exactly the same as the stem cell donor. (5)

Reading List
1. Simbolon, H. Biology Jilid 3. Jakarta, Erlangga Publishers, 1994).
2. Sambrook et al. Molecular Cloning: A Laboratory Manual. New York, CSHL Press, 1989).
3. Stainer et al. The Microbial World. New Jersey, Prentice-Hall, 1986).
4. Seno D.S and E.D. Purwakusumah. Production by L-iisin Genetic Engineering Strain Results. Research Report on FMIPA UI 1995 (not published).
5. Quoted from http://strategis.ic.gc.ca/SSG/tc00026e.html.