In the era of modernization is now a lot of technologies that help someone to communicate or to create something. In the field of biology, the technology used to improve or benefit in life is referred to as biotechnology.
One of the biotechnology that until now still exist and often cause controves one of them is cloning in humans. In short, cloning has an understanding to produce new individuals who have the same genes.
Eve was the first human being to be the result of this cloning biotechnology. Even so, it turns out Eve is still alive today and certainly in healthy condition. Then how does the cloning process happen? Here is a description of the cloning process in humans.
1. Isolation of DNA fragments
The initial process before heading to the core process of cloning in humans is the isolation stage of DNA fragments. The purpose of isolation of this DNA fragment is to separate between good and bad DNA fragments, which will be either used for grafting or paired with other good DNA. This isolation of DNA fragments commonly uses the PCR (polymerase chainreaction) technique. Note that in this process at least requires primary DNA, DNA polymeration and DNA which is a mixture of 4 deoxyribonucleotides-triphosphates comprising dATP, dCTP, dGTP and dTTP as well as MgCl2.
The stages of PCR are as follows:
Phase I - The initial stage is DNA denaturation which means that DNA must be upgraded first to a temperature of approximately 94 degrees Celsius within 1 minute for a separation between 2 duplex DNAs.
Phase II - The next stage is the attachment of primary oligonucleotides or hybridization. Where at this stage the temperature is pre-arranged to 50-60 degrees Celsius within 1 minute in order for the hybridation to be attached to the DNA.
Stage III - The third stage is the extension of the DNA chain by reheating it to 72°C in 1 minute. Keep in mind that at this stage at least repeated as many as 25-35 times.
It can be further elaborated that in each duplex DNA present in the first stage each will form two strands of the child's DNA, thus producing 4 DNA strands. Then in the second stage, the 4 DNA strands will produce 8 strands of DNA. And in the later stages of the second stage will be formed polinukleotida with a very limited length between 1 primary DNA with the other. These fragments of DNA fragments are then referred to as amplicons. The DNA fragment will be multiplied even more and will result in a very complete DNA that is the result of the initial DNA sample.
2. Insertion of DNA fragments
The process of cloning in humans the second stage is the insertion of DNA fragments or ligation is a stage of merging between DNA fragments with each other so that later will be created recombinant DNA. In general, ligation of DNA fractures by way of in vitro has 3 ways, namely:
- The first way is to use a DNA ligase enzyme derived from bacteria.
- The second way is to use DNA ligase derived from E-Coli bacteria that have been infected by T4 bacteriophage or commonly called enzyme T4 ligase.
- The third way is to give the deoxynucleotidil enzymes so that the DNA fragments can be synthesized well.
It should be remembered that in the first way it is only used to meligase the sticky end of the DNA, whereas in the second way it can be used in DNA that has a sticky or dull end. On the other hand, in the third way is used to synthesize a single homopolymeric DNA strand 3 '. Another thing to note is that about the optimum temperature of DNA. The optimum temperature of the ligase DNA will range from 37 degrees Celsius, where at that temperature hydrogen will usually form a bond by itself which can be found at the sticky end of DNA, but this makes it unstable and can cause damage to DNA because of the temperature which is too high in place of the bond. Therefore, this ligase process will usually take place at a temperature of 4-15 degrees Celsius within 24 hours.
In order for ligation to work efficiently, it can use high-concentration DNA fragmentation of more than 100 μg / ml or by using alkaline phosphatase enzymes in order to remove phosphate groups starting from the 5 'ends of the DNA molecule. Next can use the addition of linker molecules, adapter molecules for synthesizing a single homopolymeric strand 3 '. Thus the recombination DNA can be formed by the 5 'GAATTC 3' - 3'CTTAAG 5 'arrangement.
3. DNA transformation
DNA transformation is a process of transferring DNA molecules that come from donors that are outside the scope of the cell. In this transformation stage can be done with natural or artificial. If in a natural process, the DNA will form a double chain and has a very specific base chain of membrane proteins that will eventually enter the body of bacteria by passing through a hydrolyzed bacterial cell membrane. While in the artificial transformation, the bacterial cell will be converted into a competent cell and will enter the bacterial cell envelope with a permeable property, most likely the DNA will bind to the cell and enter the cytoplasm and react with the bacterial genome. What needs to be known here is that the process of entering the competent cell is done by means of shock.
The general stage of DNA transformation is as follows:
- At first there will be introduction and attachment of dsDNA from the outside to the cell wall of bacteria.
- The dsDNA then enters the wall and cell membrane through the protein channel.
- dsDNA will experience degradation in one part of the strand and leaving ssDNA which will go into the cytoplasm.
- In this cytoplasm ssDNA will later be a template that will be used in the process of synthesis of new dsDNA which will be integrated in one chromosome and become a new plasmid.
4. Selection of Cloning
The final stage of the cloning process in humans is the selection of cloning. The purpose of this selection is to prevent undesired bacterial colonies from being cut off with the help of retrictive enzymes. Such selection typically uses an X-gal system to identify recombinant plasmid parts with their complementation. The result of the cut will be electroforesized so that the DNA fragment band can be seen and can be separated.
The cloning stages after going through several stages above are as follows:
- Stem Cells - Initially prepare a stem cell that is a cell that will grow into various parts of body cells first.
- Cell Core Preparation - After the stem cells are ready, then the cell nucleus is taken in which there is genetic info.
- Egg Cells - While the stem cell nucleus is separated, prepare the egg to be used and the point must also be separated.
- Merging - The next step is to implement between the stem cell nucleus and the egg cell.
- Egg Encouragement - After both nuclei of the cell meet, the egg must be triggered in order to divide and become an embryo.
- Embryonic Cleavage - The embryo will eventually continue to divide (blastosis), then slowly will separate itself and is ready to be implemented into the woman's uterus.
- Embryo Implementation - The last stage is to implement embryo cells into the womb to develop into a fetus that has a genetic code as predetermined.
Thus the discussion about the cloning process in humans that until now its existence still cause pros and cons with some circles. Maybe this biotechnology can give a positive side to some people, but some people think this is very contrary to religious norms or other norms.
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