The Cloning of a DNA Fragment, and a Southern Blot

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Southern blotting can either be used in the determination of small fragment of a single gene or a large DNA sequence such as part of the genome of an organism. Therefore southern blotting is an invaluable tool in DNA technology. This method was discovered in the 1970s by Edward Southern

in Edinburg University and since then it has found use in various areas of science including molecular biology and DNA technology.. Annealing of the probe with a DNA of known sequence makes the determination of probe sequence obvious to the complementary nature of the double stranded DNA¹.

Gene cloning also referred to as DNA cloning in the recombinant DNA technology is the procedure of creating multiple copies of fragments of isolated DNA by in vivo or in vitro methods or reproduction of fragments of DNA.DNA cloning can either be based on cells or achieved by using polymerase chain reaction(PCR).

In the approach based on cells both the replicating molecule or the biological vehicle known as the vector and the foreign DNA fragment are cut using the same restriction enzyme(s ) to produce compatible cohesive (sticky) or blunt ends on the DNA molecules, then the foreign DNA fragment is permanently joined to the DNA of the vector using an enzyme known as DNA ligase which catalyzes the formation of a phosphodiester bond between the two DNA chains thus producing a chimera or a recombinant DNA molecule.

The replicating molecule is meant to carry the foreign DNA fragment into the host cell. Bacterial plasmids are commonly used as cloning vectors in most laboratory experiments².

DNA cloning involves the following steps;

DNA recombination-This involves the identification and isolation of the DNA fragment containing the gene of interest from the chromosomal DNA using restriction enzymes or by using the polymerase chain reaction(PCR),gel electrophoresis and sonication of DNA. The fragment of DNA isolated must be joined to a replicating molecule or vector which acts as a vehicle that transports the DNA into the host cell. Both the isolated DNA fragment and the vector are cut using restriction enzymes at their restriction sites into sizeable fragments suitable for cloning. The desired DNA fragment is inserted into the cut ends of the vector and permanently linked using the enzyme DNA ligase thus forming a recombinant DNA molecule or chimera, but in some instances if processed under in vivo conditions, the enzyme terminal transferase may be added in order to avoid free sticky ends to rejoin instead of forming a chimera since it catalyzes the addition of tails of the nucleotide to the 3ends of the DNA chains³.
Transformation-This is whereby the recombinant DNA molecule enters the host cell (which is usually a bacterium) and proliferates. The recombinant plasmid molecule also contains color selection markers which show white/blue screening on a media of X-gal.
Selective amplification-Within the host bacterium the vector multiplies producing numerous identical copies not only of itself but also of the gene that it carries.. After a large number of cell divisions, a colony or a clone of identical host cells is produced.
Isolation of desired DNA clones-Culturing of transfected cells is done. The selectable antibiotic resistance markers are used as well as the color selection markers if present in the recombinant plasmid, though further confirmation is done using PCR, DNA sequencing and restriction fragment analysis.

Results

Strain	Enzymes	Probe L	Probe M	Probe N	Probe O
A198	EcoRI	5.0	7.0	6.8	6.9
	HindIII	5.1	5.0	5.2	5.2
	EcoRI + HindIII	0.7	4.0	4.2	4.2
X1	EcoRI	2.8	No band	7.2	7.4
	HindIII	7.0	No band	3.8	3.8
	EcoRI + HindIII	2.8	No band	3.6	3.7
X2	EcoRI	4.2	7.0	7.2	7.0
	HindIII	5.8	6.0	5.9	6.0
	EcoRI + HindIII	1.4	4.2	4.2	4.2
X3	EcoRI	4.2, 1.7	7.0	7.2	6.9
	HindIII	6.4 + 5.8	6.0	5.9	6.0
	EcoRI + HindIII	1.9 + 1.4	4.2	4.2	4.2

The isolated DNA was purified and placed on electrophoretic gel and the results were as shown above. Fragments were eluted in terms of their sizes. The specific DNA fragments were then blotted on a membrane appearing as show above.

The DNA was then denatured using sodium hydroxide to separate the strands to allow or annealing with the probe. The denatured DNA strands appeared as shown above. The probe DNA was then labeled with a radioactive marker.

Discussion

Purification of the DNA fragments is important to ensure the integrity of the results. In an experiment aimed at determination of a portion of a large DNA fragment it is important to eliminate the sequences that have not been affected by specific enzymes. Consequently, the presence of these fragments may affect the clarity and the precision of the results.

Conclusion

Human cloning

The aim of this paper is to critically analyze the controversial issue of human cloning. It will begin by defining what human cloning is, the history of human cloning and finally the pros and cons of this science as presented by those for and against it.

The genesis of cloning can be traced to 1950s when scientists started cloning frogs. Human cloning refers to the production or creation of a duplicate of a human tissue, human cell or human being. There are two kinds of human cloning and they include: reproductive cloning and therapeutic cloning.

Reproductive cloning is aimed at making a human being through cloning whereas therapeutic cloning is the employment of cells from adult individuals for medical purposes. This approach is sometimes referred to as a nuclear transfer. It is feared that the same approach may be employed to clone human beings.

These cells have the ability to differentiate into all body tissues and researchers are optimistic that ability to direct these cells to develop into desired implants, which enable the treatment of many diseases. Presently the main source of these cells is discarded embryos resulting from fertility complications. Were it not for the ethical and other controversial issues surrounding human cloning, it would have provided a continuous supply of these for stem research.

Pros and cons of human cloning

The proponents of human cloning use scientific benefits that this new technology is able to give to justify their course of action. These benefits include, elimination of genetic disorders, enabling sterile couples to have children and possibility of creating donors to supply organs for transplant. Others have given the possibility to clone people who are already dead and those who are aging (Guardian, 3).⁴

The opponents of this technology and other related technologies do not overtly employ religious dogma in the justification of their opposition. Countries like America which are ruled or guided by a non-religious constitution should allow sectarian creeds to rule them. The opponents rely mainly on the belief and not on research findings.

According to the new times (2) if a research concludes that GMOs are safe for use may satisfy traditional Christians in the city of Kansas in the united states , but will not be able to Stockholms post-Christians (Christians in the modern world) from doubting Franken food (GMO).

Some key opponents of the human cloning research, like Leon Kass, claim that their business is not defending the belief of Judeo- Christians, but the dignity of mankind. Dr. Kass , one of the leading opponents of this technology in the united kingdom, claims that the book of genesis describes the special status of human beings.

According to him, this special status should be heeded because of the cosmological truth reflected by the message and not the basis of the biblical authority (New York Times, 2).

These articles by the New York Times also indicated that people from different places like California, had voted in support of the cloning using embryos and have even volunteered in support of it. According to the Christian institute Magazine (5), both therapeutic and reproductive cloning methods result in the creation of a new life.

Since life is considered sacred from the time of conception, hence the destruction of the embryos in therapeutic research for the purpose of obtaining cell for stem research is unjustifiable and amounts to killing. The prolife groups term this technological cannibalism.

when DNA was proposed by a famous pioneer in the field of criminology, Alec Jeffreys. He detailed various forensic techniques such as typing and DNA fingerprinting that was new and modern at the time. DNA sequencing developed into a profession and has assisted many law enforcers in the prosecution of offenders through the presenting evidence in a court of law.

Examples of where DNA can be obtained from include, hair, semen and skin samples. This information is stored in large databases and search queries regarding suspects can be found from them. These two fields are fast becoming the modes in which terror cell can be unraveled and terror plots against the state and its citizens unraveled.

Researchers and scholars have confirmed that there is a definite similarity in the chemical structure of deoxyribonucleic acid in every individual. The difference occurs in the series of base pairs. These differences can be detected through DNA fingerprinting. The process of fingerprinting can prevent terrorism through profiling of suspects and people placed in the most wanted list.

The state has a large database that samples frequent crime offenders such as rapists and other violent individuals. Terrorist are people who have a have a history of conducting other crimes. It is through the effort of DNA implemented by local law enforcement agencies, that we can positively identify potential terrorism and prevents any plots that they may be planning against the state.

These two instruments of forensics play a big role in the identification and arrest of suspects involved in activities that may lead to the death of innocent lives in any country. Digital forensics is used in collaboration DNA sampling to positively match individuals. There are times that DNA can be used to match a suspect who has committed electronic fraud. The fraud can be further supported through evidence acquired through Digital forensics.

Most scientists are against the use of human cloning for reproductive purposes. Among them is the British embryologist, Wilmut and Richard Gardner. Wilmut played a central role in the cloning of Dolly and Gardener was the chair of the royal society concern with human cloning. They both claimed that at present there are too many risks.

According to professor gardener, the experience with animals has given a clear indication of the possibility of producing an individual with extreme deformities never witnessed before. Many religious organizations are of the opinion that human cloning may result in ethical issues like emergence of embryo black market, designer babies and the formation of a genetic underclass.

According to Kitcher (61) human cloning should be considered as either good or bad on the basis of the ultimate goal for which it is being carried out for. For example if it is aimed at producing a human being with certain characteristics, then it is wrong. But if it is aimed for medical purposes then it is justified. He employs three examples to drive his point home.

One of these examples is the scenario of a dying child and the only treatment that could save his life is a kidney transplant with a ten year period. Unfortunately the kidneys from both parents are not compatible and it is rare to get individuals with compatible kidneys. Were it allowed to clone a brother, kidney transplant would have been very simple and very fast.

The second case is that of a widow, who has lost the husband through an accident, her daughter is in comma and she has reached menopause. The question whether this widow could be justified to take an egg from a surrogate mother and a cell from her daughter to create a clone of her dying through a surrogate mother is difficult to give a direct answer⁵.

To the present time no attempt has been undertaken to practically clone a human being due to ethical and moral issues surrounding this issue. From the view point of the researchers, if well used the human cloning will have enormous benefits mainly in the field of medicine but if misused the consequences are unimaginable.

From the perspective of the religious people and other opponents of the research practice, the approach is unacceptable and unjustifiable as it is seen as unethical under the religious believes.

Bibliography

McFarland, D. Preparation of pure cell cultures by cloning. Oxford Publishers, London, 2009.

McLaren, A. Cloning: pathways to a pluripotent future. Wiley Publishers, Perth, 2000.

Perrone, J. Government legislation designed to prevent cloning of human beings is on track. Oxford Publishers, London, 2005.

Tierney, J. Are Scientists Playing God? It Depends on Your Religion, CRC publishers, New York, 2007.

Kitcher P. There will never be another you. University of Illinois Press, Chicago,2007.

Footnotes

D. McFarland, Preparation of pure cell cultures by cloning. Oxford Publishers, London, p. 25.
P. Kitcher,. There will never be another you. University of Illinois Press, Chicago,2007, p. 56.
A. McLaren, Cloning: pathways to a pluripotent future. Wiley Publishers, Perth, 2000, P. 105.
D. McFarland, Preparation of pure cell cultures by cloning. Oxford Publishers, London, p. 25.
J. Tierney, Are Scientists Playing God? It Depends on Your Religion, CRC publishers, New York, 2007, p. 28.

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The Cloning of a DNA Fragment, and a Southern Blot

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