Polymerase Chain Reaction (PCR)

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Polymerase Chain reaction (PCR)

Principle: PCR is a process which involves taking a DNA template and amplifying distinct regions of it in vitro. To conduct PCR you need the DNA sample, DNA primers( two because one is forward and one is a reverse primer), Deoxynucleoside triphosphate bases(dNTP), DNA (Taq) polymerase, a buffer and some cations (mg2+). The reaction is carried out in a thermal cycler which fluctuates the temperature to allow progression of the amplification.

Procedure:
Initially the double helix is separated by breaking the hydrogen bonds using heat, leaving the bases exposed. This is called denaturation and it occurs at approximately 960C.
The temperature of the system is lowered (50-650C) allowing the primers to bind non-covalently
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gel electrophoresis, sequencing cloning (into a plasmid) etc.
Identifying pathogens

Limitations:
There is a possibility that a mutation can occur which will also be replicated during PCR.
The DNA sequence for the target region should be known prior to PCR so it is not useful for regions in which the sequence is unknown.

Ethical issues
Some people are of the opinion that their DNA can be acquired by others easily. Also many believe that genetic engineering is unethical as manipulating DNA is unnatural.

DNA FINGERPRINTING
Principle: Everyone has a unique DNA fingerprint so this method is used to distinguish and identify people. A similar process which uses PCR as opposed to restriction enzymes is DNA profiling or STR analysis (uses micro satellites instead of mini satellites as they have less base pairs).

Procedure:
DNA is extracted e.g.. from blood, saliva, hair etc.
Restriction enzymes cut the DNA resulting in varied lengths which are separated according to size. This is done by gel electrophoresis: the DNA is loaded into the wells which act like a filter and an electrical current is passed through the gel separating the
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In addition, it is a costly procedure so there is limited access to this treatment.

Ethical issues:
Offspring of germ line gene therapy have not provided their consent for the procedure. This process can also be used to enhance offspring i.e.. their genetic and physical features, leaving the patient with unfair advantages in certain situations.

STEM CELL THERAPY
Principles:
Stem cells can be used to generate new cells that can develop into functional cells/tissues/organs and it is therefore introduced in the patients body for this purpose.

Procedure:
First the patient is exposed to radiation. This is known as the conditioning treatment which is different depending on the ailment of the patient. The stem cells are inserted through the patients central venous allowing the cells to enter the bloodstream directly. The cells then multiply inside the patients body. However, this form of therapy comes with numerous uncomfortable side effects. An example of the use of this therapy is for bone marrow

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