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Describe How A Gene Encoding A Therapeutic Protein Could Be Cloned Into A Vector To Allow Expression

2054 words - 9 pages

A vector is a recombinant DNA carrier, all types have three generic properties; introduction to the host cell can be accomplished easily, each vector contains a replication origin enabling it to reproduce inside the cell and in order to determine which cells contain the vector a simple assay can be carried out, such as, growing the cells on agar plates. At present, there are many different types of vectors available for use the best expression system depends on the gene involved (Hartl, 2011). Examples of vectors include; Plasmids, Bacteriophages, Artificial chromosomes, bacteria, cell free systems and viruses (Klug et al., 2003). The fundamentals of molecular genetics is centred around the “Central Dogma”, this governs protein expression and demonstrates how genetic information in DNA is expressed in a polypeptide chain as show in Figure 1 below.
DNA does not code directly for proteins it does so through Ribose Nucleic Acid (RNA), a similar molecule to DNA but, the sugar it contains is called ribose, the molecule itself is single stranded and within the nucleotide base Thymine (T) is replaced with Uracil (U) which still binds to Adenine (A) in the same manner. There are three types of RNA, messenger RNA (mRNA), ribosomal RNA (rRNA) and transfer RNA (tRNA) as well as this, each gene includes a particular nucleotide sequence that initiates and stops transcription. Each of which are involved in the intermediary process of transcribing and translating DNA into a protein (Hartl, 2011).
With the use of recombinant DNA essential products can be produced on a large scale in a short period of time i.e. human insulin for type two diabetics. As well as this there are various agricultural advantages, such as, the growth rate of some animals can be genetically altered, crop plants may be able to gain resistance to certain conditions or diseases and the expression of protein is the primary goal for recombinant DNA technology. The aim of this essay is to explain in detail, the processes involved in cloning a specific protein.
In order to clone a protein in an efficient manner the gene for it must be transferred into a bacterial plasmid, this is known as subcloning and is mainly used in protein expression and to study the effects of a protein once it has entered a cell. There are five main steps to subclone a gene:
Obtaining Plasmid Maps and selecting restriction enzymes; The purpose of this is to be able to select enzymes for ligation. This enables enzymes to make a single cut but at a multiple cloning site and helps produce linear DNA. A plasmid map, such as Figure 2 below, also helps to determine which antibiotic the plasmid has resistance to.
Digestion of DNAs with restriction enzymes; this step can be divided into 3 smaller sub-steps the first of which is turning a circular plasmid linear. It is also known as using a restriction enzyme such as EcoRI or HindIII to perform the first enzyme cut. It is preferred to use two matching restriction enzymes as...

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