SCIENTIFIC PRINCIPLE ONE: GENETIC CLONING
Gene cloning is the process in which a gene of interest is located and copied (cloned) out of DNA extracted from an organism. When DNA is extracted from an organism, all of its genes are extracted at one time. This DNA contains thousands of different genes. The genetic engineer must find the one specific gene that encodes the specific protein of interest.
Since there is no way to locate a gene by visibly looking at all of the DNA, scientists make gene libraries to catalogue the organism's DNA. The gene the scientist is looking for is selected from this library.
It can be used for many different purposes, such as creating GM crops, finding a cure for disease or for gene therapy.There are two types of gene cloning: 'in vivo', which involves the use of restriction enzymes and ligases using vectors and cloning the fragments into host cells. The other type is 'in vitro' which is using the polymerase chain reaction (PCR) method to create copies of fragments of DNA.
For in vivo cloning a fragment of DNA, containing a single gene or a number of genes, is inserted into a vector that can be amplified within another host cell. A vector is a section of DNA that can incorporate another DNA fragment without losing the capacity for self-replication, and a vector containing an additional DNA fragment is known as a hybrid vector. If the fragment of DNA includes one or more genes the process is referred to as gene cloning.
Gene cloning is the process in which a gene of interest is located and copied (cloned) out of DNA extracted from an organism. When DNA is extracted from an organism, all of its genes are extracted at one time. This DNA contains thousands of different genes. The genetic engineer must find the one specific gene that encodes the specific protein of interest.
Since there is no way to locate a gene by visibly looking at all of the DNA, scientists make gene libraries to catalogue the organism's DNA. The gene the scientist is looking for is selected from this library.
It can be used for many different purposes, such as creating GM crops, finding a cure for disease or for gene therapy.There are two types of gene cloning: 'in vivo', which involves the use of restriction enzymes and ligases using vectors and cloning the fragments into host cells. The other type is 'in vitro' which is using the polymerase chain reaction (PCR) method to create copies of fragments of DNA.
For in vivo cloning a fragment of DNA, containing a single gene or a number of genes, is inserted into a vector that can be amplified within another host cell. A vector is a section of DNA that can incorporate another DNA fragment without losing the capacity for self-replication, and a vector containing an additional DNA fragment is known as a hybrid vector. If the fragment of DNA includes one or more genes the process is referred to as gene cloning.
The diagram below is an 'in vitro' method for making many copies of a specific section of DNA, without the need for vectors or host cells. The DNA to be copied – the template DNA – is mixed with forward and reverse primers complementary to the end of the template DNA, nucleotides, and a version of DNA polymerase. (This enzyme is stable under high temperatures.) The process involves the repetition of three steps:
Denaturation, which separates the two nucleotide strands of the DNA molecule.
Primer annealing, in which the primers bind to the single-stranded DNA.
Extension, in which nucleotides are added to the primers – in the 5' to 3' direction – to form a double-stranded copy of the target DNA.
Each cycle takes a few minutes, and repeated cycles can produce large amounts of a specific DNA sequence in a matter of hours rather than days.
However, this cloning method does require knowledge of some details about the nucleotide sequence to be copied, and the technique is very sensitive to small amounts of contamination.
Denaturation, which separates the two nucleotide strands of the DNA molecule.
Primer annealing, in which the primers bind to the single-stranded DNA.
Extension, in which nucleotides are added to the primers – in the 5' to 3' direction – to form a double-stranded copy of the target DNA.
Each cycle takes a few minutes, and repeated cycles can produce large amounts of a specific DNA sequence in a matter of hours rather than days.
However, this cloning method does require knowledge of some details about the nucleotide sequence to be copied, and the technique is very sensitive to small amounts of contamination.
SCIENTIFIC PRINCIPLE TWO: PROCESS AND VIRAL VECTORS USED IN SOMATIC GENE THERAPY
The process of Somatic Gene Therapy entails inserting normal genes into the cell/s of an individual with the genetic disease and, thus, eradicating the disease completely.
The diagram below outlines the simplest methods of getting genes into the person's cells using either viruses (which carry the human gene, in place of one of their own genes, into a cell) or liposomes (small molecules which can carry DNA into a cell).
In some cells, the gene/s are inserted into a chromosome in the nucleus.
The process of Somatic Gene Therapy entails inserting normal genes into the cell/s of an individual with the genetic disease and, thus, eradicating the disease completely.
The diagram below outlines the simplest methods of getting genes into the person's cells using either viruses (which carry the human gene, in place of one of their own genes, into a cell) or liposomes (small molecules which can carry DNA into a cell).
In some cells, the gene/s are inserted into a chromosome in the nucleus.
To reverse disease caused by genetic damage, researchers isolate normal DNA and package it into a vehicle known as a vector, which acts as a molecular delivery truck. Vectors composed of viral DNA sequences have been used successfully in human gene therapy trials. Doctors infect a target cell — usually from a tissue affected by the illness, such as liver or lung cells—with the vector. The vector unloads its DNA cargo, which then begins producing the proper proteins and restores the cell to normal. Problems can arise if the DNA is inserted into the wrong place in the genome. For example, in rare instances the DNA may be inserted into a regulatory gene, improperly turning it on or off, leading to cancer.
However, there are three major scientific hurdles that have to be overcome before somatic gene therapy is likely to work. The first is getting the human gene into the patient's cells (using viruses or liposomes).
The second obstacle is getting the gene into the right cells.
The final obstacle is making sure the gene is active, that is, switched on in the cell to produce the protein that the patient needs. This means it must be under the control of the sequence of DNA that is responsible for switching the gene on.
The results do not have to be perfect to produce benefits. In cystic fibrosis, animal tests have shown that if the normal gene can be transferred to only five per cent of cells in the lungs, this restores some normal function.
Viral vectors are a tool commonly used by molecular biologists to deliver genetic material into cells. Which is how somatic gene therapy works, the genomes are delivered to the cells that the viruses infect. This process can be performed inside a living organism or in cell culture. Viruses have evolved to create specialised molecular mechanisms to deliver their genomes into the cells they infect.
The diagram below shows which viral vectors there are and how often they are used (shown as a percentage of use).
However, there are three major scientific hurdles that have to be overcome before somatic gene therapy is likely to work. The first is getting the human gene into the patient's cells (using viruses or liposomes).
The second obstacle is getting the gene into the right cells.
The final obstacle is making sure the gene is active, that is, switched on in the cell to produce the protein that the patient needs. This means it must be under the control of the sequence of DNA that is responsible for switching the gene on.
The results do not have to be perfect to produce benefits. In cystic fibrosis, animal tests have shown that if the normal gene can be transferred to only five per cent of cells in the lungs, this restores some normal function.
Viral vectors are a tool commonly used by molecular biologists to deliver genetic material into cells. Which is how somatic gene therapy works, the genomes are delivered to the cells that the viruses infect. This process can be performed inside a living organism or in cell culture. Viruses have evolved to create specialised molecular mechanisms to deliver their genomes into the cells they infect.
The diagram below shows which viral vectors there are and how often they are used (shown as a percentage of use).