Recombinant DNA

Recombinant DNA, or rDNA, is a strand of DNA that is artificially created by combining two separate pieces of DNA into one DNA molecule. Often, these two pieces of DNA come from two different organisms of two unique species. There are three methods to make rDNA. These are transformation, phage introduction and non-bacterial transformation. However, transformation is the most commonly used method. Recombinant DNA can be used in several different aspects. It can be used to amplify DNA for analysis, in agricultural applications and in the pharmaceutical industry. The development and application of recombinant DNA is a fairly new process that is changing the world of science and technology.

It is important to note the difference between recombinant DNA and natural DNA recombination that results from the process of crossing-over between homologous chromosomes. This is why some scientists will refer to rDNA as chimeric DNA. Chimera refers to the combination of parts of different organisms so recombinant DNA is a DNA chimera. The technology behind this science is referred to as “Recombinant DNA Technology”.

To begin, one has to plan out how the recombinant DNA will be created. In transformation, the first step is to select a piece of DNA that will be inserted into the vector. The vector is the piece of DNA that will be carrying the piece of DNA that is of interest, also known as the donor DNA. In this situation, the vectors used are often circular DNA that is extracted from bacteria, commonly E. coli. Both the donor DNA and the vector need to be cut with a restriction enzyme. Restriction enzymes are like molecular scissors that will be discussed later in more detail. The two pieces of DNA will be combined in a solution and allowed to bond together. The bonds will then be sealed with DNA ligase, which acts like a molecular glue bottle.

This newly recombined DNA is introduced back into the bacteria and replication will begin to occur. The piece of donor DNA will contain a marker that acts as a way to double check that the donor DNA is in fact in the vector. These can be antibiotic resistant markers or color markers; essentially something that will allow the scientist to differentiate the bacteria with the desired donor DNA from those that do not contain the DNA of interest.

This newly recombined DNA is introduced back into the bacteria and replication will begin to occur. The piece of donor DNA will contain a marker that acts as a way to double check that the donor DNA is in fact in the vector. These can be antibiotic resistant markers or color markers; essentially something that will allow the scientist to differentiate the bacteria with the desired donor DNA from those that do not contain the DNA of interest.

This newly recombined DNA is introduced back into the bacteria and replication will begin to occur. The piece of donor DNA will contain a marker that acts as a way to double check that the donor DNA is in fact in the vector. These can be antibiotic resistant markers or color markers; essentially something that will allow the scientist to differentiate the bacteria with the desired donor DNA from those that do not contain the DNA of interest.

Non-bacterial transformation is very similar to transformation, but there is no bacterial host. The recombinant DNA is either injected directly into the nucleus of the cells or the cells are bombarded with microprojectiles that are coated with the recombinant DNA. Phage introduction involves the process of transfection. This is essentially equivalent to transformation, except a phage is used as the host instead of bacteria. This is done via in vitro vector packaging.

In transformation, the vectors used are circular DNA from bacteria known as a plasmid. Plasmids are independent of the bacteria’s chromosomes and therefore, can replicate independently. Plasmids are very useful in recombinant DNA because they can be opened up with a specific restriction enzyme to allow for the insertion of the donor DNA fragments. Restriction enzymes are a natural defense mechanism present in bacteria. They are used like scissors to cut up the DNA of bacteriophages, in turn inactivating them. These “scissors” do not cut randomly, though.

They have specific recognition sites that they look for and have to match up with in order to attach to the DNA. Restriction enzymes can make jagged or clean breaks in the DNA depending on the type of enzyme and recognition site. The cut ends are then called sticky ends because they are now free to bond to complementary sticky ends. When the same restriction enzyme is used on both the vector and the donor DNA they will have complimentary sticky ends and be able to bond to each other. This allows for the formation of the rDNA. DNA ligase is then introduced to seal the breaks because the complimentary bonding is not enough to hold the strand of DNA together.

The ideal restriction enzyme is one that will have only one recognition site on the vector being used. This will ensure that the vector is only broken in one place, turning it into one single strand of DNA with two sticky ends. The donor DNA will be cut into many small fragments with the same restriction enzyme, giving them complimentary sticky ends, allowing these small pieces to bind into the vectors. There may only be a small number of vectors containing the donor DNA fragment that is desired, and this is where the markers are useful and allow the correct recombinant DNA to be selected.

Another key molecular factor is the addition of expression factors. These are sequences on a molecule of DNA that signal for transcription and translation of the DNA so that the DNA can be translated into the proper proteins. There has to be a promoter, a ribosome-binding site and a terminator or the molecular machinery responsible for generating the proteins will never recognize the recombinant DNA, and therefore never replicate it which is the entire purpose of this process. These expression factors are species specific, so it is important that the expression factors added are those that are specific to the host cell and not the donor.

To carry out the process of transformation, first the DNA of both the donor and the vector need to be isolated from the organisms. There are specific protocols that, when followed properly, allow for the isolation of the necessary DNA. If the desired DNA is to be used for analysis, the genomic DNA will be used in the case of both prokaryotes and eukaryotes. The genomic DNA is the DNA that contains all of the genetic information for all of the proteins in an organism. If the desired DNA is the vector, the plasmids need to be purified out of the genomic DNA because we want the genomic DNA of the donor, not the bacterial host. This can be done via centrifugation. The prokaryote DNA is suspended in a solution and spun in the centrifuge, allowing the different sized DNA to separate. The band of DNA that contains the plasmid will then be identified and extracted from the tube.

Once the donor DNA and vector DNA have been isolated, cut with restriction enzymes, bonded together and sealed with DNA ligase, they are ready to be introduced back into the bacteria so that replication can begin. This is where the process of transformation comes in. Transformation occurs when the bacteria takes up and incorporates the DNA plasmid and begins to replicate it along with its own DNA. The bacteria containing the recombinant vector will be plated and allowed to reproduce. As the host bacteria replicates and reproduces, the DNA will be amplified. A single colony of bacteria from the host cell can contain billions of copies of the rDNA that is of interest to the scientist. This colony contains DNA clones of the original host bacteria.

Since the donor DNA was originally cut into many small pieces with the restriction enzymes, many of the colonies that grow from the bacteria will not contain the donor DNA of interest to the scientist. This is when the colonies that display the characteristics of the chosen marker are isolated from the rest. For example, if an antibiotic resistant marker was used, antibiotics can be introduced that will kill all of the colonies except for the one containing the desired recombinant DNA. They can then be plated and allowed to replicate and reproduce, endlessly copying the recombinant DNA.

Once the bacteria containing the recombinant DNA have been identified, isolated and reproduced, detailed analyses can now be performed since there is a much larger quantity of rDNA. Often, the goal is to reintroduce the cloned DNA back into the donor organism in order to achieve the desired genetic manipulations. This cloning process is also useful to amplify and recover a specific segment from a large segment of a DNA molecule.

More often than not, the recombinant DNA is used in prokaryotic cells because they are simpler than eukaryotic cells. Using rDNA in eukaryotic cells is more difficult, but sometimes necessary as the desired proteins to be created are too complex to be produced in bacterium. Recently, recombinant DNA has become useful in the agricultural and pharmaceutical industries. This technology has been used to produce stronger plants that are more likely to survive in order to compensate for the decrease of available farmland around the world. From a medical perspective, this technology can be used to create vaccines, for gene-therapy, and to prevent and cure disease like sickle cell anemia and cystic fibrosis.

A couple of decades ago, this process would seem like something out of a science fiction movie. Now it is a very useful technology with important applications that are able to save lives. There is still some controversy around the use of genetically modified organisms, but in a world where the population is growing faster than ever before and faster than the environment can handle, this technology may become more valuable than ever anticipated.

References

Griffiths, A.J.F., J.H. Miller and D.T. Suzuki. An Introduction to Genetic Analysis. 7th edition. . 2000. 2016 16-August <http://www.ncbi.nlm.nih.gov/books/NBK21881/>

Kuure-Kinsey, Matthew and Beth McCooey. The Basics of Recombinant DNA. 2000 August. 2016 16-August <https://www.rpi.edu/dept/chem-eng/Biotech-Environ/Projects00/rdna/rdna.html>

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