Table of Contents
Recombinant DNA (rDNA) technology involves combining DNA fragments from two sources.
The resulting DNA is called recombinant DNA. Naturally, DNA recombinants occur during crossing-over of homologous chromosomes. The context of recombinant DNA is the artificial or uncommon union of DNA fragments from two different sources of DNA. Some scientist also uses the term chimeric DNA. The experimental manipulation to produce rDNA is what is called now as recombinant DNA technology.
Preparation of an rDNA involves a number of steps. These are:
- DNA isolation
- DNA splicing
- DNA joining
- rDNA amplification
Before preparing the rDNA, it is important to identify the correct gene of interest depending on the target results. One of the main goals of rDNA technology is to develop species with characteristics or properties not normally found in them.
The first step in the preparation of the rDNA is to isolate both the vector and the donor DNA
For prokaryotic applications, the commonly used vector is the circular bacterial DNA plasmid. To be able to isolate the plasmid from the rest of the DNA genome, differential centrifugation by addition of cesium chloride and ethidium bromide is done. The two DNA fragments selectively bind to the ethidium bromide producing a cesium chloride gradient after centrifugation. The plasmid DNA which sinks at the bottom can be collected for the next step.
The second step involves cutting the DNA fragment using restriction enzymes
Restriction enzymes are responsible for cutting the DNA of the bacteria, in the process deactivating it. They cut at specific locations in the DNA strand which make it possible to selectively obtain desired DNA fragment. They usually recognize the specific DNA sequence where the cutting of DNA will occur.
These sequences occur as palindromes, that is the two strands of the DNA have the same sequence but in opposite directions. The restriction enzyme recognizes this sequence and cuts the fragments, opening up the circular DNA. To be able to successfully produce the rDNA, the donor fragment should also be cut by the same restriction enzymes. This is to ensure that the sticky ends (points where the cut was made) of the donor DNA are compatible with the sticky ends of the vector DNA.
The third step is the joining of the two DNA fragments
The two DNA fragments cut using the same restriction enzyme will be mixed. Since the sticky ends of both fragments are complementary, the two DNA fragments can combine to form the rDNA. To further strengthen the joining, DNA ligase is used to create phosphodiester linkages between the two fragments.
The last step involves DNA amplification
When the rDNA obtained is entered into the bacterial cell by transformation, the plasmid vector will then be replicated via natural DNA replication processes. When they are replicated, the donor DNA is also replicated and multiple copies of it are produced. Continued cell division will lead to millions of cells containing the desired DNA fragment.
In the laboratory, large DNA fragments are hard to analyze because of the amount of
nucleotide base contained in them. The use of restriction enzymes enables fragmentation of the DNA sequence to fragments of varying lengths or sizes. To be able to obtain the desired fragment, a separation technique must be employed.
In molecular biology, these DNA fragments are separated by using Gel Electrophoresis. In this technique, mixtures of DNA fragments are placed on top lanes of the gels. Electricity is then applied to separate the fragments in terms of size and charge. Smaller DNA fragments will naturally move faster than larger ones. After the separation, the DNA fragments can be visualized by adding fluorescent dyes that bind to DNA.
Bacterial transformation normally occurs when bacteria uptake DNA fragments from the environment. These DNA fragments enter the cell and can be integrated into the bacterial chromosome by nonreciprocal recombination. When recombination is successful, a stable transformation will occur and the inserted DNA fragments may be expressed by the organism. Otherwise, the DNA fragment is degraded.
Molecular cloning follows basic steps in rDNA technology which are isolation, insertion, and multiplication. The first step is the isolation of the vector DNA. The next step is to use the correct restriction endonuclease to cut the DNA in a specific location. The foreign DNA is then inserted and connected using DNA ligase. The transformation will then occur to incorporate modified DNA into the bacterial species The species containing the modified gene will be amplified via natural cell division and will exhibit different properties compared to natural species.
Host Vector System
To be able to produce multiple copies of a gene, the gene must be inserted into DNA segments which can spontaneously replicate. This DNA fragment that can replicate with the inserted DNA fragment is known as the vector or cloning vehicle. A good vector should have the origin of replication, one or more genetic markers for selection, and a cloning site where foreign DNA can be inserted.
Replication of the DNA can only be achieved inside a host which provides the enzymes and factors needed for replication. This is achieved by the successful introduction of the vector into the host.
Common hosts used are:
- E. coli
- Yeast cell
- Mammalian tissue culture cells
- Insect cells
Common vectors include:
- Bacterial and yeast artificial chromosomes
Transforming Eukaryotic Cells
Unlike in the transformation of prokaryotic cells, transformation in eukaryotic cells is more challenging. A common technology employed is the use of a gene gun. The gene gun inserts DNA by bombarding electrically charged cells with gold or tungsten particles coated with DNA.
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Cas9 enables editing of
DNA by having the Cas9 acting like a pair of molecular scissors to cut pieces of the DNA of the disease-causing organism, the organisms then later incorporate part of the invader’s DNA to its own genome so that it can recognize the invader in the future.
In this technique, scientists create a guide RNA sequence that matches the DNA they want to modify. This sequence is then added to cells together with a protein called Cas9, an RNA-guided DNA endonuclease that interrogates and cleaves foreign DNA. The Cas9 protein cuts the DNA and inserts the Guide RNA in the DNA sequence. Enzymes then repair the cuts and leave the modified DNA.
This technique finds great application in fixing genetic defects. It was already used before to treat in Duchenne Muscular Dystrophy in Mice and Huntington’s Disease.
Polymerase Chain Reaction
Polymerase chain reaction (PCR) is a technique used to produce multiple replicates of DNA fragments. The process of DNA amplification is divided into three stages namely denaturing stage, annealing stage, and extending stage. In the denaturing stage, the DNA molecule is heated up to 95º C to separate the two strands. Primers then attach to each of the two strands in the annealing stage. Extending stage then proceeds by adding more nucleotides in the growing chain. The steps are then repeated to produce more copies of the DNA.