The following points highlight the seven main stages of recombinant DNA technology. The stages are: 1. Isolation of the Genetic Material (DNA) 2. Cutting of DNA at Specific Locations 3. Isolation of Desired DNA Fragment 4. Amplification of Gene of Interest using PCR 5. Ligation of DNA Fragment into a Vector 6. Insertion of Recombinant DNA into the Host Cell/Organisms 7. Obtaining or Culturing the Foreign Gene Product.
Stage # 1. Isolation of the Genetic Material (DNA):
Nucleic acid is the genetic material, which is present in all living organisms. In majority of organisms, this is present in the form of deoxyribonucleic acid (DNA). DNA must be present into pure form, i.e., free from other macro-molecules (like proteins, RNA, enzymes, etc.) in order to cut the DNA with restrictor enzymes.
Isolation of genetic material (DNA) is carried out in the following steps:
(a) Since the DNA is enclosed within the membranes, so, in order to release DNA along with other macro-molecules such as proteins, polysaccharides and lipids, bacterial cells/plant or animal tissues are treated with the enzyme lysozyme (bacteria), cellulose (plant cells), chitinase (fungus), respectively.
(b) RNA can be removed by the treatment with ribonuclease, whereas proteins can be removed by the treatment with protease.
(c) Other molecules can be removed by appropriate treatments and ultimately purified DNA will precipitate out, after the addition of chilled ethanol. This can be seen as collection of fine threads in the suspension.
Stage # 2. Cutting of DNA at Specific Locations:
Restriction enzyme digestions are performed by incubating purified DNA molecules with the restriction enzyme. This is done at the optimal conditions for that specific enzyme.
Stage # 3. Isolation of Desired DNA Fragment:
Using agarose gel electrophoresis, the activity of the restriction enzymes can be checked. Since, the DNA is negatively charged, it moves towards the positive electrode or anode and DNA tends to separates out in this process. After that the desired DNA fragment is eluted out.
Stage # 4. Amplification of Gene of Interest using PCR:
Polymerase Chain Reaction (PCR) is best defined as the DNA replication in vitro. This techniques was developed by Kary Mullis in 1985 and received Nobel Prize for chemistry in 1993. PCR is used for the amplification of gene of interest using two set of primers.
The basic requirements of a PCR reaction are the following:
(a) DNA Template:
The double-stranded DNA that needed to be amplified.
These are chemically synthesised oligonucleotides (short segment of DNA) that are complementary to a region of DNA template.
Two enzymes are commonly used.
i. Taq Polymerase:
It is isolated from a thermophilic bacterium, i.e., Thermus aquaticus. It has a property to remain active during the high temperature which have induced denaturation of double-stranded DNA.
ii. It also helps in the amplification of a segment of DNA to approximately billion times, i.e., I billion copies are made it the process of replication of DNA is repeated many times.
iii. Vent Polymerase (isolated from Thermococcus litoralis).
Three main steps involved in PCR technique are:
The double-stranded DNA is denatured by using high temperature of 95°C for 15 seconds. Now each separated single-strand acts as a template for DNA synthesis.
Two sets of oligonucleotide primers are used to anneal (hybridise). This step is carried out at a slightly lower temperature (40-60°C) using Mg2+ and dNTPs (deoxynucleoside triphosphates), depending on the length and sequence of the primers.
The thermo-stable enzyme Taq DNA polymerase is used in this reaction, which can tolerate the high temperature of the reaction that extends the primers by adding nucleotides complementary of the template.
Mg2+ is required as a cofactor for thermo-stable DNA polymerase, e.g., Taq polymerase.
These steps are repeated many times in order to obtain several copies of desired DNA.
Stage # 5. Ligation of DNA Fragment into a Vector:
This process requires a vector DNA and a source DNA. In order to obtain sticky ends, both of these should cut with the same endonuclease. After which both are ligated by mixing vector DNA, gene of interest and enzyme DNA ligase to form the recombinant DNA/hybrid DNA.
Stage # 6. Insertion of Recombinant DNA into the Host Cell/Organisms:
This can occur by several methods, before which the recipient cells are made competent to receive the DNA. If a recombinant DNA bearing gene for resistance to an antibiotic (e.g., ampicillin) is transferred into E. coli cells, the host cells become transformed into ampicillin resistant cells.
The ampicillin resistance gene in this case is called a selectable marker. When transformed cells are grown on agar plates containing ampicillin, only transformants will grow and others will die.
Stage # 7. Obtaining or Culturing the Foreign Gene Product:
When you insert a piece of alien DNA into a cloning vector and transfer it into a bacterial cell, the alien DNA gets multiplied. The ultimate aim is to produce a desirable protein expression. The expressions of the foreign gene or genes in host cells involve understanding of many technical details.
If the protein encoded gene is expressed in the heterologous host, it is called recombinant protein. The cells harbouring cloned genes of interest are grown on small scale in the laboratory. These cell cultures are used for extracting the desired protein using various separation techniques.