1. Isolation of the genetic material (DNA)
• In order to cut the DNA with restriction enzymes, it needs to be in pure form, free from other macromolecules.
• The DNA enclosed within the membranes has to be released by breaking the nuclear and cell membranes, along with other macromolecules such as RNA, proteins, polysaccharides and also lipids.
• This can be achieved by treating the bacterial cells/ plant or animal tissue with enzymes such as lysozyme (bacteria), cellulase (plant cells), chitinase (fungus).
• Genes are present on long molecules of DNA which is interwined with proteins like histones, RNA etc. RNA can be removed by treating with ribonudease, whereas proteins can be removed by treating with protease.
• Other molecules can be removed by appropriate treatments, and purified DNA ultimately precipitates out after addition of chilled ethanol.
2. Cutting of DNA at specific locations
• Restriction enzyme digestions are performed by incubating purified DNA molecules with the restriction enzyme, at the optimal conditions for that specific enzyme.
• Agarose gel electrophoresis is employed to check the progression of a restriction enzyme digestion.
• The joining of DNA involves several processes. After having cut the source DNA as well as the vector DNA with a specific restriction enzyme, the cut out ‘gene of interest’ from the source DNA, and the cut vector, with space, are mixed, and ligase enzyme is added. This results in the formation of recombinant DNA.
3. Amplification of gene of interest using PCR
• The polymerase chain reaction (PCR) technique was invented by Kary Mullis in 1985. PCR is best defined as the DNA replication in vitro.
• It is based on the principle that a DNA molecule, when subjected to high temperature, splits into two strands due to denaturation.
• These single stranded molecules are then converted to original double stranded molecules by synthesizing new strands, in the presence of enzyme DNA polymerase.
• A double stranded molecule of DNA is duplicated in this way and multiple copies of the original DNA sequence
can be generated by repeating the process several times.
• The basic requirements of PCR are:
DNA template: The desired segment of the target DNA molecule that is to be amplified.
Two nucleotide primers: Primers, which are oligo-nudeotides, that hybridize to the target DNA region, one to each strand of the double helix.
– Two primers are required, and these primers are oriented with their ends facing each other allowing synthesis of the DNA towards one another. Enzyme: DNA polymerase, which is stable at high temperature (more than 90°C). Taq polymerase is generally used in PCR reactions which is isolated from a bacterium Thermos aquaticus.
Procedure of PCR
• The PCR operation is followed in a sequence, involving denaturation, primer annealing and primer extention.
• The target DNA is first heated to a temperature between 94 – 96°C that ensures DNA denaturation i.e., the separation of the two strands.
• Each single strand of the target DNA then acts as a template for DNA synthesis.
• The mixture is now cooled to a temperature (generally 40-60°C) that permits annealing of the primer to the complementary sequences in the DNA; these sequences are located at the 3’-ends of the two strands of the desired segment.
Primer extension (Polymerisation)
• The temperature is now so adjusted, that the DNA polymerase synthesizes the complementary strands by utilizing 3-OH of the primers.
• This reaction is the same, as that occurs in vivo, during replication of the leading strand of a DNA duplex.
• The primers are extended towards each other, so that the DNA segment lying between the two primers is copied.
• This is ensured by employing primers complementary to the 3’-ends of the segment to be amplified. The duration of primer extension is usually 2 min at 72°C.
• It has been shown that in case of longer target sequences, best results are obtained when the period of extension is kept at the rate of 1 min per Kb of Applications of PCR the target sequence and the extension is carried out
Some of the areas of application of PCR are mentioned at 68°C in the place of usual 72°C. here :
• Taq DNA polymerase synthesizes the DNA region between the primers, using DNTPs (deoxynudeoside triphosphates) and Mg2+.
• To begin the second cycle, the DNA is again heated to convert all the newly synthesized DNA into single strands, each of which can now serve as a template for synthesis of more new DNA.
• Thus, the extension product of one cycle can serve as a template for subsequent cycles, and each cycle essentially doubles the amount of DNA from the previous cycle. As a result, from a single template molecule, it is possible to generate 2n molecules after n number of cycles.
Applications of PCR
• Some of the areas of application of PCR are mentioned here :
– Detection of pathogens.
– Diagnosis of specific mutation.
– DNA fingerprinting.
– Detection of specific microorganisms.
– In prenatal diagnosis.
– Diagnosis of plant pathogens.
– In palaeontology.
– Gene therapy.
• Molecular probes are small DNA or RNA segments that are used to detect the presence of complementary sequences in nucleic acid samples.
• These are usually formed of 200-500 nucleotide sequences, but may have upto 500 nucleotides.
• These segments or probes are labelled either with radioactive or with nonradioactive compound.
• Probes are used for identification and isolation of DNA and RNA.
• Types of probes are cDNA probes, Genomic DNA probes, synthetic probes.
• The probes are being used for following purposes:
– Accurate diagnosis of pathogenic diseases
– Isolation of gene or related sequences.
– Preparation of genome maps of plants, animals, viruses and bacteria.
4. Insertion of Recombinant (rDNA) into the host cell/organism
• Both direct and indirect methods are used to introduce the ligated DNA into the competent host cells. The transformed host cells are identified with the help of selective markers e.g. antibiotic resistance genes.
5. Obtaining desirable foreign gene product
• When recombinant DNA is transferred into a bacterial, plant or animal cell, the foreign DNA is multiplied. Most of the recombinant technologies are aimed to produce a desirable protein.
• If any protein encoding gene is expressed in a heterologous host, it is known as a “recombinant protein”.
• After the cloning of the gene of interest, one has to maintain the optimum conditions to induce the expression of the target gene, and consider producing it on a large scale.
• The cells having cloned genes of interest can be grown on a small scale in the laboratory. The cultures may be used for extracting and purifying the desired protein.
• The cells can also be multiplied in a continuous system, where the used medium is passed out from one side, and fresh medium is added from the other side, in order to maintain the cells in their physiologically most active log or exponential phase.
• This type of culturing method produces a larger biomass to get higher yield of desired protein in bioreactors and fermenters.
• Bioreactors are considered as vessels in which raw materials are biologically converted into specific products by microbes, plant and animal cells or their enzymes.
• Small volume cultures cannot give large quantities of the products. To produce large quantities of these products, bioreactors are required where
large volume (100 – 1000 litres) of culture can be processed.
• Bioreactor provides the optimal conditions for obtaining the desired product by providing optimum growth conditions such as temperature, pH, substrate, vitamins, oxygen and salts.
Types of bioreactor
• The most commonly used bioreactors are of stirring type. A bioreactor (fermenter) has a provision for batch culture or continuous culture.
• In continuous culture, the culture medium is added continuously as it is utilised, and the product is taken out, while in batch culture, the nutrients and microbes are added in closed chamber and not replaced continuously.
• The most common bioreactors are of stirring type,
(i) simple, stirred tank bioreactor, (ii) spraged stirred tank bioreactor.
• A simple stirred tank bioreactor is a large stainless steel vessel, which has the main parts as cooling jacket, air inlet and filter, stirrer, sparger, nutrient, antifoam, inoculum, pH and dissolved oxygen lines, temperature sensor and control unit.
• Most fermenters are aerobic and require a large volume of sterile air through air inlet and filter.
• Cooling jacket reduces temperature because the culture generates heat during growth.
• Stirrer consists of a vertical rotating stirrer shaft and flat, vertical stirrer blades.