• Isolation of nucleic acids from different organisms is the most essential requirement for any molecular biology experiment. There are four steps in the process of extraction of nucleic acids i.e., disruption of biological samples, protection of nucleic acids from its degrading enzymes, separation of nucleic acids from other molecules and assessment of purity and quality of the isolated nucleic acids.
• Various enzymes play an important role in recombinant DNA (rDNA) technology. These are nucleases, DNA ligase, alkaline phosphatase, polynucleotide kinase, poly A polymerase, etc.
•The major task of the manipulation of DNA involves cutting and ligation of the gene of interest into the vector DNA.
• Nucleases are the enzymes that cleave nucleic acids by hydrolysing the phosphodiester bond that joins the sugar residues of adjacent nucleotides. Two major types of nuclease enzymes depending on its action on the phosphodiester bonds of polynucleotide chains have been identified, which are exonuclease and endonuclease.
• Exonuclease enzymes can remove mononucleotide either from the 3′ or 5′ end of the DNA molecule.
• Endonuclease enzymes cleave DNA molecules at a specific sequence, hence called restriction endonucleases or restriction enzymes (REs). REs are mainly categorised into three groups (i.e., Types I, II and III) based on their cofactor requirement and the position of their DNA cleavage site relative to the target sequence. Type II REs find application in rDNA technology.
• DNA ligase can join two DNA strands together by catalysing the formation of a phosphodiester bond in the duplex form.
• DNA polymerases are a group of enzymes that catalyse the synthesis of new DNA strand by using dNTPs on a template strand.
• Alkaline phosphatase is used to remove the terminal phosphate group from 5′ end of DNA strands.
• Reverse transcriptase is used to generate complementary DNA (cDNA) strand from an RNA template, a process called reverse transcription.
• In rDNA technology, the recombinant DNA is introduced (transferred) in host cells by a number of methods, such as chemical based transfection (calcium chloride, lipofection etc.) and physical transfection (electroporation, microinjection and biolistic) methods.
• Selection of transformed bacteria is the most essential step for a successful cloning experiment i.e., to identify the transformed cells having recombinant vector (with gene of interest) from a mixture of transformed and non-transformed cells. These selection methods may be direct or through insertional inactivation.
• In direct selection, the transformed cells are distinguished from non-transformed cells on the basis of expression of certain traits, such as resistance to antibiotics.
• In insertional inactivation method, a vector is used having two markers (either two antibiotic resistant genes or one antibiotic resistant gene and lacZ gene).
• Blue-white selection method is another example of insertional inactivation to select recombinant transformed cells in which the expression of lacZ gene can directly be observed in bacterial colonies.
• Blotting techniques are widely used to separate and identify DNA, RNA and proteins from a mixture of molecules.
• Southern blotting technique is used to detect specific sequence of DNA in DNA samples.
• Northern blotting technique is used to detect specific RNA molecules in a mixture of RNA.
• Western blotting is used to detect specific proteins in a sample of tissue homogenate or extract.
• Polymerase Chain Reaction (PCR) is used to amplify a small amount of DNA into thousands to millions of copies, which involves three steps i.e., denaturation, annealing and extension. The amplified product of PCR can be analysed by gel electrophoresis at the end of reaction (end point analysis).
• The latest advancement in PCR technology is real-time quantitative PCR (qPCR), in which the fluorescent markers are used that have specific binding affinity to double stranded DNA. In qPCR, gel electrophoresis is not needed as in the case of conventional PCR.
• DNA libraries are constructed by collecting DNA fragments that have been cloned into vectors so that specific DNA fragments of interest can be identified and isolated. There are basically two types of DNA libraries — genomic and cDNA library.
• A genomic library is a collection of clones of small fragments of DNA that together represent complete genome of an organism.
• The cDNA library constitutes cDNA clones of all the genes expressed in a specific cell type or tissue of an organism.
