<h5 id="31-identification-of-candidate-gene">3.1 Identification of Candidate Gene</h5> <ul> <li>Over the past decades, rDNA technology has been used to produce crops resistant to pests, diseases, herbicides, and pathogens.</li> <li>The first step in this process is identifying the candidate gene in an organism’s genome, which can be challenging due to the large size of the genome.</li> <li>Identification of a gene to be cloned depends on its significance in biomedical, economical, and evolutionary fields, which can be determined through biochemical and physiological studies.</li> <li>If the protein product of a gene is known, its mRNA sequence can be synthesized using the genetic code, or the mRNA can be used as a probe to search for the gene in its genome library.</li> <li>The candidate gene can then be cloned, which is discussed in subsequent chapters.</li> </ul> <p>======</p> <h5 id="32-isolation-of-nucleic-acids">3.2 Isolation of Nucleic Acids</h5> <ul> <li>Extraction of nucleic acids involves 4 main steps: cell membrane/wall rupture, protection from degrading enzymes, separation from other molecules, and precipitation/concentration.</li> <li>Different strategies are used to disrupt cell boundaries for releasing nucleic acids, depending on the type of organism. Animal cells have plasma membranes, while plant cells and bacteria have tough layers like cell walls.</li> <li>For isolation of DNA from plant cells, CTAB (a cationic detergent) is used to take advantage of the differential solubility of DNA and polysaccharides in varying ionic strengths.</li> <li>Nucleic acids are separated from bound proteins by decreasing interaction between proteins and nucleic acids using detergents like SDS, which makes protein molecules anionic and dissociates them from nucleic acids.</li> <li>RNA isolation is challenging due to the abundance of ribonucleases. Total RNA is extracted from biological samples using guanidinium isothiocyanate (GITC) phenol-chloroform, which disrupts hydrogen bonds, denatures proteins, and inactivates RNase enzymes.</li> </ul> <p>======</p> <h5 id="33-enzymes-used-for-recombinant-dna-technology">3.3 Enzymes used for recombinant DNA technology</h5> <ul> <li>DNA ligase is an enzyme that joins DNA strands together by catalyzing the formation of a phosphodiester bond. Bacterial DNA ligases use NAD as an energy source, while DNA ligases from bacteriophages and eukaryotic cells use ATP.</li> <li>DNA polymerases are enzymes that catalyze the synthesis of new DNA strands using mono-deoxyribonucleoside triphosphates (dNTPs) on a template strand. They synthesize new DNA strands in the 5’ → 3’ direction and require a primer with a free 3’-end hydroxyl group.</li> <li>Alkaline phosphatase is an enzyme that removes the terminal phosphate group from the 5’ end of DNA strands.</li> <li>Polynucleotide kinase is an enzyme that attaches a phosphate group to the hydroxyl (-OH) group present on the 5’ end of DNA.</li> <li>Terminal deoxynucleotidyl transferase or terminal transferase is an enzyme that adds similar nucleotide residues to form a homopolymer tail on the 3’ end of a DNA strand, without requiring a template.</li> <li>Reverse transcriptase, also known as RNA-directed DNA polymerase, is found in many retroviruses and is used to generate complementary DNA (cDNA) strands from RNA templates, a process called reverse transcription.</li> <li>Poly A polymerase is an enzyme that incorporates adenine residues to the hydroxyl group of the 3’ end of RNA.</li> </ul> <p>======</p> <h5 id="34-modes-of-dna-transfer">3.4 Modes of DNA Transfer</h5> <ul> <li>Transformation in bacteria is the direct uptake of exogenous DNA molecules from the surroundings through the cell membrane, resulting in genetic alteration of the cell.</li> <li>Transduction is a process where viruses mediate the uptake of foreign DNA into the genome of a cell. Bacteriophages infect bacteria and can take small sequences of bacterial DNA with them when they infect a new bacterial cell.</li> <li>Conjugation is the transfer of genetic material from one bacterium to another through cell-to-cell direct contact, mediated by F plasmids that carry a DNA sequence called the fertility factor.</li> <li>In rDNA technology, the rDNA is introduced (transferred) in host cells by various methods such as calcium chloride, lipofection, electroporation, microinjection, and biolistic methods.</li> <li>Chemical and physical methods for introducing foreign DNA molecules into host cells are commonly used, with the goal of creating a recombinant DNA molecule that can be replicated and expressed in the host cell.</li> </ul> <p>======</p> <h5 id="35-screening-and-selection">3.5 Screening and Selection</h5> <ul> <li>The selection of transformed bacteria with recombinant vectors is crucial for a successful cloning experiment.</li> <li>The selection is based on the principle of difference in biological traits present in hosts with recombinant DNA from those without recombinant DNA.</li> <li>There are two main types of selection procedures: (i) Direct selection of recombinants, where transformed cells are distinguished from non-transformed cells on the basis of expression of certain traits, such as antibiotic resistance. (ii) Selection of recombinants by insertional inactivation, where a vector having two markers is used, and the expression of one of the selection marker genes is disrupted when the gene of interest is inserted.</li> <li>In the insertional inactivation method, the recombinant plasmids can be identified by the loss of resistance to a particular antibiotic, such as ampicillin.</li> <li>The Blue-white selection method is another example of insertional inactivation selection method, where the enzyme $\beta$-galactosidase is not expressed in hosts containing recombinant plasmids when the lacZ gene is inactivated due to insertion of the insert DNA.</li> </ul> <p>======</p> <h5 id="36-blotting-techniques">3.6 Blotting Techniques</h5> <ul> <li>Blotting techniques are used to separate and identify DNA, RNA, and proteins.</li> <li>The molecules of interest are immobilized on nitrocellulose, nylon, or PVDF membranes.</li> <li>Detection methods include chromogenic, fluorescence, chemiluminescence, and radioactive methods.</li> <li>Three types of blotting techniques are: <ul> <li>Southern blotting: detects specific sequences in DNA samples.</li> <li>Northern blotting: detects specific RNA molecules in a mixture.</li> <li>Western blotting: detects specific proteins in a sample.</li> </ul> </li> <li>Eastern blot is used for the detection of specific post-translational modifications of proteins.</li> </ul> <p>(Note: The text does not provide any equations or formulae related to these techniques.)</p> <p>======</p> <h5 id="37-polymerase-chain-reaction-pcr">3.7 Polymerase Chain Reaction (PCR)</h5> <ul> <li>Polymerase Chain Reaction (PCR) is a technique developed by Kary B. Mullis to amplify a small amount of DNA to thousands to millions of copies.</li> <li>PCR is based on the principle of DNA replication and involves denaturation, annealing, and extension steps. It requires a heat stable DNA polymerase enzyme, such as Taq polymerase, which can make new strands of DNA on template strands at a high temperature.</li> <li>The PCR process involves repeated cycles of heating and cooling, called thermal cycling, which is carried out by a machine called a thermocycler. The number of DNA copies doubles after each cycle, and usually, 25 to 30 cycles are carried out in a typical PCR reaction.</li> <li>Real-time quantitative PCR (real-time qPCR) is a latest advancement in PCR technology that uses fluorescent markers to detect and quantitate the amount of double stranded PCR product after each PCR cycle.</li> <li>PCR has several applications in molecular biology and rDNA technology, including quantifying mRNA to assess the expression of a gene, amplifying minute DNA samples collected from crime scenes and fossils, and COVID-19 detection tests.</li> </ul> <p>======</p> <h5 id="38-dna-libaries">3.8 DNA Libaries</h5> <ul> <li>A DNA library is a collection of cloned DNA fragments that represents the complete genome of an organism.</li> <li>There are two main types of DNA libraries: genomic and cDNA libraries.</li> <li>A genomic DNA library is constructed by digesting genomic DNA into fragments of a specific size, inserting the fragments into vectors, and allowing the recombinant vectors to multiply in host bacterial cells.</li> <li>A cDNA library is constructed by isolating mRNA from a specific tissue, converting the mRNA into cDNA using reverse transcriptase, and cloning the cDNA in a suitable vector.</li> <li>Genomic and cDNA libraries have various applications in biotechnology, such as genome sequencing, identifying genes that are not expressed, understanding the evolution of species, and isolating specific genes.</li> </ul> <p>======</p>