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10. Describe briefly the following: (a) Origin of replication (b) Bioreactors (c) Downstream processing Solution: (a) Origin of replication It can be defined as a DNA sequence in a genome where replication is initiated. The process of replication initiation can either be uni-directional or bi-directional. Any piece of DNA when linked to this sequence can be made to replicate within the host cells. The sequence is also responsible to control the copy of number of the linked DNA. Hence, to recover many copies of target DNA, it should be cloned in a vector whose origin supports high copy number. (b) Bioreactor They are large vessels that are used for the large-scale production of biotechnological products from raw resources. In order to obtain the required product, these bioreactors offer optimal conditions by supplying optimum pH, temperature, vitamin, oxygen etc. They have an oxygen delivery system, a foam control system, a temperature and pH control system. Also, it consists of a sampling port to withdraw a small quantity of culture for the purpose of sampling. (c) Downstream processing It is a method of separating and purifying foreign gene products once the biosynthetic stage is completed. Then, the product is exposed to different procedures to separate and purify the product. Once the process is completed, the product is formulated and made to undergo several clinical trials for quality check and other related assessments. 11. Explain briefly (a) PCR (b) Restriction enzymes and DNA (c) Chitinase Solution: (a) PCR In molecular biology, PCR or polymerase chain reaction is a technique to amplify a gene or a fragment of DNA in order to get multiple copies. It is widely used in the gene manipulation process. The phenomena involves the in vitro synthesis of sequences with the help of a template strand, a primer and a thermostable DNA polymerase enzyme produced by a bacterium known as Thermus aquaticus. The enzyme uses the building blocks deoxynucleotides (dNTPs) in order to extend the primer. The following are the three steps in PCR: (i) Initially, the double stranded DNA molecules are heated to a high temperature to separate the two strands into a single stranded DNA molecule. This process is referred to as denaturation. (ii) This DNA molecule is then used as a template strand to synthesize a new strand by the DNA polymerase enzyme. The process is termed as annealing that leads to the replication of the original DNA molecule, and the process is carried out for multiple cycles to obtain multiple copies of the rDNA fragment. (iii) Primer is extended by Taq DNA polymerase isolated from Thermits aquatics. NCERT solutions class 12 biology chapter 11 - 4 (b) Restriction enzymes and DNA In molecular biology, restriction enzymes are molecular scissors use to cut DNA sequences from a particular site. It has a critical role to play in the gene manipulation process. These enzymes identify a particular six-base pair sequence referred to as the recognition sequence and snip the sequence at specific sites. For instance, the recognition site for the ECORI enzyme is as given below: NCERT solutions class 12 biology chapter 11 - 5 Restriction enzyme are grouped into two types: (i) Endonuclease – it is a type of restriction enzyme that cuts within the DNA at specific sites. It serves as a significant tool in genetic engineering. Typically, it is used to make a snip in the sequence to get DNA fragments possessing sticky ends. These ends are later fused by the enzyme DNA ligase. (ii) Exonuclease – this type of restriction enzyme removes the nucleotides either from 3’ or 5’ ends of the DNA molecule. (c) Chitinase It is a class of enzyme that is used for degradation of chitin, that forms the main component of the cell wall of fungi. Hence, to isolate the DNA enveloped within the cell membrane of the fungus, the Chitinase enzyme is used to break the cell to release its genetic material. 12. Discuss with your teacher and find out how to distinguish between (a) Plasmid DNA and Chromosomal DNA (b) RNA and DNA (c) Exonuclease and Endonuclease Solution: The differences are as follows: (a) Plasmid DNA and Chromosomal DNA Plasmid DNA Chromosomal DNA It is an extra chromosomal DNA molecule found in bacteria, capable of replicating and is independent of chromosomal DNA It forms the complete DNA of an entity found inside the chromosomes (b) RNA and DNA RNA DNA Single-stranded molecule Double-stranded molecule Cannot replicate by themselves Have the potential to replicate Consist of ribose sugar Consists of deoxyribose sugar Pyrimidines are uracil and adenine Pyrimidines are thymine and adenine It is a component of ribosomes It is a component of chromosomes (c) Exonuclease and Endonuclease Exonuclease Endonuclease It is a kind of restriction enzyme which removes the nucleotides from 5’ or 3’ terminals of the DNA molecule It is a kind of restriction enzyme that snips within the DNA at particular sites to produce sticky ends