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Cloning of Genes | Recombinant DNA Technology

Biotechnology deals with the application of biology and biological concepts to science and engineering for the welfare of mankind. It is the crossroad of the biological sciences with other major disciplines of science, from organic chemistry to mechanical engineering. The history of biotechnology is as old as the history of man. When the first human beings realized that they could plant their own crops and breed their own animals, they learned to use biotechnology. But the term biotechnology was introduced by the end of the 20th century. Now a days the field of biotechnology becomes a vast field, but this chapter touches a brief part of biotechnology, called genetic engineering, which deals with manipulation or alteration in the genetic material of an organism. CLONING OF GENES Gene cloning is the act of making copies, or clones, of a single gene. Once a gene is identified and cloned, it can be used in many areas of biomedical and industrial research. There are two possible ways of cloning of gene: recombinant DNA technology and polymerase chain reaction (PCR). Recombinant DNA Technology Recombinant DNA technology is a series of procedures that are used to join DNA segments from different sources. This is an in vivo (in living cells) method which is used when gene cloning is required at industrial scale. The main advantage of this method is the production of product of gene beside copies of gene. It involves the selection and isolation of desired gene (gene of interest), inserting it in a suitable vector and the transformation of a suitable host by the recombinant DNA. Components/Tools of Recombinant DNA Technology The cloning of gene through recombinant DNA technology requires gene of interest, molecular scissors, molecular carrier or vector, molecular glue and expression system. Gene of Interest The gene of interest is the gene which is to be cloned. It can be obtained by one of the three possible ways: (a) artificial gene synthesis is the process of synthesizing a gene in vitro (In glassware) without template DNA samples with the help of DNA synthesizer machine. (b) Gene of interest can also be obtained by synthesizing it from its mRNA. Synthesis of gene ham mRNA is carried out by reverse transcriptase enzymes which are naturally found in retroviruses. The DNA formed by this process is called complementary DNA (cDNA). (c) In most of the cases the gene of interest is directly cleaved from a chromosomal DNA by using particular DNA scissors called restriction endonucleases. MOLECULAR SCISSORS/RESTRICTION ENDONUCLEASES Restriction endonucleases are enzymes that cleave the phosphodiester bonds of both strands of duplex DNA at specific sequences. In 1970, the first restriction enzyme was isolated. Many different restriction endonucleases have been isolated so far. Naturally restriction enzymes are found in bacteria, where they appear to serve as host-defense role because they chop up and inactivate (“restrict”) the DNA of infecting viruses. Each ‘restriction enzyme cleaves DNA at specific sequence of DNA called recognition sites or restriction sites. These sites have palindromic sequences. A palindromic sequence is a four to eight base pairs in DNA in which nucleotides are arranged symmetrically in reverse order. Restriction enzymes either make staggered cut or blunt cut. A staggered cut is one in which the resulting duplex fragments show single stranded projected ends called sticky ends. While in blunt cut the resulting duplex, fragments do not show such sticky ends. In the fig 26.1, the recognition site is boxed in yellow and the cut sites indicated by red triangles. MOLECULAR CARRIERS OR VECTORS Vectors are another major component required to make a recombinant DNA (rDNA) molecule for gene cloning. Vectors act as a vehicle for carrying foreign DNA into a host cell for multiplication. Usually small circular DNA molecules of bacterial origin are used as cloning vectors. A DNA molecule should possess the following essential characteristics to act as a cloning vector: (a) Origin of replication site, (b) antibiotics resistant genes, (c) restriction sites of different enzymes. Example of vectors are: Plasmid, lambda phage DNA, Cosmid (combination of plasmid and phage DNA), Yeast artificial chromosomes (YACs) etc. MOLECULAR GLUE (DNA LIGASE) This enzyme is responsible for the formation of the phosphodiester linkage between two adjacent nucleotides and thus joins two double-stranded DNA fragments; therefore, it is called molecular glue. In rDNA experiments, DNA ligase is used to join two different DNA fragments (plasmid/vector and the foreign DNA) that are annealed by the sticky ends. EXPRESSION SYSTEM A suitable organism that can act as host for the recombinant vector to express (multiplication) is called expression system. Therefore, the selection of suitable expression system always depends upon the type of vector which is being used while making recombinant DNA. The most important character of an ideal expression system is its short generation time and simplicity of its genetic system. So bacterial cells can act as an ideal expression system. MECHANISM OR PROCEDURE OF RECOMBINANT DNA TECHNOLOGY Cloning of the desired gene through recombinant DNA technology involves the formation of recombinant DNA (gene of interest + vector DNA), transformation of a suitable expression system by the recombinant DNA, and the identification of transformed clones. FORMATION OF RECOMBINANT DNA The first step in the construction of a recombinant DNA, is the isolation and purification of vectors and gene of interest. First, digest the vector DNA (e.g., plasmid) with same restriction enzyme by which gene of interest is cleaved so that compatible sticky ends can be produced. Next both, vector and gene of interest are incubated together in the presence of DNA ligase which connects them by forming phosphodiester linkage. This results in the formation of recombinant DNA molecule of vector and the gene of interest. TRANSFORMATION OF EXPRESSION SYSTEM Here transformation refers to the insertion of recombinant DNA into the expression system which can be performed by putting the expression system (bacterial cells that already contain no plasmids) and recombinant plasmids into the same medium. Bacterial cells take up recombinant plasmid, especially, if they are treated with calcium chloride which make them more

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Polymerase Chain Reaction (PCR)

The technique used to amplify (clone) a single gene or a piece of DNA into thousands to millions of copies by means of in vitro replication process is called polymerase chain reaction (PCR). In this technique DNA polymerase is compelled to polymerize (polymerase reaction) a given piece of DNA repeatedly, so that multiple copies are produced, thus, the technique is known as polymerase chain reaction (PCR). A special DNA polymerase, the Taq polymerase is used in PCR technique, which is specialized temperature-tolerant enzyme isolated from Thermus aquaticus, a bacterium found in hot springs. This enzyme is stable and active at near-boiling temperatures. In order to perform PCR, template DNA (DNA to be amplified), free nucleotides (deoxyribo-nucleoside triphosphates or dNTPs), primers and Taq polymerase are dissolve in suitable buffer to make PCR mixture or reaction mixture. The PCR mixture is placed in an instrument called thermocycler or PCR machine. Thermocycler regulates the temperature during various steps of PCR reaction according to the need. Mechanism of PCR Reaction PCR cycle consists of three steps: denaturation, primer annealing, and extension or polymerization each requires a specific temperature. The time duration, temperature and sequence of the steps have to be programmed in the thermocycler. DENATURATION In the denaturation step, the template is heated to 94°C for one minute. At this high temperature the DNA undergoes complete denaturation and the double-stranded DNA (dsDNA) becomes single-stranded DNA (ssDNA). Each single ssDNA can act as the template for the in vitro DNA synthesis. PRIMER ANEALING The next step is the primer annealing. In this step the two primers, the forward primers and the backward primers, anneal or hybridize to the single-stranded template DNA at its complementary regions. Annealing is usually carried out at a lower temperature depending on the length and sequence of the primers. In standard cases it is 54°C and approximate time required for this step is 2 minutes. EXTENSION OR POLYMERIZATION The final step in each cycle is the primer extension or polymerization in which the Taq polymerase synthesizes new DNA strands to the 3′ ends of primers using dNTPs. The optimum temperature for carrying out the primer extension reaction or polymerization of dNTPs is standardized at 72°C. This step takes just one minute to be completed. At the end of first cycle one target DNA molecule is converted in to two molecules. The 2ND cycle immediately starts with the denaturation by heating at 94°C, so that all the newly synthesized u are also denatured to single strands, which again act as templates. It will again be followed by the Primer annealing and extension and thus the cycle of denaturation, primer annealing, and extension continues resulting in the amplification of selected DNA sequence at an exponential rate i.e. the number of existing DNA molecules becomes doubled after each cycle.

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Grade 9th Biology Chapter 10 Evolution Exercise MCQs & Short Questions New Biology NBF Book 2025

Section II: Short Answer Questions Explain the homologous structures in plants. Ans; Homologous structures are organs that have a similar structure but different functions. These structures are similar anatomically because they were inherited from a common ancestor. The cactus has modified its leaves into small needle-like structures to reduce water loss in dry areas with high temperatures and to protect itself from herbivores (animals that feed on plants). Why the theory of evolution was attributed to Darwin? Ans; Evolution had been proposed before, but Darwin’s really new idea was natural selection. Darwin formulated the theory of natural selection, which provides a mechanism for evolution. He proposed that organisms with traits better suited to their environment are more likely to survive and reproduce, passing those advantageous traits to the next generation. His many books provided thorough, detailed, and compelling arguments for natural selection and evolution. What is meant by descent with modification? Ans; It is an important postulate of Darwin theory of Natural selection. According to Darwin species change over time, give rise to new species and share an ancestor is known as “descent with modification,”. In simple words, “descent with modification,” means the descendants produced from ancestor will always be different/modified. Which variety was more common when there was more air pollution? Give a reason as well. In England 90% of the peppered moth during the industrial era of pollution was black. This is because Sulphur dioxide from coal burning blackened tree trunks and walls. This made the light-colored peppered moth more visible to predator birds, while the black form was better camouflaged. Over time, black moths became more common. Which variety is more common in England nowadays? Give a reason as well. After the 1950s, England industrial pollution was reduced and as a result the number of light-colored peppered moths became more common again. This is because the tree trunks became cleaner and lichen growth increased, making the light-colored peppered moth better camouflaged. Define/Describe/Explain briefly: evolution, homologous organs, speciation, natural selection, artificial selection, industrial melanism, paleontology, comparative anatomy, vestigial organs, selective breeding, Evolution The slow change in living things over time, leads to new species. It happens as organisms adapt to their environment and pass on traits to their offspring. Homologous Organs Body parts in different animals or plants that have a similar structure but do different jobs. Example: A bat’s wing and a human’s arm are homologous because they have the same bone structure but different functions. Speciation The process by which new species are formed. This happens when groups of the same species become so different that they can no longer reproduce together. Natural Selection A process where organisms with traits that help them survive (like better camouflage) are more likely to reproduce and pass those traits to their offspring. Artificial Selection When humans choose specific traits in plants or animals and breed them to produce offspring with those traits. Example: Breeding dogs for size or farming crops for sweetness. Industrial Melanism A change in the color of species (like moths) due to industrial pollution. Example: Dark-colored moths became more common during the Industrial Revolution because they were harder for predators to see on polluted, dark trees. Palaeontology The study of fossils (remains of extinct plants and animals) to learn about life from the past and how it has changed over time. Comparative Anatomy Comparing the body structures of different animals to understand how they are related. Example: Similar bone patterns in a human arm, a dogs leg, and a whale’s flipper show they share a common ancestor. Vestigial Organs Body parts that no longer have a use but were useful to ancestors. Example: The human appendix, wings of flightless birds, leaves of cacti. Selective Breeding When humans choose certain plants or animals with specific traits to breed, creating new varieties or breeds over time. Example: Growing bigger fruits or breeding cows for more milk. Differentiate between natural and artificial selection. Natural Selection Artificial selection Selection occurs when natural environmental conditions changes. It is selected by man. Varieties are produced by mutations. Varieties are produced by selective breeding. It operates in a natural population. It operates in a domesticated population. It is a slow process and takes thousands to millions of years. It is a fast process, and results are immediate (in the next generation). It is responsible for the great biological diversity. It is responsible for new varieties different from starting generation. Section III: Extensive Answer Questions What is variation? Explain the sources of variation. Ans; The term variation refers to the differences in traits displayed by members of the same species. For instance, although all domestic cats are members of the same species, they differ greatly in terms of size, coat colour, eye colour, hair length, and other characteristics. Sources of variations Genetic recombination results from crossing over during gamete development. Different chromosome combinations in gametes are formed by independentcollections of chromosomes. The process of fertilization in which one of the countless sperms’ fuses with an egg. A mutation in the gamete-forming germ cells. Gene flow, or the transfer of genes from one population of a species to another. Assess selection as a possible means of evolution. Selection is one of the main ways evolution happens. It decides which traits get passed on to the next generation. There are two main types of selection: natural selection and artificial selection. Natural Selection In nature, some organisms have traits that help them survive better in their environment. These organisms are more likely to survive, reproduce, and pass on those helpful traits to their offspring. Over many generations, these traits become more common, and the species evolves. Artificial Selection Humans choose which traits they want in plants or animals and breed only those with those traits. This changes the species over time, but it’s guided by humans, not nature. Explain the theory of evolution by natural selection with examples. Theory Natural Selection Natural selection is the process through which organisms who possess traits that are helpful for

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Cell Signaling | Class 11 Biology Notes

All the cells in our bodies communicate with each other all the time. Every cell in the multicellular organism’s body sends and receives millions of messages through chemical signalling molecules. A process by which cells in multicellular organisms send and receive messages from each other or with the external environment is called cell signalling. It is found in all multicellular organisms and is absent in unicellular. Because of cell signalling, our body acts as one unit rather than as a collection of individual cells acting independently. Ligands & ReceptorsThe cell that wants to communicate with other cells sends a signalling molecule called ligands. This molecule initiates signalling and triggers a specific response in the target cell. Ligands can be proteins, lipids, amino acids, metabolites, gas, or any other molecule. It is carried by blood or tissue fluid towards target cells. Signalling molecules or ligands pass through thousands of cells; however, they bind only with a specific protein called a receptor (located in/on the target cell). Every ligand requires specific receptors. Events of cell signalling at the target site;Events of cell signalling at the target site are divided into three stages: reception, transduction, and response.Reception: Reception is the target cell’s detection of a signalling molecule coming from outside the cell. A chemical signal is “detected” when the signalling molecule binds to a receptor protein located at the cell’s surface (or inside the cell).Transduction: The binding of the signalling molecule changes the receptor protein in some way, initiating the process of transduction. The transduction stage converts the signal to a form that can bring about a specific cellular response.Response: In the third stage of cell signalling, the transduced signal finally triggers a specific cellular response. Different ligands trigger different responses, such as catalysis by an enzyme, rearrangement of the cytoskeleton, or activation of specific genes in the nucleus. Pathway of a signal from outside to inside; Transduction sometimes occurs in a single step but more often requires a sequence of changes in a series of different molecules called the signal transduction pathways. The molecules in the pathway are called relay molecules.Protein SignalingProtein and peptide hormones are water-soluble, so they cannot pass through the plasma membrane of the cell. These hormones or environmental stimuli are the first messenger. They bind with their receptors on the plasma membrane of the target cell, starting a series of events in the cell that generate a second messenger, e.g., cAMP (cyclic adenosine monophosphate). The second messenger then triggers various changes in the cell, including activation of enzymes, gene activation, etc. Federal Board (FBISE) Islamabad | National Books Foundation Biology Notes