<h4 id="successbiotechnology-principles-and-processessuccess"><Success>Biotechnology Principles and Processes</Success></h4> <h3 id="successpart-2success"><Success>Part 2</Success></h3> <!--<div style='float: left; text-align: left; font-size: 30px; width:50%;'>--> <!--</div>--> <div style='float: right; width:0%;'> <p><img alt="image" src="https://temp-public-img-folder.s3.ap-south-1.amazonaws.com/sathee.prutor.images/subject-images/iitpal/image/biology-class-12-unit-11-chapter-02-biotechnology-principles-and-processes-lecture-2_6-rzulxvdakjk-1.jpg">

<h3 id="successgenetic-engineeringsuccess"><Success>Genetic engineering</Success></h3> <div style='float: left; text-align: left; font-size: 30px; width:50%;'> <p><strong>Tools</strong></p> <ul> <li>Polymerases</li> <li>Restriction Enzymes</li> <li>Ligase</li> <li>Vectors</li> </ul> </div> <div style='float: right; width:50%;'> <img src="https://imagedelivery.net/YfdZ0yYuJi8R0IouXWrMsA/50604b93-d64c-4977-48e7-7371c83b8400/public" width="100%"> <!---->

<div> <h3 id="successgenetic-engineeringsuccess-1"><Success>Genetic engineering</Success></h3> </div> <div style='float: left; width:100%;font-size:30px;text-align:left;'> <p><strong>Procedures</strong></p> <ul> <li>Isolation of Genomic DNA</li> <li>Polymerase chain reactions</li> <li>Restriction Digestion</li> <li>Ligation</li> <li>Competent Cells Preparation</li> <li>Transformation</li> <li>Screening of Clones</li> </ul> </div>

<h3 id="successpolymerasessuccess"><Success>Polymerases</Success></h3> <div style='float: left; text-align: left; font-size: 30px; width:50%;'> <p>The enzyme synthesize double-stranded DNA molecules (fragments) with same (identical) size and sequence</p> </div> <div style='float: right; width:50%;'> <!----> <img src="https://imagedelivery.net/YfdZ0yYuJi8R0IouXWrMsA/ee397dd9-ce1c-4e4f-9347-050e4b19f900/public" width="100%"> </div>

<h4 id="successdna-synthesis-is-linked-to-cell-division-and-growthsuccess"><Success>DNA synthesis is linked to cell division and growth</Success></h4> <div style='float: left; text-align: left; font-size: 30px; width:50%;'> <ul> <li>Plasma membrane</li> <li>Cell wall</li> <li>Duplication of chromosome</li> <li>Continued growth of cell</li> <li>Division into two cells</li> </ul> </div> <div style='float: right; width:50%;'> <img src="https://imagedelivery.net/YfdZ0yYuJi8R0IouXWrMsA/963592af-d169-4c4e-bcd6-e06b321f5b00/public" width="100%"> <!---->

<h3 id="successdna-replicationsuccess"><Success>DNA replication</Success></h3> <div style='float: left; text-align: left; font-size: 30px; width:100%;'> <p>Process of duplication of the entire genome prior to cell division</p> <p><strong>Biological significance</strong></p> <ul> <li> <p>Extreme accuracy of DNA replication is necessary in order to preserve the integrity of the genome in successive generations</p> </li> <li> <p>In eukaryotes, replication only occurs during the S phase of the cell cycle</p> </li> <li> <p>Replication rate in eukaryotes is slower resulting in a higher fidelity/accuracy of replication in eukaryotes</p> </li> </ul> </div> <div style='float: right; width:0%;'> <p><img alt="image" src="https://temp-public-img-folder.s3.ap-south-1.amazonaws.com/sathee.prutor.images/subject-images/iitpal/image/biology-class-12-unit-11-chapter-02-biotechnology-principles-and-processes-lecture-2_6-rzulxvdakjk-5.jpg"></p> </div>

<h3 id="successdna-replicationsuccess-1"><Success>DNA replication</Success></h3> <div style='float: left; text-align: left; font-size: 30px; width:100%;'> <p><strong>3 Steps event</strong></p> <p><strong>Initiation Prepare DNA for Synthesis</strong></p> <ul> <li>Proteins bind to DNA</li> <li>Opening of double helix DNA</li> </ul> <p><strong>Elongation Synthesis Phase</strong></p> <ul> <li>Proteins connect the correct sequences of nucleotides into a continuous new strand of DNA</li> </ul> <p><strong>Termination Stopping of DNA synthesis</strong></p> <ul> <li>Proteins release the replication complex</li> </ul> </div> <div style='float: right; width:0%;'> <p><img alt="image" src="https://temp-public-img-folder.s3.ap-south-1.amazonaws.com/sathee.prutor.images/subject-images/iitpal/image/biology-class-12-unit-11-chapter-02-biotechnology-principles-and-processes-lecture-2_6-rzulxvdakjk-6.jpg"></p> </div>

<h3 id="successdna-replicationsuccess-2"><Success>DNA replication</Success></h3> <div style='float: left; text-align: left; font-size: 30px; width:100%;'> <ul> <li> <p>Helicases - separates 2 strands</p> </li> <li> <p>Primase - RNA primer synthesis</p> </li> <li> <p>SSB Protein - prevent re-annealing of single strands</p> </li> <li> <p>DNA polymerase - synthesis of new strand</p> </li> <li> <p>Tethering protein - stabilises polymerase</p> </li> </ul> </div> <div style='float: right; width:0%;'> <p><img alt="image" src="https://temp-public-img-folder.s3.ap-south-1.amazonaws.com/sathee.prutor.images/subject-images/iitpal/image/biology-class-12-unit-11-chapter-02-biotechnology-principles-and-processes-lecture-2_6-rzulxvdakjk-7.jpg"></p> </div>

<h4 id="successrole-of-dna-polymerase-in-genetic-engineeringsuccess"><Success>Role of dna polymerase in genetic engineering</Success></h4> <div style='float: left; text-align: left; font-size: 30px; width:50%;'> </div> <div style='float: right; width:100%;'> <!----> <img src="https://imagedelivery.net/YfdZ0yYuJi8R0IouXWrMsA/726a59b9-4886-491b-56dc-5b6d72ba9400/public" width="60%"> </div>

<h3 id="successrestriction-methylase-systemsuccess"><Success>Restriction-methylase system</Success></h3> <div style='float: left; text-align: left; font-size: 30px; width:100%;'> <p><strong>Distinction between self Vs Foreign DNA</strong></p> <ul> <li> <p>Although the precised mechanism of distinction is not known but based on available literature</p> </li> <li> <p>In the absence of methylation a closed complex is formed and it allowes the proper activation of clevage activity of the enzyme</p> </li> <li> <p>But presence of methyl group (even hemi- methylation) on nucleotide does not allow formation of closed complex and consequently enzyme falls from the DNA</p> </li> </ul> </div> <div style='float: right; width:0%;'> <p><img alt="image" src="https://temp-public-img-folder.s3.ap-south-1.amazonaws.com/sathee.prutor.images/subject-images/iitpal/image/biology-class-12-unit-11-chapter-02-biotechnology-principles-and-processes-lecture-2_6-rzulxvdakjk-10.jpg"></p> </div>

<h3 id="successrestriction-methylase-systemsuccess-1"><Success>Restriction-methylase system</Success></h3> <div style='float: left; text-align: left; font-size: 30px; width:50%;'> <p><strong>1. Recognition of cleavage site</strong> Type II restriction enzymes have specific recognition DNA sequences to rapidly identify the site, enzyme scan the strecth of DNA through a non-specific interaction and diffusion along the length of DNA To expedite the recognition process, enzyme does not makes interaction with the bases, instead of it makes contact with DNA back bone</p> </div> <div style='float: right; width:50%;'> <img src="https://imagedelivery.net/YfdZ0yYuJi8R0IouXWrMsA/ce1904ce-78af-4daa-760a-cc7774191f00/public" width="100%"> <!----> </div>

<div> <h4 id="successrestriction-methylase-systemsuccess-2"><Success>Restriction-methylase system</Success></h4> </div> <div style='float: left; width:100%;font-size:30px;text-align:left;'> <p><strong>2. Binding to the recognition site</strong> Once the recognition site is located, enzymes makes specific interaction with the nucleotides present at the recognition site via entry through major groove Hydrogen bonding, van der waals interaction plays crucial role in this step Enzyme DNA closed complex formation induces major conformational changes in restriction enzyme</p> <p><strong>3. Cleavage reaction</strong> The closed complex activates the cleavage activity of the enzyme resulting into the introduction of DNA break on both strand to give fragment with and a 5’-phosphate group</p> </div>

<h4 id="successrestriction-methylase-systemsuccess-3"><Success>Restriction-methylase system</Success></h4> <div style='float: left; text-align: left; font-size: 30px; width:100%;'> <p><strong>Nomenclature of restriction enzymes</strong> Due to extensive search of presence of restriction enzyme in different microorganisms, a nomenclature system has been adopted. In this system, first alphabet represents name of the genus, 2nd alphabet represents species, 3rd alphabet gives information about strain and fourth is the order in which enzyme has been isolated from the particular micro-organism</p> <p><strong>For example</strong></p> <ul> <li>EcoR-1, E = <em>Escherichia</em> (genus)</li> <li>Co = <em>Coli</em> (species)</li> <li>R = RY13 (strain)</li> <li>I = it is the first restriction enzyme from this bacteria</li> </ul> </div> <div style='float: right; width:0%;'> <p><img alt="image" src="https://temp-public-img-folder.s3.ap-south-1.amazonaws.com/sathee.prutor.images/subject-images/iitpal/image/biology-class-12-unit-11-chapter-02-biotechnology-principles-and-processes-lecture-2_6-rzulxvdakjk-12.jpg"></p> </div>

<h3 id="successrestriction-methylase-systemsuccess-4"><Success>Restriction-methylase system</Success></h3> <div style='float: left; text-align: left; font-size: 30px; width:100%;'> <p><strong>Different types of restriction enzymes</strong> Restriction enzymes vary in the structure, recognition site, cutting site and cofactor requirement</p> <p><strong>TYPE-I Restriction Enzymes</strong> The recognition site of type-I enzyme consists of 3-4 nucleotides at 3’ end followed by a non-specific stretch of 6-8 nucleotide and a 4 nucleotide at 5’ The cleavage site is ~1000bp away from recognition site and it is presumed that cleavage follow a DNA translocation of enzyme</p> </div> <div style='float: right; width:0%;'> <p><img alt="image" src="https://temp-public-img-folder.s3.ap-south-1.amazonaws.com/sathee.prutor.images/subject-images/iitpal/image/biology-class-12-unit-11-chapter-02-biotechnology-principles-and-processes-lecture-2_6-rzulxvdakjk-13.jpg"></p> </div>

<div> <h3 id="successrestriction-methylase-systemsuccess-5"><Success>Restriction-methylase system</Success></h3> </div> <div style='float: left; width:100%;font-size:30px;text-align:left;'> <p><strong>Type-III Restriction Enzymes</strong> The recognition site has two separate non-palindromic sequence arranged inversely oriented The cutting site is ~20-30 base pair away from the recognition site</p> <p><strong>Type-II Restriction Enzymes</strong> The recognition site of type-II enzyme is 4-8 nucleotide long and it cuts the DNA within the specific site Due to this feature, type-II restriction enzymes have application in genetic engineering for cloning purpose</p> </div>

<h3 id="successrestriction-methylase-systemsuccess-6"><Success>Restriction-methylase system</Success></h3> <div style='float: left; text-align: left; font-size: 30px; width:50%;'> </div> <div style='float: right; width:100%;'> <img src="https://imagedelivery.net/YfdZ0yYuJi8R0IouXWrMsA/66f21570-c863-43e4-23fe-e686091de100/public" width="60%"> <!----> </div>

<h4 id="successproperties-of-type-ii-restriction-enzymessuccess"><Success>Properties of type II restriction enzymes</Success></h4> <div style='float: left; text-align: left; font-size: 30px; width:50%;'> <p><strong>Palindromic Sequence</strong> The recognition sequence of type II restriction enzyme is palindromic It means that the sequence read out will be same in forward and reverse direction For example Bam H-I has a recognition site as GGATTC and as shown below, black arrow on strand 1 and 2 will read same sequence Same is applicable to the blue arrow as well</p> </div> <div style='float: right; width:50%;'> <img src="https://imagedelivery.net/YfdZ0yYuJi8R0IouXWrMsA/a92a1adb-6fc7-458b-873d-f7b53f4a9600/public" width="100%"> <!----> </div>

<h4 id="successproperties-of-type-ii-restriction-enzymessuccess-1"><Success>Properties of type II restriction enzymes</Success></h4> <div style='float: left; text-align: left; font-size: 30px; width:50%;'> <p><strong>Sticky Ends</strong> The type II restriction enzymes cut the both DNA strand together to generates DNA with hanging DNA stretch with 4-6 nucleotide These DNA stretch containing fragments are cohesive to each other as sequence present on complex 1 will be complementary to the sequence present on complex 2</p> </div> <div style='float: right; width:50%;'> <img src="https://imagedelivery.net/YfdZ0yYuJi8R0IouXWrMsA/c2e80081-a236-49a2-a273-bcd03a4eda00/public" width="60%"> <!----> </div>

<div style='float: left;width:100%;'> <h3 id="successthank-yousuccess"><Success>Thank you</Success></h3> <div style='float: right; width:0%;'> <!----> </div>