biology-class-12-unit-11-chapter-05-biotechnology-principles-and-processes-lecture-5-6-kkxwmqfhnca

<h3 id="successgenetic-engineeringsuccess-1"><Success>Genetic engineering</Success></h3>
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<ul>
<li>Procedures
<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>
</li>
</ul>
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<h3 id="success-dna-success"><success> DNA </success></h3>
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<p>T- DNA is a nucleic acid that is composed of two complementary nucleotide building block chains</p>
<ul>
<li>The nucleotides are made up of a phosphate group, a five carbon sugar, and a nitrogen base</li>
</ul>
<p><strong>DNA has four nitrogen bases</strong></p>
<ul>
<li>
<p>Two are purines (2 ringed base) - Adenine (A), Guanine (G)</p>
</li>
<li>
<p>Two are pyrimidines (1 ringed base) - Cytosine (C), Thymine (T)</p>
</li>
<li>
<p>These four bases are linked in a repeated pattern by hydrogen bonding between the nitrogen bases</p>
</li>
<li>
<p>The linking of the two complementary strands is called hybridization</p>
</li>
</ul>
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<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-05-biotechnology-principles-and-processes-lecture-5_6-kkxwmqfhnca-3.jpg"></p>
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<h3 id="success-polymerase-chain-reaction-success"><success> Polymerase chain reaction </success></h3>
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<ul>
<li>PCR is a repeated cycle reaction that involves mechanism of DNA replication</li>
<li>It results in production of multiple copies of DNA from a single one</li>
<li>The whole process involves three main events, denaturation, annealing and elongation</li>
<li>A DNA fragment of interest is used as a template from which a pair of primers or short oligonucleotides complimentary to the both the double strands of the DNA are made to prime the DNA synthesis where the direction of synthesis or extension is from 5’ to 3’ as in DNA replication</li>
<li>The number of amplified DNA or the amplicons increases exponentially per cyele thus one molecule of DNA give rise to 2,4,8,16 and so forth Amount of amplified DNA</li>
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<h3 id="success-polymerase-chain-reactionsuccess-1"><success> Polymerase chain reaction</success></h3>
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<li><strong>Initial Denaturation</strong> Heating the PCR mixture at 94°C to 96°C for 10min to ensure complete denaturation of template DNA</li>
<li><strong>Denaturation</strong> This is the first step in which the double stranded DNA template is denatured to form two single strand by heating at 95°C for 15-30 secs</li>
<li><strong>Annealing</strong> This is the annealing step where at lower temperature (usually 50-65°C) primers are allowed to bind to template DNA, annealing time is 15-30 secs and it depends on the length and bases of the primers</li>
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<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-05-biotechnology-principles-and-processes-lecture-5_6-kkxwmqfhnca-9.jpg"></p>
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<h3 id="successanalysis-of-pcr-reactionsuccess"><Success>Analysis of PCR reaction</Success></h3>
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<p><strong>Analysis of PCR results</strong></p>
<p>Once PCR cycle is completed, the amplified product is loaded in the agarose gel and observed after ethidium bromide staining under UV light source A water blank reaction is included to monitor the cross contaminating DNA source as template The percentage of agarose gel depends on the size of DNA to be visualized Generally 0.8-1% agarose gel is used for analyzing 0.5-5 kb amplified DNA while a DNA of larger size or genomic DNA is visualized in gel as low as 0.5%</p>
<ul>
<li>Speed</li>
<li>Ease of use</li>
<li>Sensitivity</li>
<li>Robustness</li>
</ul>
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