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Biotechnology: Principles and Processes - Processes of Recombinant DNA Technology (PCR, Bioreactors)

Grade 12CBSEBiology

Review the key concepts, formulae, and examples before starting your quiz.

🔑Concepts

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Isolation of Genetic Material: DNA is released by treating cells with enzymes like LysozymeLysozyme (bacteria), CellulaseCellulase (plant cells), or ChitinaseChitinase (fungus). Purified DNA is precipitated using chilled C2H5OHC_2H_5OH (ethanol).

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PCR (Polymerase Chain Reaction): A technique for in-vitro amplification of a specific DNA segment. It involves three main steps: Denaturation, Annealing, and Extension.

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Denaturation: The double-stranded DNA (dsDNA) is heated to approximately 94∘C94^\circ C to separate the strands by breaking hydrogen bonds.

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Annealing: Two sets of oligonucleotide primers bind to the complementary regions on the single-stranded DNA (ssDNA) templates at a lower temperature (usually 50−65∘C50-65^\circ C).

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Extension: The enzyme TaqTaq polymerase (thermostable DNA polymerase from Thermus aquaticusThermus\ aquaticus) adds dNTPsdNTPs (deoxynucleoside triphosphates) to the primers at 72∘C72^\circ C, extending the DNA sequence.

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Bioreactors: Large-scale vessels (100−1000100 - 1000 liters) used to biologically convert raw materials into specific products (proteins/enzymes) under optimal conditions of pHpH, temperature, substrate, and O2O_2.

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Stirred-tank Bioreactor: A cylindrical vessel with a curved base to facilitate mixing. It contains an agitator system and an oxygen delivery system to maintain aerobic conditions.

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Sparged stirred-tank Bioreactor: A variation where sterile air is bubbled through the system. The bubbles increase the surface area for O2O_2 transfer.

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Downstream Processing: A series of processes including separation and purification of the product, followed by formulation with suitable preservatives and clinical trials for quality control.

📐Formulae

N=2nN = 2^n

Where N=Total number of DNA molecules produced\text{Where } N = \text{Total number of DNA molecules produced}

and n=Number of PCR cycles completed\text{and } n = \text{Number of PCR cycles completed}

💡Examples

Problem 1:

Starting with a single molecule of double-stranded DNA, calculate the theoretical number of DNA copies produced after 3030 cycles of PCR.

Solution:

N=230≈1.07×109N = 2^{30} \approx 1.07 \times 10^9

Explanation:

Using the exponential amplification formula 2n2^n, where n=30n = 30, the DNA is doubled in every cycle, resulting in approximately one billion copies.

Problem 2:

Identify the primary advantage of using a 'sparged' bioreactor over a simple stirred-tank bioreactor regarding gas exchange.

Solution:

Increased O2O_2 transfer area.

Explanation:

In a sparged bioreactor, air is bubbled through the medium. These bubbles significantly increase the surface area for the transfer of O2O_2 (oxygen) into the liquid culture, supporting higher cell densities.

Problem 3:

Why is TaqTaq polymerase used in PCR instead of human DNA polymerase?

Solution:

Thermostability at 94∘C94^\circ C.

Explanation:

PCR requires a high temperature (94∘C94^\circ C) for denaturation. Human DNA polymerase would denature and lose activity at this temperature, whereas TaqTaq polymerase remains stable and active throughout the repeated heating and cooling cycles.

Processes of Recombinant DNA Technology (PCR, Bioreactors) Revision - Class 12 Biology CBSE