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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.