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Polymerase Chain Reaction (PCR) Ingredients

• DNA Template: Segment of DNA targeted for amplification.
• Primers: Short nucleotide sequences that initiate DNA synthesis.
• Taq Polymerase: Heat-stable enzyme that synthesizes new DNA strands.
• Deoxynucleoside Triphosphates (dNTPs): Building blocks for new DNA strands (dATP, dCTP, dGTP, dTTP).
• Buffer Solution: Maintains optimal pH and ionic strength for the PCR reaction.
• Magnesium Ions (Mg²⁺): Cofactor essential for Taq polymerase activity.
• Water: Serves as a solvent for the PCR reaction components.

Major Steps of PCR

• Denaturation (94-98°C): DNA strands are separated by breaking hydrogen bonds, creating single-stranded DNA.
• Annealing (50-65°C): Primers attach to their complementary sequences on the single-stranded DNA templates.
• Extension (72°C): Taq polymerase synthesizes new DNA strands by adding nucleotides to the primers.

Applications of PCR

• Diagnosis of Infectious Diseases: Identifies pathogens (viruses/bacteria) in clinical samples.
• Genetic Testing: Detects genetic mutations linked to hereditary diseases.

Taq Polymerase Characteristics

• Thermal Stability: Taq polymerase can endure high temperatures (94-98°C) during the denaturation step without denaturing itself, making it ideal for PCR.

Limitations of PCR

• Contamination Sensitivity: High sensitivity to contamination from other DNA can result in misleading results.
• Prior Knowledge Requirement: Necessitates prior knowledge of target DNA sequence for primer design, limiting use for unknown sequences.

Types of DNA Libraries

• Genomic Library: Contains all DNA sequences from an organism, useful for genome mapping and studying regulatory elements.
• cDNA Library: Comprises only expressed genes (mRNA) converted to DNA, important for analyzing gene expression and protein coding sequences.

Advantages of cDNA Libraries

• Focus on Expressed Genes: Concentrates on active genes, providing relevant information.
• Absence of Introns: Facilitates easier analysis due to the lack of non-coding regions.

Variations of PCR

• Reverse Transcription PCR: Converts RNA into DNA before amplification.
• Real-time PCR: Monitors the amplification process in real-time for quantification.
• Nested PCR: Increases sensitivity and specificity through a two-step amplification process.