• Target Gene Amplification Workflow:

• Primer Synthesis: Before initiating PCR amplification, oligonucleotide primers flanking the target gene sequence must be designed and synthesized.
• Separating Two Strands of DNA (Denaturation): The double-stranded template DNA is heated to $94\text{--}95^\circ\text{C}$ to break hydrogen bonds and produce single-stranded templates.
• Annealing with Template: The reaction is cooled to $50\text{--}65^\circ\text{C}$ to allow forward and reverse primers to hybridize specifically to their complementary target sequences.
• DNA Polymerase Action (Extension): Thermostable DNA polymerase (e.g., Taq polymerase) extends the primers from $5'$ to $3'$ at $72^\circ\text{C}$ by incorporating dNTPs.
  • Correct Order: (B) $\to$ (A) $\to$ (D) $\to$ (C).

• Gene Pool: The total collection of all genes and alleles present in a breeding population at a given time is termed the gene pool.
• Related Genetic Concepts:
• Genotype: The specific allelic constitution of an individual organism at one or more loci.
• Phenotype: The observable physical, physiological, or biochemical characteristics of an individual resulting from its genotype interacting with the environment ($P = G + E$).
• Breeding Value ($A$): The value of an individual's genes judged by the mean phenotypic value of its progeny; represents the sum of the average effects of the genes it transmits to offspring.

Correct option: (1) Gene Pool

• cDNA Library Construction Sequence:

• Elution of mRNA: Total cellular RNA is isolated, and mature polyadenylated mRNA is selectively purified using oligo(dT) cellulose affinity chromatography.
• Action of Reverse Transcriptase: Reverse transcriptase synthesizes a complementary DNA strand (cDNA) from the mRNA template using an oligo(dT) primer, followed by second-strand DNA synthesis.
• Incorporate cDNA into Vector: Double-stranded cDNA is ligated with linkers/adaptors and inserted into a cloning vector (e.g., plasmid or bacteriophage $\lambda$).
• Clone cDNA: Recombinant vectors are transformed into host cells (e.g., E. coli) and propagated as individual cDNA clones.
  • Correct Chronological Sequence: (D) $\to$ (B) $\to$ (C) $\to$ (A).