Amplification methods: Which molecular technique selectively amplifies a defined DNA segment located between two designed oligonucleotide primers?

Introduction / Context:
The polymerase chain reaction (PCR) enables exponential amplification of a specific DNA region using thermostable DNA polymerase and two primers that flank the target. PCR revolutionized diagnostics, forensics, cloning, and sequencing by making tiny amounts of DNA analyzable and manipulable in hours.

Given Data / Assumptions:

• A double-stranded DNA template contains the sequence of interest.
• Two primers are complementary to opposite strands, oriented toward each other.
• Thermostable polymerase, dNTPs, buffer, and thermal cycling are available.

Concept / Approach:
PCR cycles through denaturation, annealing, and extension, doubling the number of target molecules each cycle. Specificity derives from primer–template base pairing and annealing temperature. The method amplifies only the segment between the primer binding sites, enabling downstream analysis or cloning.

Step-by-Step Solution:

Verification / Alternative check:
Gel electrophoresis shows a single amplicon of expected size; Sanger sequencing confirms identity. Negative controls verify that amplification is primer-dependent.

Why Other Options Are Wrong:

• Southern blotting: detects DNA fragments by hybridization; does not amplify.
• Northern blotting: detects RNA transcripts by hybridization; no amplification.
• In vivo replication: copies entire genomes, not targeted segments with primers.
• In situ hybridization: localizes sequences in cells or tissues without amplification (unless combined with PCR variants).

Common Pitfalls:
Primer-dimer formation, nonspecific bands, and contamination; optimize annealing temperature and design primers carefully to ensure specificity.

Final Answer:
Polymerase chain reaction