• Inverse PCR: A variant of PCR used to amplify unknown DNA flanking regions that lie on either side of a known core sequence.
• Mechanism: The target genomic DNA is digested with a restriction enzyme cutting outside the known sequence, circularized by self-ligation using T4 DNA ligase, and then amplified using primers pointing outwards (divergent primers) from the known segment.
• Other PCR Variants:
• Nested PCR: Uses two successive sets of primers (outer and inner) targeting the same region to enhance specificity and yield.
• RT-PCR: Reverse Transcription PCR, uses reverse transcriptase to synthesize cDNA from an RNA template before PCR amplification.
• Anchored PCR: Amplifies sequence when only one end has a known sequence by attaching an artificial poly-G or adaptor anchor.

Correct option: (2) Inverse PCR

• Transposase Enzyme Functions: Transposase is a site-specific recombinase encoded by DNA transposons (such as bacterial IS elements and Tn transposons) that catalyzes cut-and-paste transposition:

• Restriction / Cleavage of IS Elements: Specifically recognizes and binds terminal inverted repeats (TIRs), excising the transposon from the donor site.
• Target Site Cleavage / Restriction: Creates staggered nicks in the host genomic target DNA.
• Integration: Ligation of the excised transposon into the cleaved target site, leading to target site duplications (TSDs).
  • Function Not Performed: Formation of RNA intermediate is performed by reverse transcriptase in retrotransposons (Class I transposable elements, e.g., LINEs, SINEs, LTR retrotransposons), not by DNA transposases (Class II).

• Thermodynamic Basis of DNA Stability:
• Base pairing via hydrogen bonds (2 between A-T, 3 between G-C) provides structural fidelity, sequence specificity, and precise alignment of keto/amino tautomers.
• When single strands hybridize into an ordered double helix, conformational entropy of the polynucleotide backbone decreases ($\Delta S_{\text{chains}} \lt 0$). However, the release of ordered water molecules from non-polar surfaces during hydrophobic base stacking provides a favorable net entropic gain ($\Delta S_{\text{solvent}} \gt 0$).
• False Statement: Stating simply that hydrogen bonding "decreases the entropy" is false or misleading; the net thermodynamic stability ($\Delta G = \Delta H - T\Delta S$) is primarily driven by favorable negative enthalpy (hydrogen bonding and van der Waals stacking) and hydrophobic effect.

Correct option: (3) Decreases the entropy