5. If substitution is the only possible type of mutation, then which technique would be no more useful for detecting them?
(1) DNA sequencing
(2) PCR amplification
(3) Database searching
(4) Restriction fragment analysis

Question and Correct Answer

In scenarios where substitution is the only possible mutation type, restriction fragment analysis (option 4) becomes no more useful for detection. Substitution mutations involve single nucleotide changes that may not alter restriction enzyme recognition sites, rendering this method ineffective without accompanying insertions or deletions. Other techniques remain viable as they directly identify base changes or compare sequences.

Option-Wise Detailed Explanation

DNA Sequencing (Option 1)

DNA sequencing reads the exact nucleotide order, directly revealing substitution mutations by comparing to reference sequences. It excels at pinpointing point mutations regardless of type, making it highly useful even for substitutions alone.

PCR Amplification (Option 2)

Standard PCR amplifies DNA but does not detect mutations itself; however, modified versions like allele-specific PCR (AS-PCR), mismatch amplification, or real-time PCR with blockers selectively amplify mutant alleles containing substitutions. These adaptations make PCR useful for substitution detection in isolation.

Database Searching (Option 3)

Database searching compares query sequences against known databases to identify substitutions via alignment tools like BLAST, flagging mismatches as potential mutations. This bioinformatics approach remains effective solely for substitutions by highlighting sequence variations.

Restriction Fragment Analysis (Option 4)

This technique relies on mutations creating or destroying restriction sites, typically from insertions, deletions, or frameshifts that change fragment lengths on gels. Substitutions rarely affect sites unless they coincidentally alter enzyme recognition, so it loses utility when substitutions are the only mutation type.