Allele-specific detection — Some genetic diseases cannot be diagnosed by changes in restriction sites. What are allele-specific oligonucleotide (ASO) probes used for in such cases?

Difficulty: Easy

They are altered copies of a gene that insert a new restriction site for detection
They are mutagenized full-length genes used as functional replacements
They are short DNA sequences that hybridize only to a specific target base sequence (often distinguishing single-base variants)
They are PCR-amplified variable number tandem repeats (VNTRs)
They are antibodies used to detect protein isoforms in a Western blot

Correct Answer: They are short DNA sequences that hybridize only to a specific target base sequence (often distinguishing single-base variants)

Explanation:

Introduction:
Not all pathogenic variants create or abolish restriction enzyme sites. Allele-specific oligonucleotide (ASO) probes allow detection of known sequence variants, including single-nucleotide changes, by stringent hybridization. This question clarifies what ASO probes are and how they function in diagnostics.

Given Data / Assumptions:

ASO probes are short (typically 15–25 nt) and designed to match either the mutant or wild-type allele exactly.
Hybridization stringency can discriminate single-base mismatches.
They can be used on blots or dot blots, or combined with PCR for targeted genotyping.

Concept / Approach:
Because ASOs bind only their perfectly complementary sequence under chosen conditions, they enable yes/no detection of a particular allele regardless of nearby restriction sites. This is especially useful when the mutation does not alter an enzyme motif (i.e., is not an RFLP).

Step-by-Step Solution:

1) Design two probes: one perfectly matching the wild type, one matching the mutant.2) Hybridize each probe to DNA immobilized on a membrane or to PCR products under high-stringency conditions.3) Detect probe binding via label (radioactive, fluorescent, or enzymatic) to determine genotype.

Verification / Alternative check:
ASO-based genotyping is widely used for known point mutations (e.g., specific CFTR variants), with controls verifying stringency and specificity.

Why Other Options Are Wrong:

a) Inserting restriction sites is neither necessary nor typical for diagnostics.b) Functional gene replacement is gene therapy, not allele detection.d) VNTR analysis detects length polymorphisms, not single-base alleles by hybridization.e) Antibodies detect proteins, not DNA alleles.

Common Pitfalls:
Confusing ASO probes with restriction-based RFLP assays; using low stringency that fails to discriminate single-base differences.

Final Answer:
Short oligonucleotides that hybridize specifically to a target allele, enabling single-base variant detection.

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Allele-specific detection — Some genetic diseases cannot be diagnosed by changes in restriction sites. What are allele-specific oligonucleotide (ASO) probes used for in such cases?

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