80 To detect mutation (GAGàGTG) allele specific hybridization method is used. Four members of an affected family are investigated. DNA isolated from blood samples of parents and two offsprings are spotted on a membrane after appropriate processing and probed with either TGACTCCTGAGGAGAAGTC (first probe) or TGACTCCTGTGGAGAAGTC (second probe) after labelling. While probed with first oligonucleotide. signals are obtained for the positions where DNA are spotted from parents and offspring II. When probed with second oligonucleotide, signals are obtained at position where DNA from the parents and offspring I are spotted. Results are shown below:

	Father	Mother	Off-spring I	Off-spring II
First Probe	+	+	–	+
Second Probe	+	+	+	–

On the basis of the result, which of the following statements is correct?
(1) Parents are affected
(2) Offspring I is affected
(3) Offspring II is carrier
(2) Offspring II is affected

The GAG→GTG base change is the classic β-globin mutation for sickle hemoglobin (HbS), converting glutamic acid to valine at position 6 of β-chain. Allele-specific oligonucleotide (ASO) probes bind only to perfectly matched sequences under stringent washes, allowing separate detection of normal and mutant alleles on duplicate dot blots.

Given data

• Two ASO probes are used: first probe complementary to one allele sequence and second probe complementary to the other allele. Signals appear at the sample spots only when the corresponding allele is present in that DNA.

• Reported signals:

  • First probe: Father +, Mother +, Offspring I −, Offspring II +.

  • Second probe: Father +, Mother +, Offspring I +, Offspring II −.
    This is the classic pattern where each parent shows both alleles (both probes positive), a heterozygous state.

How ASO patterns map to genotype

• Positive with only the “normal” probe → homozygous normal (AA).

• Positive with only the “mutant” probe → homozygous mutant (SS).

• Positive with both probes → heterozygous carrier (AS).
  These readouts are a standard property of ASO dot-blot assays used for sickle mutation testing.

Interpreting each family member

• Father: + with first and + with second → carries both alleles (heterozygous carrier).

• Mother: + with first and + with second → heterozygous carrier.

• Offspring I: − with first, + with second → only the allele detected by the second probe; this pattern indicates homozygous for that allele (affected if the second probe is mutant). In sickle testing, the probe complementary to GTG detects HbS; thus Offspring I is affected (SS).

• Offspring II: + with first, − with second → only the allele detected by the first probe; this indicates homozygous for the normal allele (unaffected, AA).

Answering the options

• (1) Parents are affected: Incorrect. Both parents hybridize to both probes, indicating heterozygous carriers, not homozygous affected.

• (2) Offspring I is affected: Correct. Offspring I hybridizes only with the second probe (mutant-specific), indicating homozygous mutant (SS).

• (3) Offspring II is carrier: Incorrect. Offspring II hybridizes only with the first probe, showing homozygous normal (AA), not a carrier.

• (4) Offspring II is affected: Incorrect. Affected would require only the mutant-probe signal; Offspring II shows only the first probe.

Brief method context

ASO hybridization uses short probes to discriminate even single-base variants like GAG→GTG, a standard example from sickle-cell genetics. After stringent washing, only perfectly matched probe-target duplexes remain, enabling direct genotype calls from presence/absence of spots on duplicate membranes.

• Takeaway: The correct statement is “Offspring I is affected.”

SEO notes for educators

• Primary keyword appears in title, slug, key phrase, and early introduction.

• Contextual genetic term “sickle-cell GAG→GTG mutation” included for relevance and search intent alignment.

References in context: ASO principle and dot-blot interpretation; sickle mutation identity GAG→GTG (Glu→Val).