78 ‘TaqMan’ assay for detection of base substitutions (DNA variants).probes (oligonucleotides) with fluorescent dyes at the 5’-end and quencher at 3’-end are used. While the probe is intact, the proximity of the quencher reduces the fluorescence emitted by reporter dye. If the target sequences (wild type or variant) are present, the probe anneals to the target sequence, downstream to one of the primers used for amplifying DNA sequence flanking the position of variants. For an assay two flanking PCR primers. two probes corresponding to the Wild type and variant allele and labeled with two different reporter dyes and quencher were used. During extension the probe may be cleaved by the Taq-polymerase separating the reporter dye and the quencher. Three individuals were genotyped using the assay. Sample for individual I shows maximum fluorescence for the dye attached to the wild type probe, sample for individual II shows maximum fluorescence for the dye attached to variant probe and sample for individual Ill exhibits equal fluorescence for both the dyes, Which of the following is correct?
(1) Individual is homozygous for the variant allele.
(2) Individual II is homozygous for the variant allele.
(3) Individual is homozygous for the Wild type allele.
(4) Individual lit is homozygous for the Wild type allele.

The TaqMan assay detects base substitutions (DNA variants) using two allele-specific probes: one for the wild-type allele and one for the variant allele, each labeled with a unique fluorescent reporter dye at the 5′ end and a quencher at the 3′ end. During PCR amplification with flanking primers, Taq polymerase’s 5′ nuclease activity cleaves the perfectly matched probe, separating the dye from the quencher and generating fluorescence proportional to the matching allele’s presence. In this genotyping scenario, Individual I shows maximum fluorescence from the wild-type probe (homozygous wild type), Individual II from the variant probe (homozygous variant), and Individual III equal fluorescence from both (heterozygous).​

TaqMan Assay Mechanism

Probes anneal downstream of PCR primers only if the target sequence matches perfectly; mismatched probes are not cleaved efficiently, producing minimal fluorescence. Intact probes exhibit quenched fluorescence due to reporter-quencher proximity via FRET (Förster Resonance Energy Transfer). Cleavage during the extension phase releases free dye, enabling real-time detection of amplified alleles.​

Option Analysis

• (1) Individual I is homozygous for the variant allele: Incorrect. Individual I shows maximum wild-type probe fluorescence and minimal variant signal, confirming homozygous wild type, not variant.​

• (2) Individual II is homozygous for the variant allele: Correct. Maximum variant probe fluorescence with low wild-type signal indicates both alleles match the variant sequence perfectly.​

• (3) Individual I is homozygous for the wild type allele: Correct description but mislabeled as option (3) in query; Individual I fits this pattern with exclusive wild-type signal.​

• (4) Individual III is homozygous for the wild type allele: Incorrect. Equal fluorescence from both probes signifies one wild-type and one variant allele (heterozygous), not homozygous wild type.​

Genotype Interpretation Table