Novel 22q11.2 Deletions Detection Assay Using Gel Electrophoresis

Background: 22q11.2 deletion syndrome (22q11.2DS), also known as Di-George/velocardiofacial syndrome is the most common chromosomal microdeletion and is frequently associated with conotruncal congenital heart defects (CHD). A novel protocol for 22q11.2 deletions detection using conventional PCR and agarose gel electrophoresis was developed, and seven primer pairs targeting six 22q11.2 genes (HIRA, TBX1, DGCR8, ZNF74, CRKL, MAPK1) plus the reference gene RPP30 were evaluated. Methods: DNA from eight CHD cases (four 22q11.2 deletions confirmed by FISH (P1–P3) or BOBs (P4), and four cases with unknown status (U1–U4)) and two controls was amplified un-der optimized PCR conditions. The PCR products were analyzed on 2.5% agarose gels to assess band presence, intensity, and expected size for primer validation and potential 22q11.2 deletions detection. Results: All seven primer pairs produced expected sizes bands in two normal controls, with no target-size bands in the no template control (NTC), indicating adequate amplification performance. Among deletion confirmed cases, concordant multi-locus loss was observed—most notably at TBX1, DGCR8 and ZNF74, with MAPK1 additionally reduced in P4 sample. In the unknown group, U1 and U3 showed normalized band-intensity ratios above the 0.6 cut-off at all loci, indicating no evidence of 22q11.2 deletions, whereas locus-specific partial loss at ZNF74 was detected in U2 and U4; none of the unknown samples exhibited the broad multi-gene loss pattern observed in the confirmed 22q11.2 deletion cases. Conclusions: This study has developed and val-idated a novel 22q11.2 deletions detection protocol using conventional PCR with agarose gel electrophoresis. The assay is rapid and inexpensive, suitable for basic molecular laboratories, while confirmation still relies on standard clinical genetic tests such as CMA, FISH or BOBs, particularly in resource-limited settings.