The use of direct PCR has revolutionised DNA analysis of biological specimens over the last decades. However, information regarding the storage and amplification capabilities of these specimens over longer periods is limited, which is crucial for research and diagnostic studies. Our recent study addresses this gap by examining the effect of storage temperature and duration on direct PCR amplification of avian samples.

Whole blood, dried blood spots (DBS), and feathers from chickens were stored for five years at different temperatures: room temperature (~25°C), 4°C, and -20°C. After various durations of storage, samples underwent crude DNA extraction by diluting them in PBS buffer and heating at 98°C. Surprisingly, just 10 minutes of incubation at 98°C was sufficient for crude DNA extraction from any type of sample in PBS buffer.

Our findings revealed that storage temperature and duration had little impact on amplification success over the five-year period. We successfully amplified 96 samples using routine PCR reagents within 3.5–6.0 hours, demonstrating the feasibility of cost-effective and rapid genetic analysis of avian samples even after prolonged storage at room temperature.

Furthermore, we developed a protocol for DNA barcoding and molecular sexing using crude DNA from DBS and plucked feathers. PBS emerged as the most efficient dilution reagent for crude DNA extraction, enabling successful PCR amplification of specific targets from mitochondrial and nuclear genomes. We successfully PCR-amplified a segment of the 16S rRNA gene in 60 different avian species and performed DNA barcoding through Sanger’s sequencing. Additionally, molecular sexing of feathers was achieved through high-resolution melting (HRM) of PCR products amplified from a selected part of sex chromosomes.

In conclusion, our study suggests that PBS is a suitable choice as a dilution reagent for crude DNA extraction from blood and feathers for avian genetic analysis. These findings advance DNA barcoding and molecular sexing practices in avian research and provide valuable insights for conservation efforts.

MARYUM ASLAM AND DR. MUHAMMAD IMRAN,

Lahore.