DNA ultra-sensitive quantification, a technology for HIV linear DNA

Unintegrated HIV DNA represents between 20% and 35% of the total viral DNA in infected patients. Only the linear forms (unintegrated linear DNAs [ULDs]) can be substrates for integration and for the completion of a full viral cycle. In quiescent cells, these ULDs may be responsible for pre-integrative latency. However, their detection remains difficult due to the lack of specificity and sensitivity of existing techniques. We developed an ultra-sensitive, specific, and high-throughput technology for ULD quantification called DUSQ (DNA ultra-sensitive quantification) combining linker-mediated PCR and next-generation sequencing (NGS) using mo-lecular barcodes. Studying cells with different activity levels, we determined that the ULD half-life goes up to 11 days in resting CD4+ T cells. Finally, we were able to quantify ULDs in samples from patients infected with HIV-1, providing a proof of concept for the use of DUSQ in vivo to track pre-integrative latency. DUSQ can be adapted to the detection of other rare DNA molecules.

Automated summary

A new technology called DUSQ was developed to quantitate unintegrated HIV DNA molecules with high sensitivity, specificity, and throughput. By combining linker-mediated PCR and next-generation sequencing (NGS) with molecular barcodes, DUSQ can detect and quantify unintegrated linear DNAs (ULDs). The research revealed that the half-life of ULDs in resting CD4+ T cells can be as long as 11 days. Moreover, DUSQ successfully quantified ULDs in samples from HIV-1 infected patients, demonstrating its potential for tracking pre-integrative latency in vivo. DUSQ technology can also be adapted for the detection of other rare DNA molecules.