We developed a new approach for isolation of circulating cell-free DNA (cfDNA) from liquid biopsies (plasma, serum or urine). Our technology is based on using the proprietary bi-functional substance (SubX®) that binds DNA under physiological conditions (i.e. directly in biological liquids), followed by adsorption of [DNA-SubX®] complex on a solid phase matrix, e.g. silica coated magnetic beads. Our protocol allows scaling up or down the procedure to large or extra low volumes of starting material, as well as automation. Separation of cfDNA from the bulk of proteins occurs in a single vortex-spin step thus speeding-up isolation procedure and reducing the costs. The whole procedure can be completed within an hour and cfDNA is eluted in small volume of TE buffer. In combination with cfDNA isolation, one could analyze both genetic (DNA and RNA) as well as protein markers present in the same liquid biopsy.
Unique features of SubX® technology
* SubX® binds DNA under physiological conditions with high affinity via its phosphate groups and eliminate bias related to both AT/GC content and DNA size.
* No dilution of starting material (and cfDNA).
* No Proteinase K treatment is required
* Removes histones and other DNA binding proteins from the DNA-Protein nucleosome complex.
* Both ends of SubX® molecule display phospholipid binding groups. This feature allows each molecule of SubX® to anchor two exosomes (i.e. dimerize and further oligomerize).
* Easy precipitated in a brief 14K x g centrifugation step.
* A specially designed buffer allows for reconstitution of the pelleted exosomes.
* In combination with cfDNA isolation, one could analyze both genetic (DNA and RNA) as well as protein markers present in the same liquid biopsy.
* Different degrees of affinity of SubX® to DNA (strong) and exosome phospholipid complexes (mild). Exosomes are easily recovered in monomer format by special reconstitute buffer while tightly bound [SubX®-cfDNA-beads] complex remains in pellet and cfDNA further isolated by supplied solutions.