Method, composition and kit for size selective enrichment of nucleic acids

Provided is a method for isolating and concentrating nucleic acids of selected target sizes (e.g., in increments less than 1000 base pairs) from a biological fluid mixture comprising combining the biological fluid mixture and a first aqueous two-phase system (ATPS) formed from a first phase forming polymer or surfactant component dissolved in a first phase solution, and a second phase solution, such that target nucleic acid fragments below a desired target size partition to said second phase solution and contaminants partition to the first phase solution, extracting and mixing the second phase solution with a second ATPS formed from a second phase forming polymer or surfactant component dissolved in a third phase solution and a fourth phase solution, such that the target nucleic acid fragments partition to and concentrate in the third phase solution, and recovering the concentrated target nucleic acid fragments from the third phase solution. A composition and kit for isolating and concentrating nucleic acids of selected target sizes as described above are also provided.