Many biosensors, DNA arrays, and next-generation DNA sequencing technologies need common methods for end modification of random DNA sequences generated from a sample of DNA. Surface immobilization of chemically modified DNA is often the first step in creating appropriate sensing platforms. We describe a simple technique for efficient heterobifunctional modification of arbitrary double-stranded DNA fragments with chosen chemical groups. The modification requires the use of short (10-20 base pairs) synthetic adaptors having desired terminal functional groups and installs known sequences, which can be used for hybridization of primers in the sequencing-by-synthesis approaches. The method, based on ligation under optimized conditions, is selective and provides high yields of the target heterobifunctional DNA product. An additional two-step procedure can be applied to select further for the desired bifunctionalized product using PCR amplification with a chemically modified primer. Both functional groups in the modified DNA are chemically active and can be used in surface immobilization of the DNA strands to create the surface of a biosensor or sequencing chip.