Large DNA constructs can be synthesized from smaller oligonucleotides using the polymerase chain reaction. The set of oligonucleotides should be designed so that the melting temperature amongst oligonucleotide hybridization pairs do not vary greatly and the length of each oligonucleotide should not exceed 50 nucleotides. A near optimal oligonucleotide set is calculated using reliable gradient optimization methods. This was accomplished by defining a set of discrete arrays that is used to determine the melting temperature of a subset of the larger DNA sequence, depending on the subset start and end positions. These arrays were then incorporated into an objective function, which was optimized using the Broyden-Fletcher-Goldfarb-Shanno method. This method is adjusted slightly to incorporate explicit length and temperature constraints. Experimental results confirmed that the method performs better than similar software programs for the cases investigated and produces suitable oligonucleotide sets for DNA assembly. (C) 2010 American Institute of Chemical Engineers AIChE J, 57: 1912-1918, 2011