H.O. Smith, K.W. Wilcox, and T.J. Kelley, working at Johns Hopkins University in 1968, isolated and characterized the first restriction nuclease whose functioning depended on a specific DNA nucleotide sequence. Working with Haemophilus influenzae bacteria, this group isolated an enzyme, called HindII, that always cut DNA molecules at a particular point within a specific sequence of six base pairs. This sequence is:
- 5' G T ( pyrimidine: T or C) ( purine: A or G) A C 3'
- P3' C A ( purine: A or G) ( pyrimidine: T or C) T G 5'
They found that the HindII enzyme always cuts directly in the center of this sequence. Wherever this particular sequence of six base pairs occurs unmodified in a DNA molecule, HindII will cleave both sugar-phosphate backbones of the DNA between the 3rd and 4th base pairs of the sequence. Moreover, HindII will only cleave a DNA molecule at this particular site. For this reason, this specific base sequence is known as the "recognition sequence" for HindII.