Klenow fragment polymerase ([exo.sup.-]), Nt.BbvCI enzyme, and a mixture of deoxynucleoside triphosphates (dNTPs)  were purchased from New England Biolabs.
The 15mer thrombin aptamer appears to have a dissociation constant ([K.sub.d]) from 25 to 200 nmol/L for thrombin (26), whereas the Klenow fragment polymerase has a [K.sub.d] of 7.9 nmol/L for DNA (27).
The other heating block was set at 30[degrees]C, the optimal temperature for the Klenow fragment
The ends of the annealed OLs were filled-in by means of the Klenow fragment
of DNA polymerase and random primer kit solutions (Life Technologies, Inc., Rockville, MD).
Protease-mediated allele-specific extension (PrASE) is a flexible assay and can be performed at lower temperatures using thermolabile enzymes, such as the Klenow fragment
, or at increased temperatures using thermostable DNA polymerases, such as Taq polymerase.
The fragments were subsequently labeled with biotin, using the exonuclease-free Klenow fragment
of Escherichia coli DNA polymerase I to fill in the recessive ends with dGTP, dCTP, dTTP, and biotin-14-dATP.