Klenow fragment polymerase ([exo.sup.-]), Nt.BbvCI enzyme, and a mixture of deoxynucleoside triphosphates (dNTPs) [2] 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 of E.
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.