Direct Monitoring Kinetic Studies of DNA Polymerase Reactions on a DNA-Immobilized Quartz-Crystal Microbalance

Abstract

Catalytic reactions of DNA polymerase I from E. coli (Klenow fragment, KF) were monitored directly with a template/primer (40/25- or 75/25-mer)-immobilized 27-MHz quartz-crystal microbalance (QCM). The 27-MHz QCM is a very sensitive mass-measuring device in aqueous solution, as the frequency decreases linearly with increasing mass on the QCM electrode at the nanogram level. Three steps in polymerase reactions which include 1) binding of DNA polymerase to the primer on the QCM (mass increase); 2) elongation of complementary nucleotides along the template (mass increase); and 3) release of the enzyme from the completely polymerized DNA (mass decrease), could be monitored continuously from the time dependencies of QCM frequency changes. The binding constant (Ka) of KF to the template/primer DNA was 10(8)M(-1) (k(on) = 10(5)M(-1)s(-1) and k(off)= 10(-3)s(-1)), and decreased to 10(6)M(-1) (k'on = 10(4)M(-1)s(-1) and k'off = 10(-2)s(-1)) for completely polymerized DNA. This is due to the 10-fold decrease in binding rate constant (k(on)) and 10-fold increase in dissociation rate constant (k(off)) for completed DNA strands. Ka values depended slightly on the template and primer sequences. The kinetic parameters in the elongation process (k(cat) and Km) depended only slightly on the DNA sequences. The repair process during the elongation catalyzed by KF could also be monitored in real time as QCM frequency changes.

Influence of PCR Reagents on DNA Polymerase Extension Rates Measured on Real-Time PCR Instruments

“…Other methods have been used to measure polymerase kinetics, including atomic force microscopy (1 ), light microscopy (2 ), single molecule optical trapping (3 ), quench flow (4 ), stopped flow (4 -7 ), and quartz crystal microbalance (8 ). Each of these requires instrumentation not found in most laboratories and has relatively low throughput.…”

Detection of Olfactory Receptor I7 Self‐Assembled Multilayer Formation and Immobilization Using a Quartz Crystal Microbalance

“…One of the proposed methods accomplishes this conversion by a complex calibration of the oscillating QC in the working solution to compensate for unwanted effects, i.e. interfacial liquid properties, thin film viscoelasticity, electrode morphology, and the mechanism of acoustic coupling [Matsuno et al, 2001, Stengel et al, 2005. Another method, more widely accepted, is acoustic coupling.…”

A Novel Tool for Detecting Amyloid Deposits in Systemic Amyloidosis In Vitro and In Vivo

“…Figure 3 indicates the affinity of BSB for TTR, FAP amyloid fibrils, or DRA amyloid fibrils. QCM is a new tool for measuring the affinity of a protein for a protein, a protein for a DNA, and/or a protein for a chemical compound (Matsuno et al, 2001;Okahata et al, 1998). Our results confirmed the strong affinity of BSB for amyloid fibrils but not for the amyloidogenic protein.…”

“…DRA amyloid fibrils were purified from an intestinal sample from a patient as described by Naiki et al (1989). The affinity of BSB or Congo red for amyloid fibrils was examined by using a highly sensitive 27-MHz QCM (Affinix Q; Iitium Company, Tokyo, Japan) as described (Matsuno et al, 2001;Okahata et al, 1998). Briefly, 2 l of 100 g/ml purified amyloid fibrils, 2 l of 100 g/ml purified wild-type TTR, or the variant TTR (ATTR Val30Met) was directly immobilized on a QCM plate, the plate was soaked in 6 ml of a buffered solution [10 mM Tris-HCl, 10% dimethyl sulfoxide (DMSO), pH 7.4] at 25°C, and the resonance frequency of the QCM was defined as the 0 position after equilibrium.…”

“…To determine the usefulness of the compound in vivo, we also examined BSB reactivity in amyloid deposits in mice in which AA amyloidosis had been induced. In addition, we investigated the affinity of BSB for amyloid fibrils and the inhibitory effect of BSB on formation of transthyretin (TTR) amyloid fibrils in vitro by means of a highly sensitive 27-MHz quartz crystal microbalance (QCM) (Matsuno et al, 2001;Okahata et al, 1998) and electron microscopy, respectively. …”