2. One concentration of polymerase in the 1Â Reaction Buffer is
then injected into the fluidic system and the fluorescence variation recorded to retrieve the k ON (Fig. 1b—association).
3. Without regeneration of the chip, a second injection is directly
performed with a 1Â Reaction Buffer containing the same
concentration of polymerase (see Note 3) and an oversaturated
concentration of the four dNTP mixed nucleotides. The
increase in fluorescence signal is recorded over time to retrieve
the polymerization rate signal (Fig. 1b—polymerization, see
Note 4).
4. A last injection of 1Â Reaction Buffer completed with heparin
(see Note 5) on the non-regenerated chip is used to retrieve the
k OFF (Fig. 1b—dissociation).
3.4 Experimental
Design
1. The experimental workflow is designed using switchBUILD
software by making successive program blocks.
(a) In the first block, channel number, buffer type (P40), and
biochip (ENZ-80-1) are designed.
(b) A “Passivation” step is added as a second block (see Note
6).
(c) The third block is a “Kinetics” step, where the ligand is the
P36T80 oligonucleotide, and the analyte is a defined
concentration of polymerase (see Note 7), the measurement mode set as “Static Mode” (see Note 8). Following
parameters are also defined in this block: temperature,
electrode number (one of the six available in each channel), association time (see Note 9), flow rate (see Note
10), uncheck option for “regeneration.” If it is desired to
change any condition in the experiment (temperature,
polymerase concentration, etc.), a new block of this type,
with the corresponding modified parameter, should be
inserted next. It is also possible to add a blank-run
block, where the analyte is replaced with 1Â Reaction
Buffer alone: resulting curves could be used for baseline
correction if a significant drift of the fluorescence signal is
observed.
(d) A last “Standby” block is used at the end of the experiment (see Note 11).
(e) Once completed, the switchBUILD file is saved, and sample vials are disposed as requested in the “Autosampler”
window of the software.
2. The previous file is loaded in the switchCONTROL software.
If no other modifications are needed (see Note 12), the experiment could then start (typically 1 h 30 min for a complete cycle
with one polymerase concentration). Examples of data generated are provided on Fig. 2.
Analysis of a DNA Polymerase Using switchSENSE Approach
149
then injected into the fluidic system and the fluorescence variation recorded to retrieve the k ON (Fig. 1b—association).
3. Without regeneration of the chip, a second injection is directly
performed with a 1Â Reaction Buffer containing the same
concentration of polymerase (see Note 3) and an oversaturated
concentration of the four dNTP mixed nucleotides. The
increase in fluorescence signal is recorded over time to retrieve
the polymerization rate signal (Fig. 1b—polymerization, see
Note 4).
4. A last injection of 1Â Reaction Buffer completed with heparin
(see Note 5) on the non-regenerated chip is used to retrieve the
k OFF (Fig. 1b—dissociation).
3.4 Experimental
Design
1. The experimental workflow is designed using switchBUILD
software by making successive program blocks.
(a) In the first block, channel number, buffer type (P40), and
biochip (ENZ-80-1) are designed.
(b) A “Passivation” step is added as a second block (see Note
6).
(c) The third block is a “Kinetics” step, where the ligand is the
P36T80 oligonucleotide, and the analyte is a defined
concentration of polymerase (see Note 7), the measurement mode set as “Static Mode” (see Note 8). Following
parameters are also defined in this block: temperature,
electrode number (one of the six available in each channel), association time (see Note 9), flow rate (see Note
10), uncheck option for “regeneration.” If it is desired to
change any condition in the experiment (temperature,
polymerase concentration, etc.), a new block of this type,
with the corresponding modified parameter, should be
inserted next. It is also possible to add a blank-run
block, where the analyte is replaced with 1Â Reaction
Buffer alone: resulting curves could be used for baseline
correction if a significant drift of the fluorescence signal is
observed.
(d) A last “Standby” block is used at the end of the experiment (see Note 11).
(e) Once completed, the switchBUILD file is saved, and sample vials are disposed as requested in the “Autosampler”
window of the software.
2. The previous file is loaded in the switchCONTROL software.
If no other modifications are needed (see Note 12), the experiment could then start (typically 1 h 30 min for a complete cycle
with one polymerase concentration). Examples of data generated are provided on Fig. 2.
Analysis of a DNA Polymerase Using switchSENSE Approach
149