available thermocyclers implement a 'hot-plate' design. That is a metal
plate which is designed to accept a 96-well micro-strip. This plate is
actively heated and cooled to cycle through the appropriate temperatures
(MJ Research) While such designs have
proven reliable they do suffer from a few fundamental limitations. First,
sample volume must be above some minimum (usually approx 10µl) for
the reactions to proceed reliably. Second, in the case of the MJ, thermal
cycling is achieved by heating and cooling a massive metal block. The heat
capacity of the block slows the temperature ramping time.
The design which we have chosen
to implement requires few moving parts and avoids the time lags
associated with changing the temperature of large masses. It is
based on a device built by Scott Hunicke-Smith in which thermal
cycling is achieved, not by changing the temperature of a block,
but by moving the sample through regions of the appropriate temperature.
In this conceptually simple
design we assemble three copper blocks, each with 1/16" diameter holes
drilled through in a standard 96-well format, in a stack such that the
arrays of holes co-align. Each of the blocks is maintained at extension,
annealling, or denaturing temperature, and the blocks are well insulated
from each other. Each hole is threaded through with a teflon tube through
which the samples will move. One end of each tube is extended above the
block assembly to connect to the syringe needles of a Robbins
Scientific Hydra-96 pipettor. The other end extends below the assembly
for loading the samples from a standard 96-well plate.
By activating the Robbins
pipettor we can easily load samples, and move them through all three temperature
stages. The thermocycler is designed to interface easily into our automated
system; the plates are stored in a cassette and carousel facility, and
may be loaded to the thermocycler using our standard server-arm
Thermal stability of the copper
blocks is maintained by using commercially available temperature controllers
(Newport Electronics). Each block
has heating elements (Ni-chrome wire) on the top face, bottom face, and
around the perimeter, and each heater has a dedicated controller. This
use of three temperature controllers to regulate the temperature at each
face ensures maximum homogeneity of the thermal profile.
The Teflon tubes have several
desirable properties: smooth surfaces allow smooth sample movement, hydrophobic
surfaces ensure that the sample slug stays intact, and the inherent chemical
stability of Teflon virtually eliminates the possiblity of contamination.
The Robbins Scientific pipettor
allows precise sample movement and its 96-channel format is ideal for our
application. We modified our instrument to allow computer control through
a serial port and we have written routines in LabView to control the movement
and timing for processing sequencing reactions.
We have modified the current
implementation to include the use of another servo motor which will move
the entire thermal block assembly. In this upgraded design (shown on the
figure) we load the sample into the tubes and then move the temperature
stages past the samples, while holding the samples in place in the Teflon
tube. This modification allows highly precise positioning of the samples
relative to the thermal blocks, and faster cycling times because the speed
of movement for the blocks is not limited in the same way as is movement
of the fluid slugs.