Patent Application
20090275034
This disclosure relates to temperature control devices for chemical and biological analyses, particularly, in the context of automated DNA sequencing machines and systems.
Traditional nucleic acid sequencing-by-synthesis was commonly performed using various forms of a gel-based method developed by Sanger. Next generation sequencing technologies have sought to move beyond Sanger sequencing and into the realm of more rapid, high-throughput methods at decreased cost. Among these next-generation technologies, are single molecule methods in which individual nucleic acid duplex is observed on a surface and template-dependent base incorporation is recorded for each specific duplex. Single molecule techniques hold promise for rapid sequencing of entire genomes at low cost.
Single molecule technologies generally require sensitive optical detection and the ability to operate at multiple temperatures simultaneously in different parts of the instrument. The present invention solves the problem of thermal control in high-throughput sequencing reactions.
The present invention relates to temperature control devices and methods for using them in sequencing reactions. Essentially, the invention provides a temperature control unit in liquid communication with a cooling element and/or a heating element which, in turn are used to regulate temperature of sequencing substrates and objective lenses for optical detection of sequencing reactions.
In a preferred embodiment, the invention provides temperature control for a sequencing apparatus comprising two stages. Each of the two stages comprises a substrate for sequencing that contains a plurality of nucleic acid duplex molecules attached thereto. The duplex comprises a template nucleic acid hybridized to a primer that is extendable at its 3′ end. Sequencing-by-synthesis takes place as follows. The surface is exposed to a polymerase and a nucleotide comprising a detectable label under conditions that allow template-dependent incorporation into the primer. After incorporation, the surface is rinsed to remove unincorporated nucleotides. Then, the surface is ready for imaging of the incorporated nucleotides. This process is repeated multiple times with each of the four nucleotide bases (A, T, C, and G) in order to build a sequence for each template over time as nucleotides are incorporated in each cycle. A detailed description of single molecule sequencing-by-synthesis is found in U.S. Pat. No. 7,282,337, incorporated by reference herein.
In a preferred mode of operation, a sequencing apparatus comprises a plurality of substrates such that sequencing chemistry and imaging can be performed at the same time. In one case, two adjacent substrates are positioned so that chemistry operations are taking place on one, while imaging of incorporated nucleotides is taking place on an adjacent substrate. For single molecule sequencing, it is preferred that the substrates are microfluidic flow cells to which duplex molecules are covalently attached (typically by attachment of the primer portion of the duplex to which the template portion is hybridized). Sequencing chemistry takes place in the microfluidic channels of one of the flow cells and then a stage on which both flow cells are mounted moves to be in proximity of a microscope objective for imaging. An example of a dual flow cell component of a sequencing system is shown in
In such dual flow-cell formats, it is necessary to conduct sequencing chemistry at a temperature that is higher than the temperature at which optimal imaging is done. This is especially true when fluorescent labels are used to detect incorporated nucleotides. Also, it is important to maintain the microscope objective at an optimal imaging temperature, regardless of the temperature of the flow cells. Accordingly, the invention provides a temperature control apparatus for maintaining appropriate temperature independently in each flow cell and in the objective. The temperature controller, or temperature control apparatus, provides temperature control in the flow cells over a range of temperatures necessary for sequencing and imaging; and is able to switch between temperatures as required when the stage shifts from chemistry to imaging.
A preferred configuration for a temperature controller according to the invention comprises two separate thermal control devices in liquid communication with conduits that carry a liquid to each flow cell, or flow cell mounting chuck, and convey temperature thereto. Separately, the invention contemplates a chiller device to keep the temperature of the microscope objective constant. In one embodiment, the invention comprises a reservoir for storing fluid and a conduit for conveying the fluid to the flow cell or chuck, the reservoir being capable of tunable heating and cooling. The invention contemplates configurations in which each flow cell or chuck is heated/cooled by a separate temperature control module, as well as configurations in which a single control module separately provides temperature control to each flow cell/chuck. Finally, the controller can control temperature to the objective, which typically is the same as the imaging flow cell temperature, or objective temperature control can be done separately.
The optimal temperature for sequencing reactions is about 37° C., however, sequencing can be done at any temperature that is optimal under the desired sequencing protocol. For example, if a melt step is required, the temperature of the “sequencing” flow cell must be raised, preferably to about 70° C. Optimal imaging temperatures are lower than sequencing temperatures, and preferably are about 23° C., but can range between about 17° C. and about 32° C. The temperature of the objective should be the same or about the same as the imaging temperature. For a total internal reflection objective, the optimal temperature is about 23° C.
An example of an apparatus 200 that can be used to perform the processes described above is shown in
Flow cells can be used individually, or optionally two or more flow cells can be combined together to analyze even more samples simultaneously. As described above, using a dual flow cell assembly allows the apparatus 200 to perform the sequencing chemistry in one flow cell, while at the same time performing the imaging operation in the other flow cell.
Performing these two operations simultaneously increases throughput of the apparatus 200 by analyzing twice as many samples, but this also requires maintaining several separate components at different temperatures. For example, the optimal temperature for sequencing reactions is about 37° C., however, sequencing can be done at any temperature that is optimal under the desired sequencing protocol. Alternatively, if a melt step is required, the temperature of the “sequencing” flow cell must be raised, preferably to about 70° C. Optimal imaging temperatures are lower than sequencing temperatures, and preferably are about 23° C., but can range between about 17° C. and about 32° C. The temperature of the objective should be the same or about the same as the imaging temperature. For a total internal reflection objective, the optimal temperature is about 23° C.
Referring now to
Each side of the flow chuck 490 includes an inlet 496 and an outlet 498 fluidly coupled to a chamber 491 (
Referring now to
As shown, the temperature control system 600 is only delivery heated and cools fluid to one side of the flow chuck 490. Additional pumps, valves and conduits can be included to circulate the heated and cooled fluids from the heating control device 192 and the cooling control device 193 to both sides of the flow chuck 490 since both sides need to be maintained at different temperatures. Alternatively two separate temperature control systems 600 can optionally be used.
Although not shown, the cooling control device 193 is also in fluid communication with the optics section to keep the temperature of the microscope objective constant during imaging. The temperature of the microscope objective is typically the same as the imaging flow cell temperature. Alternatively, the objective temperature control can be done separately.
In an alternative exemplary embodiment, the temperature control system 400 comprises a reservoir for storing fluid and a conduit for transporting the fluid to the flow cell 110 or chuck 490, the reservoir being capable of tunable heating and cooling. The invention contemplates configurations in which each flow cell or chuck is heated/cooled by a separate temperature control module, as well as configurations in which a single control module separately provides temperature control to each flow cell/chuck.
In yet another alternative exemplary embodiment, thermoelectric heating and cooling can be used to control the temperature of each of the flow cells and/or the microscope objective. Thermoelectric heating/cooling uses the Peltier effect to create a heat flux between the junction of two different types of materials. A Peltier cooler, heater, or thermoelectric heat pump is a solid-state active heat pump which transfers heat from one side of the device to the other side against the temperature gradient (from cold to hot), with consumption of electrical energy. Such an instrument is also called a Peltier device, Peltier diode, Peltier heat pump, solid state refrigerator, or thermoelectric cooler (TEC). Because heating can be achieved more easily and economically by many other methods, Peltier devices are mostly used for cooling. However, when a single device is to be used for both heating and cooling, a Peltier device may be desirable. Simply connecting the device to a DC voltage will cause one side to cool, while the other side warms. The effectiveness of the pump at moving the heat away from the cold side is totally dependent upon the amount of current provided and how well the heat from the hot side can be removed.
While certain embodiments according to the invention are shown and described, other embodiments are within the scope of this disclosure and are considered to be part hereof. The invention is not to be limited just to certain embodiments shown and/or described.
This application claims priority to U.S. provisional application No. 61/034,131, filed on Mar. 5, 2008, the entire content of which is hereby incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| 61034131 | Mar 2008 | US |
