US-12623226-B2 - PCR sample block temperature uniformity

Abstract

A sample plate for a thermal cycler suitable for performing a polymerase chain reaction (PCR) procedure includes a base plate and a number of reaction vessels extending upward from the base plate. The sample plate further includes a vertical wall surrounding an outer perimeter defined by the reaction vessels. The vertical wall can be a continuation vertical wall, an intermittent vertical wall, or a perforated vertical wall. The intermittent vertical wall can include a plurality of wall portions, each of which plurality of wall portions is separated from other wall portions via a plurality of gaps.

Inventors

• Garret Bautista

Assignees

• BIO-RAD LABORATORIES, INC.

Dates

Publication Date

20260512

Application Date

20210609

Claims (20)

1. 1 . A sample plate for a thermal cycler, the sample plate comprising: a base plate; a plurality of reaction vessels extending upward from the base plate, the reaction vessels defining an outer perimeter; and a vertical wall attached to the base plate and extending upward from the base plate and surrounding the reaction vessels, wherein the vertical wall is spaced from the outer perimeter of the reaction vessels to form an air gap therebetween, and wherein the air gap has a predetermined lateral width defined by an inner surface of the vertical wall and the outer perimeter of the reaction vessels, wherein the vertical wall and the air gap control temperature variation at perimeter reaction vessels of the plurality of reaction vessels to maintain thermal uniformity across the plurality of reaction vessels during a thermal cycle.
2. 2 . The sample plate of claim 1 , wherein the sample plate is monolithic.
3. 3 . The sample plate of claim 2 , wherein the sample plate comprises aluminum.
4. 4 . The sample plate of claim 3 , further comprising thermal insulation surrounding and in contact with the vertical wall.
5. 5 . The sample plate of claim 4 , wherein the thermal insulation comprises a polymer.
6. 6 . The sample plate of claim 1 , wherein the vertical wall has a same height as the reaction vessels.
7. 7 . The sample plate of claim 1 , wherein the vertical wall is continuous.
8. 8 . The sample plate of claim 1 , wherein the vertical wall is intermittent.
9. 9 . The sample plate of claim 8 , wherein the intermittent vertical wall comprises a plurality of wall portions separated by a plurality of gaps.
10. 10 . The sample plate of claim 8 , wherein the reaction vessels are arranged to create a plurality of corners, and wherein a wall portion of the vertical wall extends around each of the plurality of corners.
11. 11 . The sample plate of claim 1 , wherein the vertical wall is perforated.
12. 12 . The sample plate of claim 11 , wherein the reaction vessels are arranged to create a plurality of corners, and wherein the perforations are located away from the corners.
13. 13 . A thermal cycling device for performing a polymerase chain reaction (PCR) procedure, the thermal cycling device comprising: a heat sink; one or more thermoelectric devices configured to produce a temperature differential in response to electric currents passing through the thermoelectric devices, wherein the one or more thermoelectric devices are in thermal contact with the heat sink; and a sample plate in thermal contact with the one or more thermoelectric devices, wherein the sample plate comprises a base plate; a plurality of reaction vessels extending upward from the base plate, the reaction vessels defining an outer perimeter; and a vertical wall extending upward from the base plate and surrounding the outer perimeter of the reaction vessels, wherein the vertical wall is spaced from the outer perimeter of the reaction vessels to form an air gap therebetween, and wherein the air gap has a predetermined lateral width defined by an inner surface of the vertical wall and the outer perimeter of the reaction vessels, wherein the vertical wall and the air gap control temperature variation at perimeter reaction vessels of the plurality of reaction vessels to maintain thermal uniformity across the plurality of reaction vessels during a thermal cycle.
14. 14 . The thermal cycling device of claim 13 , further comprising a controller configured to cycle the temperature of the reaction vessels according to a predetermined schedule, by controlling the electric currents passing through the thermoelectric devices.
15. 15 . The thermal cycling device of claim 14 , further comprising a base that houses the heat sink.
16. 16 . The thermal cycling device of claim 15 , further comprising a lid that is openable and closeable to provide access to the reaction vessels.
17. 17 . The thermal cycling device of claim 16 , wherein the lid is heated during a PCR procedure.
18. 18 . The thermal cycling device of claim 17 , wherein when the thermoelectric devices are controlled to maintain the sample plate at a nominal temperature of 95° C., a variation of temperature between the reaction vessels of the sample plate is less than 1° C.
19. 19 . A method, comprising: providing a PCR thermal cycler device as in claim 6 ; receiving a reagent mixture into the reaction vessels; and controlling a thermoelectric devices to bring the sample plate to a nominal temperature of 95° C., wherein when the sample block is held at a nominal temperature of 95° C., a variation of temperature between the reaction vessels of the sample plate reaches a value of less than 1° C.
20. 20 . The method of claim 19 , further comprising controlling the thermoelectric devices as needed to cycle the temperature of the sample block in accordance with a PCR procedure.

Description

CROSS-REFERENCES TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63/039,090, entitled “PCR SAMPLE BLOCK TEMPERATURE UNIFORMITY”, and filed on Jun. 15, 2020, the entirety of which is hereby incorporated by reference herein. BACKGROUND The polymerase chain reaction (PCR) provides a way of replicating or “amplifying” small quantities of DNA, so that sufficient quantities are available for further study. Millions or billions of copies of a DNA sample can be made in a few hours. Since its invention in 1983, PCR has revolutionized the field of molecular biology, and finds broad application in disease diagnosis, forensics, research, and other fields. To perform PCR, a reagent mixture is typically placed in reaction vessels in small quantities, for example 10-200 μL per reaction vessel. While only a single reaction vessel may be used, often an array of reaction vessels is used, including dozens or even hundreds of vessels. The reagent mixture may include the DNA to be replicated, a DNA polymerase, two DNA primers complementary to the ends of the DNA target strand, a buffer solution, and other materials. After some initialization steps, the reagent mixture is subjected to repeated temperature cycling. For example, in each thermal cycle, the reagent mixture is held for a first period of time at about 94-96° C. to “melt” the DNA into two single-stranded DNA molecules, and then held for a second period of time at a temperature of about 68° C. to anneal the primers to each of the single-stranded DNA templates, and then held for a third period of time at a temperature of about 72° C. to “elongate” the DNA strands, creating new double-stranded DNA molecules. Each thermal cycle nominally doubles the amount of the target DNA present. In a typical PCR procedure, about 20-40 thermal cycles may be performed, taking a total of a few minutes to a few hours. Devices have been developed for performing the thermal cycling automatically, and are often based on the thermoelectric effect. Ideally, the various reaction vessels undergo the as nearly the same temperature profiles as possible. However, prior system have not achieved desired levels of temperature uniformity. BRIEF SUMMARY According to one aspect, a sample plate for a thermal cycler comprises a base plate and a number of reaction vessels extending upward from the base plate. The reaction vessels define an outer perimeter, and the sample plate further comprises a vertical wall surrounding the reaction vessels. According to another aspect, a thermal cycling device for performing a polymerase chain reaction (PCR) procedure comprises a heat sink and one or more thermoelectric devices in thermal contact with the heat sink. The thermoelectric devices are configured to produce a temperature differential in response to electric currents passing through the thermoelectric devices. The thermal cycling device further comprises a sample plate in thermal contact with the one or more thermoelectric devices. The sample plate comprises a base plate and a number of reaction vessels extending upward from the base plate. The reaction vessels define an outer perimeter, and the sample plate further comprises a vertical wall surrounding the outer perimeter of the reaction vessels. According to another aspect, a method comprises providing the PCR thermal cycler, receiving a reagent mixture into the reaction vessels, and controlling the thermoelectric devices to bring the sample plate to a nominal temperature of 95° C. When the sample block is held at a nominal temperature of 95° C., the variation of temperature between the reaction vessels of the sample plate reaches a value of less than 1° C. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a simplified schematic drawing of a PCR thermal cycler. FIG. 2 shows an exploded view of some components of the PCR thermal cycler of FIG. 1. FIG. 3 shows sample block of the thermal cycler of FIG. 1, including reaction vessels. FIG. 4 shows a sample block in accordance with embodiments of the invention. FIG. 5 shows thermal modeling results of a sample block without a vertical wall. FIG. 6 shows thermal modeling results of a sample block with a vertical wall, in accordance with embodiments of the invention. FIG. 7 shows an experimental sample block, constructed according to embodiments of the invention. FIG. 8 shows a system for measuring the performance of sample blocks in accordance with embodiments of the invention. FIG. 9 illustrates the sample block of FIG. 4, with added insulation 901 in accordance with embodiments of the invention FIG. 10 shows an exploded view of the arrangement of FIG. 9. FIG. 11 shows the results of a thermal modeling analysis of the performance of a sample block with a vertical wall and added insulation, in accordance with embodiments of the invention. FIG. 12 shows a sample block in accordance with other embodiments of the invention. FIG. 13 shows a sample block in accordance with oth