Apparatus and method for performing automated amplification of nucleic acid sequences and assays using heating and cooling steps

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BACKGROUND OF THE INVENTION

The invention pertains to the field of chain reactions for amplifying DNA or RNA (nucleic acids), and, more particularly, to the field of machines for automatically performing this process through temperature cycling.

Methods described in the past for synthesizing nucleic acid sequences from an existing sequence, for example, the phosphodiester and phosphotriester methods [Narang et al., Meth. Enzymol. 68, 90 (1979); and Brown et al., Meth. Enzymol. 68, 109 (1979), respectively], are not practical to produce large amounts of nucleic acid sequences. Such methods are laborious and time-consuming, require expensive equipment and reagents, and have a low overall efficiency.

There are methods for producing nucleic acid sequences in large amounts from small amounts of an existing sequence. Such methods involve cloning of a nucleic acid sequence in an appropriate host system, and culturing the host, wherein the vector in which the nucleic acid sequence has been inserted is replicated, resulting in copies of the vector and hence the sequence. See T. Maniatis, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, pp. 390-401 (1982); and U.S. Pat. Nos. 4,416,988 and 4,403,036. The original sequence can also be organically synthesized before insertion in a vector. See U.S. Pat. No. 4,293,652.

A method, described by Saiki et al., Science, 230, 1530-1534 (1985), has been devised for amplifying one or more specific nucleic acid sequences or a mixture thereof using primers, nucleotide triphosphates, and an agent for polymerization, such as DNA polymerase. The extension product of one primer, when hybridized to the other, becomes a template for the production of the desired specific nucleic acid sequence, and vice versa. The process is repeated as often as necessary to produce the desired amount of the sequence.

This method is especially useful for performing clinical tests on the DNA or RNA from a fetus or other donor where large amounts of the DNA or RNA are not readily available and more DNA or RNA must be manufactured to have a sufficient amount to perform tests. The presence of diseases which have unique DNA or RNA signatures can be detected by amplifying a nucleic acid sample from a patient and using various probe procedures to assay for the presence of the nucleic acid sequence being detected in the test. Such test might be prenatal diagnosis of sickle cell anemia, as described by Saiki et al., supra, where the amplification of specific .beta.-globin target sequences in genomic DNA resulted in the exponential increase (220,000 times) of target DNA copies, increasing sensitivity and speed while reducing the complexity of diagnosis. Another test is the diagnosis of the AIDS virus, which is thought to alter the nucleic acid sequence of its victims.

Five patent applications which describe the amplification process are copending U.S. patent application Ser. No. 818,127, filed Jan. 10, 1986, copending U.S. Ser. No. 716,982, filed Mar. 28, 1985, copending U.S. Ser. No. 791,308, filed Oct. 25, 1985, copending U.S. Ser. No. 828,144, filed Feb. 7, 1986, and copending U.S. Ser. No. 839,331, filed Mar. 13, 1986, the disclosures of all of which are incorporated herein by reference.

The amplification method bears some similarity to the molecular cloning methods described above, but does not involve propagation of a host organism, avoiding the hazards and inconvenience therein involved. In addition, the amplification method does not require synthesis of nucleic acid sequences unrelated to the desired sequence, and thereby obviates the need for extensive purification of the product from a complicated biological mixture. Finally, the amplification is more efficient than the alternative methods for producing large amounts of nucleic acid sequences from a target sequence and for producing such sequences in a comparatively short period of time.

At first, the amplification procedure described above was carried out by hand in the laboratories. The manual process involves a great deal of repetitive liquid handling steps and incubations at controlled temperatures. This is not only time-consuming and tedious, but it is also subject to error caused by human operator attention span drift. Such errors could result in a misdiagnosis of a genetic birth defect and an unnecessary abortion or the lack of an abortion where a birth defect exists. Further, such errors could result in misdiagnosis of sickle cell anemia or other genetic disorders.

Further, certain nucleic acids amplify more efficiently than others, so some nucleic acid sequence amplifications require more amplification cycles than others. Because the cost of laboratory labor can be high, and the risks to which a laboratory is subjected are high in case of error in erroneously performing amplification, there has arisen a need for a system which can automate the amplification process.

Such a machine is described in copending U.S. application Ser. No. 833,368 filed Feb. 25, 1986, which is the parent application of the present application. This machine utilizes a liquid handling system under computer control to make liquid transfers of enzyme stored at a controlled temperature in a first receptacle into a second receptacle whose temperature is controlled by the computer to conform to a certain incubation profile. The second receptacle stores the nucleic acid sequence to be amplified plus certain reagents. The computer includes a user interface through which a user can enter process parameters which control the characteristics of the various steps in the sequence such as the times and temperatures of incubation, the amount of enzyme to transfer on each cycle into the second receptacle from the first receptacle, as well as the number of cycles through the amplification sequence that the user desires the machine to perform.

While the above-described machine increases the amount of nucleic acid sequence which can be amplified per unit of labor, thereby decreasing the possibility of error, it involves liquid handling, where reagents must be continuously transferred at various cycles. There is a need for a machine which not only automates the amplification process, but also makes it faster and more convenient. This can be accomplished using an enzyme which is thermostable, i.e., will not break down when subjected to heat.

SUMMARY OF THE INVENTION

This invention utilizes a temperature-cycling instrument for implementing the amplification process when a thermostable enzyme is employed. The use of a thermostable enzyme avoids the need for liquid transferring of the enzyme, which is necessitated when the enzyme is stable in the presence of heat.

More specifically, the invention herein relates to an apparatus for performing automated amplification of at least one specific nucleic acid sequence comprising:

a heat conducting container for holding a reaction mixture comprising a thermostable enzyme, said nucleic acid sequence(s) to be amplified, four different nucleotide triphosphates, and one oligonucleotide primer for each different specific sequence being amplified, wherein each primer is selected to be substantially complementary to different strands of each specific sequence, such that the extension product synthesized from one primer, when it is separated from its complement, can serve as a template for synthesis of the extension product of the other primer;

means for heating, cooling, and maintaining said container to or at any of a plurality of predetermined (user-defined) temperatures and having an input for receiving a control signal controlling which of said predetermined temperatures at or to which said container is heated, cooled, or maintained; and

a computer means, coupled to the input of said means for heating and cooling to generate the proper control signals to control the temperature levels, temperature rate-of-change ramps, and timing of the incubations at certain temperature levels.

This invention also provides an apparatus for performing automated amplification of at least one specific nucleic acid sequence comprising:

a first means for holding a reaction mixture comprising said nucleic acid sequence(s) to be amplified, four different nucleotide triphosphates, a thermostable enzyme, and one oligonucleotide primer for each different specific sequence being amplified, wherein each primer is selected to be substantially complementary to different stands of each specific sequence, such that the extension product synthesized from one primer, when it is separated from its complement, can serve as a template for synthesis of the extension product of the other primer, said holding being carried out at any selected temperature or plurality of temperatures; and

a second means for automatically performing a predetermined sequence of steps including causing said first means to heat its contents for a first period and to cool its contents for a second period.

In yet another embodiment, the invention herein provides an apparatus for performing an assay including heating and cooling steps as part of the sequence of steps of the assay comprising:

means for performing the sequence of steps wherein heating and cooling steps would be beneficial; and

means in said means for performing for causing said heating and cooling steps to be performed at the proper point in the sequence of steps comprising the assay.

In another embodiment, this invention provides a method for amplifying at least one specific nucleic acid sequence comprising the steps of:

using a computer-directed machine to heat to a predetermined temperature for a predetermined time a sample of the nucleic acid sequence(s) to be amplified, four different nucleotide triphosphates, a thermostable enzyme, and one oligonucleotide primer for each different specific sequence being amplified, wherein each primer is selected to be substantially complementary to different strands of each specific sequence, such that the extension product synthesized from one primer, when it is separated from its complement, can serve as a template for synthesis of the extension product of the other primer (hereafter the mixture); and

using a computer-directed machine to chill the mixture to a predetermined temperature.

In still another embodiment, this invention provides a method of amplifying at least one specific nucleic acid sequence comprising the steps of:

a) using a computer-directed machine to issue a heat signal to a heating apparatus to cause a reaction chamber to be heated for a predetermined time to and/or at a predetermined temperature, wherein said reaction chamber contains the mixture described above;

b) using a computer-directed machine to issue a cool signal to a cooling apparatus to cause said reaction chamber to be cooled for a predetermined time to and/or at a predetermined temperature; and

c) using a computer-directed machine to repeat the cycle consisting of steps a through c when the elapsed time for the active cooling signal equals a user-defined time if the number of cycles performed thus far is less than a user-defined number of cycles.

The apparatus herein also generally contains a power supply for operation, a structural system to contain all the elements of the apparatus, and a keyboard and display panel to allow control of the apparatus by an operator.

The receptacle which holds the reagents where the reaction occurs has its temperature controlled by a computer to conform to a certain incubation profile defined by the user. Three circulating fluid reservoirs and solenoid operated valves, or any other method, may be employed to control temperature. The Peltier heat pumps available from Materials Electronics Products Corporation in Trenton, N.J. may also be used, as well as a water heat exchanger or any other heating and cooling system which may be controlled by a computer.

If solenoid-operated valves are employed, they are coupled to the computer such that the proper temperature fluid can be directed through the supported structure for the heat-conducting receptacle at the proper times in the amplification process under computer control. The receptacle is switched under computer control between two temperatures by the transmission of a control signal to the solenoid-operated valves at the proper time in the sequence to gate either the hot fluid or the cold fluid through the support structure of the receptacle. A temperature sensor coupled to the reaction chamber and the computer is used to provide a signal indicating the actual temperature. The computer compares the actual temperature to the desired temperature. An error signal is generated in this fashion which is used to control the apparatus which heats and cools the reaction chambers. The computer also keeps track of the elapsed time at particular temperatures to implement the incubation periods in the protocol.

The basic process that the machine performs to implement the amplification protocol after the starting materials are loaded into the reaction well, in one embodiment using water baths, is as follows.

The computer signals the solenoid-operated valves to gate the hot fluid through the supporting structure for the reaction chamber thereby heating the contents of the reaction well to the temperature of the hot fluid.

The amount of time the hot fluid is gated "on" is measured by an elapsed time counter.

The computer compares the elapsed time the hot fluid has been gated "on" to a variable set in memory. In the preferred embodiment, this variable can be changed by the user through the user interface. In other embodiments, it may be fixed.

When the elapsed time matches the variable for the hot incubation, the computer sends a signal to the solenoid-operated valves to stop the hot fluid flow and gate the cold fluid flow through the supporting structure for the reaction vessel.

In embodiments using temperature control feedback instead of empirically determined "on" times for the hot and cold fluids, a temperature profile versus time for the reaction chamber is programmed into the computer via the user interface. This causes the computer to control the reaction or reagent vessel temperature in the sequence required by the particular amplification reaction parameters. Such an embodiment uses a thermistor or other temperature sensor to monitor the temperature of the reaction chamber and generates an error signal derived by comparing the actual temperature of the reaction chamber to the user-defined temperature profile. The error signal is used to control a heat pump or other heating and cooling apparatus to maintain the desired temperature profile during the high temperature heat-up and high temperature incubation and during the chill-down and low-temperature incubation.

On either temperature feedback or empirically determined time embodiments, the computer starts a timer and compares the elapsed time for hot or cold fluid flow or the elapsed time at a particular temperature to a user-defined variable stored in memory for each segment or leg in the temperature profile. These variables can be set by the user in the preferred embodiment through the user interface. In embodiments where no temperature sensor is used, the variable for proposed time of hot or cold fluid flow is empirically determined by the user as the time it takes to heat or cool the reaction vessel to a predetermined temperature from the starting temperature plus the desired incubation time.

The above temperature profile control apparatus and methods for embodiments using hot and cold fluid reservoirs and solenoid-operated valves are equally applicable to embodiments using Peltier heat pumps or other forms of heating and cooling apparatus coupled to the reaction chamber or chambers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a general block diagram of a machine which can perform the amplification process using the thermostable enzyme and Peltier heat pumps to cycle the temperature of the reaction vessels.

FIG. 2 is a general block diagram of a machine which can perform the thermostable enzyme amplification process herein using water baths to cycle the temperature of the reaction vessels.

FIG. 3 is a diagram of a solid state heat pump and reaction chamber heat exchanger structure.

FIG. 4 is a schematic diagram of the interface unit for a solid state heat pump.

FIG. 5 is a diagram of a typica