Device and method for the automated cycling of solutions between two or more temperatures

• US 5,270,183 A
• Filed: 02/08/1991
• Issued: 12/14/1993
• Est. Priority Date: 02/08/1991
• Status: Expired due to Fees

• Current Assignee: Beckman Research Institute of the City of Hope (City of Hope)
• Original Assignee: Beckman Research Institute of the City of Hope (City of Hope)
• Inventors: Corbett, John M., Riggs, Arthur D., Reed, Kenneth C.
• Primary Examiner(s): Wityshyn, Michael G.
• Assistant Examiner(s): Beisner, William H.
• Application Number:
  US07/653,659
• Time in Patent Office: 1,040 Days
• Field of Search: 435/3, 435/6, 435/91, 435/287, 435/289, 435/290, 435/291, 435/311, 435/316, 435/819, 435/809, 435/288, 935/17, 935/85, 935/86, 935/88, 935/87, 165/65, 165/66, 165/120, 422/109, 422/188, 422/189, 422/198, 422/200
• US Class Current: 435/91.2
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00495 Means for heating or cooling the reaction vessels
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: B01L 7/525 with physical movement of samples between temperature zones
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 60/14 for creating libraries

Abstract

The present invention provides an apparatus and method for the amplification of a particular sequence(s) of DNA in a sample using polymerased chain reaction. The method of the present invention involves the injection of a reaction mixture into a stream of carrier fluid. The carrier fluid then passes through a plurality of temperature zones in which the polymerase chain reactions take place. The method of the present invention allows the sequential processing of a number of samples.

Claims

1. A method of amplifying an amount of DNA present in a sample using a polymerase chain reaction comprising the following steps:

i) adding oligonucleotide primers, deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), deoxythymidine triphosphate (dTTP), and at least one of a thermostable DNA polymerase, enzymatically active fragments thereof, an enzymatically active derivative thereof and a reverse transcriptase to a DNA containing sample to form a first reaction mixture;

ii) injecting the reaction mixture into a stream of carrier fluid in which the reaction mixture is immiscible;

iii) passing the stream of carrier fluid containing the reaction mixture through a plurality of different temperature zones, the temperature of the different temperature zones and the time taken for the carrier fluid containing the reaction mixture to pass through the individual temperature zones being selected such that the following reactions take place in the reaction mixture;

(a) denaturation of the DNA into its component strands;

(b) annealing of the oligonucleotide primers to complementary sequences in the DNA; and

(c) synthesis of new DNA strands;

iv) repeating step iii) until a desired level of amplification has been achieved; and

v) recovering the reaction mixture from the carrier fluid.

2. A method as claimed in claim 1 in which prior to step v) at least one of an extent or specificity of amplification of the DNA in the reaction mixture is assessed by in-line analysis means.

3. A method as claimed in claim 1 in which there are a first, a second and a third temperature zone at temperatures of about 94°

C., 60°

C. and 73°

C., respectively, and the carrier fluid cyclically passes through the three temperature zones.

4. A method as claimed in claim 1 in which there are a first and a second temperature zone at temperatures of about 94°

C. and about 70°

C., respectively, and the carrier fluid cyclically passes through the two temperature zones.

5. A method as claimed in claim 1 in which a second reaction mixture is injected into the stream of carrier fluid upstream of the first reaction mixture, the two reaction mixtures being separated by the carrier fluid.

6. A method as claimed in claim 5 in which a purging reagent destructive to DNA is injected into the stream of carrier fluid between the two reaction mixtures.

7. A method as claimed in claim 1 in which, prior to step (v), the carrier fluid passes through a cooling zone such that the reaction mixture is cooled.

8. An apparatus for use in amplifying an amount of DNA present in a sample using a polymerase chain reaction, the apparatus comprising a tube constructed so as to carry a stream of carrier fluid, the tube being provided with an inlet port to enable injection of reaction mixtures comprising a DNA containing sample, oligonucleotide primers, deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), deoxythymidine triphosphate (dTTP), and at least one of a thermostable DNA polymerase, enzymatically active fragments thereof, an enzymatically active derivative thereof and a reverse transcriptase into the carrier fluid;

pump means constructed so as to maintain a flow of the stream of carrier fluid through the tube;

a plurality of different temperature zones through which the tube containing the carrier fluid cyclically passes, the different temperatures and the time taken for the carrier fluid containing the reaction mixtures to pass through the individual zones being selected such that the following reactions take place in the reaction mixtures;

(a) denaturation of the DNA in the sample into its component strands,

(b) annealing of the oligonucleotide primers with complementary sequences in the DNA, and

(c) synthesis of new DNA strands;

recovery means constructed so as to allow removal of the reaction mixtures from the carrier fluid following amplification of the DNA present in the sample; and

an in-line analysis means constructed so as to assess at least one of an extent and specificity of amplification in the reaction mixtures the in-line analysis means including means to detect at least the presence of DNA in the tube, the in-line analysis means being positioned between the different temperature zones and the recovery means.

9. An apparatus as claimed in claim 8 in which there are a first, a second and a third temperature zone at temperatures of about 94°

C., 60°

C. and 73°

C., respectively, and the tube cyclically passes through the three temperature zones.

10. An apparatus as claimed in claim 8 in which there are a first and a second temperature zone at temperatures of about 94°

C. and 70°

C., respectively, and the tube cyclically passes through the two temperature zones.

11. An apparatus as claimed in claim 8 in which the apparatus includes a cooling zone through which the tube passes prior to reaching the recovery means.

12. A method of amplifying an amount of DNA present in a sample using a ligation amplification reaction comprising the following steps:

(i) adding oligonucleotides, deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), deoxythymidine triphosphate (dTTP), and at least one of a thermostable DNA ligase, an enzymatically active fragment thereof, and an enzymatically active derivative thereof to a DNA containing sample to form a first reaction mixture;

(ii) injecting the reaction mixture into a stream of carrier fluid, the reaction mixture being immiscible in the carrier fluid;

(iii) passing the stream of carrier fluid containing the reaction mixture through a plurality of different temperature zones, the temperature of the different temperature zones and the time taken for the carrier fluid containing the reaction mixture to pass through the individual temperature zones being selected such that the following reactions take place sequentially in the reaction mixture;

(a) denaturation of the DNA into its component strands, (b) annealing of the oligonucleotides to complementary sequences in the DNA, and (c) ligation of adjacent oligonucleotides;

(iv) repeating step (iii) until a desired level of amplification has been achieved; and

(v) recovering the reaction mixture from the carrier fluid.

13. A method as claimed in claim 12 in which prior to step (v) at least an extent or specificity of amplification of the DNA in the reaction mixture is assessed by in-line analysis means.

14. A method as claimed in claim 12 in which there are a first, a second, and a third temperature zone at temperatures of about 94°

C., 60°

C. and 73°

C., respectively, and the carrier fluid cyclically passes through the three temperature zones.

15. A method as claimed in claim 12 in which there are a first and a second temperature zone at temperatures of about 94°

C. and 70°

C., respectively, and the carrier fluid cyclically passes through the two temperature zones.

16. A method as claimed in claim 12 in which a second reaction mixture is injected into the stream of carrier fluid upstream of the first reaction mixture.

17. A method as claimed in claim 16 in which a purging reagent destructive to DNA is injected into the stream of carrier fluid between the two reaction mixtures, being separated from each other by the carrier fluid.

18. A method as claimed in claim 12 in which, prior to step (v), the carrier fluid passes through a cooling zone such that the reaction mixture is cooled.

19. An apparatus for use in amplifying an amount of DNA present in a sample using a ligation amplification reaction, the apparatus comprising a tube constructed so as to carry a stream of carrier fluid, the tub being provided with an inlet port to enable injection of a reaction mixture comprising a DNA containing sample, oligonucleotides, deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), deoxythymidine triphosphate (dTTP), and at least one of a thermostable DNA ligase, an enzymatically active fragment thereof, and an enzymatically active derivative thereof into the carrier fluid;

pump means constructed so as to maintain a flow of the stream of carrier fluid through the tube;

a plurality of different temperature zones through which the tube containing the carrier fluid cyclically passes, the different temperatures and the time taken for the carrier fluid containing the reaction mixture to pass through the individual zones being selected such that the following reactions take place in the reaction mixture;

(a) denaturation of the DNA into its component strands,

(b) annealing of the oligonucleotides with complementary sequences in the DNA, and

(c) ligation of adjacent oligonucleotides;

recovery means adapted to allow removal of the reaction mixture from the tube following amplification of the DNA present in the sample; and

in-line analysis means constructed so as to assess at least one of an extent and specificity of amplification in the reaction mixture, the in-line analysis means comprising means to at least detect the presence of DNA in the tube, the in-line analysis means being positioned between the different temperature zones and the recovery means.

20. An apparatus as claimed in claim 19 in which there are a first, a second, and a third temperature zone at temperatures of about 94°

C., 60°

C., and 73°

C., respectively, and the tube cyclically passes through the three temperature zones.

21. An apparatus as claimed in claim 19 in which there are a first and a second temperature zone at temperatures of about 94°

C. and about 70°

C., respectively, and the tube cyclically passes through the two temperature zones.

22. An apparatus as claimed in claim 19 in which the apparatus includes a cooling zone through which the tube passes prior to reaching the recovery means.