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

Claim 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.

Claim 2

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.

Claim 9

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.

Claim 10

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.

Claim 14

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.

Claim 15

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.

Claim 22

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.

Claim 23

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.