Nucleic acid amplification method
OwnerStephen Bustininventor
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Abstract
This invention relates to methods for amplifying target nucleic acid. A target nucleic acid is contacted with specific primers in the presence of DNA polymerase and deoxyribonucleotide triphosphates to produce an amplification solution with a volume of 5 pl or less. The amplification solution is subjected to a denaturation temperature of 75° C.-85° C. for 1.5 second or less; and then to a polymerisation temperature of 25° C.-45° C. for 1 second or less. These denaturation and polymerisation steps are repeated to amplify the target nucleic acid. Devices and systems for performing such methods are also provided.
Claims55
A method for amplifying a target nucleic acid comprising:
- (i) contacting a target nucleic acid with a forward and reverse amplification primers specific for the target nucleic acid in the presence of DNA polymerase and deoxyribonucleotide triphosphates to produce an amplification solution,
- wherein the amplification solution has a volume of 5 μl or less,
- (ii) subjecting the amplification solution to a denaturation temperature of 75° C.-85° C. for 1.5 second or less, such that nucleic acid in the amplification solution is denatured,
- (iii) subjecting the amplification solution to a polymerisation temperature of 25° C.-45° C. for 1 second or less,
- such that the primers hybridise to the target nucleic acid and are extended by the polymerase,
- (iv) repeating steps (ii) and (iii) one or more times, thereby amplifying the target nucleic acid.
The method of any preceding claim, wherein steps (ii) and (iii) are repeated 5 or more times, 10 or more times, 15 or more times, 20 or more times, 25 or more times or 30 or more times.
The method of any preceding claim, wherein steps (ii) to (iv) are performed in 2 or less minutes to amplify the target nucleic acid.
The method of any preceding claim, wherein the forward and reverse amplification primers have a melting temperature of between 40° C.-50° C.
The method of any preceding claim, wherein the target nucleic acid is 50-60 bases in length.
The method of any preceding claim, wherein the melting temperature of the target nucleic acid is around 70° C.
The method of any preceding claim, wherein the concentration of each of the forward amplification primer and reverse amplification primer in the amplification solution is between 5 μM and 15 μM.
The method of any preceding claim, wherein the DNA polymerase is present at a concentration of 50 U/mL
The method of any preceding claim, wherein the DNA polymerase is a thermostable polymerase, preferably Taq polymerase.
The method of any preceding claim, wherein the volume of the amplification solution is 1 μL or less
The method of any preceding claim, wherein the amplification solution further comprises a probe to determine the amount of amplified target nucleic acid.
The method of any preceding claim, wherein the amplification solution is thermally cycled between a denaturation region held at the denaturation temperature and a polymerisation region held at the polymerisation temperature.
The method of any preceding claim, wherein the amplification solution is substantially free of a PCR enhancer.
The method of any preceding claim, wherein the forward and reverse amplification primers are contacted with a sample nucleic acid that comprises the target nucleic acid.
The method of any preceding claim, wherein the sample nucleic acid is a deoxyribonucleic acid produced by a method comprising providing a sample ribonucleic acid and reverse transcribing the sample ribonucleic acid to produce the sample nucleic acid.
The method of any preceding claim, wherein the sample ribonucleic acid is transcribed by subjecting the sample comprising a target ribonucleic acid and a reverse transcriptase to a reverse transcription temperature of between 20° C.-25° C. for 1-10 minutes.
A nucleic acid amplification device comprising;
- a denaturation region,
- a polymerisation region,
- a heater to heat the denaturation region to between 75° C.-85° C.,
- a heater to heat the polymerisation region to between 25° C.-45° C., and
- an actuator to move an amplification vessel for containing an amplification solution between the denaturation region and the polymerisation region.
The device of any preceding claim, wherein the actuator to move the amplification vessel between the denaturation region and the polymerisation region is configured to move the amplification vessel from the denaturation region to the polymerisation region or from the polymerisation region to the denaturation region in 2 seconds or less.
The device of any preceding claim, wherein the device is configured to adjust the length of time for which the amplification solution is subjected to the denaturation temperature and/or polymerisation temperature.
The device of any preceding claim, wherein the device is configured to adjust the denaturation temperature and/or polymerisation temperature.
The device of any preceding claim, wherein the device is configured to adjust the length of time taken for the actuator to move the amplification vessel from the denaturation region to the polymerisation region or from the polymerisation region to the denaturation region.
The device of any preceding claim, wherein the actuator to move the amplification vessel between the denaturation region and the polymerisation region is a robotic arm.
The device of any preceding claim, wherein the amplification vessel for containing an amplification solution is a plate or tube.
The device of any preceding claim, wherein the denaturation region and/or the polymerisation region is a water bath.
The system of any preceding claim, wherein the target nucleic acid is 50-60 bases in length.
The system of any preceding claim, wherein the melting temperature of the target nucleic acid is around 70° C.
The system of any preceding claim, wherein the concentration of the forward amplification primer and reverse amplification primer in the amplification solution is between 5 μM and 15 μM.
The system of any preceding claim, wherein the system further comprises a probe to determine the amount of amplified target nucleic acid.
The system of any preceding claim, wherein the final concentration of the probe is between 100 nM to 200 nM.
The system of any preceding claim, wherein the DNA polymerase is present at a concentration of 50 U/mL.
The system of any preceding claim, wherein the DNA polymerase is a thermostable polymerase, preferably Taq polymerase.
Dependencies are inferred from the claim text.