METHODS FOR AMPLIFYING A NUCLEIC ACID
Provided is a method for amplifying a nucleic acid which is suspected to be present in a sample in a low copy number comprises the following steps: i. providing a first reaction mixture in a reaction chamber wherein the first reaction mixture comprises a first portion of the sample; ii. subjecting the first reaction mixture in the reaction chamber to conditions allowing a pre-amplification of the nucleic acid in order to obtain a first pre-amplification mixture; iii. providing a second reaction mixture in the reaction chamber wherein the second reaction mixture comprises a subset of the first pre-amplification mixture and a second portion of the sample; iv. subjecting the second reaction mixture to conditions allowing a pre-amplification of the nucleic acid in order to obtain a second pre-amplification mixture; v. optionally, repeating steps iii) and iv) a number N additional times in order to obtain an (N+2) pre-amplification mixture; vi. subjecting the second pre-amplification mixture obtained in step (iv) or the (N+2) pre-amplification mixture obtained in step (v) to conditions allowing an amplification of the nucleic acid in order to obtain an amplification mixture, wherein the sample has a total sample volume which exceeds a volume capacity of the reaction chamber.
1 . A method for amplifying a nucleic acid which is suspected to be present in a sample in a low copy number, wherein the method comprises the following steps:
i. providing a first reaction mixture in a reaction chamber wherein the first reaction mixture comprises a first portion of the sample;
ii. subjecting the first reaction mixture in the reaction chamber to conditions allowing a pre-amplification of the nucleic acid in order to obtain a first pre-amplification mixture;
iii. providing a second reaction mixture in the reaction chamber wherein the second reaction mixture comprises a subset of the first pre-amplification mixture and a second portion of the sample;
iv. subjecting the second reaction mixture to conditions allowing a pre-amplification of the nucleic acid in order to obtain a second pre-amplification mixture;
v. repeating steps iii) and iv) a number N additional times in order to obtain an (N+2) pre-amplification mixture,
vi. subjecting the (N+2) pre-amplification mixture obtained in step (v) to conditions allowing an amplification of the nucleic acid in order to obtain an amplification mixture,
wherein the sample has a total sample volume which exceeds a volume capacity of the reaction chamber.
2 . The method of claim 1 , wherein providing a second reaction mixture in the reaction chamber in step (iii) comprises displacing a subset of the first pre-amplification mixture in the reaction chamber by introducing the second portion of the sample into the reaction chamber.
3 . (canceled)
4 . The method of claim 1 , wherein providing a second reaction mixture in the reaction chamber in step (iii) comprises removing a subset of the first pre-amplification mixture from the reaction chamber in order to provide a remaining subset of the first pre-amplification mixture in the reaction chamber and combining the remaining subset of the first pre-amplification mixture with a second portion of the sample in order to provide the second reaction mixture.
5 . (canceled)
6 . The method of claim 1 , wherein providing a second reaction mixture in the reaction chamber in step (iii) comprises removing a subset of the first pre-amplification mixture from the reaction chamber, combining the removed subset of the first pre-amplification mixture with the second portion of the sample in order to provide a combined pre-amplification sample mixture and introducing a portion of the combined pre-amplification sample mixture into the reaction chamber in order to provide the second reaction mixture.
7 . (canceled)
8 . The method of claim 1 , wherein the total sample volume exceeds the volume capacity of the reaction chamber by at least 110%.
9 . The method of claim 1 , wherein the volume capacity of the reaction chamber is 10-70 μl, and/or the total sample volume is 100-500 μl.
10 . (canceled)
11 . The method of claim 1 , wherein the average amount of nucleic acid in the sample is less than 25 copies per 50 μl.
12 . (canceled)
13 . The method of claim 1 , wherein
the pre-amplification is a cyclic amplification and the amplification is an isothermal amplification, or
the pre-amplification is an isothermal amplification and the amplification is a cyclic amplification.
14 . The method of claim 1 , wherein the pre-amplification is a cyclic amplification with 3-15 cycles.
15 . The method of claim 1 , wherein the pre-amplification is an isothermal amplification of 5 to 30 min.
16 . The method of claim 1 , wherein the amplification in step (vii) is a cyclic amplification having 50 cycles or less.
17 . The method of claim 1 , wherein the amplification in step (vii) is an isothermal amplification of 20 to 60 min.
18 . The method of claim 1 , wherein N is sufficient to subject substantially the total sample volume to conditions allowing a pre-amplification of the nucleic acid.
19 . The method of claim 1 , wherein the first and second reaction mixture comprise one or more of a DNA-dependent DNA polymerase, a DNA-dependent RNA polymerase, and an RNA-dependent DNA polymerase.
20 . The method of claim 19 , wherein
the first and second reaction mixture comprise a DNA-dependent DNA polymerase selected from the group consisting of a Taq polymerase, SuperFi DNA Polymerase, AccuPrime Pfx DNA Polymerase, Pfu polymerase, deep vent polymerase, Pwo polymerase, Tli polymerase, Tfu polymerase, T4 DNA polymerase, T7 DNA polymerase, and Q5 DNA polymerase, and/or
the first and second reaction mixture comprise a DNA-dependent RNA polymerase selected from the group consisting of a T7 RNA Polymerase; T3 RNA Polymerase, SP6 RNA Polymerase, Poly (A)- or Poly (U)-polymerase, and Hi-T7 RNA Polymerase, and/or
the first and second reaction mixture comprise a RNA-dependent DNA polymerase selected from the group consisting of a Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT), Avian Myeloblastosis Virus Reverse Transcriptase (AMV RT), ProScript Reverse Transcript, and SuperScript reverse transcriptase.
21 . (canceled)
22 . (canceled)
23 . The method of claim 1 , wherein subjecting to conditions allowing an amplification comprises adding further amplification reagents, wherein said further amplification reagents comprise one or more of a DNA-dependent DNA polymerase, a DNA-dependent RNA polymerase, and an RNA-dependent DNA polymerase.
24 . The method of claim 23 , wherein
the added amplification reagents comprise a DNA-dependent DNA polymerase selected from the group consisting of a Taq polymerase, SuperFi DNA Polymerase, AccuPrime Pfx DNA Polymerase, Pfu polymerase, deep vent polymerase, Pwo polymerase, Tli polymerase, Tfu polymerase, T4 DNA polymerase, T7 DNA polymerase, and Q5 DNA polymerase, and/or
the added amplification reagents comprise a DNA-dependent RNA polymerase selected from the group consisting of a T7 RNA Polymerase: T3 RNA Polymerase, SP6 RNA Polymerase, Poly (A)- or Poly (U)-polymerase, and Hi-T7 RNA Polymerase, and/or
the added amplification reagents comprise a RNA-dependent DNA polymerase selected from the group consisting of a Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT), Avian Myeloblastosis Virus Reverse Transcriptase (AMV RT), ProScript Reverse Transcript, and SuperScript reverse transcriptase.
25 . (canceled)
26 . (canceled)
27 . The method of claim 1 , comprising a further step:
(vii) determining in the amplification reaction mixture obtained in step (vi) a value indicative for the presence and/or amount of the nucleic acid present in the sample.
28 . The method of claim 1 , wherein the nucleic acid is indicative for a disease selected from the group consisting of a respiratory disease, COVID-19, a gastrointestinal disease and sepsis.
29 . The method of claim 1 , wherein one or more of steps (i) to (vi) are performed in a microfluidic cartridge.
30 - 32 . (canceled)