DIRECT NUCLEIC ACID AMPLIFICATION KIT, REAGENT AND METHOD
The present invention relates to compositions, methods and kits which can be used to amplify nucleic acids with the advantage of decreasing user time and possible contamination. The dried reagent composition of the invention can be used for easy processing and amplification of nucleic acid samples.
1 . A dried reagent composition for nucleic acid amplification comprising a sequestering reagent, a polymerase and a deoxyribonucleotide triphosphate (dNTP).
2 . The dried reagent composition of claim 1 , wherein said sequestering agent is a cyclodextrin.
3 . The dried reagent composition of claim 2 , wherein said cyclodextrin is selected from a group consisting of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, 6-O-α-D-Maltosyl-β cyclodextrin, hydroxyethyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin and 2-hydroxypropyl-β-cyclodextrin and derivatives thereof.
4 . The dried reagent composition of claim 2 , wherein the cyclodextrin is α-cyclodextrin.
5 . The dried reagent composition of claim 1 , further comprising at least one primer.
6 . The dried reagent composition of claim 1 , further comprising an excipient mix.
7 . The dried reagent composition of claim 6 , wherein the excipient mix comprises 10×PCR buffer, Ficoll 70, Ficoll 400, Melezitose and nuclease free water.
8 . The dried reagent composition of claim 1 , further comprising an exchange buffer.
9 . The dried reagent composition of claim 1 , further comprising bovine serum albumin (BSA).
10 . The dried reagent composition of claim 1 , wherein said polymerase is OmniKlen Taq (OKT).
11 . The dried reagent composition of claim 1 , comprising α-cyclodextrin, at least one primer, a polymerase, dNTP, BSA, an excipient mix and an exchange buffer.
12 . A method for producing a dried reagent composition for nucleic acid amplification comprising the steps:
i) combining a polymerase with a sequestering reagent and dNTP, to provide a mixture thereof; and
ii) drying said mixture to form a dried reagent composition.
13 . The method of claim 12 , wherein the mixture of step i) further comprises at least one primer.
14 . The method of claim 12 , wherein the mixture of step i) further comprises an excipient mix.
15 . The method of claim 12 , wherein the mixture of step i) further comprises an exchange buffer.
16 . The method of claim 12 , wherein said drying step is achieved by lyophilizing.
17 . The method of claim 12 , further comprising freezing said composition prior to said drying step.
18 . A method for amplification of nucleic acid comprising the steps:
i) incubating a solution containing nucleic acid with the dried reagent composition of claim 1 ; and
ii) amplifying said nucleic acid.
19 . The nucleic acid amplification method of claim 18 , wherein the amplification method is a polymerase chain reaction.
20 . The nucleic acid amplification method of claim 18 , wherein the amplification method comprises reverse transcription polymerase chain reaction or isothermal amplification.
21 . The method of claim 18 , further comprising a step prior to step i) wherein said solution is formed by adding water to the nucleic acid.
22 . The method of claim 18 , wherein the nucleic acid is immobilised on a solid support.
23 . The method of claim 22 , wherein said solid support is a cellulose based matrix.
24 . The method of claim 22 , wherein the solid support is in the form of a pre punched disc.
25 . The method of claim 22 , wherein the solid support is in the form of an FTA pre punched disc.
26 . The method of claim 22 , wherein a lysis reagent is embedded onto said solid matrix.
27 . The method of claim 18 , wherein the amplification is carried out in a single reaction vessel.
28 . A method of detecting and/or quantifying amplified nucleic acid using a detection system comprising the steps:
i) amplifying nucleic acids using the method of claim 18 to produce amplified nucleic acid;
ii) detecting said amplified nucleic acid; and
iii) optionally quantifying the amplified nucleic acid.
29 . The method of claim 28 , wherein said detection system is a PCR imaging system.
30 . A kit for amplifying the nucleic acid comprising the dried reagent of claim 1 and instructions for use thereof.