RECOMBINASE POLYMERASE AMPLIFICATION REAGENTS AND KITS
This disclosure describes kits, reagents and methods for Recombinase Polymerase Amplification (RPA) of a target DNA that exploit the properties of recombinase and related proteins, to invade double-stranded DNA with single stranded homologous DNA permitting sequence specific priming of DNA polymerase reactions. The disclosed kits, reagents and methods have the advantage of not requiring thermocycling or thermophilic enzymes, thus offering easy and affordable implementation and portability relative to other amplification methods.
1 - 41 . (canceled)
42 . A freeze-dried pellet for a recombinase polymerase amplification process of DNA amplification of a target nucleic acid molecule, said freeze-dried pellet comprising:
a recombinase;
a recombinase loading protein;
a single-stranded DNA binding protein;
a strand-displacing DNA polymerase;
trehalose;
a first primer;
a second primer; and
Tris buffer,
wherein potassium acetate is not present in said pellet.
43 . The freeze-dried pellet of claim 42 , further comprising dNTPs, DTT, and ATP;
and/or polyethylene glycol.
44 . The freeze-dried pellet of claim 42 , further comprising a nuclease.
45 . The freeze-dried pellet of claim 44 , wherein the nuclease is selected from the group consisting of exonuclease III (exoIII), endonuclease IV (Nfo), and 8-oxoguanine DNA glycosylase (fpg).
46 . The freeze-dried pellet of claim 42 , wherein said strand-displacing DNA polymerase is Bsu polymerase or Sau polymerase.
47 . The freeze-dried pellet of claim 42 , comprising 25 mM Tris buffer.
48 . The freeze-dried pellet of claim 42 , comprising 240 μM dNTPs.
49 . The freeze-dried pellet of claim 42 , comprising 5 mM DTT.
50 . The freeze-dried pellet of claim 42 , comprising 2.5 mM ATP.
51 . The freeze-dried pellet of claim 42 , further comprising 50 mM phosphocreatine and 100 ng/μL creatine kinase.
52 . The freeze-dried pellet of claim 42 , wherein said freeze-dried pellet is freeze-dried onto the bottom of a tube.
53 . The freeze-dried pellet of claim 42 , wherein said freeze-dried pellet is freeze-dried in a well of a multi-well container.
54 . The freeze-dried pellet of claim 42 , wherein said freeze-dried pellet is attached to a mobile solid support.
55 . The freeze-dried pellet of claim 54 , wherein said mobile solid support is a bead or a strip.
56 . The freeze-dried pellet of claim 42 , wherein said freeze-dried pellet is attached to a well.
57 . The freeze-dried pellet of claim 42 , wherein the recombinase is uvsX recombinase.
58 . The freeze-dried pellet of claim 42 , wherein the recombinase loading protein is uvsY.
59 . The freeze-dried pellet of claim 42 , wherein the single-stranded DNA binding protein is gp32.
60 . The freeze-dried pellet of claim 42 , wherein the recombinase is bacteriophage T6 uvsX H66S, the recombinase loading protein is Rb69 uvsY, and/or the single-stranded DNA binding protein is Rb69 gp32.
61 . The freeze-dried pellet of claim 42 , wherein the trehalose is present at 2.5%-7.5% weight/lyophilization mixture volume.