Use of DNA polymerases
Use of a DNA polymerase enzyme as a single stranded RNA exoribonuclease.
1 . A method using a DNA polymerase enzyme as a single stranded RNA exoribonuclease.
2 . A method according to claim 1 wherein the DNA polymerase has a 3′-5′ exonuclease coding sequence or 3′:5′ exonuclease domain, which sequence or domain has a fold containing a ribonuclease H-like structure motif.
3 . A method according to claim 1 , wherein the DNA polymerase is T7 DNA polymerase, T4 DNA polymerase or Phi29 DNA polymerase.
4 . (canceled)
5 . (canceled)
6 . A method according to claim 1 in a method for in situ detection of RNA.
7 . A method according to claim 6 wherein the method is for in situ detection of an RNA loci or species, a single nucleotide polymorphism (SNP), a splice variant, or a deletion or insertion in the RNA.
8 .- 19 . (canceled)
20 . A process for hydrolysing a single stranded RNA sequence comprising contacting the RNA sequence with a DNA polymerase enzyme having exoribonuclease activity.
21 . A process for polynucleotide production from a target region within an RNA sequence comprising:
a) forming a padlock probe-target region hybrid;
b) adding a DNA polymerase enzyme;
c) allowing the enzyme to hydrolyse the RNA sequence; and
d) allowing the enzyme to act as a polymerase to produce the polynucleotide in the presence of dNTPs,
wherein the process is conducted in the absence of an exogenous primer.
22 . A process according to claim 20 wherein the enzyme is contacted with the RNA sequence in the presence of a divalent metal ion.
23 . A process according to claim 21 wherein the enzyme is contacted with the RNA sequence in the presence of a divalent metal ion.
24 . A process according to claim 22 wherein the divalent metal ion is selected from Mg2+, Mn2+ and Co2+.
25 . A process according to claim 23 wherein the divalent metal ion is selected from Mg2+, Mn2+ and Co2+.
26 .- 29 . (canceled)
30 . A process according to claim 21 wherein step a) is performed in the presence of Rec A protein.
31 .- 35 . (canceled)
36 . A process according to claim 20 wherein the DNA polymerase has a 3′-5′ exonuclease coding sequence or 3′-5′ exonuclease domain, which sequence or domain has a fold containing a ribonuclease H-like structure motif.
37 . A process according to claim 21 wherein the DNA polymerase has a 3′-5′ exonuclease coding sequence or 3′-5′ exonuclease domain, which sequence or domain has a fold containing a ribonuclease H-like structure motif.
38 . A process according to claim 20 wherein the DNA polymerase is T7 DNA polymerase, T4 DNA polymerase or Phi29 DNA polymerase.
39 . A process according to claim 21 wherein the DNA polymerase is T7 DNA polymerase, T4 DNA polymerase or Phi29 DNA polymerase.
40 . A process according to claim 20 wherein the RNA sequence is an mRNA.
41 . A process according to claim 21 wherein the RNA sequence is an mRNA.
42 .- 54 . (canceled)
55 . A process for polynucleotide production from a target region within an RNA sequence comprising:
a) forming a padlock probe-target region hybrid;
b) adding a phi29 DNA polymerase enzyme;
c) allowing the enzyme to hydrolyse the RNA sequence; and
d) allowing the enzyme to act as a polymerase to produce the polynucleotide in the presence of dNTPs,
wherein the process is conducted in the absence of an exogenous primer.
56 . A kit for polynucleotide production from a target region within an RNA sequence comprising:
a) a padlock probe or a padlock probe precursor
b) a phi29 DNA polymerase
wherein the kit does not contain a further primer other than the probe or probe precursor.