NUCLEIC ACID DETECTION METHOD
The present invention relates to methods for the detection of nucleic acids of defined sequence and kits and devices for use in said methods. The methods employ restriction enzymes, polymerase and oligonucleotide primers to produce an amplification product in the presence of a target nucleic acid, which is contacted with oligonucleotide probes to produce a detector product.
1 . A method for detecting the presence of a single stranded target nucleic acid of defined sequence in a sample comprising:
a) contacting the sample with:
i. a first oligonucleotide primer and a second oligonucleotide primer wherein said first primer comprises in the 5′ to 3′ direction one strand of a restriction enzyme recognition sequence and cleavage site and a region that is capable of hybridising to a first hybridisation sequence in the target nucleic acid, and said second primer comprises in the 5′ to 3′ direction one strand of a restriction enzyme recognition sequence and cleavage site and a region that is capable of hybridising to the reverse complement of a second hybridisation sequence upstream of the first hybridisation sequence in the target nucleic acid;
ii. a strand displacement DNA polymerase;
iii. dNTPs;
iv. one or more modified dNTP;
v. a first restriction enzyme that is not a nicking enzyme but is capable of recognising the recognition sequence of the first primer and cleaving only the first primer strand of the cleavage site when said recognition sequence and cleavage site are double stranded, the cleavage of the reverse complementary strand being blocked due to the presence of one or more modifications incorporated into said reverse complementary strand by the DNA polymerase using the one or more modified dNTP; and
vi. a second restriction enzyme that is not a nicking enzyme but is capable of recognising the recognition sequence of the second primer and cleaving only the second primer strand of the cleavage site when said recognition sequence and cleavage site are double stranded, the cleavage of the reverse complementary strand being blocked due to the presence of one or more modifications incorporated into said reverse complementary strand by the DNA polymerase using the one or more modified dNTP;
to produce, without temperature cycling, in the presence of said target nucleic acid, amplification product;
b) contacting the amplification product of step a) with:
i. a first oligonucleotide probe which is capable of hybridising to a first single stranded detection sequence in at least one species within the amplification product and which is attached to a moiety that permits its detection; and
ii. a second oligonucleotide probe which is capable of hybridising to a second single stranded detection sequence upstream or downstream of the first single stranded detection sequence in the same strand of said at least one species within the amplification product and which is attached to a solid material or to a moiety that permits its attachment to a solid material;
where hybridisation of the first and second probes to said at least one species within the amplification product produces a detector species; and
c) detecting the presence of the detector species produced in step b) wherein the presence of the detector species indicates the presence of the target nucleic acid in said sample.
2 . A method according to claim 1 wherein one of the first and second oligonucleotide probes is blocked at the 3′ end from extension by the DNA polymerase and is not capable of being cleaved by either the first or second restriction enzymes.
3 . A method according to claim 2 wherein the blocked oligonucleotide probe is rendered not capable of being cleaved by either the first or second restriction enzymes due to the presence of one or more sequence mismatch and/or one or more modifications such as a phosphorothioate linkage.
4 . A method according to claim 2 wherein the blocked oligonucleotide probe is contacted with the sample simultaneously to the performance of step a).
5 . A method according to claim 2 to wherein the blocked oligonucleotide probe comprises an additional region such that the 3′ end of the species within the amplification product to which the blocked oligonucleotide probe hybridises can be extended by the strand displacement DNA polymerase.
6 . A method according to claim 1 wherein the sample additionally is contacted in step a) with: (A) a third oligonucleotide primer which third primer comprises in the 5′ to 3′ direction one strand of the recognition sequence and cleavage site for the first restriction enzyme and a region that is capable of hybridising to the first hybridisation sequence in the target nucleic acid and wherein said third primer is blocked at the 3′ end from extension by the DNA polymerase; and/or (B) a fourth oligonucleotide primer which fourth primer comprises in the 5′ to 3′ direction one strand of the recognition sequence and cleavage site for the second restriction enzyme and a region that is capable of hybridising to the reverse complement of the second hybridisation sequence in the target nucleic acid and wherein said fourth primer is blocked at the 3′ end from extension by the DNA polymerase.
7 . (canceled)
8 . A method according to claim 1 wherein the one or more modified dNTP is an alpha thiol modified dNTP.
9 . A method according to claim 1 wherein the first and second restriction enzyme are the same restriction enzyme.
10 . (canceled)
11 . A method according to claim 1 wherein step a) is performed at a temperature of not more than 50° C.
12 . (canceled)
13 . A method according to claim 1 wherein the moiety that permits the detection of the first oligonucleotide probe is a colorimetric or fluorometric dye or a moiety that is capable of attachment to a colorimetric or fluorometric dye.
14 . A method according to claim 1 wherein the detector species is detected by a change in electrical signal.
15 . A method according to claim 1 wherein the moiety that permits the detection of the first oligonucleotide probe is an enzyme that yields a detectable signal following contact with a substrate.
16 . A method according to claim 1 wherein the moiety that permits the attachment of the second oligonucleotide probe to a solid material is a single stranded oligonucleotide.
17 . (canceled)
18 . A method according to claim 1 wherein in step c) the presence of the detector species is detected by nucleic acid lateral flow.
19 . A method according to claim 18 wherein the nucleic acid lateral flow utilises one or more nucleic acids that is capable of sequence specific hybridisation to the moiety that permits the attachment of the second oligonucleotide probe to a solid material.
20 . A method according to claim 1 wherein step c) produces a colorimetric or electrochemical signal using carbon or gold.
21 . A method according to claim 1 wherein the first and/or second oligonucleotide primers comprise a stabilising sequence upstream of the restriction enzyme recognition sequence and cleavage site.
22 . A method according to claim 1 wherein the hybridising region of the first and/or second oligonucleotide primers is between 9 and 16 bases in length.
23 . (canceled)
24 . A method according to claim 1 wherein the first and second hybridisation sequences in the target nucleic acid are separated by 0 to 15 bases.
25 . A method according to claim 1 wherein the first and second hybridisation sequences in the target nucleic acid are separated by 3 to 15 bases.
26 . A method according to claim 1 wherein in step b) either the first or second single stranded detection sequence in the at least one species within the amplification product includes at least 3 bases of the sequence corresponding to the 3 to 15 bases defined in claim 24 .
27 . (canceled)
28 . A method according to claim 1 wherein the target nucleic acid is selected from the group consisting of single stranded RNA, including single stranded RNA derived from double stranded RNA and single stranded RNA derived from double stranded DNA, and single stranded DNA, including single stranded DNA derived from single stranded RNA and single stranded DNA derived from double stranded DNA including single stranded DNA derived from double stranded DNA by strand invasion.
29 . (canceled)
30 . A method according to claim 28 wherein said single stranded DNA is derived from double stranded DNA by use of a nuclease or derived from single stranded RNA by use of reverse transcriptase.
31 . A method according to claim 1 wherein the presence of two or more different target nucleic acids of defined sequence are detected in the same sample.
32 . A method according to claim 1 wherein the sample is selected from the group consisting of a nasal or nasopharyngeal swab or aspirate, blood or a sample derived from blood, and urine.
33 . A method according to claim 1 wherein the target nucleic acid is viral or derived from viral nucleic acid material, is bacterial or derived from bacterial nucleic acid material, is circulating, cell-free DNA released from cancer cells or foetal cells, is micro RNA or derived from micro RNA.
34 . A method according to claim 1 wherein the detection of the target nucleic acid is used for the diagnosis, prognosis or monitoring of a disease or a diseased state.
35 . A method according to claim 34 wherein said disease is selected from the group consisting of an infectious disease, including human immunodeficiency virus (HIV), influenza, respiratory syncytial virus (RSV), Rhinovirus, norovirus, tuberculosis, human papillomavirus (HPV), meningitis, hepatitis, methicillin-resistant Staphylococcus aureus (MRSA), Ebola, Clostridium difficile , Epstein-Barr virus, malaria, plague, polio, chlamydia, herpes, gonorrhoea, measles, mumps, rubella, cholera or smallpox; and a cancer, including colorectal cancer, lung cancer, breast cancer, pancreatic cancer, prostate cancer, liver cancer, bladder cancer, leukaemia, esophageal cancer, ovarian cancer, kidney cancer, stomach cancer or melanoma.
36 - 49 . (canceled)