Nucleic acid detection method
AssigneeSense Biodetection Limited
InventorsHenry John Lamble, David Lloyd, Eryk Dunski, Steven Watson
Life of this patent
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Abstract
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.
Claims74
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 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.
A method according to any of the preceding claims 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.
A method according to any of the preceding claims wherein the one or more modified dNTP is an alpha thiol modified dNTP.
A method according to any of the preceding claims wherein the first and second restriction enzyme are the same restriction enzyme.
A method according to any of the preceding claims wherein two or more of steps a), b) and c) are performed simultaneously.
A method according to any of the preceding claims wherein step a) is performed at a temperature of not more than 50° C.
A method according to any of the preceding claims wherein the temperature is increased during the performance of step a), such as an increase from an ambient starting temperature, e.g. in the range of 15-30° C. up to a temperature in the range of 40-50° C.
A method according to any of the preceding claims 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 such as biotin.
A method according to any of the preceding claims wherein the detector species is detected by a change in electrical signal.
A method according to any of the preceding claims wherein the moiety that permits the detection of the first oligonucleotide probe is an enzyme that yields a detectable signal, such as a colorimetric or fluorometric signal, following contact with a substrate.
A method according to any of the preceding claims wherein the moiety that permits the attachment of the second oligonucleotide probe to a solid material is a single stranded oligonucleotide.
A method according to any of the preceding claims wherein in step c) the presence of the detector species is detected by nucleic acid lateral flow.
A method according to any of the preceding claims wherein step c) produces a colorimetric or electrochemical signal using carbon or gold, preferably carbon.
A method according to any of the preceding claims wherein the first and/or second oligonucleotide primers comprise a stabilising sequence upstream of the restriction enzyme recognition sequence and cleavage site, e.g. of 5 or 6 bases in length.
A method according to any of the preceding claims wherein the hybridising region of the first and/or second oligonucleotide primers is between 9 and 16 bases in length.
A method according to any of the preceding claims wherein one of the first and second oligonucleotide primers is provided in excess of the other.
A method according to any of the preceding claims wherein the first and second hybridisation sequences in the target nucleic acid are separated by 0 to 15 bases.
A method according to any of the preceding claims wherein the first and second hybridisation sequences in the target nucleic acid are separated by 3 to 15 bases.
A method according to any of the preceding claims wherein the level of the target nucleic acid in said sample is quantified in step c).
A method according to any of the preceding claims wherein the target nucleic acid is single stranded RNA, including single stranded RNA derived from double stranded RNA and single stranded RNA derived from double stranded DNA, or single stranded DNA, including single stranded DNA derived from single stranded RNA and single stranded DNA derived from double stranded DNA.
A method according to any of the preceding claims wherein the presence of two or more different target nucleic acids of defined sequence are detected in the same sample.
A method according to any of the preceding claims wherein the sample is a biological sample. such as a nasal or nasopharyngeal swab or aspirate, blood or a sample derived from blood, or urine.
A method according to any of the preceding claims 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.
A method according to any of the preceding claims wherein the detection of the target nucleic acid is used for the diagnosis, prognosis or monitoring of a disease or a diseased state.
A method according to any of the preceding claims wherein the detection of said target nucleic acid is used for human genetic testing, prenatal testing, blood contamination screening, pharmacogenomics or pharmacokinetics.
A method according to any of the preceding claims wherein the sample is a human sample, a forensic sample, an agricultural sample, a veterinary sample, an environmental sample or a biodefence sample.
A kit comprising:
- a) a first oligonucleotide primer and a second oligonucleotide primer wherein said first primer comprises in the 5′ to 3′ direction a restriction enzyme recognition sequence and cleavage site and a region that is capable of hybridising to a first hybridisation sequence in a single stranded target nucleic acid of defined sequence, and said second primer comprises in the 5′ to 3′ direction 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;
- b) a first restriction enzyme that is not a nicking enzyme and is capable of recognising the recognition sequence of and cleaving the cleavage site of the first primer and a second restriction enzyme that is not a nicking enzyme and is capable of recognising the recognition sequence of and cleaving the cleavage site of the second primer;
- c) a strand displacement DNA polymerase;
- d) dNTPs;
- e) one or more modified dNTP;
- f) a first oligonucleotide probe which is capable of hybridising to a first single stranded detection sequence in at least one species in amplification product produced in the presence of said target nucleic acid and which is attached to a moiety which permits its detection;
- and
- g) 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 said at least one species in amplification product and which is attached to a solid material or to a moiety which permits its attachment to a solid material.
A substrate for capturing a detector nucleic acid having an immobilised oligonucleotide capture probe comprising a single stranded hybridisation region of at least 8 bases in length which comprises 3 or more repeat copies of a 2 to 4 base DNA sequence motif wherein the copies of the sequence motif are contiguous or separated by one base.
The substrate according to any one of the preceding claims wherein the capture probe may comprise one or more modified base and/or modified internucleotide linkage.
The substrate according to any one of the preceding claims wherein the capture probe additionally comprises a thymidine region 5′ or 3′ of the hybridisation region.
The substrate according to any of the preceding claims wherein the DNA sequence motif is selected from AG, AT, CA, AAG. AAT, AGT, CCA, AAT, CCT, CAA, AAC, GAC, AACT, TACC, CAGT, GACT, TAGT, GAGT, AAGT, TCAT, ATCT, TAGA, TACA, CACT, CCAA, CACT and AGAC or the reverse complement of any one thereof.
The substrate according to any one of the preceding claims having two or more spatially distinct immobilised capture probes each comprising a different hybridisation region for capturing a different detector nucleic acid.
The substrate according to any of the previous claims wherein the capture probe is immobilized to a test zone and the substrate may additionally comprise a sample pad and/or conjugate pad upstream of the test zone.
The substrate according to any one of the preceding claims which additionally comprises a control zone.
The substrate according to any one of the preceding claims wherein the substrate comprises a nitrocellulose membrane.
The substrate according any one of the preceding claims wherein the capture probe is immobilised by UV cross-linking.
The substrate according to any one of the preceding claims which is a lateral flow strip.
An oligonucleotide probe comprising a single stranded hybridisation region of at least 8 bases in length which comprises 3 or more repeat copies of a 2 to 4 base DNA sequence motif wherein the copies of the sequence motif are contiguous or separated by one base.
Dependencies are inferred from the claim text.