Use of Mass Labelled Probes to Detect Target Nucleic Acids Using Mass Spectrometry
First Claim
1. A method of detecting the presence of a target nucleic acid in a sample, which method comprises a) contacting the sample, under hybridizing conditions, with a probe for said target nucleic acid, wherein said probe comprises two terminal nucleic acid target recognition sequences that are complementary to and capable of hybridizing to two neighbouring regions of the target sequence, and wherein the probe is linked to a tag that is identifiable by mass spectrometry;
- b) covalently connecting the ends of the hybridized probe with each other to form a circularized-probe, which interlocks with the target strand through catenation;
c) cleaving the mass tag from the circularized probe; and
d) detecting the mass tag by mass spectrometry.
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Abstract
The invention relates to the use of mass labelled probes to characterise nucleic acids by mass spectrometry. Thus the invention provides methods of detecting the presence of a target nucleic acid in a sample, using a circularising probe in which a mass tag is present in the probe. Further methods of detecting the presence of a target nucleic acid are provided, which in contrast use a probe detection sequence in the circularising probe, wherein the probe detection sequence is detected with a probe attached to a mass tag. Methods for determining a genetic profile from the genome of an organism also form part of the invention.
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Citations
32 Claims
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1. A method of detecting the presence of a target nucleic acid in a sample, which method comprises
a) contacting the sample, under hybridizing conditions, with a probe for said target nucleic acid, wherein said probe comprises two terminal nucleic acid target recognition sequences that are complementary to and capable of hybridizing to two neighbouring regions of the target sequence, and wherein the probe is linked to a tag that is identifiable by mass spectrometry; -
b) covalently connecting the ends of the hybridized probe with each other to form a circularized-probe, which interlocks with the target strand through catenation;
c) cleaving the mass tag from the circularized probe; and
d) detecting the mass tag by mass spectrometry. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 30, 32)
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10. A method of detecting the presence of a target nucleic acid in a sample, which method comprises
a) contacting the sample, under hybridizing conditions, with a probe for said target nucleic acid, wherein said probe comprises two terminal nucleic acid target recognition sequences that are complementary to and capable of hybridizing to two neighbouring regions of the target sequence, and wherein the probe comprises a probe identification sequence; -
b) covalently connecting the ends of the hybridized probe with each other to form a circularized-probe, which interlocks with the target strand through catenation;
c) hybridizing a probe detection oligonucleotide to the probe identification sequence present in the said probe, where the probe detection oligonucleotide is cleavably linked to a mass tag;
d) cleaving the mass tag from the probe detection oligonucleotide; and
e) detecting the mass tag by mass spectrometry. - View Dependent Claims (11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21)
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22. A method of detecting the presence of a target nucleic acid in a sample, which method comprises
a) contacting the sample, under hybridizing conditions, with a probe for said target nucleic acid, wherein said probe comprises two terminal nucleic acid target recognition sequences that are complementary to and capable of hybridizing to two neighbouring regions of the target sequence, and wherein the probe further comprises a probe identification sequence and a pair of primer binding sequences; -
b) covalently connecting the ends of the hybridized probe with each other to form a circularized-probe, which interlocks with the target strand through catenation;
c) cleaving the circularized probe such that the opened probe has the primer binding sequences oriented to enable polymerase chain reaction amplification of the probe identification sequence;
d) hybridizing a probe detection oligonucleotide to the probe identification sequence present in the said probe, where the probe detection oligonucleotide is cleavably linked to a mass tag;
e) performing a primer extension reaction by providing a primer capable of hybridizing to the primer binding sequence upstream of the probe identification sequence and extending said primer with a polymerase having 5′
exonuclease activity, so as to cleave the mass tag from the probe detection oligonucleotide; and
f) detecting the mass tag by mass spectrometry. - View Dependent Claims (23, 24, 25, 26, 27, 28)
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29. A method of detecting the presence of a target nucleic acid in a sample, which method comprises
a) contacting the sample, under hybridizing conditions, with a probe for said target nucleic acid, wherein said probe comprises two terminal nucleic acid target recognition sequences that are complementary to and capable of hybridizing to two neighbouring regions of the target sequence, and wherein the probe further comprises a probe identification sequence and a pair of primer binding sequences; -
b) covalently connecting the ends of the hybridized probe with each other to form a circularized-probe, which interlocks with the target strand through catenation;
c) contacting one primer binding sequence with a complementary primer under conditions for rolling circle replication to occur, to provide a linear extension product;
d) contacting the linear extension product with a primer having the sequence of the second primer binding sequence, under conditions to provide for hyper-branching rolling circle replication;
e) hybridizing a probe detection oligonucleotide to the probe identification sequence present in the said probe, where the probe detection oligonucleotide is cleavably linked to a mass tag; and
f) detecting the mass tag by mass spectrometry. - View Dependent Claims (31)
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Specification