Use of mass labeled probes to detect target nucleic acids using mass spectrometry

Abstract

The invention relates to the use of mass labeled 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.

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 neighboring 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.   
     
     
         2 . The method according to  claim 1 , wherein the two neighboring regions of the target sequence are immediately adjacent to each other. 
     
     
         3 . The method according to  claim 1 , wherein the two neighboring regions of the target sequence are separated by a gap, and wherein covalent connection of the sequences is performed by providing an oligonucleotide capable of hybridizing to the sequence between the neighboring regions of the target sequence, and ligating said oligonucleotide to the terminal nucleic acid recognition sequences. 
     
     
         4 . The method according to  claim 1 , wherein the two neighboring regions of the target sequence are separated by a gap, and wherein covalent connection of the sequences is performed by providing a gap-filling polymerase and one or more nucleotide triphosphates to extend the 3′ terminal nucleic acid target recognition sequence of the probe to fill the gap, and ligating the terminal nucleic acid recognition sequences. 
     
     
         5 . The method according to  claim 1 , wherein the sample is contacted with two or more different probes capable of binding different alleles of the target sequence, under conditions which a probe complementary for an allele present in the sample will form a circularized probe and a probe not complementary for an allele present in the sample will not form a circularized probe, wherein each probe comprises a different mass tag, and wherein the method includes the step of separating circularized probes from non-circularized probes. 
     
     
         6 . The method according to  claim 5 , wherein circularized probe is separated from non-circularized probe by digesting non-circularized probe with an exonuclease. 
     
     
         7 . The method according to  claim 5 , wherein the probes are captured onto a solid support and cleaved such that only circularized probes retain the tagged portion on the solid support. 
     
     
         8 . The method according to  claim 1 , wherein the sample is contacted with two or more sets of probes, each probe set comprising one or more probes for one or more alleles of a target sequence. 
     
     
         9 . The method according to  claim 8 , wherein each probe in a set comprises a tandem mass tag having a mass tag component and a mass normalization component such that the sum of the masses of the two components are the same for each member of the set. 
     
     
         10 . The method according to  claim 1 , wherein the probe further comprises a microarray address sequence. 
     
     
         11 . A method for determining a genetic profile from the genome of an organism, said method comprising:
 a) providing a microarray which has an array of microarray address sequence complements at discrete locations on said array;   b) performing the method of  claim 10  so as to detect the presence of one or more mass tags at one or more locations of the microarray; and   c) correlating the presence of a mass tag at a location with the presence of a target sequence in the organism.   
     
     
         12 . 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 neighboring 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.   
     
     
         13 . The method according to  claim 12 , wherein the two neighboring regions of the target sequence are immediately adjacent to each other. 
     
     
         14 . The method according to  claim 12 , wherein the two neighboring regions of the target sequence are separated by a gap, and wherein covalent connection of the sequences is performed by providing an oligonucleotide capable of hybridizing to the sequence between the neighboring regions of the target sequence, and ligating said oligonucleotide to the terminal nucleic acid recognition sequences. 
     
     
         15 . The method according to  claim 12 , wherein the two neighboring regions of the target sequence are separated by a gap, and wherein covalent connection of the sequences is performed by a providing a gap-filling polymerase and one or more nucleotide triphosphates to extend the 3′ terminal nucleic acid target recognition sequence of the probe to fill the gap, and ligating the terminal nucleic acid recognition sequences. 
     
     
         16 . The method according to  claim 12 , wherein the sample is contacted with two or more different probes capable of binding different alleles of the target sequence, under conditions which a probe complementary for an allele present in the sample will form a circularized probe and a probe not complementary for an allele present in the sample will not form a circularized probe, wherein each probe comprises a different probe identification sequence, and wherein the method includes the step of separating circularized probes from non-circularized probes. 
     
     
         17 . The method according to  claim 16 , wherein circularized probe is separated from non-circularized probe by digesting non-circularized probe with an exonuclease. 
     
     
         18 . The method according to  claim 16 , wherein the circularized probes comprise a primer binding site, and said probes are contacted with a rolling circle primer under conditions for rolling circle replication to occur, to provide a linear extension product. 
     
     
         19 . The method according to  claim 18 , wherein said rolling circle primer is attached to a solid support. 
     
     
         20 . The method according to  claim 18 , wherein said rolling circle primer is attached to an affinity ligand that allows the replication product to be captured onto a solid support derivatized with the corresponding ligand for the affinity ligand. 
     
     
         21 . The method according to  claim 18 , wherein the probe detection oligonucleotide is hybridized to the probe identification sequence present in the linear extension product. 
     
     
         22 . The method according  claim 12 , wherein the sample is contacted with two or more sets of probes, each probe set comprising one or more probes for one or more alleles of a target sequence. 
     
     
         23 . The method according to  claim 22 , wherein each probe in a set is detected with a probe detection oligonucleotide attached to a tandem mass tag having a mass tag component and a mass normalization component such that the sum of the masses of the two components are the same for each member of the set. 
     
     
         24 . The method according to  claim 12 , wherein the probe further comprises a microarray address sequence. 
     
     
         25 . A method for determining a genetic profile from the genome of an organism, said method comprising:
 a) providing a microarray which has an array of microarray address sequence complements at discrete locations on said array;   b) performing the method of  claim 24  so as to detect the presence of one or more mass tags at one or more locations of the microarray; and   c) correlating the presence of a mass tag at a location with the presence of a target sequence in the organism.   
     
     
         26 . 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 neighboring 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.   
     
     
         27 . The method according to  claim 26 , wherein prior to detection of the mass tag the probe is hybridized to a microarray at a location having a nucleotide sequence complementary to the microarray address sequence of the probe. 
     
     
         28 . The method according to  claim 26 , wherein the probe further comprises a microarray address sequence. 
     
     
         29 . A method for determining a genetic profile from the genome of an organism, said method comprising:
 a) providing a microarray which has an array of microarray address sequence complements at discrete locations on said array;   b) performing the method of  claim 28  so as to detect the presence of one or more mass tags at one or more locations of the microarray; and   c) correlating the presence of a mass tag at a location with the presence of a target sequence in the organism.