Methods of screening nucleic acids using mass spectrometry

Abstract

Methods for screening nucleic acids for mutations by analyzing nonrandomly fragmented nucleic acids using mass spectrometric techniques and to procedures for improving mass resolution and mass accuracy of these methods of detecting mutations. Kits for performing the methods are provided.

We claim:  
     
         1 . A method of detecting mutations in a target nucleic acid comprising: 
 obtaining a target nucleic acid in single-stranded form;    hybridizing the single-stranded target nucleic acid to one or more sets of fragmenting probes to form hybrid target nucleic acid/fragmenting probe complexes comprising at least one double-stranded region and at least one single-stranded region;    nonrandomly fragmenting the target nucleic acid by cleaving the hybrid target nucleic acid/fragmenting probe complexes at every single-stranded region with at least one single-strand-specific cleaving reagent to form a set of nonrandom length fragments (NLFs); and    determining masses of the members of the set using mass spectrometry.    
     
     
         2 . The method of  claim 1 , wherein at least one member of the set of single-stranded NLFs optionally has one or more nucleotides replaced with mass-modified nucleotides.  
     
     
         3 . The method of  claim 2 , wherein the determining step optionally further comprises utilizing internal self-calibrants to provide improved mass accuracy.  
     
     
         4 . The method of  claim 1 , wherein the set of fragmenting probes leaves single-strand gaps between double-stranded regions formed by hybridization of the set of fragmenting probes to the target nucleic acid.  
     
     
         5 . The method of  claim 4 , wherein the hybridizing step further comprises: 
 providing two sets of single-stranded target nucleic acid and    separately hybridizing a first set of fragmenting probes to a first set of single-stranded target nucleic acid and a second set of fragmenting probes to a second set of single-stranded target nucleic acid, wherein the members of the second set of fragmenting probes comprise at least one single-stranded nucleotide sequence complementary to regions of the target nucleic acid that are not complementary to any nucleotide sequences in any members of the first set of fragmenting probes.    
     
     
         6 . The method of  claim 5 , wherein the members of the first set of fragmenting probes comprise nucleotide sequences that overlap with nucleotide sequences of the members of the second set of fragmenting probes.  
     
     
         7 . The method of  claim 1 , wherein the single-strand-specific cleaving reagent is a single-strand-specific endonuclease.  
     
     
         8 . The method of  claim 1 , wherein the single-strand-specific cleaving reagents are single-strand specific chemical cleaving reagents.  
     
     
         9 . The method of  claim 8 , wherein the single-strand specific chemical cleaving reagents are selected from the group consisting of hydroxylamine, hydrogen peroxide, osmium tetroxide and potassium permanganate.  
     
     
         10 . The method of  claim 1 , further comprising after the nonrandomly fragmenting step: 
 hybridizing one or more of the NLFs to one or more capture probes, wherein the capture probes comprise a single-stranded region complementary to at least one of the NLFs and a first binding moiety,    binding the first binding moiety to a second binding moiety attached to a solid support, wherein the binding occurs either before or after the hybridizing of the NLFs to one or more capture probes, isolating a set of single-stranded NLFs.    
     
     
         11 . The method of  claim 1 , wherein the fragmenting probes comprise a single-stranded nucleotide sequence and a first binding moiety, further comprising: 
 after the nonrandomly fragmenting step, binding the first binding moiety to a second binding moiety attached to a solid support; and    isolating the set of single-stranded NLFs.    
     
     
         12 . A method of detecting mutations in a target nucleic acid comprising: 
 obtaining a target nucleic acid in single-stranded form;    nonrandomly fragmenting the target nucleic acid with one or more restriction endonucleases to form a set of nonrandom length fragments (NLFs);    hybridizing one or more of the set of NLFs or a subset thereof to one or more oligonucleotide probes, wherein each of the oligonucleotide probes comprises a nucleic acid comprising a single-stranded region and a first binding moiety, binding the first binding moiety to a second binding moiety attached to a solid support either before or after the hybridizing step;    isolating the set or subset of single-stranded NLFs; and    determining masses of the members of the set using mass spectrometry.    
     
     
         13 . The method of  claim 12 , wherein all of the oligonucleotide probes consist of one of either full-length positive or full-length negative single strands of the target nucleic acid and a first binding moiety.  
     
     
         14 . The method of  claim 12 , wherein the binding between the first binding moiety and the second binding moiety is a covalent attachment.  
     
     
         15 . The method of  claim 12 , wherein one binding moiety is a member selected from the group consisting of an antibody, a hormone, an inhibitor, a co-factor portion, a binding ligand and a polynucleotide sequence, and the other binding moiety is a corresponding member selected from the group consisting of an antigen capable of recognizing the antibody, a receptor capable of recognizing the hormone, an enzyme capable of recognizing the inhibitor, a cofactor enzyme binding site capable of recognizing the co-factor portion, a substrate capable of recognizing the binding ligand and a complementary polynucleotide sequence.  
     
     
         16 . The method of  claim 12 , wherein the isolating further comprises: 
 washing the set of NLFs bound to the solid support with a solution comprising volatile salts selected from the group consisting of ammonium bicarbonate dimethyl ammonium bicarbonate and trimethyl ammonium bicarbonate.    
     
     
         17 . A method of detecting mutations in a target nucleic acid comprising: 
 obtaining a target nucleic acid in single-stranded form;    hybridizing the single-stranded target nucleic acid to one or more restriction site probes to form hybridized target nucleic acids having double-stranded regions, wherein the restriction site probes have hybridized to the single-stranded target nucleic acid and at least one single-stranded region;    nonrandomly fragmenting the hybridized target nucleic acids by contacting it with one or more restriction endonucleases that cleave at restriction sites within the double-stranded regions to produce a set of single-straneded nonrandom length fragments (NLFs); and    determining masses of the members of the set using mass spectrometry.    
     
     
         18 . The method of  claim 17 , further comprising after the nonrandomly fragmenting step, 
 hybridizing the NLFs to one or more capture probes, wherein the capture probes comprise a single-stranded region complementary to at least one or the NLFs and a first binding moiety, binding the first binding moiety to a second binding moiety attached to a solid support, wherein the binding occurs either before or after the hybridizing of the NLFs to one or more capture probes, isolating a set of single-stranded NLFs.    
     
     
         19 . The method of  claim 18 , wherein the cleaved restriction site probes comprise a single-stranded region complementary to half of a restriction endonuclease site and a first binding moiety; and further comprising, after the nonrandomly fragmenting step, binding the first binding moiety to a second binding moiety attached to a solid support, and isolating a set of single-stranded NLFs.  
     
     
         20 . A method of detecting mutations in a target nucleic acid comprising: 
 obtaining a target nucleic acid in single-stranded form;    exposing the target nucleic acid to conditions permitting folding of the single-stranded target nucleic acid to form a three-dimensional structure having intramolecular secondary and tertiary interactions;    nonrandomly fragmenting the folded target nucleic acid with at least one structure-specific endonuclease to form a set of single-stranded nonrandom length fragments (NLFs);    modifying either the target nucleic acid or the set of single-stranded NLFs such that members of the set of single-stranded NLFs comprise a single-stranded nucleotide sequence and at least one first binding moiety;    binding the first binding moiety to a second binding moiety attached to a solid support;    isolating the set of single-stranded NLFs; and    determining masses of the members of the set using mass spectrometry.    
     
     
         21 . The method of  claim 20 , wherein the isolated set of single-stranded NLFs comprise any NLFs having a 5′ end of the target nucleic acid.  
     
     
         22 . A method of detecting mutations in a target nucleic acid comprising: 
 obtaining a target nucleic acid in single-stranded form;    exposing the nucleic acid to conditions permitting folding of the single-stranded target nucleic acid to form a three-dimensional structure having intramolecular secondary and tertiary interactions;    nonrandomly fragmenting the folded target nucleic acid with at least one structure-specific endonuclease to form a set of single-stranded nonrandom length fragments (NLFs);    hybridizing one or more of the set of NLFs to one or more capture probes, wherein the capture probes comprise a single-stranded nucleotide sequence and a first binding moiety;    binding the first binding moiety to a second binding moiety attached to a solid support either before or after the hybridizing step;    isolating a set of single-stranded NLFs; and    determining masses of the members of the set using mass spectrometry.    
     
     
         23 . The method of  claim 22  wherein the isolated set of single-stranded NLFs comprise any NLFs having a 5′ end of the target nucleic acid.  
     
     
         24 . The method of  claim 22 , wherein the structure-specific endonuclease is selected from the group consisting of: 
 T4 endonuclease VII, RuvC, MutY and the endonucleolytic activity from the 5′-3′ exonuclease subunit of thermo-stable polymerases.    
     
     
         25 . A method of detecting mutations in a target nucleic acid comprising: 
 obtaining a target nucleic acid in single-stranded form;    hybridizing the single-stranded target nucleic acid to one or more wild type probes;    nonrandomly fragmenting the target nucleic acid with one or more mutation-specific cleaving reagents that specifically cleave at any regions of nucleotide mismatch that form between the target nucleic acid and any of the wild type probes to form a set of single-stranded nonrandom length fragments (NLFs); and    determining masses of the members of the set using mass spectrometry    
     
     
         26 . The method of  claim 25 , wherein the nonrandomly fragmenting step further comprises: 
 digesting the first set of nonrandom length fragments with one or more restriction endonucleases or    cleaving the first set of nonrandom length fragments with one or more single-strand-specific cleaving reagents.    
     
     
         27 . The method of  claim 25 , wherein members of the set of single-stranded NLFs comprise a single-stranded region and at least one first binding moiety, further comprising, after the nonrandomly fragmenting step, binding the first binding moiety to a second binding moiety attached to a solid support; and isolating a set of single-stranded NLFs.  
     
     
         28 . The method of  claim 25 , wherein the obtaining step further comprises: 
 hybridizing members of the set of NLFs to one or more capture probes, 
 wherein the capture probes comprise a single-stranded nucleotide sequence and at least one first binding moiety, binding the first binding moiety to a second binding moiety attached to a solid support; and isolating a set of single-stranded NLFs.  
   
     
     
         29 . The method of  claim 25 , wherein the obtaining step further comprises: 
 isolating a set of single-stranded NLFs comprising any NLFs having a 5′ end of the target nucleic acid.    
     
     
         30 . A method of detecting mutations in a target nucleic acid comprising: 
 nonrandomly fragmenting the target nucleic acid with one or more restriction endonucleases to form a set of double-stranded nonrandom length fragments (NLFs), wherein the nonrandomly fragmenting further comprises including volatile salts in the restriction buffer; and    determining masses of the members of the set of double-stranded NLFs, wherein the determining does not involve sequencing of the target nucleic acid.    
     
     
         31 . A method of detecting mutations in a double-stranded target nucleic acid comprising: 
 nonrandomly fragmenting the target nucleic acid using one or more restriction endonucleases to form a first set of nonrandom length fragments (NLFs);    hybridizing members of the first set of NLFs to a set of wild type probes;    nonrandomly fragmenting one or more members of the set of NLFs with one or more mutation-specific cleaving reagents that specifically cleave at any regions of nucleotide mismatch that form between members of the first set of NLFs and complementary members of the set of wild type probes, wherein the nonrandomly fragmenting step forms a second set of NLFs; and    determining masses of members of the second set of NLFs using mass spectrometry, wherein the determining does not require sequencing of the target nucleic acid.    
     
     
         32 . The method of  claim 31  further comprising: 
 obtaining the set of wild type probes by nonrandomly fragmenting a wild type target nucleic acid using the same restriction endonucleases used to form the first set of NLFs.  
 
     
     
         33 . The method of  claim 32 , wherein the steps of nonrandomly fragmenting of the target nucleic acid and obtaining the set of wild type fragmenting probes are performed simultaneously in a single solution.  
     
     
         34 . The method of  claim 32  further comprising, before the determining step, 
 isolating the second set of NLFs wherein the members of the second set comprise double-stranded nucleotide sequences and a first binding moiety; and binding the first binding moiety to a second binding moiety attached to a solid support.  
 
     
     
         35 . The method of  claim 32  further comprising before the determining step, 
 isolating the second set of NLFs wherein the isolating comprises hybridizing members of the second set of NLFs to one or more capture probes, wherein the capture probes comprise a single-stranded nucleotide sequence and a first binding moiety, binding the first binding moiety to a second binding moiety attached to a solid support.  
 
     
     
         36 . A method of detecting mutations in a target nucleic acid comprising: 
 nonrandomly fragmenting the target nucleic acid, using a solution comprising one or more volatile salts to form a set of nonrandom length fragments (NLFs); and    determining masses of members of the set of NLFs using mass spectrometry, wherein the determining does not involve sequencing of the target nucleic acid.    
     
     
         37 . A kit for detecting mutations in one or more target nucleic acids in a sample comprising: 
 (a) one or more sets of fragmenting probes, wherein the fragmenting probes are complementary to a sequence of one or more of the target nucleic acids;    (b) a single-strand specific cleaving regent;    (c) a solid support for isolating single-stranded target nucleic acids that have been nonrandomly fragmented into single-stranded nonrandom length fragments; and    (d) matrix for performing mass spectrometry analyses.    
     
     
         38 . The kit of  claim 37 , wherein the single-strand specific cleaving reagent is a single-strand-specific chemical cleaving reagent selected from the group consisting of hydroxylamine, hydrogen peroxide, osmium tetroxide and potassium permanganate.  
     
     
         39 . The kit of  claim 37 , wherein the single-strand specific cleaving reagent is a nuclease selected from the group consisting of Mung bean nuclease, Nuclease S1 and RNase A.  
     
     
         40 . A kit for detecting mutations in one or more target nucleic acids in a sample comprising: 
 (a) one or more sets of restriction site probes, wherein the probes comprise a single-stranded sequence capable of hybridizing to a sequence of the one or more target nucleic acids;    (b) one or more restriction endonucleases that cleave at restriction sites within the restriction site probes; and    (c) a solid support for isolating single-stranded target nucleic acids that have been nonrandomly fragmented into single-stranded nonrandom length fragments.    
     
     
         41 . The kit of  claim 40 , wherein the restriction endonuclease is a Class IIS restriction endonuclease.  
     
     
         42 . The kit of  claim 40 , wherein the restriction site probe comprises two regions, a first region that is single-stranded and complementary to a specific sequence within the target nucleic acid, and a second region that is double-stranded and contains a restriction recognition site for a Class IIS restriction endonuclease.  
     
     
         43 . The kit of  claim 40 , further comprising matrix for performing mass spectrometry analyses.