Methods, Kits and Compositions Pertaining to the Suppression of the Detectable Probe Binding to Randomly Distributed Repeat Sequences Genomic Nucleic Acid

Abstract

This invention is directed to methods, kits, non-nucleotide probes as well as other compositions pertaining to the suppression of binding of detectable nucleic acid probes to undesired nucleotide sequences of genomic nucleic acid in assays designed to determine target genomic nucleic acid.

1 - 17 . (canceled) 
     
     
         18 . A mixture of two or more peptide nucleic acid probes of different nucleobase sequence, wherein the two or more probes are 16 to 50 nucleobases in length, and each probe comprises at least sixteen consecutive nucleobases that are greater than ninety percent homologous to a fraction of SEQ ID NO: 1 or SEQ ID NO: 2. 
     
     
         19 - 36 . (canceled) 
     
     
         37 . A composition comprising:
 a) genomic nucleic acid comprising one or more segments of randomly distributed repeat sequence; and   b) two or more peptide nucleic acid probes of different nucleobase sequence hybridized to at least a fraction of the one or more segments of randomly distributed repeat sequence of the genomic nucleic acid, wherein each of the two or more probes is 16 to 50 nucleobases in length and each comprises at least sixteen consecutive nucleobases that are greater than eighty percent homologous to a fraction of SEQ ID NO: 1 or SEQ ID NO: 2.   
     
     
         38 . A composition comprising:
 a) a detectable nucleic acid probe of at least 100 nucleobases that has been derived from genomic nucleic acid and that contains one or more segments of randomly distributed repeat sequence; and   b) two or more peptide nucleic acid probes of different nucleobase sequence hybridized to at least a fraction of the one or more segments of randomly distributed repeat sequence of the detectable nucleic acid probe, wherein each of the two or more probes is 16 to 50 nucleobases in length and each comprises at least sixteen consecutive nucleobases that are greater than eighty percent homologous to a fraction of SEQ ID NO: 1 or SEQ ID NO: 2.   
     
     
         39 - 90 . (canceled) 
     
     
         91 . A reagent kit comprising:
 a) a mixture of two or more peptide nucleic acid probes of different nucleobases sequence, wherein the two or more probes are 16 to 50 nucleobases in length, and wherein each probe comprises at least sixteen consecutive nucleobases that are at least eighty percent homologous to a fraction of SEQ ID NO: 1 and SEQ ID NO:2; and   b) reagents and compositions for performing an assay to determine hybridization of one or more detectable nucleic acid probes to one or more targets in genomic nucleic acid of a sample.   
     
     
         92 - 96 . (canceled) 
     
     
         97 . A kit comprising:
 a) a mixture of two or more peptide nucleic acid probes of different nucleobases sequence, wherein the two or more probes are 16 to 50 nucleobases in length, and wherein each probe comprises a segment of at least ten consecutive nucleobases that are at least eighty percent homologous to a sequence selected from the group consisting of: SEQ ID NOS: 3-13 and 15-25; and   b) reagents and compositions for performing an assay to determine hybridization of one or more detectable nucleic acid probes to one or more targets in genomic nucleic acid of a sample.   
     
     
         98 - 102 . (canceled) 
     
     
         103 . A combination of two or more peptide nucleic acid probes of differing sequence each consisting of a nucleobase sequence from 16 to 50 nucleobases in length wherein each probe comprises at least sixteen consecutive nucleobases that are at least eighty percent homologous to one or more sequences selected from the group consisting of SEQ ID NOS: 3-13. 
     
     
         104 . A peptide nucleic acid probe, comprising one or more of the nucleobase sequences selected from the group consisting of SEQ ID NOS: 3, 5-9, 11-13 and 15-25, wherein the peptide nucleic acid probe is 16 to 50 nucleobases in length. 
     
     
         105 . The peptide nucleic acid probe of  claim 104 , wherein the peptide nucleic acid subunits of the peptide nucleic acid probe have the formula: 
       
         
           
           
               
               
           
         
         wherein: 
         each J is the same or different and is selected from the group consisting of: H, R 1 , OR′, SR 1 , NHR 1 , NR 1   2 , F, CI, Br and I; 
         each K is the same or different and is selected from the group consisting of: O, S, NH and NR 1 ; 
         each R 1  is the same or different and is an alkyl group comprising one to five carbon atoms that may optionally contain a heteroatom or a substituted or unsubstituted aryl group; 
         each A is selected from the group consisting of a single bond, a group of the formula; —(CJ 2 ) s - and a group of the formula; —(CJ 2 ) s C(O)—, wherein J is defined above and each s is an integer from one to five; 
         each t is 1 or 2; 
         each u is 1 or 2; and 
         each L is the same or different and is selected from the group consisting of: J, dabcyl, fluorescein, adenine, cytosine, guanine, thymine, uridine, 5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, p seudoisocyto sine, 2-thiouracil, 2-thiothymidine, other naturally occurring nucleobase analogs, other non-naturally occurring nucleobases and substituted and unsubstituted aromatic moieties. 
       
     
     
         106 . The peptide nucleic acid probe of  claim 104 , wherein the peptide nucleic acid subunits of the peptide nucleic acid probe comprise a naturally or nonnaturally occurring nucleobase attached to an aza nitrogen of an N-[2-(aminoethyl)]glycine backbone through a methylene carbonyl linkage. 
     
     
         107 . A composition, comprising:
 a) one or more detectable nucleic acid probes; and   b) the peptide nucleic acid probe of  claim 104 .   
     
     
         108 . The composition of  claim 107 , wherein at least one detectable nucleic acid probe is at least 100 nucleobases in length. 
     
     
         109 . A composition, comprising:
 a) genomic nucleic acid; and   b) the peptide nucleic acid probe of  claim 104 .   
     
     
         110 . The composition of  claim 109 , wherein the genomic nucleic acid comprises one or more segments of randomly distributed repeat sequence. 
     
     
         111 . A kit, comprising:
 a) the peptide nucleic acid probe of  claim 110 ; and   b) reagents and compositions for performing an assay to determine hybridization of one or more detectable nucleic acid probes to one or more targets in genomic nucleic acid of a sample.   
     
     
         112 . A mixture of about 5 to about 50 peptide nucleic acid probes of different nucleobase sequence, wherein each peptide nucleic acid probe is at least sixteen subunits in length and comprises a nucleobase sequence of at least ten consecutive nucleobases that are at least eighty percent homologous to SEQ ID NO: 1 or SEQ ID NO: 2, without regard to whether the subunits containing the at least ten consecutive nucleobases are separated by one or more linkers. 
     
     
         113 . The mixture of peptide nucleic acid probes of  claim 112 , wherein the peptide nucleic acid subunits of at least one peptide nucleic acid probe have the formula: 
       
         
           
           
               
               
           
         
         wherein: 
         each J is the same or different and is selected from the group consisting of: H, R 1 , OR′, SR 1 , NHR 1 , NR 1   2 , F, CI, Br and I; 
         each K is the same or different and is selected from the group consisting of: O, S, NH and NR 1 ; 
         each R 1  is the same or different and is an alkyl group comprising one to five carbon atoms that may optionally contain a heteroatom or a substituted or unsubstituted aryl group; 
         each A is selected from the group consisting of a single bond, a group of the formula; —(CJ 2 ) s - and a group of the formula; —(CJ 2 ) s C(O)—, wherein J is defined above and each s is an integer from one to five; 
         each t is 1 or 2; 
         each u is 1 or 2; and 
         each L is the same or different and is selected from the group consisting of: J, dabcyl, fluorescein, adenine, cytosine, guanine, thymine, uridine, 5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, pseudoisocytosine, 2-thiouracil, 2-thiothymidine, other naturally occurring nucleobase analogs, other non-naturally occurring nucleobases and substituted and unsubstituted aromatic moieties. 
       
     
     
         114 . The mixture of peptide nucleic acid probes of  claim 112 , wherein the peptide nucleic acid subunits of at least one peptide nucleic acid probe comprise a naturally or non-naturally occurring nucleobase attached to an aza nitrogen of an N42-(aminoethyl)]glycine backbone through a methylene carbonyl linkage. 
     
     
         115 . A composition, comprising:
 a) one or more detectable nucleic acid probes; and   b) the mixture of peptide nucleic acid probes of  claim 112 .   
     
     
         116 . The composition of  claim 115 , wherein at least one detectable nucleic acid probe is at least 100 nucleobases in length. 
     
     
         117 . A composition, comprising:
 a) genomic nucleic acid; and   b) the mixture of peptide nucleic acid probes of  claim 112 .   
     
     
         118 . The composition of  claim 117 , wherein the genomic nucleic acid comprises one or more segments of randomly distributed repeat sequence. 
     
     
         119 . A kit, comprising:
 a) the mixture of peptide nucleic acid probes of  claim 112 ; and   b) reagents and compositions for performing an assay to determine hybridization of one or more detectable nucleic acid probes to one or more targets in genomic nucleic acid of a sample.   
     
     
         120 . A mixture of about 5 to about 50 peptide nucleic acid probes of different nucleobase sequence, wherein each peptide nucleic acid probe is at least sixteen subunits in length and comprises a nucleobase sequence of at least ten consecutive nucleobases that are at least eighty percent homologous to a sequence selected from the group consisting of: SEQ ID NOS: 3-26, without regard to whether the subunits containing the at least ten consecutive nucleobases are separated by one or more linkers. 
     
     
         121 . The mixture of peptide nucleic acid probes of  claim 120 , wherein the peptide nucleic acid subunits of at least one peptide nucleic acid probe have the formula: 
       
         
           
           
               
               
           
         
         wherein: 
         each J is the same or different and is selected from the group consisting of: H, R 1 , OR′, SR 1 , NHR 1 , NR 1   2 , F, CI, Br and I; 
         each K is the same or different and is selected from the group consisting of: O, S, NH and NR 1 ; 
         each R 1  is the same or different and is an alkyl group comprising one to five carbon atoms that may optionally contain a heteroatom or a substituted or unsubstituted aryl group; 
         each A is selected from the group consisting of a single bond, a group of the formula; —(CJ 2 ) s - and a group of the formula; —(CJ 2 ) s C(O)—, wherein J is defined above and each s is an integer from one to five; 
         each t is 1 or 2; 
         each u is 1 or 2; and 
         each L is the same or different and is selected from the group consisting of: J, dabcyl, fluorescein, adenine, cytosine, guanine, thymine, uridine, 5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, pseudoisocytosine, 2-thiouracil, 2-thiothymidine, other naturally occurring nucleobase analogs, other non-naturally occurring nucleobases and substituted and unsubstituted aromatic moieties. 
       
     
     
         122 . The mixture of peptide nucleic acid probes of  claim 120 , wherein the peptide nucleic acid subunits of at least one peptide nucleic acid probe comprise a naturally or non-naturally occurring nucleobase attached to an aza nitrogen of an N-[2-(aminoethyD]glycine backbone through a methylene carbonyl linkage. 
     
     
         123 . A composition, comprising:
 a) one or more detectable nucleic acid probes; and   b) the mixture of peptide nucleic acid probes of  claim 120 .   
     
     
         124 . The composition of  claim 123 , wherein at least one detectable nucleic acid probe is at least 100 nucleobases in length. 
     
     
         125 . A composition, comprising:
 a) genomic nucleic acid; and   b) the mixture of peptide nucleic acid probes of  claim 121 .   
     
     
         126 . The composition of  claim 125 , wherein the genomic nucleic acid comprises one or more segments of randomly distributed repeat sequence. 
     
     
         127 . A kit, comprising:
 a) the mixture of peptide nucleic acid probes of  claim 121 ; and   b) reagents and compositions for performing an assay to determine hybridization of one or more detectable nucleic acid probes to one or more targets in genomic nucleic acid of a sample.   
     
     
         128 . A mixture of about 5 to about 50 peptide nucleic acid probes of different nucleobase sequence, wherein each peptide nucleic acid probe is at least sixteen subunits in length and comprises a nucleobase sequence selected from the group consisting of: SEQ ID NOS: 3-14, without regard to whether the subunits containing the at least sixteen nucleobases are separated by one or more linkers. 
     
     
         129 . The mixture of peptide nucleic acid probes of  claim 128 , wherein each peptide nucleic acid probe comprises a nucleobase sequence at least sixteen nucleobases in length selected from the group consisting of: SEQ ID NOS: 3-13 and the sequence TT(k)TTTTT(k)TTTLysOLysOTTT(k)TTTTT(k)TT, wherein each k is a D-lysine; Lys is L-lysine; and each 0 is 8-amino-3,6-dioxaoctanoic acid, without regard to whether the subunits containing the at least sixteen nucleobases are separated by one or more linkers. 
     
     
         130 . The mixture of peptide nucleic acid probes of  claim 128 , wherein the peptide nucleic acid subunits of at least one peptide nucleic acid probe have the formula: 
       
         
           
           
               
               
           
         
         wherein: 
         each J is the same or different and is selected from the group consisting of: H, R 1 , OR′, SR 1 , NHR 1 , NR 1   2 , F, CI, Br and I; 
         each K is the same or different and is selected from the group consisting of: O, S, NH and NR 1 ; 
         each R 1  is the same or different and is an alkyl group comprising one to five carbon atoms that may optionally contain a heteroatom or a substituted or unsubstituted aryl group; 
         each A is selected from the group consisting of a single bond, a group of the formula; —(CJ 2 ) s - and a group of the formula; —(CJ 2 ) s C(O)—, wherein J is defined above and each s is an integer from one to five; 
         each t is 1 or 2; 
         each u is 1 or 2; and 
         each L is the same or different and is selected from the group consisting of: J, dabcyl, fluorescein, adenine, cytosine, guanine, thymine, uridine, 5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, pseudoisocytosine, 2-thiouracil, 2-thiothymidine, other naturally occurring nucleobase analogs, other non-naturally occurring nucleobases and substituted and unsubstituted aromatic moieties. 
       
     
     
         131 . The mixture of peptide nucleic acid probes of  claim 128 , wherein the peptide nucleic acid subunits of at least one peptide nucleic acid probe comprise a naturally or non-naturally occurring nucleobase attached to an aza nitrogen of an N42-(aminoethyl)]glycine backbone through a methylene carbonyl linkage. 
     
     
         132 . A composition, comprising:
 a) one or more detectable nucleic acid probes; and   b) the mixture of peptide nucleic acid probes of  claim 128 .   
     
     
         133 . The composition of  claim 132 , wherein at least one detectable nucleic acid probe is at least 100 nucleobases in length. 
     
     
         134 . A composition, comprising:
 a) genomic nucleic acid; and   b) the mixture of peptide nucleic acid probes of  claim 128 .   
     
     
         135 . The composition of  claim 134 , wherein the genomic nucleic acid comprises one or more segments of randomly distributed repeat sequence. 
     
     
         136 . A kit, comprising:
 a) the mixture of peptide nucleic acid probes of  claim 128 ; and   b) reagents and compositions for performing an assay to determine hybridization of one or more detectable nucleic acid probes to one or more targets in genomic nucleic acid of a sample.