Applications with and methods for producing selected interstrand cross-links in nucleic acids

Abstract

The invention relates to a method for distinguishing at least two target bio-organic molecules with dyes selected from a pool of at least two dyes, the method comprising: (a) providing a first set of at least two probes, wherein each probe recognizes a target bio-organic molecule in a first set of target bio-organic molecules, and wherein each probe is distinctly-labelled with primary labels that are distinct from one another due to the presence of dyes in distinct ratios; (b) providing a second set of probes distinctly-labelled with said primary labels described in step (a), wherein each probe in said second probe set recognizes a target bio-organic molecule in a second set of target bio-organic molecules; wherein each probe in said first or second probe set is further labelled with the same first binary label, wherein said first binary label is distinct from said primary labels; and (c) contacting said at least two target bio-organic molecules with said probe sets, wherein said target bio-organic molecules are distinguished.

1 . A method for providing at least one selected sequence in a nucleic acid with interstrand cross-links comprising hybridising at least one selected single strand sequence with a complementary single strand nucleic acid or a functional analogue thereof, wherein said selected sequence or said complementary nucleic acid or both comprise a cross-linking agent.  
     
     
         2 . A method according to  claim 1 , wherein said functional analogue of said complementary single strand nucleic acid comprises peptide nucleic acid.  
     
     
         3 . A method according to  claim 1  or  claim 2 , wherein said selected interstrand cross-links hamper further hybridisation and/or replication of said selected sequences.  
     
     
         4 . A method for the generation of a probe wherein at least one selected sequence in said probe is at least in part prevented from functioning as a probe through providing said selected sequence with interstrand cross-links by a method according to anyone of claims  1 - 3 .  
     
     
         5 . A method according to  claim 3  or  claim 4  wherein at least one of said selected sequence comprises at least one repetitive sequence.  
     
     
         6 . A method according to anyone of the claims  1 - 5  wherein the cross-linking agent comprises a transition metal, preferably platinum.  
     
     
         7 . A method according to anyone of the claims  1 - 6  wherein the cross-linking agent is trans-dichlorodiammineplatinum(II).  
     
     
         8 . A method for the selected amplification of certain amplifiable sequences from a pool of amplifiable sequences comprising producing a selected interstrand cross-linked nucleic acid or probe, obtainable by a method according to anyone of the claims  1 - 7 , to decrease the amount of amplification of a subset of amplifiable sequences and subjecting said pool to an amplification reaction.  
     
     
         9 . A method according to  claim 8  wherein a single stranded nucleic acid is prevented from taking part in said amplification through disabling the primer extension function of said single stranded nucleic acid, preferably through modification of the 3′-hydroxy group.  
     
     
         10 . A method according to  claim 8  or  claim 9 , wherein said pool is selected from sequences present in a chromosome.  
     
     
         11 . A collection of amplified sequences obtainable by a method according anyone of the claims  8 - 10 .  
     
     
         12 . A probe for the detection of nucleic acid comprising a collection of labelled amplified sequences according to  claim 11 .  
     
     
         13 . A method referred to as COBRA for the labelling of a set of at least two bio-organic molecules with a set of at least two colours, comprising generating said set of colours through combining ratio labelling with binary labelling.  
     
     
         14 . A method according to  claim 13 , wherein the total number of distinguishable colours of said combination can, at least in the case wherein two fluorophores are simultaneously used per target, be calculated according to the formula I  
         No . of colours=( n +(( r×n !)/(2×( n− 2)!)))×2 m   I:  
       wherein n is the number of fluorophores used for ratio labelling, m is the number of fluorophores used to binary label the same target, and r is the number of ratios that is resolved by ratio labelling.  
       with: 
 2≦n≦∞,  
 0≦r≦∞ 
 0≦m≦∞ 
 
     
     
         15 . A method according to  claim 13  or  claim 14 , wherein at least one of said bio-organic molecules comprises nucleic acid, protein, carbohydrate and/or lipid.  
     
     
         16 . A method for simultaneous identification of sequences of at least one chromosome or part thereof, through the use of at least one probe according to  claim 12 , wherein said probe is labelled according to a COBRA method for doubling the number of identifiable labels obtainable by ratio labelling, comprising adding to a first set of fluorophores, used for the ratio labelling of a first set of probes, a novel fluorophore and labelling a second set of probes.  
     
     
         17 . The use of a probe according to  claim 12  for the detection of chromosomes or parts thereof.  
     
     
         18 . The use of selected interstrand cross-links for decreasing the amount of amplified product of certain amplifiable sequences.  
     
     
         19 . The identification of a disease through the typing of at least one chromosome wherein at least one chromosome is labelled, with at least one probe labelled according to anyone of the claims  13 - 15 , or by a method according to  claim 16  or a probe according to  claim 12 .  
     
     
         20 . A kit for the detection of nucleic acid comprising at least one probe according to  claim 12 .  
     
     
         21 . A kit for performing a method according to anyone of claims  13 - 16  comprising at least one probe labelled with a COBRA method.  
     
     
         22 . A kit for generating a probe according to  claim 12 , comprising at least a cross-linking agent, preferably linked to a single stranded nucleic acid.  
     
     
         23 . A molecule comprising at least two parts, cross-linked with a cross-linking agent, wherein said cross-linking agent comprises a transition metal, preferably platinum, wherein at least two of said parts comprise a protein.