Homologous pairing capture assay and related methods and applications

Abstract

A Homologous Pairing Capture Assay is described which enables detection of coalignment between homologous DNA sequences. The assay involves ligating closely positioned homologous sequences to each other thereby generating head-to-head ligation products or inverted repeats. DNA fragments containing an inverted repeat are then converted into hairpin DNA molecules. The hairpin DNA molecules can then be readily separated from DNA molecules free of inverted repeats. Also described are various diagnostic applications and kits relating to the assay.

What is claimed is: 
     
         1 . A kit for detecting pairing between homologous nucleic acid (DNA) sequences in DNA species, the kit comprising:
 at least one crosslinking agent, wherein upon reacting the homologous DNA sequence with the at least one crosslinking agent, pairing interactions are formed;   at least one agent for forming head-to-head ligation products from the stabilized pairing interactions, wherein head-to-head ligation products formed from closely juxtaposed pairs of homologous DNA sequences are inverted repeats; and   at least one agent for forming hairpin molecules from inverted repeats.   
     
     
         2 . The kit of  claim 1  wherein the crosslinking agent is selected from the group consisting of formaldehyde, dimethyl adipimidate, disuccinimidyl suberate, dithiobis succinimidyl propionate, ethylene glycolbis[succinimidyl succinate], DNA intercalating agents linked to biotinstreptavidin, psoralen, and difunctional alkylating agents. 
     
     
         3 . The kit of  claim 1  wherein the at least one agent for forming head-to-head ligation products is selected from the group consisting of T4 DNA ligase and  E. coli  DNA ligase. 
     
     
         4 . The kit of  claim 1  wherein the at least one agent for forming hairpin molecules is an agent used in one or more of heat denaturation followed by rapid reannealing or isothermal hybridization approaches. 
     
     
         5 . The kit of  claim 1  further comprising a prepacked column comprising a charged resin. 
     
     
         6 . The kit of  claim 5  wherein the charged resin is benzoylated napthoylated diethylaminoethyl cellulose. 
     
     
         7 . The kit of  claim 1  further comprising:
 at least one single strand specific nuclease for eliminating or separating single stranded DNA species from the hairpin molecules. 
 
     
     
         8 . A kit for detecting pairing between homologous nucleic acid (DNA) sequences in DNA species, the kit comprising:
 at least one crosslinking agent, wherein upon reacting the homologous DNA sequence with the at least one crosslinking agent, pairing interactions are formed;   at least one agent for forming head-to-head ligation products from the stabilized pairing interactions, wherein head-to-head ligation products formed from closely juxtaposed pairs of homologous DNA sequences are inverted repeats; and   at least one agent for forming hairpin molecules from inverted repeats by rapid instrastrand annealing.   
     
     
         9 . The kit of  claim 8  wherein the crosslinking agent is selected from the group consisting of formaldehyde, dimethyl adipimidate, disuccinimidyl suberate, dithiobis succinimidyl propionate, ethylene glycolbis[succinimidyl succinate], DNA intercalating agents linked to biotinstreptavidin, psoralen, and difunctional alkylating agents. 
     
     
         10 . The kit of  claim 8  wherein the at least one agent for forming head-to-head ligation products is selected from the group consisting of T4 DNA ligase and  E. coli  DNA ligase. 
     
     
         11 . The kit of  claim 8  wherein the at least one agent for forming hairpin molecules is an agent used in one or more of heat denaturation followed by rapid reannealing or isothermal hybridization approaches. 
     
     
         12 . The kit of  claim 8  further comprising a prepacked column comprising a charged resin. 
     
     
         13 . The kit of  claim 8  wherein the charged resin is benzoylated napthoylated diethylaminoethyl cellulose. 
     
     
         14 . The kit of  claim 8  further comprising:
 at least one single strand specific nuclease for eliminating or separating single stranded DNA species from the hairpin molecules. 
 
     
     
         15 . The kit of  claim 8  wherein rapid instrastrand annealing is performed by denaturation followed by renaturation. 
     
     
         16 . The kit of  claim 15  wherein denaturation and renaturation are carried out under conditions that (i) promote intramolecular annealing over intermolecular annealing, and that (ii) allow annealing between homologous sequences but discourage annealing between non-homologous sequences. 
     
     
         17 . A kit for detecting pairing between homologous nucleic acid (DNA) sequences in DNA species, the kit comprising:
 at least one crosslink agent, wherein upon reacting the homologous DNA sequence with the at least one crosslink agent, pairing interactions are formed;   at least one agent for forming head-to-head ligation products from the stabilized pairing interactions, wherein head-to-head ligation products formed from closely juxtaposed pairs of homologous DNA sequences are inverted repeats; and   at least one agent for eliminating supercoiled ligation products;   at least one agent for forming hairpin molecules from inverted repeats by rapid instant intrastrand annealing.   
     
     
         18 . The kit of  claim 17  wherein the agent for eliminating supercoiled ligation products is selected from the group consisting of topoisomerase, single strand nicking enzyme, and pre-cast two dimensional gel.