Compositions and Methods for Intramolecular Nucleic Acid Rearrangement

Abstract

Aspects of the present invention are drawn to processes for moving a region of interest in a polynucleotide from a first position to a second position with regard to a domain within the polynucleotide, also referred to as a “reflex method”. In certain embodiments, the reflex method results in moving a region of interest into functional proximity to specific domain elements present in the polynucleotide (e.g., primer sites and/or MID). Compositions, kits and systems that find use in carrying out the reflex processes described herein are also provided.

1 - 20 . (canceled) 
     
     
         21 . A kit for analyzing a nucleic acid sample, comprising:
 (a) a first forward primer comprising, from 5′ to 3′:
 (i) a first primer site; 
 (ii) a reflex sequence; and 
 (iii) a region that that hybridizes to a first sequence in a genome; 
   (b) a first reverse primer that hybridizes to a second sequence in said genome, wherein said forward primer and said reverse primer are characterized in that they amplify a target fragment when employed in a polymerase chain reaction (PCR) using said genome or cDNA copied from mRNA encoded by said genome as a template;   (c) a second reverse primer comprising:
 (i) a 3′ region that is complementary to a first site in the top strand of said target fragment and that can prime template dependent nucleic acid synthesis therefrom; and 
 (ii) a 5′ region that comprises at least part of the reflex sequence of the first forward primer of (a); and 
   (d) a third reverse primer comprising:
 (i) a 3′ region that is complementary to a second site in the top strand of said target fragment and that can prime template dependent nucleic acid synthesis therefrom; and 
 (ii) a 5′ region that comprises at least part of the reflex sequence of the first forward primer of (a). 
   
     
     
         22 . The kit of  claim 21 , further comprising:
 (e) a second forward primer that hybridizes to the complement of the primer site of (a)(i).   
     
     
         23 . The kit of  claim 21 , further comprising:
 (f) a third forward primer, comprising:
 (i) a 3′ region that hybridizes to a first position in the bottom strand of said target fragment, wherein the complement of said first position is upstream from the first site of (c)(i); and 
 (ii); a 5′ region comprising a second primer site; and 
   (g) a fourth forward primer, comprising:
 (i) a 3′ region that hybridizes to a second position in the bottom strand of said target fragment, wherein the complement of said second site is upstream from the second site of (d)(i); and 
 (ii) a 5′ region comprising said second primer site. 
   
     
     
         24 . The kit of  claim 23 , wherein the distance between complement of the first site of said target fragment of (c)(i) is separated from the first position of said target fragment of (f)(i) by 400 bases in length or less. 
     
     
         25 . The kit of  claim 23 , wherein the distance between complement of the second site of said target fragment of (d)(i) is separated from the second position of said target fragment of (g)(i) by 400 bases in length or less. 
     
     
         26 . The kit of  claim 21 , wherein said first forward primer of (a) further comprises a multiplex identifier (MID) between said first primer site and said reflex sequence. 
     
     
         27 . The kit of  claim 26 , wherein kit comprises multiple first forward primers in separate containers, wherein multiple first forward primers have different multiplex identifiers relative to one another. 
     
     
         28 . The kit of  claim 21 , wherein said genome is a mammalian genome. 
     
     
         29 . The kit of  claim 28 , wherein said mammalian genome is a human genome. 
     
     
         30 . The kit of  claim 21 , wherein said genome is a viral genome. 
     
     
         31 . The kit of  claim 21 , wherein said target fragment is 100 bases to 30 kb in length. 
     
     
         32 . The kit of  claim 21 , wherein said reflex sequence is 5 to 200 nucleotides in length. 
     
     
         33 . The kit of  claim 21 , wherein said first primer site is 10 to 50 nucleotides in length. 
     
     
         34 . The kit of  claim 21 , wherein the 5′ regions of said second and third reverse primers comprise at least 10 contiguous nucleotides of the reflex sequence of the first forward primer of (a).