Method for target specific rna transcription of dna sequences

Abstract

Disclosed herein are methods of long range target specific amplification and sequencing using an RNA intermediate synthesized directly from the target including using hairpin adaptors having a double stranded promoter and an overhang which hybridizes with a reverse-complementary overhang on a target nucleic acid. RNA transcription eliminates clonal amplification of early synthesis errors. Approaches allow for the identification of target-adjacent sequence, such as sequence adjacent to a repeat element target. Also disclosed herein are compositions and kits for amplification and sequencing.

What is claimed is: 
     
         1 . A method of determining a sequence adjacent to a region of known sequence of a nucleic acid molecule, the method comprising:
 a) attaching a hairpin nucleic acid fragment to the nucleic acid molecule, wherein the hairpin nucleic acid fragment comprising i) a double stranded promoter segment, and ii) an overhanging single stranded portion that anneals to a part of the known region of the nucleic acid molecule; and   b) contacting the hairpin nucleic acid fragment to an RNA polymerase directed by the promoter, thereby synthesizing an RNA transcript comprising a portion of the sequence adjacent to the known region of a nucleic acid molecule.   
     
     
         2 . The method of  claim 1 , wherein synthesizing comprises synthesizing a plurality of the RNA transcripts. 
     
     
         3 . The method of  claim 1 , further comprising sequencing the RNA transcript. 
     
     
         4 . The method of  claim 2 , wherein the plurality of RNA transcripts comprises a consensus sequence. 
     
     
         5 . The method of  claim 4 , wherein the consensus sequence comprises a nucleic acid sequence synthesized directly from the nucleic acid molecule. 
     
     
         6 . The method of  claim 4 , wherein the consensus sequence is at least 10 kilobases in length. 
     
     
         7 . The method of  claim 1 , wherein the attaching comprises inserting the hairpin nucleic acid fragment at the known region of the nucleic acid molecule. 
     
     
         8 . The method of  claim 1 , wherein the attaching comprises hybridizing the hairpin nucleic acid fragment at the region of known sequence of the nucleic acid molecule. 
     
     
         9 . The method of  claim 1 , comprising, prior to attaching the hairpin nucleic acid fragment, cleaving the region of known sequence of the nucleic acid molecule. 
     
     
         10 . The method of  claim 9 , wherein the cleaving is sequence-specific, and the sequence-specific cleavage comprises contacting the known region of the nucleic acid molecule to a nucleic acid sequence guided nuclease. 
     
     
         11 . The method of  claim 8 , wherein the attaching comprises ligating the hairpin nucleic acid fragment to the nucleic acid molecule. 
     
     
         12 . The method of  claim 1 , wherein the hairpin nucleic acid fragment comprises a viral promoter, bacterial promoter or a eukaryotic promoter. 
     
     
         13 . The method of  claim 1 , wherein the known region of a nucleic acid molecule comprises a repetitive element. 
     
     
         14 . The method of  claim 13 , wherein the repetitive element comprises a mobile insertion element. 
     
     
         15 . The method of  claim 13 , wherein the repetitive element comprises at least one of a LINE element, a SINE element, an Alu repeat, a transposon, a retrotransposon, a centromeric repeat, and a telomeric repeat. 
     
     
         16 . A nucleic acid library comprising a nucleic acid fragment coupled with a hairpin nucleic acid fragment, wherein the nucleic acid fragment comprises a first nucleic acid sequence adjacent to a second nucleic acid sequence that is a priori known sequence, wherein the hairpin nucleic acid fragment comprises (i) a double stranded promoter sequence and (ii) an overhanging single stranded portion that anneals to a portion of the second nucleic acid. 
     
     
         17 . The nucleic acid library of  claim 16 , wherein the second nucleic acid sequence comprises a mobile element.