Methods of sequencing nucleic acids in mixtures and compositions related thereto

Abstract

This disclosure relates to analyzing the end-to-end sequence and the relative distributions in heterogeneous mixtures of polynucleotides and methods and enabling reagents related thereto. In certain embodiments this method relates to the complete full length sequencing and quantitative profiling of mRNAs present in the transcriptomes of cells or tissues of, but not limited to, higher multicellular organisms that possess interrupted genes subject to complex post-transcriptional RNA processing

We claim: 
     
         1 . A plurality of nucleic acid markers,
 each nucleic acid marker comprising   a sequence identifiable region, wherein the sequence identifiable region comprises a series of random bases interspersed between single invariant bases, and   a first invariant sequence comprising invariant bases flanking the 5′ end of the sequence identifiable region, a second invariant sequence comprising invariant bases flanking the 3′ end of the sequence identifiable region, or both,   wherein each nucleic acid marker further comprises a polymerase chain reaction (PCR) primer sequence or its complement adjacent that first invariant sequence or the second invariant sequence, and   wherein the single invariant bases, the first invariant sequence, and the second invariant sequence, do not vary within the plurality of nucleic acid markers.   
     
     
         2 . The plurality of nucleic acid markers of  claim 1 , wherein each nucleic acid marker comprises a recognition sequence for one or more restriction endonucleases adjacent to the first invariant sequence or the second invariant sequence. 
     
     
         3 . A plurality of nucleic acid molecules, each comprising a nucleic acid marker of the plurality of nucleic acid markers of  claim 1 ,
 wherein the marker has a 5′ end and a 3′ end, and wherein each nucleic acid molecule comprises a) a first rare consensus binding and cleavage site for one restriction enzyme at the 5′ flank of the marker, and b) a second rare consensus binding and cleavage site for a second restriction enzyme at the 3′ flank of the marker.   
     
     
         4 . The plurality of nucleic acid molecules of  claim 3 , each further comprising a 3′ poly dT tail. 
     
     
         5 . A plurality of single stranded polynucleotides each comprising consecutive domains in 5′ to 3′ order: an optional 5′ poly dT tail, a first nucleic acid marker of the plurality of nucleic acid markers of  claim 1 , an intervening loop, a second nucleic acid marker of the plurality of nucleic acid markers of  claim 1 , and an optional 3′ poly dA tail, wherein the intervening loop comprises a binding site for a primer or its complement. 
     
     
         6 . The plurality of single stranded polynucleotides of  claim 5 , comprising the 5′ poly dT tail. 
     
     
         7 . The plurality of single stranded polynucleotides of  claim 5 , wherein the second nucleic acid marker is the base complement of the first nucleic acid marker, and wherein the first marker block and the second marker block create identical copies of the sequence identifiable region in the same 5′ to 3′ orientation when copied into double stranded DNA. 
     
     
         8 . The plurality of single stranded polynucleotides of  claim 5 , wherein the second nucleic acid marker is not the base complement of the first nucleic acid marker, and wherein when the first nucleic acid marker and the second nucleic acid marker hybridize, a duplex is formed with an unpaired loop. 
     
     
         9 . The plurality of single stranded polynucleotides of  claim 5 , each comprising the poly A tail and the poly T tail, wherein the first nucleic acid marker and the second nucleic acid marker form a duplex in solution, and wherein invariant bases on the 3′ end of the sequence identifiable region form a consensus sequence for a rare restriction enzyme, such that, when copied into double stranded cDNA, the restriction enzyme can be used to delete the intervening loop from the double stranded DNA. 
     
     
         10 . The plurality of single stranded polynucleotides of  claim 9 , each comprising a site for priming on the 5′ side of the first nucleic acid marker and the 3′ side of the second nucleic acid marker. 
     
     
         11 . The plurality of single stranded polynucleotides of  claim 5 , wherein the first nucleic acid marker is an exact duplicate of the second nucleic acid marker. 
     
     
         12 . The plurality of nucleic acid markers of  claim 1 , wherein the sequence identifiable region comprises 18 random bases, and wherein the random bases are present in the sequence identifiable region as triplets between single invariant bases.