Isolation and immobilization of nucleic acids and uses thereof

Abstract

Disclosed is a novel technique of directly isolating nucleic acids from a biological sample and use of the isolated nucleic acid complexes for various applications and assays such as biobanking and sequencing.

1 .- 14 . (canceled) 
     
     
         15 . A method of generating a cDNA library from a biological sample, comprising:
 obtaining a plurality of isolated nucleic acid fragments bound to a solid support;   hybridizing one or more primers to the plurality of the isolated nucleic acid fragments;   conducting primer extension using the plurality of the isolated nucleic acid fragments as templates to synthesize cDNA fragments; and   eluting the synthesized cDNA fragments from the solid support to generate the cDNA library,   wherein the plurality of the isolated nucleic acid fragments is obtained by contacting a biological sample containing the plurality of the nucleic acid fragments with a reaction mix comprising a template-independent DNA or RNA polymerase, and one or more NTPs, dNTPs or ddNTPs to obtain a tagged nucleic acid, wherein at least a fraction of the one or more NTPs, dNTPs or ddNTPs are modified by an affinity tag;   binding the tagged nucleic acid fragments to a surface of a solid support; and   washing the solid support to obtain the bound nucleic acid fragments isolated from the biological sample.   
     
     
         16 . The method of  claim 15 , wherein the primer is a sense strand primer. 
     
     
         17 . The method of  claim 15 , wherein the primer is an anti-sense strand primer. 
     
     
         18 . The method of  claim 15 , wherein the primer is a specific primer. 
     
     
         19 . The method of  claim 15 , wherein the primer is a random primer. 
     
     
         20 . The method of  claim 15 , wherein the one or more primers bind to different nucleic acid fragments. 
     
     
         21 . The method of  claim 15 , wherein the one or more primers bind to different locations of a nucleic acid fragment. 
     
     
         22 . The method of  claim 15 , wherein the one or more primers bind to the same location of different nucleic acid fragments. 
     
     
         23 . The method of  claim 15 , further comprising:
 annealing the eluted sense strand cDNA fragments and anti-sense strand cDNA fragments; and   extending the annealed cDNA fragments to fill in the gap thereby to obtain double stranded cDNA fragments.   
     
     
         24 . A captured nucleic acid complex comprising:
 a solid support; and   a plurality of nucleic acid fragments, each fragment having a 3′ end bound to a polymeric tail, and each polymeric tail is bound to a surface of the solid support.   
     
     
         25 - 31 . (canceled) 
     
     
         32 . A biobank comprising a plurality of the complexes of  claim 24 . 
     
     
         33 . The biobank of  claim 32 , wherein the plurality of the complexes comprises the DNA of the entire genome. 
     
     
         34 . The biobank of  claim 32 , wherein the plurality of the complexes comprises the DNA of substantially the entire genome. 
     
     
         35 . The method of  claim 15 , wherein the biological sample is selected from the group consisting of blood, plasma, serum, urine, saliva, exosome, a lysed formalin-fixed, paraffin-embedded (FFPE) tissue sample, and cells. 
     
     
         36 . The method of  claim 15 , wherein the nucleic acid fragments are DNA fragments, RNA fragments, or a mixture of both. 
     
     
         37 . The method of  claim 15 , wherein the nucleic acid fragments are double-stranded, single-stranded, or a mixture of both. 
     
     
         38 . The method of  claim 37 , wherein the double-stranded nucleic acid fragments are denatured to single-stranded nucleic acid fragments before or after contacting the biological sample with the reaction mix. 
     
     
         39 . The method of  claim 15 , wherein the template-independent DNA or RNA polymerase is terminal transferase (TdT). 
     
     
         40 . The method of  claim 15 , wherein the affinity tag is biotin, and the surface of the solid support is coated with avidin, streptavidin, or neutravidin. 
     
     
         41 . The method of  claim 15 , wherein the reaction mix comprises biotinylated dUTP or biotinylated ddUTP. 
     
     
         42 . The method of  claim 15 , wherein a polymeric tail comprising the unmodified and modified NTPs, dNTPs or ddNTPs is attached to a 3′ end of the nucleic acid fragments. 
     
     
         43 . The method of  claim 15 , wherein the solid support is a bead, a plate, or a column.