Selective amplification using blocking oligonucleotides

Abstract

Disclosed herein include methods and compositions for selectively amplifying and/or extending nucleic acid target molecules in a sample. The methods and compositions can, for example, reduce the amplification and/or extension of undesirable nucleic acid species in the sample, and/or allow selective removal of undesirable nucleic acid species in the sample.

What is claimed is: 
     
         1 . A method of selective amplification, comprising:
 providing a plurality of sample molecules comprising a plurality of nucleic acid target molecules and one or more undesirable nucleic acid species;   providing one or more amplification primers;   providing a blocking oligonucleotide that specifically binds to at least one of the one or more undesirable nucleic acid species within 100 nt of the 5′ end of the one or more undesirable nucleic acid species; and   amplifying the plurality of sample molecules in the presence of the blocking oligonucleotide and the one or more amplification primers to generate a plurality of amplicons, whereby the amplification of the undesirable nucleic acid species is reduced by the blocking oligonucleotide not being able to function as a primer for a polymerase.   
     
     
         2 . The method of  claim 1 , wherein the nucleic acid target molecules comprise DNA molecules, RNA molecules, genomic DNA molecules, cDNA molecules, mRNA molecules, rRNA molecules, mtDNA, siRNA molecules, or any combination thereof 
     
     
         3 . The method of  claim 1 , wherein the sample molecules comprise whole transcriptome amplification (WTA) products. 
     
     
         4 . The method of  claim 1 , wherein the one or more undesirable nucleic acid species comprise rRNA, mtRNA, genomic DNA, intronic sequence, high-abundance sequence, or any combination thereof. 
     
     
         5 . The method of  claim 1 , wherein the one or more undesirable nucleic acid species amount to about 50%, 60%, 70%, or 80% of the nucleic acid content of the plurality of sample molecules. 
     
     
         6 . The method of  claim 1 , comprising providing blocking oligonucleotides that specifically bind to two or more undesirable nucleic acid species in the plurality of sample molecules. 
     
     
         7 . The method of  claim 1 , wherein the blocking oligonucleotide specifically binds to within 50 nt of the 5′ end of the one or more undesirable nucleic acid species. 
     
     
         8 . The method of  claim 1 , wherein the blocking oligonucleotide is 10 nt to 50 nt long. 
     
     
         9 . The method of  claim 1 , wherein the blocking oligonucleotide has a T m  of at least 60° C. 
     
     
         10 . The method of  claim 1 , wherein the one or more amplification primers add sequencing adaptors to the plurality of extension products 
     
     
         11 . The method of  claim 10 , further comprising sequencing the plurality of amplicons, or products thereof. 
     
     
         12 . The method of  claim 1 , further comprising removing a hybridized complex formed between the blocking oligonucleotide and the undesirable nucleic acid species,
 wherein the removing comprises immobilizing the hybridized complex formed between the blocking oligonucleotide and the undesirable nucleic acid species on a solid support,   wherein the blocking oligonucleotide comprises an affinity moiety, and   wherein the solid support comprises a binding partner of the affinity moiety.   
     
     
         13 . A kit for selective amplification of a plurality of nucleic acid target molecules in a sample, comprising:
 one or more blocking oligonucleotides that specifically bind to at least one of one or more undesirable nucleic acid species in the sample within 100 nt of the 5′ end of the one or more undesirable nucleic acid species, wherein the one or more blocking oligonucleotides are unable to function as a primer for a reverse transcriptase or a polymerase.   
     
     
         14 . The kit of  claim 13 , further comprising a reverse transcriptase, a polymerase, a ligase, a nuclease, a plurality of particles each comprising a plurality of oligonucleotide probes, or any combination thereof. 
     
     
         15 . The kit of  claim 13 , further comprising a solid support,
 wherein the one or more blocking oligonucleotides comprise an affinity moiety,   wherein the solid support comprises a binding partner of the affinity moiety, and   wherein the affinity moiety comprises biotin, streptavidin, heparin, an aptamer, a click-chemistry moiety, digoxigenin, primary amine(s), carboxyl(s), hydroxyl(s), aldehyde(s), ketone(s), or any combination thereof.   
     
     
         16 . The kit of  claim 13 , wherein the undesirable nucleic acid species comprise rRNA, mtRNA, genomic DNA, intronic sequence, high-abundance sequence, or any combination thereof. 
     
     
         17 . The kit of  claim 13 , wherein the one or more blocking oligonucleotides specifically bind to two or more undesirable nucleic acid species in the sample. 
     
     
         18 . The kit of  claim 13 , wherein the one or more blocking oligonucleotides comprise a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a DNA, an LNA/PNA chimera, an LNA/DNA chimera, a PNA/DNA chimera, or any combination thereof. 
     
     
         19 . The kit of  claim 13 , wherein the one or more blocking oligonucleotides specifically bind to within 50 nt of the 5′ end of the one or more undesirable nucleic acid species. 
     
     
         20 . The kit of  claim 13 , wherein the one or more blocking oligonucleotides has a T m  of at least 60° C.