Methods and compositions for nucleic acid sample preparation

1. A method of producing a population of linear single stranded nucleic acids, the method comprising:

providing a double-stranded genomic DNA, cDNA, or DNA concatamer;

cleaving the genomic DNA, the cDNA, or the concatemer, thereby producing linear double-stranded nucleic acids;

ligating exonuclease-sensitive tags to the 5′ ends of the first strands of the linear double-stranded nucleic acids and ligating exonuclease-resistant tags to the 5′ ends of the second strands of the linear double-stranded nucleic acids, thereby generating double-stranded fragments from the genomic DNA, the cDNA, or the concatamer, wherein first strands of the fragments are exonuclease-sensitive strands and second strands of the fragments are exonuclease-resistant strands; and

eliminating the first strands from the double-stranded fragments by digesting the first strands with an exonuclease, thereby producing the population of linear single-stranded nucleic acids.

2. The method of claim 1 , wherein the genomic DNA, the cDNA, or the concatamer is derived from an eukaryote.

3. The method of claim 1 , wherein said cleaving the genomic DNA, the cDNA, or the concatamer comprises one or more of: enzymatic digestion, sonication, mechanical shearing, electrochemical cleavage, or nebulization of the genomic DNA, the cDNA, or the concatamer.

4. The method of claim 1 , wherein the exonuclease-sensitive tags and the exonuclease-resistant tags comprise one or more moieties selected from: a ligand, a fluorescent label, a blocking group, a phosphorylated nucleotide, a phosphorothioated nucleotide, a biotinylated nucleotide, a methylated nucleotide, a nucleotide analog, a uracil, a sequence capable of forming a secondary structure, an oligonucleotide hybridization site, a restriction site, a DNA promoter, an RNA promoter, a sample or library identification sequence, and a cis regulatory sequence.

5. A method of preparing closed single-stranded nucleic acid loops, the method comprising:

providing a double-stranded genomic DNA, cDNA, or DNA concatamer;

cleaving the genomic DNA, the cDNA, or the concatemer, thereby producing linear double-stranded nucleic acids;

ligating exonuclease-sensitive tags to the 5′ ends of the first strands of the linear double-stranded nucleic acids and ligating exonuclease-resistant tags to the 5′ ends of the second strands of the linear double-stranded nucleic acids, thereby generating double-stranded fragments from the genomic DNA, the cDNA, or the concatamer, wherein first strands of the fragments are exonuclease-sensitive strands and second strands of the fragments are exonuclease-resistant strands;

separating the first strands of the fragments from the second strands by digesting the first strands of the double-stranded fragments with an exonuclease, thereby producing single-stranded fragments; and

circularizing the single-stranded fragments, thereby producing the closed single-stranded nucleic acid loops.

6. The method of claim 5 , wherein the genomic DNA, the cDNA, or the concatamer is derived from an eukaryote.

7. The method of claim 5 , wherein said cleaving the genomic DNA, the cDNA, or the concatamer comprises one or more of: enzymatic digestion, sonication, mechanical shearing, electrochemical cleavage, or nebulization of the genomic DNA, the cDNA, or the concatamer.

8. The method of claim 5 , wherein the tags comprise one or more moieties selected from: a ligand, a fluorescent label, a blocking group, a phosphorylated nucleotide, a phosphorothioated nucleotide, a biotinylated nucleotide, a methylated nucleotide, a nucleotide analog, a uracil, a sequence capable of forming a secondary structure, an oligonucleotide hybridization site, a restriction site, a DNA promoter, an RNA promoter, a sample or library identification sequence, and a cis regulatory sequence.

9. The method of claim 5 , wherein said circularizing the single-stranded fragments comprises:

annealing single-stranded nucleic acid splints to the single-stranded fragments, wherein first ends of the splints comprise a nucleotide sequence complementary to a nucleotide sequence at the first ends of the single-stranded fragments, and wherein second ends of the splints comprise a nucleotide sequence complementary to a nucleotide sequence at the second ends of the single-stranded fragments, such that the first and the second ends of each of the single-stranded fragments are brought into proximity to one another;

ligating the first and the second ends of the single-stranded fragments to one another; and

removing the splints, thereby producing the closed single-stranded nucleic acid loops.

10. The method of claim 9 , wherein said removing the splints comprises digesting the splints with an exonuclease.