Methods and compositions for nucleic acid sample preparation

• US 8,236,499 B2
• Filed: 03/27/2009
• Issued: 08/07/2012
• Est. Priority Date: 03/28/2008
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Patel, Pranav, Bjornson, Keith, Travers, Kevin, Heiner, Cheryl
• Primary Examiner(s): Lu, Frank W
• Application Number:
  US12/383,855
• Publication Number:
  US 20090280538A1
• Time in Patent Office: 1,229 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/183, 435/6.1, 435/6.11, 435/91.52, 436/94, 436/501, 536/23.1, 536/24.3, 536/24.33, 536/25.3
• US Class Current: 435/6.1
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/65 using markers enzymes used as markers C12N15/52
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease

Abstract

Provided are methods and compositions for the production of linear single-stranded nucleic acids, which can be used as templates in high-throughput sequencing systems. Also provided are methods and compositions for the production of closed single-stranded nucleic acid loops, which can be used as templates in high-throughput sequencing systems.

Claims

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.