METHODS AND COMPOSITIONS FOR NUCLEIC ACID SAMPLE PREPARATION

• US 20120309650A1
• Filed: 06/12/2012
• Published: 12/06/2012
• 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
• Granted Patent: US 8,535,886 B2
• Time in Patent Office: Days
• US Class Current: 506/16
• 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 nucleic acid loops, the method comprising:

providing a genomic DNA, a cDNA, or a DNA concatamer;

generating double-stranded nucleic acid fragments from the genomic DNA, the cDNA, or the concatamer, which fragments comprise a first strand that comprises a gap site; and

circularizing the double-stranded nucleic acid fragments to produce nucleic acid loops that comprise first non-contiguous strands and second contiguous strands.

2. The method of claim 1, further comprising using the second contiguous strands as template strands in a sequencing reaction.

3. The method of claim 1, further comprising removing the non-contiguous strands from the double-stranded loops, thereby producing a population of closed single-stranded nucleic acid loops.

4. The method of claim 3, wherein removing the non-contiguous strands from the double-stranded loops comprises digesting the non-contiguous strands with an exonuclease, thereby producing the population of closed single-stranded nucleic acid loops.

5. The method of claim 3, further comprising sequencing the closed single-stranded nucleic acid loops.

6. The method of claim 1, wherein generating the double-stranded fragments that comprise the gap site comprises:

cleaving the genomic DNA, the cDNA, or the concatamer to produce linear double-stranded nucleic acids, and;

ligating tags, which tags comprise the gap site, to the ends of the linear double-stranded nucleic acids to produce the double-stranded fragments that comprise the gap site.

7. The method of claim 6, wherein cleaving the genomic DNA, the cDNA, or the concatamer comprises:

enzymatic digestion, sonication, mechanical shearing, electrochemical cleavage, or nebulization of the genomic DNA, the cDNA, or the concatamer to produce the linear double-stranded nucleic acids.

8. The method of claim 6, 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 1, wherein generating the double-stranded fragments that comprise the gap site comprises:

annealing a population of primer pairs, which primers comprise the gap site, to subsequences of the genomic DNA, the cDNA, or the concatamer; and

,

extending the primers with a polymerase, thereby producing the generating the double-stranded fragments that comprise the gap site.

10. The method of claim 9, wherein the primer pairs 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.

11. The method of claim 1, wherein the gap site comprises an unphosphorylated 5′

end.

12. The method of claim 1, wherein the gap site comprises a sequence recognized by a nicking enzyme.

13. The method of claim 12, wherein the nicking enzyme is uracil-DNA N-glycosylase (UNG), uracil-DNA glycosylase (UDG) or a site-specific restriction endonuclease that has been engineered to cleave the gap site.

14. The method of claim 1, wherein the gap site comprises an unphosphorylated 5′

and wherein circularizing the double-stranded nucleic acid fragments to produce double-stranded nucleic acid loops that comprise first non-contiguous strands and second contiguous strands comprises ligating first ends of the fragments to second ends of the fragments, thereby forming the population of nucleic acid loops that comprise first non-contiguous strands and second contiguous strands.

15. The method of claim 1, wherein the gap site comprises a sequence recognized by a nicking enzyme and wherein circularizing the double-stranded nucleic acid fragments to produce double-stranded nucleic acid loops that comprise the first non-contiguous strands and the second contiguous strands comprises:

ligating first ends of the fragments to second ends of the fragments, thereby forming a population of double-stranded loops; and

,

cleaving the strands of the loops that comprise the gap site with a nicking enzyme that specifically cleaves the gap site, thereby forming the population of nucleic acid loops that comprise first non-contiguous strands and second contiguous strands

16. A composition, comprising a plurality of double-stranded nucleic acid loops that comprise overlapping sequences of a genomic DNA, a cDNA, or a DNA concatamer, wherein the loops comprise first non-contiguous strands and second contiguous strands.

17. The composition of claim 16, wherein the loops comprise tags, which tags include 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 uracil, a nucleotide analog, 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.

18. The composition of claim 16, wherein the composition comprises an exonuclease.

19. A composition comprising a plurality of closed double-stranded nucleic acid loops, wherein the loops comprise overlapping fragments of a genomic DNA, a cDNA, or a DNA concatamer and wherein the loops comprise a sequence that is specifically recognized by a nicking enzyme.

20. The composition of claim 19, wherein the loops comprise tags, which tags include 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 uracil, a nucleotide analog, a sequence capable of forming a secondary structure, an oligortucleotide hybridization site, a restriction site, a DNA promoter, an RNA promoter, a sample or library identification sequence, and a cis regulatory sequence.

21. The composition of claim 19, wherein the composition comprises a nicking enzyme.