SYSTEM AND METHOD FOR PRODUCING FUNCTIONALLY DISTINCT NUCLEIC ACID LIBRARY ENDS THROUGH USE OF DEOXYINOSINE

• US 20120077716A1
• Filed: 09/15/2011
• Published: 03/29/2012
• Est. Priority Date: 09/29/2010
• Status: Abandoned Application

• Current Assignee: 454 Life Sciences Corporation (Roche Holding AG)
• Original Assignee: 454 Life Sciences Corporation (Roche Holding AG)
• Inventors: Godwin, Brian Christopher, Mealmaker, Craig Elder
• Application Number:
  US13/233,653
• Publication Number:
  US 20120077716A1
• Time in Patent Office: Days
• US Class Current: 506/26
• CPC Class Codes: C12N 15/1079 Screening libraries by altering the phenotype or phenotypic trait of the host reporter assays C12N15/1086

Abstract

An embodiment of a nucleic acid adaptor is described that comprises a double stranded nucleic acid element that comprises a plurality of deoxyinosine species positionally located in a spaced relationship from each other on a first strand and base pair to an A, T, or G nucleotide species on a second strand, where a first end of the double stranded nucleic acid element is constructed and arranged to preferentially ligate to each end of a double stranded target nucleic acid molecule.

Claims

1. A nucleic acid adaptor, comprising:

a double stranded nucleic acid element that comprises a plurality of deoxyinosine species positionally located in a spaced relationship from each other on a first strand and base pair to an A, T, or G nucleotide species on a second strand, wherein a first end of the double stranded nucleic acid element is constructed and arranged to preferentially ligate to each end of a double stranded target nucleic acid molecule.

2. The nucleic acid adaptor of claim 1, wherein:

the first strand comprises a 3′

single base overhang and a second strand comprises a 5′

phosphate group that preferentially ligates the first end of the double stranded nucleic acid element to each end of the double stranded target nucleic acid molecule.

3. The nucleic acid adaptor of claim 1, wherein:

the first strand comprises a detectable moiety at the 5′

terminus.

4. The nucleic acid adaptor of claim 1, wherein:

the second strand comprises a detectable moiety at the 3′

terminus.

5. The nucleic acid adaptor of claim 1, wherein:

the deoxyinosine species are spaced 4 or more sequence positions away from each other.

6. The nucleic acid adaptor of claim 1, wherein:

the deoxyinosine species are spaced no closer than 6 sequence positions from an end of the double stranded nucleic acid element arranged to ligate to a double stranded target nucleic acid molecule.

7. The nucleic acid adaptor of claim 1, wherein:

the first and the second strands comprise a plurality of phophorothioates at each end.

8. The nucleic acid adaptor of claim 7, wherein:

one or more of the phophorothioates at each end of the first strand are positionally located in a complementary relationship to one or more of the phosphorothioates at each end of the second strand.

9. The nucleic acid adaptor of claim 8, wherein:

the first and the second strands comprise at least 4 phosphorothioates at each end, wherein the phosphorothioates are interspaced by a natural nucleotide species.

10. A kit comprising the nucleic acid adaptor of claim 1.

11. A method for preparing a nucleic acid library, comprising the steps of:

ligating a fully complementary double stranded nucleic acid adaptor to each end of a double stranded nucleic acid target molecule to produce a ligated double stranded adaptor-target-adaptor molecule, wherein the fully complementary double stranded nucleic acid adaptor comprises a plurality of deoxyinosine species positionally located in a spaced relationship from each other on a first adaptor strand that base pair to an A, T, or G nucleotide species on a second adaptor strand;

denaturing the double stranded adaptor-target-adaptor molecule to produce two single stranded adaptor-target-adaptor molecules, wherein the single stranded adaptor-target-adaptor molecules comprise a first region from the first adaptor strand at a first end, a middle region from the nucleic acid target molecule, and a second region from the second adaptor strand at a second end;

annealing a first primer to the second region of a first strand of the single stranded adaptor-target-adaptor molecules;

extending the first primer to produce a second strand complementary to the first strand, wherein the second strand comprises a third region at the second end comprising C nucleotide species base paired to the deoxyinosine species in the first region of the first strand;

annealing a second primer to the third region of second strand; and

extending the second primer to produce a third strand complementary to the second strand, wherein the second and third strands comprise semi-complementary ends resistant to hybridization to each other.

12. The method of claim 11, comprising:

the second strand comprises sequence composition from the first primer at a first end, a middle region from the nucleic acid target molecule, and the third region at a second end.

13. The method of claim 11, comprising:

the third strand comprises second region at a first end, a middle region from the nucleic acid target molecule, and sequence composition from the second primer at a second end.

14. The method of claim 11, comprising:

the second strand and the third strand are each sequencable products.

15. The method of claim 11, further comprising the step of:

clonally amplifying the second strand to produce a population of substantially identical copies; and

sequencing the population of substantially identical copies to produce a sequence composition of the second strand.

16. The method of claim 11, further comprising the step of:

clonally amplifying the third strand to produce a population of substantially identical copies; and

sequencing the population of substantially identical copies to produce a sequence composition of the third strand.

17. The nucleic acid adaptor of claim 11, wherein:

the first adaptor strand comprises a 3′

single base overhang and the second adaptor strand comprises a 5′

phosphate group that preferentially ligates a first end of the the fully complementary double stranded nucleic acid adaptor to each end of the double stranded target nucleic acid molecule.

18. The nucleic acid adaptor of claim 11, wherein:

the deoxyinosine species are spaced 4 or more sequence positions away from each other.

19. The nucleic acid adaptor of claim 11, wherein:

the deoxyinosine species are spaced no closer than 6 sequence positions from an end of the fully complementary double stranded nucleic acid adaptor arranged to ligate to the double stranded target nucleic acid molecule