OLIGONUCLEOTIDE REPLACEMENT FOR DI-TAGGED AND DIRECTIONAL LIBRARIES

• US 20160090591A1
• Filed: 12/08/2015
• Published: 03/31/2016
• Est. Priority Date: 01/28/2011
• Status: Active Application

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: GORYSHIN, IGOR, BAAS, BRADLEY, VAIDYANATHAN, RAMESH, MAFFITT, MARK
• Granted Patent: US 10,287,574 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12N 9/1241 Nucleotidyltransferases (2.7.7)
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 2521/507 Recombinase
• CPC Class Codes: C40B 50/06 Biochemical methods, e.g. using enzymes or whole viable microorganisms

Abstract

Transposomes and oligonucleotide replacement methods to make DNA libraries that have distinct 5′ and 3′ tags, and to make directional libraries that are enriched for a desired strand.

Claims

1. A method for adding a tag to the double-stranded product of a tagmentation reaction, comprising the steps of:

(a) providing a double-stranded target nucleic acid and a transposome having a transposase with two transposon end sequences;

a “

transferred strand” and

a “

non-transferred strand”

, wherein the transferred strand comprises the ME sequence, and wherein the ME sequence is not CGTTGTGTGGACGAGACAG (SEQ ID NO;

5);

(b) allowing the transposome to fragment the target nucleic acid, whereby the transferred strand is covalently transferred to a first strand of the fragment, and the non-transferred strand remains hybridized to the transferred strand;

(c) removing the non-transferred strand from the transferred strand;

(d) providing a replacement oligonucleotide that comprises a tag sequence, to hybridize to the transferred strand; and

(e) ligating the replacement oligonucleotide to the second strand of the fragment;

thereby generating a tagmentation product having a transferred strand and a replacement oligonucleotide.

2. The method of claim 1, wherein one strand of the target nucleic acid is chemically modified.

3. The method of claim 2, wherein the chemical modification is conversion of cytosines to uracils.

4. The method of claim 1, wherein the transposome comprises a Mu transposase

5. The method of claim 1, wherein the transferred strand comprises the ME sequence

6. The method of claim 5, wherein the transferred strand further comprises a tag sequence.

7. The method of claim 1, wherein the non-transferred strand comprises

8. The method of claim 1, wherein the non-transferred strand is selected from the group consisting of SEQ ID NO:

9, SEQ ID NO;

10, SEQ ID NO;

11, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, and SEQ ID NO;

17.

9. The method of claim 1, wherein step (e) further comprises an extension step.

10. The method of claim 2, further comprising the step of

(f) enriching for a desired strand.

11. The method of claim 10, wherein step (f) comprises treating the fragment with an enzyme that preferentially cleaves a nondesired strand of the fragment.

12. A nucleic acid comprising one or two copies of the transposase end sequences

13. The nucleic acid of claim 12, wherein the transposase end sequence is selected from the group consisting of

14. A library of different nucleic acids of claim 12.

15. A method for making a transposome, comprising the steps of

(a) providing a transposase;

(b) providing a transferred end sequence comprising the nucleic acid of claim 12 under conditions whereby the transposase binds to the transferred end sequence.

16. A transposome comprising a transposase and the nucleic acid of claim 12.

17. A tagmentation method comprising the steps of:

(a) providing target nucleic acids;

(b) providing the transposomes of claim 16;

(c) allowing the transposomes to fragment the target nucleic acids and tag at least transposase end sequences to the ends of the fragments;

thereby generating a library of tagged fragments.