Use of transposase and Y adapters to fragment and tag DNA

Patent metadata

Field	Value
Publication number	US-11535889-B2
Application number	US-201916295838-A
Country	US
Kind code	B2
Filing date	Mar 7, 2019
Priority date	Mar 31, 2016
Publication date	Dec 27, 2022
Grant date	Dec 27, 2022

Abstract

Described herein is an adapter comprising a population of first oligonucleotides, a second oligonucleotide and a third oligonucleotide, wherein the first oligonucleotides, the second oligonucleotide and the third oligonucleotide are hybridized together to produce a complex that comprises: (i) a first end comprising a transposase recognition sequence, (ii) a central single-stranded region of variable sequence and (iii) a second end comprising sequences that are non-complementary. A method, as well as a kit for practicing the method, are also provided.

First claim

The invention claimed is:

1. A method for tagmenting a sample, comprising:
contacting a sample comprising double-stranded DNA with a transposase loaded with adapters;
wherein the adapters comprise a population of first oligonucleotides, a second oligonucleotide and a third oligonucleotide, wherein the first oligonucleotides, the second oligonucleotide and the third oligonucleotide are hybridized together to produce a complex that comprises: (i) a first end comprising a transposase recognition sequence, (ii) a central single-stranded region of variable sequence and (iii) a second end comprising sequences that are non-complementary; and
filling in and sealing the central single-stranded region of the adaptor using a polymerase and ligase, thereby producing a population of DNA fragments that are tagged at both ends by a Y adaptor each comprising the variable sequence of a first oligonucleotide on both strands.

2. The method of claim 1 , wherein the method is done by combining, in a single reaction vessel, the sample, the transposase loaded with the adaptors, the polymerase and the ligase.

3. The method of claim 1 , wherein the filling in is done by Sulfolobus DNA polymerase IV, at a temperature less than 50 degrees Celsius.

4. The method of claim 1 , wherein the method further comprises amplifying the population of DNA fragments using primers that target a non-complementary sequence of the Y adaptor.

5. The method of claim 4 , wherein the amplifying is done in solution.

6. The method of claim 4 , wherein the amplifying is done by bridge PCR.

7. The method of claim 1 , further comprising sequencing at least some of the tagged DNA fragments.

8. The method of claim 1 , wherein the variable sequence has a complexity of at least 1,000.

9. The method of claim 1 , wherein the first, second, and third oligonucleotides are formed by allowing self-annealing of a single oligonucleotide molecule of greater than 60 nucleotides, followed by cleavage of cleavable sites located between the first and third oligonucleotides and between the second and third oligonucleotide.

10. The method of claim 1 , wherein the transposase recognition sequence is a recognition sequence for a Vibhar transposase.

11. The method of claim 1 , wherein:
a) the population of first oligonucleotides comprise a 5′ region, a 3′ region, and a region of variable sequence between the 5′ region and the 3′ region;
b) the second oligonucleotide is complementary to and hybridizes with the 3′ region of the first oligonucleotides to form the transposase recognition sequence; and
c) the third oligonucleotide comprises a 5′ end that is complementary to and is hybridized with the 3′ end of the 5′ region of the first oligonucleotide and comprises a 3′ tail that is non-complementary to the 5′ end of the 5′ region of the first oligonucleotide.

12. The method of claim 11 , wherein the 5′ end of the first oligonucleotide and the 3′ tail of the third oligonucleotide are in a single molecule and joined together by a cleavable region.

13. The method of claim 11 , wherein the 3′ tail of the third oligonucleotide comprises a modification rendering the 3′ end resistant to digestion by a 3′-5′ exonuclease activity.

Assignees

• Agilent Technologies Inc

Inventors

• Ach Robert A
• Sampas Nicholas M
• Peter Brian Jon

Classifications

• C12Q1/6869

  Methods for sequencing · CPC title

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q1/6855 Primary

  Ligating adaptors · CPC title

• C12Q1/6848

  characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction · CPC title

• C12Q1/6874

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title