METHOD FOR CONTROLLED DNA FRAGMENTATION

• US 20160177359A1
• Filed: 11/17/2015
• Published: 06/23/2016
• Est. Priority Date: 02/03/2014
• Status: Active Grant

• Current Assignee: Thermo Fisher Scientific Baltics, UAB (Thermo Fisher Scientific Incorporated)
• Original Assignee: Thermo Fisher Scientific Baltics, UAB (Thermo Fisher Scientific Incorporated)
• Inventors: UKANIS, Mindaugas, LUBYS, Arvydas, TAMOSEVICIUS, Romas, GAIDAMAUSKAS, Ervinas
• Granted Patent: US 10,731,152 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/1082 Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/507 Recombinase
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 50/04 using dynamic combinatorial chemistry techniques
• CPC Class Codes: C40B 70/00 Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or bar codes
• CPC Class Codes: C40B 80/00 Linkers or spacers specially adapted for combinatorial chemistry or libraries, e.g. traceless linkers or safety-catch linkers

Abstract

A composition and method for controlled in vitro fragmentation of nucleic acids. A transposase forms catalytically active complexes with a modified transposon end that contains within its end sequence degenerate, apurinic/apyrimidinic sites, nicks, or nucleotide gaps, to fragment or shear a target nucleic acid sample in a controlled process. This method yields desired average nucleic acid fragment sizes. The inventive composition and method may be applied for generation of DNA fragments containing shortened transposon end sequences to facilitate subsequent reactions, for production of asymmetrically tailed DNA fragments, etc.

Claims

1. A method for fragmenting nucleic acids from an initial nucleic acid sample in an in vitro reaction, comprising:

a) providing a plurality of transpososome complexes, which include (i) a plurality of transposases, (ii) a first transposon end sequence, wherein the first transposon end sequence is capable of binding to a transposase from the plurality of transposases and wherein the first transposon end sequence contains at least one nick, gap, apurinic site or apyrimidinic site, (iii) a second transposon end sequence, wherein the second transposon end sequence is capable of binding to a transposase from the plurality of transposases and wherein the second transposon end sequence contains at least one nick, gap, apurinic site or apyrimidinic site;

b) contacting, in a single reaction mixture, the plurality of transpososome complexes with the nucleic acids, under conditions that are suitable for transposing the first and second transposon end sequences into the nucleic acids and fragmenting the nucleic acids, where the nucleic acids includes a first nucleic acid molecule; and

c) producing at least one fragmented tagged DNA molecule having a first end joined to the first transposon end sequence and a second end joined to the second transposon end sequence, by transposing the first transposon end sequences into the first nucleic acid molecule at a first position and fragmenting and tagging the first nucleic acid molecule, and by transposing the second transposon end sequences into first nucleic acid molecule at a second position and fragmenting and tagging the first nucleic acid molecule, wherein the at least one fragmented tagged DNA molecules includes the first transposon end sequence having at least one nick, gap, apurinic site or apyrimidinic site, and a second end having at least one nick, gap, apurinic site or apyrimidinic site.

2. The method of claim 1, wherein the initial nucleic acid sample includes RNA or DNA.

3. The method of claim 1, wherein the initial nucleic acid sample includes genomic DNA or cell-free DNA.

4. The method of claim 1, wherein the plurality of transpososome complexes comprises a plurality of homo-transpososome complexes or a mixture of hetero-transpososome complexes.

5. The method of claim 4, wherein the mixture of hetero-transpososome complexes includes 2-100 different transpososome complexes.

6. The method of claim 1, further comprising:

appending at least one universal adaptor sequence to the first end of the fragmented tagged DNA molecule which is joined to the first transposon end sequence.

7. The method of claim 1, further comprising:

appending at least one universal adaptor sequence to the second end of the fragmented tagged DNA molecule which is joined to the second transposon end sequence.

8. The method of claim 6, wherein the universal adaptor sequence comprises an amplification primer sequence, a sequencing primer sequence and/or a barcode sequence.

9. The method of claim 6, further comprising:

amplifying the at least one fragmented tagged DNA molecule to generate tagged DNA amplicons.

10. The method of claim 9, further comprising:

sequencing the tagged DNA amplicons to generate a plurality of sequencing reads.

11. The method of claim 10, further comprising:

aligning the sequencing reads to at least one reference sequence.

12. The method of claim 10, wherein at least one sequencing read contains one or more mutations, including point mutations, deletions, insertions, or substitutions of one or more nucleotides, inversions, rearrangements, fusions, truncations, transversions, transitions, non-sense mutations, translocations, duplications, sequence repeats, fusion sequences, single nucleotide polymorphism (SNP), copy number variation (CNV) and/or variant or abnormal splice junction sequences.

13. The method of claim 10, further comprising:

quantifying the number of sequencing reads that correspond to a first target sequence of interest from the initial nucleic sample to obtain a first number.

14. The method of claim 10, wherein the sequencing comprises a massively parallel sequencing reaction.

15. The method of claim 14, wherein the massively parallel sequencing reaction comprises incorporating a nucleotide.

16. The method of claim 14, wherein the massively parallel sequencing reaction comprises providing a surface having an array of a plurality of reaction sites, and the reaction site is operatively linked to a sensor.

17. The method of claim 16, wherein the sensor detects a change in ions, hydrogen ions, protons, phosphate groups, including pyrophosphate groups.

18. The method of claim 16, wherein the sensor detects at least one byproduct or cleavage product of a nucleotide incorporation reaction.

19. The method of claim 18, wherein the byproduct or cleavage product of a nucleotide incorporation reaction includes ions (e.g., hydrogen ions), protons, phosphate groups, including pyrophosphate groups.

20. The method of claim 18, wherein the sensor comprises an ISFET.