# US 10,308,978 B2 · Transposon nucleic acids comprising a calibration sequence for DNA sequencing

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- Number: US 10,308,978 B2
- Document ID: US10308978B2
- Status: Granted
- Kind: B2
- Application number: 15/791,740
- Filed: 2017-10-24
- Published: 2018-02-08
- Granted: 2019-06-04
- Earliest claimed: 2011-07-20
- Assignees: Thermo Fisher Scientific Baltics UAB
- Applicants: Thermo Fisher Scientific Baltics Uab
- Inventors: Ian Kavanagh; Laura-Leena Kiiskinen; Heli Haakana
- Web page: https://patentgrep.com/patent/US10308978B2

## CPC

- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12N 15/1093: {General methods of preparing gene libraries, not provided for in other subgroups}
- C40B 40/08: Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
- C40B 50/06: Biochemical methods, e.g. using enzymes or whole viable microorganisms
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60)

## Abstract

Transposon nucleic acids comprising a transposon end sequence and a calibration sequence for DNA sequencing in the transposon end sequence. In one embodiment, the transposon end sequence is a Mu transposon end. A method for the generation of DNA fragmentation library based on a transposition reaction in the presence of a transposon end with the calibration sequence providing facilitated downstream handling of the produced DNA fragments, e.g., in the generation of sequencing templates.

## Claims

### Claim 1

A method for in vitro assembly of a plurality of transpososome complexes, comprising:
contacting under a condition for forming a transpososome complex (i) a plurality of MuA transposase enzymes, and (ii) a plurality of modified MuA transposon end sequences each comprising the nucleotide sequence of SEQ ID NO:5, thereby forming a plurality of transpososome complexes.

### Claim 2 (depends on 1)

The method of claim 1, wherein the condition for forming the plurality of transpososome complexes is devoid of magnesium.

### Claim 3 (depends on 1)

The method of claim 1, further comprising contacting the plurality of transpososome complexes with a plurality of target nucleic acids in the presence of magnesium.

### Claim 4 (depends on 3)

The method of claim 3, further comprising: incubating the plurality of transpososome complexes and plurality of target nucleic acids under a condition for performing a transposition reaction, wherein the transposition reaction results in fragmentation of the plurality of target nucleic acids and incorporation of a MuA transposon end sequence from the plurality of MuA transposon end sequences into the ends of the fragmented target nucleic acids, thereby generating a plurality of target nucleic acid fragments attached at both ends to a modified MuA transposon end sequence.

### Claim 5 (depends on 4)

The method of claim 4, wherein the modified MuA transposon end sequences which are attached to both ends of the target nucleic acid fragments further comprise a cleavage site.

### Claim 6 (depends on 5)

The method of claim 5, further comprising: incubating the plurality of target nucleic acid fragments with an enzyme that specifically cleaves the cleavage site so that the modified MuA transposon end sequences which are attached to both ends of the target nucleic acid fragments are cleaved at the cleavage site.

### Claim 7 (depends on 6)

The method of claim 6, wherein the enzyme that cleaves the cleavage site comprises an N-glycosylase or a restriction enzyme.

### Claim 8 (depends on 7)

The method of claim 7, wherein the N-glycosylase is uracil-N-glycosylase.

### Claim 9 (depends on 7)

The method of claim 7, wherein the restriction enzyme is a methylation specific restriction enzyme.

### Claim 10 (depends on 4)

The method of claim 4, further comprising: contacting a DNA polymerase having 5′-3′ exonuclease or strand displacement activity with the plurality of target nucleic acid fragments attached at both ends to the modified MuA transposon end sequence to generate fully double-stranded nucleic acid molecules.

### Claim 11 (depends on 10)

The method of claim 10, further comprising: denaturating the fully double-stranded nucleic acid molecules to produce single stranded nucleic acids, and amplifying the single stranded nucleic acids.

### Claim 12 (depends on 4)

The method of claim 4, further comprising: amplifying the plurality of target nucleic acid fragments in an amplification reaction to generate amplification products using oligonucleotide primers which include sequences that are complementary to the modified MuA transposon end sequence, and the oligonucleotide primers further include a 5′ adaptor tail which is not complementary to the modified MuA transposon end sequence.

### Claim 13 (depends on 12)

The method of claim 12, wherein the 5′ adaptor tail of the oligonucleotide primers comprise a tag selected from the group consisting of an amplification tag, a sequencing tag, and a detection tag.

### Claim 14 (depends on 13)

The method of claim 13, wherein the detection tag comprises a sample-specific barcode tag.

### Claim 15 (depends on 13)

The method of claim 13, wherein the 5′ adaptor tail of the oligonucleotide primers comprise a sequencing tag, and wherein the method further comprises denaturating the amplification products to produce single-stranded nucleic acid and annealing the single-stranded nucleic acid to a solid support coated with an oligonucleotide which is complementary to the sequencing tag thereby immobilizing the single-stranded nucleic acid to the solid support.

### Claim 16 (depends on 15)

The method of claim 15, further comprising: sequencing single-stranded nucleic acid which is immobilized to the solid support.

### Claim 17 (depends on 16)

The method of claim 16, wherein the sequencing includes sequencing the modified MuA transposon end sequence.

### Claim 18 (depends on 16)

The method of claim 16, wherein the sequencing includes sequencing the target nucleic acid sequence.

### Claim 19 (depends on 16)

The method of claim 16, wherein the sequencing includes sequencing together multiple samples of target nucleic acid, each sample identified with a different barcode sequence.