# US 11,028,426 B2 · Nucleic acid sequence amplification method

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- Number: US 11,028,426 B2
- Document ID: US11028426B2
- Status: Granted
- Kind: B2
- Application number: 15/553,091
- Filed: 2016-02-23
- Published: 2018-02-15
- Granted: 2021-06-08
- Earliest claimed: 2015-02-23
- Assignees: Kyoto University
- Applicants: Kyoto University
- Inventors: Mitinori Saitou; Tomonori Nakamura; Yukihiro Yabuta
- Web page: https://patentgrep.com/patent/US11028426B2

## CPC

- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/68: involving nucleic acids
- C12Q 1/6851: Quantitative amplification
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- 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

## Abstract

The invention provides a method of preparing a nucleic acid population suitable for RNA sequencing. The method involves amplifying a double-stranded DNA and a poly T sequence by using the DNA constituted of any additional nucleic acid sequence X, poly T sequence, mRNA sequence isolated from a biological sample, poly A sequence and any additional nucleic acid sequence Y in this order as a template, a first primer containing any additional nucleic acid sequence X having amine added to the 5′-terminal (and a poly T sequence), and a second primer containing any additional nucleic acid sequence Y (and a poly T sequence), followed by fractionalizing the DNA, phosphorylating the DNA, preparing cDNA by using the DNA as a template and a third primer, adding adenine (A) to the cDNA, linking a DNA, and amplifying the DNA by using the DNA as a template, a fourth primer, and a fifth primer.

## Claims

### Claim 1

A method of preparing a nucleic acid population comprising an amplification product maintaining a relative relationship of gene expression level in a biological sample, which method comprising
(a) a step of amplifying a double-stranded DNA comprising a sense strand and an antisense strand, wherein the double-stranded DNA is constituted of (1) any additional nucleic acid sequence X, (2) poly T sequence, (3) cDNA sequence prepared using mRNA sequence isolated from a biological sample as a template, (4) poly A sequence and (5) any additional nucleic acid sequence Yin this order in the orientation from 5′ to 3′ of the sense strand by using the double-stranded DNA as a template, a first primer comprising any additional nucleic acid sequence X having amine added to the 5′-terminus, and optionally further comprising a poly T sequence at the downstream thereof, and a second primer comprising any additional nucleic acid sequence Y, and optionally further comprising a poly T sequence at the downstream thereof, wherein the additional nucleic acid sequence X is different from the additional nucleic acid sequence Y, such that the first primer cannot hybridize to the 3′-terminus of the sense strand of the double-stranded DNA,
(b) a step of fragmenting the double-stranded DNA obtained in step (a),
(c) a step of phosphorylating the 5′-termini of the fragmented double-stranded DNA obtained in step (b),
(d) a step of preparing cDNA by using the double-stranded DNA obtained in step (c) and having a phosphorylated 5′-termini as a template, and a third primer comprising any additional nucleic acid sequence Z and said additional nucleic acid sequence Y in this order, and optionally further comprising a poly T sequence at the downstream thereof, and adding adenine (A) to the 3′-termini of the cDNA,
(e) a step of linking a double-stranded DNA containing any sequence V having 3′-overhang thymine (T) to the double-stranded DNA obtained in step (d), and
(f) a step of amplifying the double-stranded DNA by using the double-stranded DNA obtained in step (e) as a template, a fourth primer comprising the sequence V, and a fifth primer comprising the additional nucleic acid sequence Z, and optionally further comprising the additional nucleic acid sequence Y downstream thereof,
thereby selectively amplifying a fragment of the double stranded DNA comprising the 3′-terminus of the sense strand of the cDNA generated in step (b).

### Claim 2 (depends on 1)

The method according to claim 1, wherein the double-stranded DNA used in step (a) is prepared by a method containing the following steps:
(i) a step of preparing a primary stranded cDNA by reverse transcription using an mRNA isolated from a biological sample as a template, and a sixth primer composed of the additional nucleic acid sequence Y and the poly T sequence,
(ii) a step of preparing a double-stranded DNA, which is a secondary strand, including subjecting the primary stranded cDNA obtained in step (i) to a poly A tailing reaction, and using the primary stranded cDNA as a template, and a seventh primer composed of the additional nucleic acid sequence X and the poly T sequence, and
(iii) a step of amplifying the double-stranded DNA obtained in step (ii) by using an eighth primer comprising the additional nucleic acid sequence X, and optionally further comprising a poly T sequence at the downstream thereof, and a ninth primer comprising the additional nucleic acid sequence Y, and optionally further comprising a poly T sequence at the downstream thereof.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the fragmenting in step (b) is performed by sonication.

### Claim 4 (depends on 1)

The method according to claim 1, wherein smoothing of the termini is performed simultaneously with phosphorylation of the 5′-termini in step (c).

### Claim 5 (depends on 1)

The method according to claim 1, wherein step (c) further comprises a step of selecting a fragmented double-stranded DNA with a size of 200 bases to 250 bases.

### Claim 6 (depends on 1)

The method according to claim 1, wherein the amplification in step (a) is performed by 2 to 8 cycles of PCR.

### Claim 7 (depends on 1)

The method according to claim 1, wherein the amplification in step (f) is performed by 5 to 20 cycles of PCR.

### Claim 8 (depends on 2)

The method according to claim 2, wherein the amplification in step (iii) is performed by 5 to 30 cycles of PCR.

### Claim 9 (depends on 1)

The method according to claim 1, wherein the fifth primer used in step (f) further comprises a barcode sequence.

### Claim 10 (depends on 1)

The method according to claim 1, wherein the biological sample is one to several cells.

### Claim 11 (depends on 10)

The method according to claim 10, wherein the biological sample is one cell.

### Claim 12 (depends on 1)

A method of measuring an amount of mRNA in cells for preparing a nucleic acid population, comprising measuring, by a next-generation sequencer, the amount of the amplified double-stranded DNA in the nucleic acid population prepared by the method according to claim 1.