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CN-122003509-A - Method for degrading 5' -terminal adenylated single-stranded DNA, method for ligating 3' -linker to nucleic acid, method for preparing NGS library, NGS library preparation kit, and 3' -linker for preparing nucleic acid library

CN122003509ACN 122003509 ACN122003509 ACN 122003509ACN-122003509-A

Abstract

A method for degrading 5 '-terminal adenylated single-stranded DNA, which comprises degrading 5' -terminal adenylated single-stranded DNA with RecJ, wherein the base sequence of the 5 '-terminal adenylated single-stranded DNA except for the 5' -terminal adenylate is a specific base sequence.

Inventors

  • NISHIDA RYUSUKE
  • Hojo Kojiro
  • OTOMO RYO

Assignees

  • 爱科来株式会社

Dates

Publication Date
20260508
Application Date
20241007
Priority Date
20231013

Claims (13)

  1. 1. A method for degrading 5 '-terminal adenylated single-stranded DNA, which comprises degrading 5' -terminal adenylated single-stranded DNA with RecJ, wherein the base sequence of the 5 '-terminal adenylated single-stranded DNA excluding the 5' -terminal adenylate is any one of the base sequences shown in Table A, TABLE 1 。
  2. 2. The method for degrading a 5 '-terminal adenylated single-stranded DNA according to claim 1, wherein the 5' -terminal base sequence is any one selected from the group consisting of the base sequences of numbers 1 to 118 shown in Table A.
  3. 3. The method for degrading 5 '-terminal adenylated single-stranded DNA according to claim 1 or claim 2, wherein the enzyme other than RecJ maintains the 5' -terminal adenylated single-stranded DNA in an adenylated state in the presence of the enzyme other than RecJ in the degradation.
  4. 4. A method of ligating a 3' linker to a nucleic acid comprising: ligating 5' -terminal adenylated single-stranded DNA as a 3' -linker to the 3' -terminal of the nucleic acid, and After ligation of the 5 'terminal adenylated single stranded DNA, the remaining 5' terminal adenylated single stranded DNA is degraded by the degradation method of claim 1 or claim 2.
  5. 5. A method of preparing an NGS library, comprising: Ligating 5' -terminal adenylated single-stranded DNA as a 3' -linker to the 3' -terminal of each of a plurality of species of RNAs differing in sequence; after ligation of the 5 'terminal adenylated single stranded DNA, degrading the remaining 5' terminal adenylated single stranded DNA by the degradation method of claim 1 or claim 2; ligating a single-stranded RNA as a 5 'linker to the 5' end of each of the plurality of species of RNA; synthesizing cDNA from the plurality of species of RNA linked to the 3 'linker and the 5' linker by reverse transcription reaction, and The cDNA obtained was amplified by PCR.
  6. 6. The NGS library preparation method of claim 5, wherein the enzyme other than RecJ maintains the 5' terminal adenylated single stranded DNA in an adenylated state in the presence of the enzyme other than RecJ in the degradation.
  7. 7. The NGS library preparation method of claim 5, wherein the plurality of species of RNAs are small RNAs.
  8. 8. An NGS library preparation kit comprising a 5' -terminal adenylated single-stranded DNA contained in a first container and RecJ contained in a second container, wherein the base sequence of the 5' -terminal adenylated single-stranded DNA excluding the 5' -terminal adenylate is any one of the base sequences shown in Table A below, TABLE 2 。
  9. 9. The NGS library preparation kit of claim 8, wherein the 3 '-terminal nucleotide of the 5' -terminal adenylated single stranded DNA is modified with a compound having a modification structure capable of inhibiting nucleic acid binding.
  10. 10. The NGS library preparation kit of claim 8 or claim 9, wherein at least one selected from the group consisting of a 5' linker, a primer, a reverse transcriptase, a reverse transcription primer, a PCR enzyme, and a PCR primer is further included in separate containers, respectively.
  11. 11. A3 '-adaptor for preparing a nucleic acid library, which is a single-stranded DNA having an adenylated nucleotide at the 5' -end, wherein the base sequence of the 5 '-end of the single-stranded DNA excluding the adenylate at the 5' -end is any one of the base sequences shown in Table A, TABLE 3 。
  12. 12. The 3 'linker for nucleic acid library preparation according to claim 11, wherein the base sequence at the 5' end is any one selected from the group consisting of the base sequences numbered 1 to 118 shown in Table A.
  13. 13. The 3 'linker for nucleic acid library preparation according to claim 11 or claim 12, wherein a nucleotide at the 3' end of the single-stranded DNA is modified with a compound having a modification structure capable of inhibiting nucleic acid binding.

Description

Method for degrading 5' -terminal adenylated single-stranded DNA, method for ligating 3' -linker to nucleic acid, method for preparing NGS library, NGS library preparation kit, and 3' -linker for preparing nucleic acid library Technical Field The present disclosure relates to a method for degrading 5' -terminal adenylated single-stranded DNA, a method for ligating a 3' -linker to a nucleic acid, a method for preparing NGS library, a NGS library preparation kit, and a 3' -linker for preparing a nucleic acid library. Background In recent years, nucleic acid comprehensive quantitative analysis represented by Next-generation sequencing (Next-Generation Sequencing (hereinafter also referred to as "NGS") has been used for discriminating diseases. In library preparation for NGS, ligation is performed by attaching base sequences called a 5 'linker and a 3' linker to the 5 'end and the 3' end of a target base sequence to be sequenced, respectively. However, in the above ligation operation, a side reaction occurs in which the remaining 5 'linker not ligated to the target base sequence is bound to the remaining 3' linker to generate a linker dimer. Furthermore, PCR of the above-mentioned linker dimer also occurs along with the polymerase chain reaction (Polymerase Chain Reaction (hereinafter also referred to as PCR) of the target base sequence. That is, sequencing reads from the adaptor dimer will also be obtained in subsequent sequencing, and thus the sequencing efficiency (i.e., the number of reads per unit data amount) will be reduced. As a method for suppressing the generation of a linker dimer and improving sequencing efficiency, there is known a method in which after a 3' linker is ligated to a target base sequence, the remaining 3' linker is degraded using a desadenylase called TAP and an exonuclease called RecJ, and then a 5' linker is ligated (for example, refer to patent document 1). Patent document 1 International publication No. 2011/056866 Disclosure of Invention Problems to be solved by the invention The nucleotide at the 5' -end of the 3' -linker is subjected to an adenylation modification required for ligation to the 3' -end of the target base sequence. According to patent document 1, a method is adopted in which, since the 3' linker modified by adenylation cannot be directly degraded by RecJ, the remaining 3' linker is subjected to a deadenonylation treatment after the ligation of the 3' linker, and then degraded by RecJ. However, the method of patent document 1 requires a multistage enzyme treatment, and simplification of the steps is desired. In view of the above, an object of the present disclosure is to provide a method for degrading 5' -terminal adenylated single-stranded DNA, a method for ligating 3' -terminal to nucleic acid, a method for preparing NGS library, a NGS library preparation kit, and a 3' -adaptor for preparing nucleic acid library, which can rapidly and easily prepare NGS library using 5' -terminal adenylated single-stranded DNA as a 3' -adaptor. Means for solving the problems Specific means for solving the above problems include the following means. <1> A method for degrading 5 '-terminal adenylated single-stranded DNA, comprising degrading 5' -terminal adenylated single-stranded DNA with RecJ, wherein the base sequence of the 5 '-terminal adenylated single-stranded DNA excluding the 5' -terminal adenylate is any of the base sequences shown in Table A below. TABLE 1 <2> The method for degrading a5 '-terminal adenylated single stranded DNA according to <1>, wherein the 5' -terminal base sequence is any one selected from the group consisting of the base sequences of numbers 1 to 118 shown in Table A. <3> The method for degrading a 5 '-terminal adenylated single stranded DNA according to <1> or <2>, wherein the enzyme other than RecJ keeps the 5' -terminal adenylated single stranded DNA in an adenylated state when the enzyme other than RecJ is also present in the degradation. <4> A method for ligating a3 '-adaptor to a nucleic acid, comprising ligating a 5' -terminal adenylated single-stranded DNA as a3 '-adaptor to the 3' -terminal of the nucleic acid, and degrading the remaining 5 '-terminal adenylated single-stranded DNA by the degradation method of any one of <1> to <3> after ligating the 5' -terminal adenylated single-stranded DNA. <5> A method for producing an NGS library, comprising ligating 5' -terminally adenylated single-stranded DNAs as 3' -linkers to 3' -ends of respective RNAs of a plurality of species having different sequences, degrading remaining 5' -terminally adenylated single-stranded DNAs by the degradation method of any one of <1> to <3> after ligating the 5' -terminally adenylated single-stranded DNAs, ligating single-stranded RNAs as 5' -linkers to the 5' -ends of respective RNAs of the plurality of species, synthesizing cdnas from the plurality of species having the 3' -linkers and the 5' -linkers attached thereto by reverse transcription reaction, and amplify