阅读说明：本技术 一种将寡聚核苷酸逆转录成cDNA的方法及寡聚核苷酸检测方法 (Method for reverse transcription of oligonucleotide into cDNA and oligonucleotide detection method ) 是由 赵利峰 秦少伟 李才林 于 2020-04-21 设计创作，主要内容包括：本发明一种将寡聚核苷酸逆转录成cDNA的方法及寡聚核苷酸检测方法,包括如下步骤：(1)设计单链DNA,单链DNA的中间部分与目标寡聚核苷酸碱基互补；(2)根据目标寡聚核苷酸选择上游辅助RNA和下游辅助RNA,且上游辅助RNA的3’端含有羟基,下游辅助RNA的5’端含有单磷酸基团；单链DNA的两端分别与上游辅助RNA和下游辅助RNA碱基互补；(3)利用T4DNA连接酶将目标寡聚核苷酸两端分别与上游辅助RNA和下游辅助RNA连接起来；(4)将单链DNA消化掉,得到中间部分为目标寡聚核苷酸的待逆转录单链RNA；(5)在待逆转录单链RNA的3’端设计引物,将待逆转录单链RNA逆转录成cDNA。不仅能够检测10nt以上的核苷酸,特别是能够检测6-10nt长的小片段核苷酸。(The invention relates to a method for reverse transcribing oligonucleotide into cDNA and an oligonucleotide detection method, comprising the following steps: (1) designing single-stranded DNA, wherein the middle part of the single-stranded DNA is complementary with the base of the target oligonucleotide; (2) selecting an upstream auxiliary RNA and a downstream auxiliary RNA according to the target oligonucleotide, wherein the 3 'end of the upstream auxiliary RNA contains hydroxyl, and the 5' end of the downstream auxiliary RNA contains a monophosphate group; the two ends of the single-stranded DNA are respectively complementary with the basic groups of the upstream auxiliary RNA and the downstream auxiliary RNA; (3) connecting two ends of the target oligonucleotide with upstream auxiliary RNA and downstream auxiliary RNA respectively by using T4DNA ligase; (4) digesting the single-stranded DNA to obtain a single-stranded RNA to be reverse-transcribed, the middle part of which is the target oligonucleotide; (5) designing a primer at the 3' end of the single-stranded RNA to be reverse-transcribed, and reverse-transcribing the single-stranded RNA to be reverse-transcribed into cDNA. Not only can detect more than 10nt of nucleotide, but also can detect small fragment nucleotide with length of 6-10 nt.)

1. A method of reverse transcribing an oligonucleotide to cDNA comprising the steps of:

(1) designing a single-stranded DNA, wherein the middle part of the single-stranded DNA is complementary with a target oligonucleotide base;

(2) selecting an upstream helper RNA and a downstream helper RNA according to a target oligonucleotide, wherein the 3 'end of the upstream helper RNA contains monohydroxy, and the 5' end of the downstream helper RNA contains monophosphate group; the two ends of the single-stranded DNA are respectively complementary with the upstream auxiliary RNA and the downstream auxiliary RNA;

(3) connecting two ends of the target oligonucleotide with the upstream auxiliary RNA and the downstream auxiliary RNA respectively;

(4) digesting the single-stranded DNA to obtain a single-stranded RNA to be reverse-transcribed, the middle part of which is the target oligonucleotide;

(5) designing a primer at the 3' end of the single-stranded RNA to be reverse-transcribed, and reverse-transcribing the single-stranded RNA to be reverse-transcribed into cDNA.

2. The method of claim 1, wherein in step (3): and (3) connecting the two ends of the target oligonucleotide with the upstream auxiliary RNA and the downstream auxiliary RNA respectively by using ligase.

3. The method of claim 2, wherein in step (3): the ligase is T4DNA ligase.

4. The method of claim 1, wherein in step (1): the single-stranded DNA was digested with DNase I.

5. The oligonucleotide detection method is characterized by comprising the following steps:

(A) the method of any one of claims 1 to 4, wherein the oligonucleotide is reverse transcribed into cDNA;

(B) qPCR quantitative detection, high-throughput sequencing detection or gene chip detection is carried out on the cDNA, so that qPCR quantitative detection, high-throughput sequencing detection or gene chip detection can be carried out on the oligonucleotide generated in vivo.

6. The method for detecting oligonucleotides as claimed in claim 5, wherein in step (B), the qPCR quantitative detection method is as follows:

the first method comprises the following steps: designing an upstream Primer F and a downstream Primer R according to the cDNA, wherein the upstream Primer F is the same as the upstream end of the single-stranded RNA to be subjected to reverse transcription, the downstream Primer R is complementary with the downstream end base of the single-stranded RNA to be subjected to reverse transcription, and then carrying out real-time quantitative PCR detection;

the second method comprises the following steps: and designing a Taqman-MGB probe, covering the target oligonucleotide and a plurality of base sequences at two ends of the target oligonucleotide in the single-stranded RNA to be subjected to reverse transcription by the Taqman-MGB probe, and detecting the content of the target oligonucleotide by using a high-specificity Taqman probe PCR method.

7. The method for detecting oligonucleotide of claim 6, wherein in step (B): the high-throughput sequencing detection method comprises the following steps:

(B1-1) designing an upstream Primer F and a downstream Primer R from the cDNA, and then performing a first round PCR on the cDNA using the upstream Primer F and the downstream Primer R;

(B1-2) designing an upstream Primer comprising Primer F, comprising a p5 sequence for binding to and cluster formation with an illumina sequencer and an i5 sequence for sequencing of read1, the i5 sequence being located between the p5 sequence and Primer F; simultaneously designing a Primer R-containing downstream Primer which comprises a p7 sequence used for combination and cluster formation with an illumina sequencer, an i7 sequence used for sequencing of read2 and a barcode sequence positioned between the p7 sequence and the i7 sequence, wherein an i7 sequence is positioned between the barcode sequence and the Primer R; the barcode sequence was used to distinguish samples.

8. The method for detecting oligonucleotide of claim 5, wherein in step (B), the method for detecting gene chip is as follows: designing restriction endonuclease EcoR V restriction sites on the upstream auxiliary RNA and the downstream auxiliary RNA, after completing PCR amplification, processing PCR products by the EcoR V to generate DNA fragments containing target oligonucleotide sequences, then designing corresponding probes, and finally detecting.

Technical Field

The invention relates to the technical field of RNA detection. In particular to a method for reverse transcribing oligonucleotide into cDNA and an oligonucleotide detection method.

Background

Oligonucleotides with fragment lengths of less than 10nt, such as Abortive Transcript (AT) generated AT the start of transcription and small fragment nucleotides generated after degradation of long fragment nucleotides, occur naturally in organisms. The existing findings suggest that the small fragment nucleotides may have important biological effects in vivo, but the small fragment nucleotides below 10nt can not be qualitatively and quantitatively detected.

Disclosure of Invention

Therefore, the present invention is to provide a method for reverse transcription of oligonucleotide into cDNA and a method for detecting oligonucleotide, which can detect not only nucleotide of 10nt or more, but also small fragment nucleotide of 6-10 nt.

In order to solve the technical problems, the invention provides the following technical scheme:

a method of reverse transcribing an oligonucleotide to cDNA comprising the steps of:

(1) designing a single-stranded DNA, wherein the middle part of the single-stranded DNA is complementary with a target oligonucleotide base;

(2) selecting an upstream helper RNA and a downstream helper RNA according to a target oligonucleotide, wherein the 3 'end of the upstream helper RNA contains monohydroxy, and the 5' end of the downstream helper RNA contains monophosphate group; the two ends of the single-stranded DNA are respectively complementary with the upstream auxiliary RNA and the downstream auxiliary RNA;

(3) connecting two ends of the target oligonucleotide with the upstream auxiliary RNA and the downstream auxiliary RNA respectively;

(4) digesting the single-stranded DNA to obtain a single-stranded RNA to be reverse-transcribed, the middle part of which is the target oligonucleotide;

(5) designing a primer at the 3' end of the single-stranded RNA to be reverse-transcribed, and reverse-transcribing the single-stranded RNA to be reverse-transcribed into cDNA.

The above method for reverse transcription of an oligonucleotide into cDNA, in step (3): and (3) connecting the two ends of the target oligonucleotide with the upstream auxiliary RNA and the downstream auxiliary RNA respectively by using ligase.

The above method for reverse transcription of an oligonucleotide into cDNA, in step (3): the ligase is T4DNA ligase.

The method for reverse transcription of an oligonucleotide into cDNA as described above, wherein in the step (1): the single-stranded DNA was digested with DNase I.

The oligonucleotide detection method comprises the following steps:

(A) the method of any one of claims 1 to 6, wherein the oligonucleotide is reverse transcribed into cDNA;

(B) qPCR quantitative detection, high-throughput sequencing detection or gene chip detection is carried out on the cDNA, so that qPCR quantitative detection, high-throughput sequencing detection or gene chip detection can be carried out on the oligonucleotide generated in vivo.

In the oligonucleotide detection method, in the step (B), the qPCR quantitative detection method is as follows:

the first method comprises the following steps: designing an upstream Primer F and a downstream Primer R according to the cDNA, wherein the upstream Primer F is the same as the upstream end of the single-stranded RNA to be subjected to reverse transcription, the downstream Primer R is complementary with the downstream end base of the single-stranded RNA to be subjected to reverse transcription, and then carrying out real-time quantitative PCR detection;

the second method comprises the following steps: and designing a Taqman-MGB probe, covering the target oligonucleotide and a plurality of base sequences at two ends of the target oligonucleotide in the single-stranded RNA to be subjected to reverse transcription by the Taqman-MGB probe, and detecting the content of the target oligonucleotide by using a high-specificity Taqman probe PCR method.

The method for detecting an oligonucleotide as described above, wherein in the step (B): the high-throughput sequencing detection method comprises the following steps:

(B1-1) designing an upstream Primer F and a downstream Primer R from the cDNA, and then performing a first round of PCR on the cDNA using the upstream Primer F and the downstream Primer R;

(B1-2) designing an upstream Primer comprising Primer F, comprising a p5 sequence for binding to and cluster formation with an illumina sequencer and an i5 sequence for sequencing of read1, the i5 sequence being located between the p5 sequence and Primer F; simultaneously designing a Primer R-containing downstream Primer which comprises a p7 sequence used for combination and cluster formation with an illumina sequencer, an i7 sequence used for sequencing of read2 and a barcode sequence positioned between the p7 sequence and the i7 sequence, wherein an i7 sequence is positioned between the barcode sequence and the Primer R; the barcode sequence was used to distinguish samples.

In the above oligonucleotide detection method, in the step (B), the gene chip detection method is as follows: designing restriction endonuclease EcoR V restriction sites on the upstream auxiliary RNA and the downstream auxiliary RNA, after completing PCR amplification, processing PCR products by the EcoR V to generate DNA fragments containing target oligonucleotide sequences, then designing corresponding probes, and finally detecting.

The technical scheme of the invention achieves the following beneficial technical effects: the invention designs a single-stranded DNA, the middle part of which is complementary with oligonucleotide, and the two ends of which are respectively complementary with upstream auxiliary RNA and downstream auxiliary RNA; and the upstream helper RNA3 'end has a hydroxyl group and the downstream helper RNA 5' end has a phosphate group. Then T4DNA ligase is used to connect the oligonucleotide and the auxiliary RNA, DNase I is used to digest the DNA chain, then a primer is designed at the 3' end of the newly connected RNA, and the newly connected RNA chain is reversely transcribed into cDNA, thereby carrying out real-time quantitative PCR detection, sequencing detection or chip detection on the oligonucleotide with more than 6nt generated in vivo. The nucleotide with the length of more than 10nt can be detected by the method in the prior art, and the invention is characterized in that the oligonucleotide with the length of 6-10nt can be detected.

Drawings

FIG. 1 is a schematic diagram of DNA/RNA base stacking hybridization;

FIG. 2T 4DNA ligase ligation;

FIG. 3 DNase I digestion of chain A;

FIG. 4 cDNA Synthesis;

FIG. 5 Taqman-MGB probe real-time quantitative PCR detection;

FIG. 6 detection of 6-10nt oligonucleotides by chip method;

FIG. 7 is a schematic representation of the MUC 168 nt AT probe;

FIG. 8 is a schematic reverse transcription diagram;

FIG. 9 electrophoretogram in example 2;

FIG. 10 Sanger sequencing results.

Detailed Description