Methods for quantitative detection of mRNA 3'UTR length

Abstract

This application relates to the field of mRNA detection technology, specifically to a method for quantitatively detecting the length of the 3'UTR of mRNA. This application provides a CDS probe that binds to the CDS region and a 3'UTR probe that binds to the 3'UTR region, as well as a first fluorescent probe that recognizes the CDS probe and a second fluorescent probe that recognizes the 3'UTR region. The first and second fluorescent probes carry different fluorescent groups. By coupling standard DNA or mRNA containing 3'UTR sequences of different lengths to magnetic beads, magnetic bead standards containing 3'UTR regions of different lengths are obtained. A standard curve is constructed based on the magnetic bead standards to obtain a standard equation. The length of the 3'UTR in the test sample is determined by comparing the fluorescence value of the test sample with the standard equation, thereby achieving quantitative detection.

Description

Technical Field

[0001] This application relates to the field of mRNA detection technology, specifically to a method for quantitatively detecting the length of the 3'UTR of mRNA. Background Art

[0002] mRNA, also known as messenger RNA, is RNA that plays a role in transmitting genetic information in cells. The 3' untranslated region (3'UTR) of mRNA contains binding sites for many trans-acting elements such as miRNAs (microRNAs) and RNA-binding proteins, thereby regulating mRNA stability, localization, and translation efficiency. The length of the mRNA 3'UTR depends on the use of polyadenylation cleavage sites (PAS) during precursor mRNA maturation. Over 70% of the genes in the mammalian genome contain multiple PASs, which can lead to various mutants with different 3'UTR lengths in the transcribed mRNA, despite these isoforms having identical coding sequences.

[0003] Therefore, detecting the length of the 3'UTR of mRNA is of great significance for analyzing trans-acting elements such as gene miRNA binding sites and RNA-binding proteins. Currently, traditional methods for detecting 3'UTR length include Northern blotting, PCR, and high-throughput sequencing. These methods can qualitatively analyze changes in the 3'UTR length of mRNA, but cannot quantitatively analyze its length. Furthermore, these traditional methods require sample or cell lysis before detection, leading to the loss of mRNA localization information. Summary of the Invention

[0004] This application provides a CDS probe that binds to the CDS region and a 3'UTR probe that binds to the 3'UTR region. The CDS probe and the 3'UTR probe are each equipped with a fluorescent group. They are hybridized with magnetic beads coupled to standard DNA or mRNA by single fluorescence in situ hybridization, respectively, and the fluorescence signal is collected. The length of the 3'UTR of the mRNA is quantitatively detected based on the fluorescence signal.

[0005] Therefore, the embodiments of this application disclose at least the following technical solutions:

[0006] This embodiment provides a method for quantitatively detecting the 3'UTR length of mRNA. The method includes: preparing multiple magnetic beads coupled with single-stranded nucleic acids, wherein the single-stranded nucleic acids contain a target gene CDS sequence, or contain a target gene CDS sequence and 3'UTR sequences of different lengths; preparing a CDS probe that binds to the CDS region and a 3'UTR probe that binds to the 3'UTR region, wherein the CDS probe carries a first fluorescent group and the 3'UTR probe carries a second fluorescent group, and the first and second fluorescent groups are different; simultaneously performing a first mixed single-stranded hybridization reaction with each magnetic bead coupled with the CDS probe and the 3'UTR probe; reading the first fluorescence value of the first mixed single-stranded hybridization reaction; constructing a standard curve based on the 3'UTR sequence length in the single-stranded nucleic acid and the first fluorescence value; preparing magnetic beads coupled with the target mRNA; simultaneously performing a second mixed single-stranded hybridization reaction with the CDS probe and the 3'UTR probe; reading the second fluorescence value of the second mixed single-stranded hybridization reaction; and determining the 3'UTR length of the target mRNA based on the fluorescence curve and the second fluorescence value.

[0007] The method provided in this embodiment, by combining a CDS probe to the CDS region and a 3'UTR probe to the 3'UTR region, can hybridize with target DNAs of different 3'UTR sequence lengths and establish a correlation between the fluorescence signal generated during hybridization and the 3'UTR sequence length. Based on this correlation, the 3'UTR sequence length can be calculated from the fluorescence signal generated by hybridization with the 3'UTR sequence of an unknown mRNA. This method can accurately detect the 3'UTR sequence length of mRNA, has high reproducibility, and is widely applicable. Attached Figure Description

[0008] Figure 1 The schematic diagram shows the principle of the method for quantitative detection of mRNA 3'UTR length provided in the example.

[0009] Figure 2 This is a schematic flowchart of a method for quantitatively detecting the 3'UTR length of mRNA provided in an example.

[0010] Figure 3 The method flowchart for steps S701 to S706 provided in the embodiment is shown.

[0011] Figure 4 This is a schematic diagram of the method flow for step S701 provided in the embodiment.

[0012] Figure 5 This is a schematic diagram of the method flow for step S100 provided in the embodiment.

[0013] Figure 6 The standard curve and standard equation for detecting the length of HeLa cell mRNA are provided in the examples. Detailed Implementation

[0014] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Reagents not specifically described in detail herein are all conventional reagents and are commercially available; methods not specifically described in detail are all conventional experimental methods and can be learned from the prior art.

[0015] like Figure 1 As shown, this application provides a CDS probe and a 3'UTR probe capable of base pairing and hybridization with all or part of the CDS region and its downstream 3'UTR region of a target gene, as well as a first fluorescent probe for recognizing the CDS probe and a second fluorescent probe for recognizing the 3'UTR probe. The first fluorescent probe carries a first fluorescent group, and the second fluorescent probe carries a second fluorescent group different from the first fluorescent group. This application first uses the CDS probe and the 3'UTR probe to perform base pairing and hybridization with the CDS region and 3'UTR region of the target sequence, respectively, to capture the target sequence from the sample. Then, the first fluorescent probe performs base pairing and hybridization with the CDS probe, simultaneously generating fluorescent information. The second fluorescent probe performs base pairing and hybridization with the 3'UTR probe, simultaneously producing different fluorescent information. By collecting this fluorescent information, the length of the 3'UTR of the target sequence is determined, achieving quantitative detection.

[0016] like Figure 2 As shown, the embodiment provides a method for quantitatively detecting the length of the mRNA 3'UTR. The method includes:

[0017] S100: Prepare magnetic beads coupled with a single-stranded nucleic acid to be tested, which contains the CDS sequence and 3'UTR sequence of the target gene;

[0018] S200: Provides a CDS probe that binds to the CDS region and a 3'UTR probe that binds to the 3'UTR region, as well as a first fluorescent probe that recognizes the CDS probe and a second fluorescent probe that recognizes the 3'UTR probe. The first fluorescent probe has a first fluorescent group, and the second fluorescent probe has a second fluorescent group that is different from the first fluorescent group.

[0019] S300: The magnetic beads coupled with the single-stranded nucleic acid to be tested are simultaneously subjected to the first mixed single-stranded hybridization reaction with the CDS probe and the 3'UTR probe.

[0020] S400: The product of the first mixed single-strand hybridization reaction is simultaneously subjected to a second mixed single-strand hybridization reaction with the first fluorescent probe and the second fluorescent probe.

[0021] S500: Read the first fluorescence information generated by the reaction of the product of the first mixed single-chain hybridization reaction with the first fluorescent probe based on the first fluorescent group; read the second fluorescence information generated by the reaction of the product of the first mixed single-chain hybridization reaction with the second fluorescent probe based on the second fluorescent group.

[0022] S600: Determine the test fluorescence value corresponding to the single-stranded nucleic acid to be tested based on the first fluorescence information and the second fluorescence information;

[0023] S700: Determine the length of the 3'UTR sequence based on the standard equation and the fluorescence value to be measured.

[0024] like Figure 3 As shown, in some embodiments of the method, the method further includes the following steps before step S700:

[0025] S701: Prepare multiple magnetic beads coupled with standard single-stranded nucleic acids, which contain the target gene CDS sequence, or contain the target gene CDS sequence and 3'UTR sequences of different lengths;

[0026] S702: Each magnetic bead coupled with a single-stranded nucleic acid is simultaneously subjected to a first mixed single-stranded hybridization reaction with both the CDS probe and the 3'UTR probe;

[0027] S703: The product of the first mixed single-strand hybridization reaction is simultaneously subjected to a second mixed single-strand hybridization reaction using the first fluorescent probe and the second fluorescent probe.

[0028] S704: Read the first fluorescence information generated by the reaction of the product of the first mixed single-strand hybridization reaction with the first fluorescent probe based on the first fluorescent group; read the second fluorescence information generated by the reaction of the product of the first mixed single-strand hybridization reaction with the second fluorescent probe based on the second fluorescent group.

[0029] S705: Based on the first fluorescence information and the second fluorescence information, determine multiple standard fluorescence values ​​corresponding to single-stranded nucleic acids without 3'UTR sequences and single-stranded nucleic acids with 3'UTR sequences of different lengths, respectively;

[0030] S706: A standard equation is determined based on multiple standard fluorescence values ​​and the length of the 3'UTR sequence in single-stranded nucleic acid. The standard equation characterizes the relationship between the standard fluorescence value and the length of the 3'UTR sequence in single-stranded nucleic acid.

[0031] The method provided in this embodiment, using a CDS probe and a 3'UTR probe, can hybridize with target DNAs of different 3'UTR sequence lengths. Further hybridization using a first fluorescent probe and a second fluorescent probe generates and amplifies fluorescent signals, establishing a correlation between the fluorescent signals generated during hybridization and the 3'UTR sequence length. Based on this correlation, the 3'UTR sequence length of an unknown mRNA can be calculated from the fluorescent signals generated by hybridization with the 3'UTR sequence of that mRNA. This method accurately detects the 3'UTR sequence length of mRNA, exhibits high reproducibility, and has wide applicability.

[0032] like Figure 4 As shown, in some embodiments of the method, step S701 includes:

[0033] S711: The first double-stranded DNA containing the CDS sequence of the target gene and the second double-stranded DNA containing the CDS sequence of the target gene and a 3'UTR sequence of a defined length are cloned into the basic plasmid to obtain the first vector and the second vector.

[0034] S721: Synthesize biotin-labeled forward and reverse primers, and use the first and second vectors as templates respectively to amplify biotin-labeled first double-stranded DNA and biotin-labeled second double-stranded DNA by PCR.

[0035] S731: Biotin-labeled first double-stranded DNA and biotin-labeled second double-stranded DNA are incubated with magnetic beads containing avidin to obtain magnetic beads coupled with first double-stranded DNA and magnetic beads coupled with second double-stranded DNA.

[0036] S741: Treat magnetic beads coupled with first double-stranded DNA and magnetic beads coupled with second double-stranded DNA with alkaline solution to obtain magnetic beads coupled with first single-stranded DNA and magnetic beads coupled with second single-stranded DNA, respectively.

[0037] In some embodiments, a 3' UTR sequence of a certain length is determined, and its length can be 10bp, 20bp, 30bp, 40bp, 50bp, 100bp, 300bp, 400bp, 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 1000bp, 1200bp, 1300bp, 1400bp, 1500bp, 1600bp, 1800bp, 2000bp, 3000bp, or 4000bp. Specifically, there are multiple second double-stranded DNAs, and the length of the additional 3' UTR sequence of each second double-stranded DNA compared to the first double-stranded DNA is in an arithmetic progression. For example, one first double-stranded DNA and three second double-stranded DNAs (corresponding to three second single-stranded DNAs and three second double-stranded DNAs) are synthesized, and the three second double-stranded DNAs are successively 1000bp, 2000bp, and 3000bp longer than the first double-stranded DNA sequence.

[0038] In some embodiments, the amplification products in step S721 are further separated by agarose gel electrophoresis, and the biotin-labeled first double-stranded DNA and biotin-labeled second double-stranded DNA are obtained after gel recovery.

[0039] In some embodiments, step S741 further includes treating the beads with an alkaline solution (e.g., a 20 mM sodium hydroxide solution) for 1 hour, removing the supernatant by magnetic separation, and washing the separated magnetic beads with TE buffer.

[0040] like Figure 5 As shown, step S100 provided in some embodiments specifically includes:

[0041] S101: The single-stranded nucleic acid to be tested, containing the CDS sequence and 3'UTR sequence of the target gene, was cloned into the basic plasmid (pBluescript II-KS(+), Nanjing Genscript Biotech Co., Ltd.) to obtain the cloning vector;

[0042] S102: Synthesize biotin-labeled forward and reverse primers, use the cloning vector as a template, and PCR amplify the biotin-labeled double-stranded DNA to be tested.

[0043] S103: Incubate biotin-labeled double-stranded DNA to be tested with magnetic beads containing avidin to obtain magnetic beads coupled with the double-stranded DNA to be tested.

[0044] S104: Treat the magnetic beads coupled with the double-stranded DNA to be tested with an alkaline solution to obtain magnetic beads coupled with the single-stranded DNA to be tested.

[0045] In step S200 of some embodiments, the CDS probe has a nucleotide sequence that is identical or complementary to a portion of the CDS sequence of the target gene, and the 3'UTR probe has a nucleotide sequence that is identical or complementary to a portion of the 3'UTR sequence of the target gene. Optionally, there are multiple CDS probes and multiple 3'UTR probes. For example, in a 3'UTR length detection process for human NUDT21, there are 12 CDS probes (as shown in SEQ ID NO: 1-12 in sequence) and 20 3'UTR probes (as shown in SEQ ID NO: 13-32 in sequence). In step S300, a magnetic bead coupled with the single-stranded nucleic acid to be tested is simultaneously subjected to a first mixed single-stranded hybridization reaction with these 12 CDS probes and 20 3'UTR probes.

[0046] In some embodiments, the S600 step is provided, where the first fluorescence information is the fluorescence value generated by the reaction of the product of the first mixed single-stranded hybridization reaction with the first fluorescent probe, and the second fluorescence information is the fluorescence value generated by the reaction of the product of the first mixed single-stranded hybridization reaction with the first fluorescent probe. The difference between the first fluorescence value and the second fluorescence value is taken as the fluorescence value of the single-stranded nucleic acid to be tested.

[0047] In the above embodiments, the first fluorescent group is selected from FAM (carboxyfluorescein), TET (tetrachloro-6-carboxyfluorescein), JOE (2,7-dimethyl-4,5-dichloro-6-carboxyfluorescein), TAMRA (carboxytetramethylrhodamine), ROX (carboxy-X-rhodamine), Cy3, Cy5, HEX (hexachlorofluorescein), TexasRed, VIC, Alexa594, and Alexa488. The second fluorescent group is selected from FAM (carboxyfluorescein), TET (tetrachloro-6-carboxyfluorescein), JOE (2,7-dimethyl-4,5-dichloro-6-carboxyfluorescein), TAMRA (carboxytetramethylrhodamine), ROX (carboxy-X-rhodamine), Cy3, Cy5, HEX (hexachlorofluorescein), TexasRed, VIC, Alexa594, and Alexa488.

[0048] In some embodiments, the S700 step uses a standard equation to characterize the mathematical relationship between the difference between the first and second fluorescence values ​​and the 3'UTR probe. In some embodiments, this standard equation represents a linear mathematical relationship.

[0049] The present application will be described below with reference to more specific embodiments, but this does not constitute a limitation on the implementation of the present application.

[0050] 1. Design the first double-stranded DNA, the second double-stranded DNA, the CDS probe, the 3'UTR probe, the first fluorescent probe, and the second fluorescent probe.

[0051] Taking the human NUDT21 gene as an example, based on the coding region (CDS, 684 bp) and the 3' untranslated region (3'UTR, 3579 bp) sequence of its mRNA (NCBI sequence number: NM_007006.3), Alexa594-labeled CDS probes and Alexa488-labeled 3'UTR probes were designed respectively.

[0052] Sequences of the first single-stranded DNA and the first double-stranded DNA: CDS sequence (NM_007006.3)

[0053] The sequences of the second single-stranded DNA and the second double-stranded DNA are: CDS+1000bp 3'UTR, SEQ ID NO:33; or CDS+2000bp 3'UTR, SEQ ID NO:34; or CDS+3000bp 3'UTR, SEQ ID NO:35.

[0054] The nucleotide sequences of the CDS probe set are shown in SEQ ID NO:1-12.

[0055] The nucleotide sequences of the 3'UTR probe group are shown in SEQ ID NO:13-32.

[0056] First fluorescent probe: 5' end Alexa594 labeled, used to identify the CDS probe set, Molecular Instruments.

[0057] Second fluorescent probe: 5' end Alexa488 labeled, used to identify the 3'UTR probe set, Molecular Instruments.

[0058] 2. Step S701

[0059] A specific S701 step includes:

[0060] The first double-stranded DNA containing the target gene CDS sequence and the second double-stranded DNA containing the target gene CDS sequence and a 3'UTR sequence of a defined length were cloned into the basic plasmid (pBluescript II-KS(+), Nanjing Genscript Biotech Co., Ltd.) to obtain a first vector carrying the CDS sequence (NM_007006.3) and three second vectors carrying SEQ ID NO:33-35 respectively.

[0061] Biotin-labeled forward and reverse primers were synthesized, and biotin-labeled first double-stranded DNA and biotin-labeled second double-stranded DNA were obtained by PCR amplification using the first and second vectors as templates, respectively.

[0062] The primer pairs used to amplify the first vector include:

[0063] The 5' biotin-labeled forward primer is: 5'-ATGTCTGTGGTACCGCCCAA-3', SEQ ID NO:36; the reverse primer (3'UTR-0bp) is: 5'-TCAAACAATAGAAAGGTGGC-3', SEQ ID NO:37, and the product is first-stranded DNA.

[0064] The primer pairs used to amplify the second vector include:

[0065] Reverse primer (3'UTR-1000bp): 5'-ACATCTGGCAAGATTCCTTT-3', SEQ ID NO:38, the product is a second double-stranded DNA containing CDS and a 1000bp 3'UTR sequence.

[0066] Reverse primer (3'UTR-2000bp): 5'-AAACAGTGCCCTTATACCCT-3', SEQ ID NO:39, the product is second double-stranded DNA, the product contains CDS and a 2000bp 3'UTR sequence.

[0067] Reverse primer (3'UTR-3000bp): 5'-CAACAACCACTAAGCCTTCT-3', SEQ ID NO:40, the product is a second double-stranded DNA, the product contains CDS and a 3000bp 3'UTR sequence.

[0068] The PCR reaction system is shown in Table 1, and the PCR reaction procedure is shown in Table 2.

[0069] Table 1

[0070] Volume (μl) 2×TaqPlusMasterMix (purchased from Novizan Biosciences Co., Ltd.) 25 Forward primer (10 μM) 2 Reverse primer (10 μM) 2 template 1 Sterile water 20 Total volume 50

[0071] Table 2

[0072] Temperature (°C) time 95 3 minutes 95 15 seconds 60 20 seconds 35 cycles 72*(*The extension time depends on the length of the product and can be 60 seconds / kb) 45 seconds to 3 minutes and 40 seconds 72 10 minutes

[0073] The recovered biotin-labeled first and second double-stranded DNA were used as standards and mixed with avidin-conjugated Dynabead magnetic beads (purchased from Thermo) at a ratio of 20 μg DNA / 1 mg magnetic beads. The mixture was incubated at room temperature for 1 hour to conjugate the DNA to the magnetic beads. The mixture was washed twice with 100 μL TE buffer (10 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 1 mM EDTA) and twice with 100 μL double-distilled water. The double-stranded DNA standard-magnetic bead complex was mixed with 100 μL NaOH (20 mM) solution and incubated at room temperature for 1 hour on a rotary mixer. The test tube was then placed on a magnet to separate the magnetic beads from the supernatant. The supernatant was discarded, and the mixture was washed three times with TE buffer to obtain magnetic beads conjugated with the first and second single-stranded DNA.

[0074] 3. Steps S702 to S706

[0075] In some embodiments, steps S702 to S706 specifically include:

[0076] S702: Simultaneously perform a first mixed single-stranded hybridization reaction with magnetic beads coupled with the first single-stranded DNA and CDS probe and 3'UTR probe; simultaneously perform a first mixed single-stranded hybridization reaction with magnetic beads coupled with the second single-stranded DNA and CDS probe and 3'UTR probe.

[0077] S703: The magnetic beads coupled with the first single-stranded DNA are reacted to obtain the first mixed single-stranded hybridization product, while the first fluorescent probe and the second fluorescent probe are used to perform the second mixed single-stranded hybridization reaction; The magnetic beads coupled with the second single-stranded DNA are reacted to obtain the first mixed single-stranded hybridization product, while the first fluorescent probe and the second fluorescent probe are used to perform the second mixed single-stranded hybridization reaction.

[0078] S704: Read the first fluorescence information generated by the magnetic bead reaction coupled with the first single-stranded DNA according to the Alexa594 of the first fluorescent probe, and read the second fluorescence information generated by the magnetic bead reaction coupled with the second single-stranded DNA according to the Alexa488 of the second fluorescent probe.

[0079] S705: Obtain the first standard fluorescence value of the magnetic bead coupled with the first single-stranded DNA and the three second standard fluorescence values ​​of the magnetic bead coupled with the second single-stranded DNA.

[0080] S706: The standard equation is determined based on four standard fluorescence values ​​and the length of the 3'UTR sequence in single-stranded nucleic acid. The standard equation characterizes the relationship between the standard fluorescence value and the length of the 3'UTR sequence in single-stranded nucleic acid.

[0081] The conditions for the first and second mixed single-stranded hybridization reactions were as follows: Magnetic beads were washed twice with 2×SSC buffer, hybridization buffer was added, and the mixture was placed on a rotary mixer and pre-hybridized at 37°C for 30 minutes. The magnetic beads were then resuspended in hybridization buffer containing CDS and 3'UTR probes and incubated on a rotary mixer at 37°C for 12 hours. The magnetic beads were then washed four times with washing buffer, once with 5×SSCT buffer, resuspended in amplification buffer, and pre-amplified at room temperature for 30 minutes. Take equal volumes of the second fluorescent probe labeled with Alexa488 (for recognizing the 3'UTR probe group, Molecular Instruments) and the first fluorescent probe labeled with Alexa594 (for recognizing the CDS probe group, Molecular Instruments), mix them well, heat shock at 95°C for 90 seconds, and cool at room temperature in the dark for at least 30 minutes. Add 50 μL of amplification buffer to the Hairpin B1 and B2 probe mixture, resuspend the magnetic beads, place them on a rotary mixer, and incubate overnight at room temperature in the dark for signal amplification. Discard the amplification buffer, wash the magnetic beads 5 times with 5×SSCT, resuspend the magnetic beads with TE buffer, and add them to a 96-well plate.

[0082] The Synergy H1 multi-functional microplate reader (BioTek, USA) was used to read the fluorescence intensity of each well. Excitation / emission settings were configured as follows: excitation 485nm / emission 528nm (detection of the 3' UTR probe), and excitation 594nm / emission 650nm (detection of the CDS probe). Endpoint detection was used. A scatter plot was created using the 528nm fluorescence readings of calibrated single-stranded DNA standards against the 3' UTR lengths (0, 1000bp, 2000bp, 3000bp). A standard curve was then plotted to obtain the regression equation.

[0083] The 2×SSC buffer is an aqueous solution containing 3M sodium chloride and 0.3M sodium citrate. The 5×SSCT buffer is a 5×SSC buffer containing 0.1% Tween-20. The hybridization buffer is a 5×SSC buffer containing 30% formamide, 9mM citrate, 0.1% Tween-20, 50μg / ml heparin, 1×Denhardt's solution, and 1% dextran sulfate. The washing buffer is a 5×SSC buffer containing 30% formamide, 9mM citrate, 0.1% Tween-20, 50μg / ml heparin, and 1×Denhardt's solution. The amplification buffer is a 5×SSC buffer containing 0.1% Tween-20 and 2.5% dextran sulfate.

[0084] Figure 6 An embodiment is shown with the standard equation Y = 0.1956 × X - 11.44, R 2 =0.9662.

[0085] 3. Step S100

[0086] In one embodiment, the culture dish was placed on ice, HeLa cells were scraped off and collected in a 1.5 ml centrifuge tube, centrifuged to obtain a cell pellet; the cells were resuspended in lysis buffer, centrifuged again, and the supernatant was retained; oligodT magnetic beads (purchased from Thermo) were added to the cell lysis buffer, and the tube was placed on a rotary mixer and incubated at room temperature for 30 minutes; the tube was placed on a magnet to separate the magnetic beads from the supernatant, the supernatant was discarded, and the magnetic beads coupled with the mRNA to be tested were obtained.

[0087] 4. Steps S200 to S700

[0088] In some embodiments, steps S200 to S700 specifically include:

[0089] S200: Provides CDS probes as shown in SEQ ID NO:1 to 12 and 3'UTR probes as shown in SEQ ID NO:13 to 32, a first fluorescent probe labeled with Alexa594 and a second fluorescent probe labeled with Alexa488;

[0090] S300: The magnetic beads coupled with the mRNA to be tested are simultaneously subjected to a first mixed single-stranded hybridization reaction with the CDS probes shown in SEQ ID NO:1-12 and the 3'UTR probes shown in SEQ ID NO:13-32.

[0091] S400: The product of the first mixed single-strand hybridization reaction is simultaneously subjected to a second mixed single-strand hybridization reaction with the first fluorescent probe and the second fluorescent probe.

[0092] S500: Read the fluorescence information generated by the second mixed single-chain hybrid reaction based on the Alexa594 of the first fluorescent probe;

[0093] S600: Calculates the fluorescence value to be tested based on the fluorescence information to be tested;

[0094] S700: Input the fluorescence value to be tested into the standard equation to determine the length of the 3'UTR sequence.

[0095] The specific steps S200 to S700, including the first mixed single-strand hybridization reaction, the second mixed single-strand hybridization reaction, and the fluorescence value reading, are as described in the above embodiments.

[0096] In one embodiment, the 3'UTR length detection results of the NUDT21 mRNA in the sample to be tested are shown in Table 3. The 3'UTR length of the two extracted samples was detected, and their lengths were 1421bp and 1124bp, respectively.

[0097] Table 3

[0098]

[0099] The above are merely preferred embodiments of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application.

Claims

1. A method for quantitatively detecting the length of mRNA 3'UTR, comprising: Magnetic beads were prepared and coupled with a single-stranded nucleic acid to be tested, which contained the CDS sequence and 3'UTR sequence of the target gene. It provides a CDS probe that binds to the CDS region and a 3'UTR probe that binds to the 3'UTR region, as well as a first fluorescent probe that recognizes the CDS probe and a second fluorescent probe that recognizes the 3'UTR probe. The first fluorescent probe has a first fluorescent group, and the second fluorescent probe has a second fluorescent group that is different from the first fluorescent group. The magnetic beads coupled with the single-stranded nucleic acid to be tested were simultaneously subjected to the first mixed single-stranded hybridization reaction with the CDS probe and the 3'UTR probe. The product of the first mixed single-strand hybridization reaction is simultaneously subjected to a second mixed single-strand hybridization reaction with the first fluorescent probe and the second fluorescent probe. The first fluorescence information generated by the reaction of the product of the first mixed single-strand hybridization reaction with the first fluorescent probe is read according to the first fluorescent group, and the second fluorescence information generated by the reaction of the product of the first mixed single-strand hybridization reaction with the second fluorescent probe is read according to the second fluorescent group. The fluorescence value corresponding to the single-stranded nucleic acid to be tested is determined based on the first fluorescence information and the second fluorescence information; the first fluorescence information is the fluorescence value generated by the reaction of the product of the first mixed single-stranded hybridization reaction with the first fluorescent probe, and the second fluorescence information is the fluorescence value generated by the reaction of the product of the first mixed single-stranded hybridization reaction with the second fluorescent probe. The difference between the first fluorescence value and the second fluorescence value is taken as the fluorescence value corresponding to the single-stranded nucleic acid to be tested. Prepare multiple magnetic beads coupled with standard single-stranded nucleic acids, which contain the target gene CDS sequence or contain the target gene CDS sequence and 3'UTR sequences of different lengths; Each magnetic bead coupled with a single-stranded nucleic acid was simultaneously subjected to a first mixed single-stranded hybridization reaction with both a CDS probe and a 3'UTR probe. The product of the first mixed single-strand hybridization reaction is simultaneously subjected to a second mixed single-strand hybridization reaction with the first fluorescent probe and the second fluorescent probe. The first fluorescence information generated by the reaction of the product of the first mixed single-strand hybridization reaction with the first fluorescent probe is read according to the first fluorescent group, and the second fluorescence information generated by the reaction of the product of the first mixed single-strand hybridization reaction with the second fluorescent probe is read according to the second fluorescent group. Based on the first fluorescence information and the second fluorescence information, multiple standard fluorescence values ​​were determined corresponding to single-stranded nucleic acids without 3'UTR sequences and single-stranded nucleic acids with 3'UTR sequences of different lengths, respectively. The standard equation is determined based on multiple standard fluorescence values ​​and the length of the 3'UTR sequence in single-stranded nucleic acid. The standard equation characterizes the relationship between the standard fluorescence value and the length of the 3'UTR sequence in single-stranded nucleic acid. The length of the 3'UTR sequence was determined based on the standard equation and the fluorescence value to be measured.

2. The method according to claim 1, preparing a plurality of magnetic beads coupled with a standard single-stranded nucleic acid, wherein the standard single-stranded nucleic acid contains a target gene CDS sequence, or contains a target gene CDS sequence and 3' UTR sequences of different lengths, comprising: The first double-stranded DNA containing the target gene CDS sequence and the second double-stranded DNA containing the target gene CDS sequence and a 3'UTR sequence of a defined length were cloned into the basic plasmid to obtain the first vector and the second vector, respectively. Biotin-labeled forward and reverse primers were synthesized, and biotin-labeled first double-stranded DNA and biotin-labeled second double-stranded DNA were obtained by PCR amplification using the first and second vectors as templates, respectively. Biotin-labeled first double-stranded DNA and biotin-labeled second double-stranded DNA were incubated with magnetic beads containing avidin to obtain magnetic beads coupled with first double-stranded DNA and magnetic beads coupled with second double-stranded DNA, respectively. Magnetic beads coupled with first double-stranded DNA and magnetic beads coupled with second double-stranded DNA were treated with alkaline solution to obtain magnetic beads coupled with first single-stranded DNA and magnetic beads coupled with second single-stranded DNA, respectively.

3. The method according to claim 2, wherein the length of the 3' UTR sequence is selected from 10bp, 20bp, 30bp, 40bp, 50bp, 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 1000bp, 1200bp, 1300bp, 1400bp, 1500bp, 1600bp, 1800bp, 2000bp, 3000bp, and 4000bp.

4. The method according to claim 2, wherein there are multiple second double-stranded DNAs, and the length of the additional 3'UTR sequence of each second double-stranded DNA is in an arithmetic progression with that of the first double-stranded DNA.

5. The method according to claim 1, preparing magnetic beads coupled with a single-stranded nucleic acid to be tested, wherein the single-stranded nucleic acid to be tested comprises a target gene CDS sequence and a 3' UTR sequence, including: The single-stranded nucleic acid to be tested, containing the CDS sequence and 3'UTR sequence of the target gene, is cloned into a basic plasmid to obtain a cloning vector; Biotin-labeled forward and reverse primers were synthesized, and biotin-labeled double-stranded DNA was obtained by PCR amplification using a cloning vector as a template. Biotin-labeled double-stranded DNA to be tested was incubated with magnetic beads containing avidin to obtain magnetic beads conjugated with the double-stranded DNA to be tested. Magnetic beads coupled with the double-stranded DNA to be tested were treated with an alkaline solution to obtain magnetic beads coupled with the single-stranded DNA to be tested.

6. The method of claim 1, comprising the steps of providing a CDS probe that binds to the CDS region and a 3'UTR probe that binds to the 3'UTR region, and a first fluorescent probe that recognizes the CDS probe and a second fluorescent probe that recognizes the 3'UTR probe, wherein the first fluorescent probe has a first fluorescent group and the second fluorescent probe has a second fluorescent gene different from the first fluorescent group, The CDS probe has a nucleotide sequence complementary to a portion of the CDS sequence of the target gene, and the 3'UTR probe has a nucleotide sequence complementary to a portion of the 3'UTR sequence of the target gene.

7. The method according to claim 1, wherein the first fluorescent group is selected from FAM (carboxyfluorescein), TET (tetrachloro-6-carboxyfluorescein), JOE (2,7-dimethyl-4,5-dichloro-6-carboxyfluorescein), TAMRA (carboxytetramethylrhodamine), ROX (carboxy-X-rhodamine), Cy3, Cy5, HEX (hexachlorofluorescein), TexasRed, VIC, Alexa594, Alexa488, and the second fluorescent group is selected from FAM (carboxyfluorescein), TET (tetrachloro-6-carboxyfluorescein), JOE (2,7-dimethyl-4,5-dichloro-6-carboxyfluorescein), TAMRA (carboxytetramethylrhodamine), ROX (carboxy-X-rhodamine), Cy3, Cy5, HEX (hexachlorofluorescein), TexasRed, VIC, Alexa594, Alexa488.

8. A method for quantitatively detecting the length of mRNA 3'UTR, comprising: Prepare a human NUDT21 The first single-stranded DNA magnetic bead containing the gene CDS sequence, and three second single-stranded DNA magnetic beads containing SEQ ID NO:33~35; Provide a CDS probe as shown in SEQ ID NO:1~12 and a 3'UTR probe as shown in SEQ ID NO:13~32. Perform a first mixed single-stranded hybridization reaction with magnetic beads coupled with the first single-stranded DNA and the CDS probe and the 3'UTR probe simultaneously. Perform a first mixed single-stranded hybridization reaction with magnetic beads coupled with the second single-stranded DNA and the CDS probe and the 3'UTR probe simultaneously. Provides a first fluorescent probe labeled with Alexa594 and a second fluorescent probe labeled with Alexa488. The magnetic beads coupled with the first single-stranded DNA are reacted to obtain a first mixed single-stranded hybridization product, which is then reacted with the first and second fluorescent probes to perform a second mixed single-stranded hybridization reaction. The magnetic beads coupled with the second single-stranded DNA are reacted to obtain a first mixed single-stranded hybridization product, which is then reacted with the first and second fluorescent probes to perform a second mixed single-stranded hybridization reaction. The first fluorescence value generated by the magnetic bead reaction coupled with the first single-stranded DNA was read according to the Alexa594 of the first fluorescent probe, and the second fluorescence value generated by the magnetic bead reaction coupled with the second single-stranded DNA was read according to the Alexa488 of the second fluorescent probe. The difference between the second fluorescence value and the first fluorescence value of each magnetic bead coupled with the second single-stranded DNA was calculated to obtain three standard fluorescence values; The standard equation is determined based on three standard fluorescence values ​​and the length of the 3'UTR sequence in single-stranded nucleic acid. The standard equation characterizes the relationship between the standard fluorescence value and the length of the 3'UTR sequence in single-stranded nucleic acid. Preparation of magnetic beads coupled with target mRNA; The magnetic beads coupled with the mRNA to be tested were simultaneously subjected to a first mixed single-stranded hybridization reaction with the CDS probes shown in SEQ ID NO:1~12 and the 3'UTR probes shown in SEQ ID NO:13~32. The product of the first mixed single-strand hybridization reaction is simultaneously subjected to a second mixed single-strand hybridization reaction with the first fluorescent probe and the second fluorescent probe. The fluorescence information of the second mixed single-chain hybrid reaction is read based on the Alexa594 of the first fluorescent probe. The fluorescence value to be tested is calculated based on the fluorescence information to be tested; The fluorescence value to be tested is input into the standard equation to determine the length of the 3'UTR sequence.

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