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Reverse transcription obstruction probe for rapidly removing target RNA in RNA library construction and application thereof

A reverse transcription and probe technology, applied in the direction of DNA/RNA fragment, recombinant DNA technology, microbial determination/inspection, etc., can solve the problems that hinder the development of RNA next-generation sequencing technology, industrial automation development application, sample source and RNA abundance High requirements, difficult storage of reagents, etc., to achieve low cost, short time-consuming, and complete effects

Active Publication Date: 2021-04-09
YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

These methods have defects such as high cost, complicated operation (about 20-40 steps), long time-consuming (about 2 hours), high requirements on sample source and RNA abundance, and difficult storage of reagents, which seriously hinder the development of RNA next-generation sequencing library construction technology. The development of industrial automation and its application in the field of rapid diagnosis of diseases

Method used

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  • Reverse transcription obstruction probe for rapidly removing target RNA in RNA library construction and application thereof
  • Reverse transcription obstruction probe for rapidly removing target RNA in RNA library construction and application thereof
  • Reverse transcription obstruction probe for rapidly removing target RNA in RNA library construction and application thereof

Examples

Experimental program
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Embodiment 1

[0041] The features and advantages of the present invention can be further understood through the following detailed description in conjunction with the accompanying drawings. The examples provided are only illustrative of the method of the present invention and do not limit the rest of the present disclosure in any way. The sequences and modifications of the probes and primers used in this example are shown in Table 1. N is a random base, that is, any base in A, T, C, and G, and RandomHexamers is a universal primer for reverse transcription.

[0042] figure 1 and figure 2 The reaction mechanism of the present invention is demonstrated: the reverse transcription hindering probe (such as LNA, PNA, etc., and the 3' end of the probe is blocked by NH2C6) is modified with a base with strong binding ability, so that the reverse transcription hindering probe can preferentially lock the target RNA, preventing reverse transcriptase from blocking the target RNA site locked by the pro...

specific Embodiment approach

[0050] refer to figure 2 In this example, 5.8S-specific probes with different modifications (No. 1-5 in Table 1) were designed to inhibit the reverse transcription process of 5.8S rRNA. The Total RNA used in the examples was extracted from cultured Human293FT cells (GM-070006H, purchased from Jimon Biotechnology (Shanghai) Co., Ltd.) according to conventional methods, the same below. The specific implementation is as follows:

[0051] Table 2

[0052] components Dosage Total RNA 1 μg 1 μM 5.8S modified probe (any one of No. 1-5 in Table 1) 1 μL 1 μM 5.8S rRNA RT primer 1 μL 10 mM dNTPs mix 1 μL Supplement DEPC H2O to 13 μL

[0053] Mix and spin away. 85 °C for 5 min, then immediately place on ice for 5 min.

[0054] table 3

[0055] components Dosage 5X Reverse transcriptase buffer 4 μL Murine RNase Inhibitor (40 U / µL, Yeasen, 10603ES05) 2 μL Hifair® V Reverse Transcriptase (200 U / μL, Yeasen,...

Embodiment 2

[0061] Example 2: The inhibitory effect of the distribution position of LNA in the probe on the reverse transcription of target RNA

[0062] In this example, 5.8S-specific probes (numbers 6-8 in Table 1) of different LNA modification positions were designed to inhibit the reverse transcription process of 5.8SrRNA. With reference to example 1 for the specific embodiment, the quantitative results are shown in Figure 4 .

[0063] It can be seen from the quantitative results that when the position of the LNA in the probe is close to the left end of the probe (ie, the 5' end), the effect of hindering the reverse transcription of the target RNA is higher than that when the position of the LNA in the probe is close to the right end of the probe (ie, the 5' end of the probe). 3' end).

[0064] Table 5: Inhibition efficiency of target RNA reverse transcription by LNA distribution position in the probe

[0065] Distribution of LNA modification positions in probes Left ...

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Abstract

The invention provides a reverse transcription obstruction probe for rapidly removing target RNA in RNA library construction and application thereof. The probe has a length of 20-40 nt, is strictly complementarily paired with a target RNA sequence, and has no complementary pairing continuously exceeding 15 nt with other non-target RNAs, the Tm value of the probe is not lower than 80 DEG C, the self complementary value is less than 5, and 3'-OH of the probe is sealed by modified deoxyribonucleotide. According to the probe, a reverse transcription inhibition probe method is utilized to realize high-efficiency and high-specificity inhibition on reverse transcription of the target RNA during synthesis of one strand of cDNA, so that the target RNA is prevented from participating in a downstream library construction process. The probe has the advantages of simplicity in operation (one-step operation), extremely short consumed time (2 minutes), completeness in retention of the non-target RNAs, high specificity, high efficiency, low cost, high gene detection number and the like, and is very suitable for the fields of industrial automatic library building and rapid disease diagnosis.

Description

technical field [0001] The patent of the invention relates to a reverse transcription hindering probe used for rapidly removing target RNA in RNA library construction and its application, belonging to the field of biotechnology. Background technique [0002] In recent years, with the rapid development of next-generation sequencing technology, various RNA library construction and sequencing technologies have emerged and been iteratively upgraded, greatly reducing the difficulty and cost of RNA library construction and sequencing, which also makes RNA next-generation sequencing technology a An important research tool in the fields of life progression and disease diagnosis. However, the proportion of RNA species in samples from different sources is extremely heterogeneous. For example, ribosomal rRNA accounts for about 90%-95% of total RNA in normal cells, and globulin mRNA accounts for about 76% of total mRNA in blood. % or more, the presence of these RNAs occupies most of th...

Claims

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Application Information

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IPC IPC(8): C12Q1/6806C12N15/11
CPCC12Q1/6806C12Q2525/121C12Q2537/163C12Q2521/107
Inventor 江翱卢瑶曹振宋东亮
Owner YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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