Hairpin-like adapter, construct and method for characterizing double-stranded target polynucleotide

A target polynucleotide, double-stranded nucleic acid technology, applied in the field of biological analysis and detection, can solve the problems of increasing the complexity of library preparation, increasing the complexity of preparation process, reducing the efficiency of library preparation, etc., and achieves high-reliability orthogonal correction reading ability, improved sequencing accuracy, and low synthesis difficulty

Active Publication Date: 2021-10-01
QITAN TECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method requires chemical modification of the nanopore, which increases the complexity of the preparation process; when constructing the library, it is necessary to connect two sets of adapters and perform two purifications respectively, which increases the complexity of library preparation and reduces the cost of library preparation. s efficiency
[0007] Although the prior art discloses a method for simultaneously sequencing the template strand and the complementary strand of dsDNA, each method has its own defects, so it is still necessary to develop a better method to realize simultaneous real-time sequencing of the template strand and complementary strand of dsDNA. Serial sequencing to balance the translocation rate of both strands of a double-stranded target polynucleotide and improve sequencing accuracy

Method used

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  • Hairpin-like adapter, construct and method for characterizing double-stranded target polynucleotide
  • Hairpin-like adapter, construct and method for characterizing double-stranded target polynucleotide
  • Hairpin-like adapter, construct and method for characterizing double-stranded target polynucleotide

Examples

Experimental program
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Effect test

Embodiment 1

[0208] Example 1: Preparation of hairpin-like adapters

[0209] (1) 3'-5'-5'-3'( figure 1 From right to left in ) the synthesis of the second single strand

[0210] Will 3'-5' ( figure 1 From right to left in the first segment (SEQ ID NO: 2, 5' end has Azide (marked with N3) modification) and 5'-3' ( figure 1 From right to left in ) the second segment (SEQ ID NO:3, 5' end is modified with DBCO (marked by DB)) in a solution of 10mM Tris, 50mM NaCl, 0.1mM EDTA at a ratio of 3:1, react A click chemical reaction occurred at a temperature of 30° C. for 12 hours, and the obtained product was purified by PAGE to obtain a 3′-5′-5′-3′ second single chain.

[0211] (2) Synthesis of DNA containing a hairpin-like structure

[0212] The 5'-3' first single strand (SEQ ID NO: 1) and the 3'-5'-5'-3' second single strand formed in step (1) were lowered from 95°C by 0.1°C every 10s. Annealed at 4°C to form a hairpin-like structure DNA, such as figure 1 shown.

[0213] (3) Preparation of ...

Embodiment 2

[0226] Example 2: Preparation of constructs

[0227] (1) Preparation of Y adapter

[0228] Y-Top-1 (SEQ ID NO:7), Y-Top-2 (SEQ ID NO:8) and Y-Bottom (SEQ ID NO:9) were annealed under the condition of decreasing 0.1°C every 10s from 95°C to 4°C to form the Y adapter.

[0229] (2) Preparation of enzyme-loaded Y adapter

[0230] The T4 Dda helicase was loaded onto the Y adapter by the method of steps (2)-(3) of Example 1.

[0231] (3) Preparation of constructs

[0232] An approximately 2700 base pair segment of plasmid DNA (SEQ ID NO: 6) was amplified using PCR primers containing restriction sites as defined below for the preparation of constructs containing the double-stranded target polynucleotide sequence.

[0233] BsaI forward primer: 5'-TCGCC ATTCA GGCTG CGC-3' (SEQ ID NO: 11)

[0234] BsaI reverse primer: 5'-GCTTA GAGAC CTGTG CGG-3' (SEQ ID NO: 12)

[0235] The fragment of about 2700bp was successively digested with a dA tail (NEB Cat#E7442) and BsaI restriction endon...

Embodiment 3

[0242] Example 3: Methods for Sequencing Double-Stranded Target Polynucleotides of Constructs Containing Hairpin-Like Adapters and Not Containing Hairpin-Like Adapters, respectively

[0243] Materials and methods:

[0244] Melt sequencing buffer (20mM HEPES pH 8.0, 50mM ATP, 50 mM MgCl 2 , 500 mMKCl), the first tether (SEQ ID NO:4, located at Figure 6 D) and the second tether (SEQ ID NO: 10 in image 3 FLT_1D on the left). Add 4ul of the above-mentioned tether (1uM) to 196ul of the sequencing buffer and mix well, then add it to the QNome-9604 sequencer of Qitan Technology and incubate for 20 minutes. The structure of the sequencer QNome-9604 is as follows Figure 12 As shown, it includes fluidic chip, signal processing circuit and sequencing software. Then, prepare the loading library, and the components are shown in Table 1 and Table 2.

[0245] Table 1: Preparation of Construct Sequencing Libraries Containing Hairpin-Like Adapters

[0246]

[0247] Table 2: Prepar...

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Abstract

The present invention provides a hairpin-like adaptor, a construct and a method for characterizing a double-stranded target polynucleotide. The hairpin-like adaptor comprises a first single strand and a second single strand which are respectively connected with a template strand and a complementary strand of the double-strand target polynucleotide, and the first single strand and the second single strand form at least one double-strand nucleic acid region which is used for enabling the complementary strand to be close to a transmembrane pore after the template strand passes through the transmembrane pore. Also provided are a construct comprising the hairpin-like adapter, as well as a method, a kit, and the like for characterizing double-stranded target polynucleotides. The characterization method disclosed by the invention has many advantages, such as balancing the translocation speed of the template strand and the complementary strand of the target polynucleotide, improving the sequencing accuracy and the like.

Description

technical field [0001] The application belongs to the technical field of biological analysis and detection, and specifically relates to a hairpin-like adapter, a construct and a method for characterizing double-stranded target nucleotides such as sequencing using transmembrane pores. Background technique [0002] At present, nucleic acid sequencing technology has application requirements in many scenarios. Existing sequencing technologies require complex processing of samples to be sequenced in the early stage, and the sequencing cycle is long, which cannot meet the needs of rapid and convenient nucleic acid sequencing technologies in clinical and other application scenarios. [0003] Transmembrane pores such as nanopores have great potential as biosensors for the development of new nucleic acid sequencing technologies. When a voltage is applied to both sides of the nanopore, the current will drop when the analyte (such as nucleotide, polypeptide) passes through the nanopor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869C12N15/11
CPCC12Q1/6869C12Q2565/631C12Q2525/301C12Q2565/607C12Q2521/513C12Q2521/319
Inventor 章益苗卉孙继国张周刚卓远
Owner QITAN TECH LTD
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