Rapid single-stranded library building method

A single-chain and fast technology, applied in chemical library, library creation, combinatorial chemistry, etc., can solve the problems of cumbersome and low-efficiency single-chain library construction technology

Pending Publication Date: 2020-02-14
深圳易倍科华生物科技有限公司
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Problems solved by technology

[0005] The purpose of the present invention is to provide a fast single-chain library building method, aiming to solve th

Method used

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  • Rapid single-stranded library building method

Examples

Experimental program
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Embodiment 1

[0060] This embodiment builds a library for 0.5ng cfDNA, including the following steps:

[0061] (1) Add 0.5ng of cfDNA standard (Horizon HD 780) to the PCR reaction tube, treat with PNK, react for 15 minutes, heat to 95°C, insert it on ice immediately, and let it stand for 3-5 minutes to obtain a single-stranded template.

[0062] (2) Carry out 3' end adapter ligation: add 3 μL 600U / μL T4 DNA ligase in the same reaction tube, add T4 DNA ligase buffer, add A3 adapter adapter (final concentration: 10 μM) to supplement H 2 O, react at 20°C for 15min.

[0063] (3) Then add AMPure XP (Beckman Coulter) magnetic beads or equivalent purified magnetic beads to the same reaction tube at a ratio of 1:2 (volume ratio) for purification. After washing with 80% ethanol, air dry and add 13 μL deionized water directly, and react at 95°C for 5 minutes. Immediately after the completion of the reaction, insert the DNA mixture containing magnetic beads into ice and incubate for 5 minutes.

[0...

Embodiment 2

[0081] In this embodiment, the methylation library construction for 1ng lambda DNA includes the following steps:

[0082] (1) After 10ng lambda DNA (λDNA) was treated with EZ DNA methylation Gold Kit, 10μL H 2 O was dissolved, and 1 μL was used for library construction. It was first treated with PNK, heated to 95°C after reacting for 15 minutes, immediately inserted into ice, and left to stand for 3-5 minutes to obtain a single-stranded template.

[0083] (2) Carry out 3' end adapter ligation: add 3 μL 600U / μL T4 DNA ligase in the same reaction tube, add T4 DNA ligase buffer, add A3 adapter adapter (final concentration: 10 μM) to supplement H 2 O, react at 20°C for 15 minutes.

[0084] (3) Then add AMPure XP (Beckman Coulter) magnetic beads or equivalent purified magnetic beads to the same reaction tube at a ratio of 1:2 (volume ratio) for purification. After washing with 80% ethanol, air dry and add 13 μL deionized water directly, and react at 95°C for 5 minutes. Immediat...

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Abstract

The invention belongs to the technical field of gene sequencing libraries, and particularly relates to a rapid single-stranded library building method which comprises the following steps: providing sample DNA, and performing phosphorylation treatment on the sample DNA to obtain a single-stranded DNA template; connecting the 3' end of the single-stranded DNA template with a first joint, and then performing purification with a first magnetic bead to obtain a first connection product containing the first magnetic bead; connecting the 5' end of the first connection product with a second joint, andperforming purification with a second magnetic bead to obtain a second connection product containing the first magnetic bead and the second magnetic bead; performing PCR amplification on the second connection product by using universal primers to obtain a library; wherein the first joint and the second joint are double-stranded joints. According to the rapid single-stranded DNA library building method, the library building efficiency is improved, the library building time is saved, and library building of the single-stranded DNA can be achieved through the method so as to meet the requirements of a second-generation sequencing platform and even a third-generation sequencing platform.

Description

technical field [0001] The invention belongs to the technical field of gene sequencing libraries, and in particular relates to a rapid single-strand library construction method. Background technique [0002] The current second-generation and third-generation gene sequencing technologies require library construction of template DNA strands. Building a library is to add a fixed sequence to both ends of the template DNA chain. The fixed sequence at both ends is called an adapter, and the DNA containing the adapter and template strand is called a library. Due to the adapter portion, the library can be captured, identified and sequenced by a sequencer. Therefore, adding adapters is a necessary and core operation for library construction. [0003] Common library construction is usually performed on double-stranded DNA. It usually includes the following steps: 1. Add A for end repair, 2. Ligate a part of the adapter or the full length of the adapter, 3. PCR amplify the introduct...

Claims

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Application Information

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IPC IPC(8): C40B50/06
CPCC40B50/06
Inventor 徐飞岳
Owner 深圳易倍科华生物科技有限公司
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