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Building method of DNA large-fragment library for Pacbio platform and assay kit

A library construction and large fragment technology, which is applied to the construction of large DNA fragment libraries. The kit field of this library construction method can solve the problems of high cost of library construction and sequencing, and limited application range, so as to improve the purity and reduce the cost of library construction. Cost, flexible and convenient effect

Inactive Publication Date: 2018-03-13
BGI GENOMICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the Pacbio nucleic acid sequencing platform still has some deficiencies. For example, due to technical monopoly, the Pacbio nucleic acid sequencing platform has relatively high library construction and sequencing costs, resulting in a relatively limited application range.

Method used

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  • Building method of DNA large-fragment library for Pacbio platform and assay kit
  • Building method of DNA large-fragment library for Pacbio platform and assay kit
  • Building method of DNA large-fragment library for Pacbio platform and assay kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0048] 1. Main reagents and materials

[0049] 1. Reagents

[0050] The main reagents in this example include Agencourt AMPure XP Magnetic Bead Purification Kit purchased from BeckMan Company, ExoVII enzyme purchased from NEB Company, 5×Reaction Buffer, Damage Repair Mix, Damage Repair Buffer, dNTPs, 10×NAD + , PNK Buffer, DNA Polymerase, PNK, Klenow Fragment, T4DNA Ligase, Ligase Buffer, ExoIII, Cutsmart Buffer.

[0051] 2. Connector

[0052] In this example, a linker sequence is designed for the Pacbio platform, such as the sequence shown in Seq ID No.1,

[0053] Seq ID No. 1: 5'-atctctctcttttcctcctcctccgttgttgttgttgagagagatt-3'.

[0054] The linker was synthesized by Shanghai Sangong.

[0055] 3. DNA samples

[0056] In this example, two DNA samples were used for library construction, and the two DNA samples were derived from the commercial items pepper and tea tree respectively.

[0057] 2. DNA large fragment library construction

[0058] In this example, two DNA sa...

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Abstract

The application discloses a building method of DNA large-fragment library for Pacbio platform and an assay kit. The building method includes subjecting broken DNA large fragments to VII enzyme treatment, sequentially carrying out primary DNA damage repairing and terminal repairing treatment, linker splicing treatment, exonuclease ExoIII and ExoVII double-digest treatment, subjecting products obtained by double-digest treatment to fragment separation through nucleic acid electrophoresis and a fragment recovery system, subjecting products obtained by the fragment separation to secondary DNA damage repairing, so as to obtain the DNA large-fragment library adaptable to the Pacbio platform. According to the building method of the DNA large-fragment library, the whole building process is carriedout without Pacbio library-building assay kit, library building cost is lowered greatly, and a material foundation is laid for promotion and application of the third-generation sequencing and Pacbionucleic acid sequencing platform.

Description

technical field [0001] This application relates to the field of nucleic acid sequencing, in particular to a method for constructing a DNA large fragment library for the Pacbio platform, and a kit especially for the method for constructing a library. Background technique [0002] The Pacbio platform, that is, the Pacbio nucleic acid sequencing platform, uses the third-generation sequencing technology, that is, single molecule real-time sequencing (Single Molecule Real-Time). The sequencing of the Pacbio platform is based on two key technologies. First, a nanopore (zero-modewaveguides, abbreviated as ZMW) with a diameter of only tens of nanometers invented by Pacific Biosciences, in which only single molecule nucleic acid can be allowed to pass through the DNA. The reaction is carried out under the action of polymerase, and the four fluorescently labeled deoxynucleotides of A, T, C, and G in the synthesis process in the nanopore are detected to realize nucleic acid sequencing;...

Claims

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Application Information

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IPC IPC(8): C40B50/06C12Q1/6869
CPCC12Q1/6869C40B50/06C12Q2561/113C12Q2563/107C12Q2563/159
Inventor 马倩倩钟宏彬梁浩唐玉婧安莹
Owner BGI GENOMICS CO LTD
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