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Method for designing and connecting amplification primers of DNA (Deoxyribose Nucleic Acid) molecules

A DNA molecule and primer pair technology, applied in the field of biological sequencing, can solve the problem of not being able to make full use of the enzyme read length advantage of the Pacbio platform, and achieve the effect of improving data utilization and ensuring data quality.

Pending Publication Date: 2022-07-29
BGI TECH SOLUTIONS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is to provide a DNA molecule amplification primer design and connection method to overcome the defect that the prior art for the existing amplification product library construction method cannot make full use of the advantages of the Pacbio platform enzyme read length, A scheme of end-to-end library construction is designed, and the PCR products are connected end-to-end through sticky ends, thereby increasing the average length of the library, so that more effective data can be obtained after sequencing

Method used

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  • Method for designing and connecting amplification primers of DNA (Deoxyribose Nucleic Acid) molecules
  • Method for designing and connecting amplification primers of DNA (Deoxyribose Nucleic Acid) molecules
  • Method for designing and connecting amplification primers of DNA (Deoxyribose Nucleic Acid) molecules

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Embodiment 1

[0033] Embodiment 1 uses the linking library building technology of the present invention to build a library

[0034] 1. PCR amplification

[0035] 1.1 Two-strand synthesis

[0036] Take 20ng of the PCR product captured by TCR / BCR and prepare the following PCR system:

[0037] Reagent name Dosage Captured PCR product 20ng KAPA HiFi HotStart Uracil+ReadyMix(2×) 50μl Nuclease-free water (NF water) Make up 94μl

[0038] Divide the PCR system into 4 tubes, add the following 4 pairs of primers to each tube, and add 0.75 μl to each primer (10mM):

[0039]

[0040] The PCR reaction program is as follows:

[0041]

[0042] 1.2 Sample purification

[0043] 1) 4 tubes of PCR products were quantified by Qubit and mixed with equal amounts.

[0044] 2) Take 1 volume of AMPure XP magnetic beads and add it to the 1.5mL centrifuge tube containing the PCR mixture, mix well and detach. Incubate for 5 min at room temperature.

[0045] 3) The cent...

Embodiment 2

[0081] Embodiment 2 uses conventional library building technology to carry out library building

[0082] 1. PCR amplification

[0083] Take 20ng of the PCR product captured by TCR / BCR and prepare the following PCR system:

[0084] Reagent name Dosage Captured PCR product 20ng KAPA HiFi HotStart ReadyMix (2×) 50μL Forward primer (10mM) 3μL Reverse primer (10mM) 3μL Nuclease-free water (NF water) Make up 100 μL

[0085] The primer information is as follows:

[0086] primer name Primer sequence 5'-3' forward primer PHO-CTACACGACGCTCTTCCGATCT reverse primer PHO-AAGCAGTGGTATCAACGCAGAG

[0087] The PCR reaction program is as follows:

[0088]

[0089] Qubit dsDNA HS Assay kit detects the concentration of PCR products, and the concentration should be greater than 20ng / μL. 0.8 volumes (80 μL) of AMPure XP magnetic beads were added to the tube for purification, and finally redissolved in 27 μL of el...

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Abstract

The invention discloses a method for designing and connecting amplification primers of DNA (deoxyribonucleic acid) molecules, and particularly relates to a library building method for increasing the effective data volume of Pacbio amplicon sequencing. Comprising the following steps: designing different primer pairs aiming at DNA molecules, and carrying out PCR amplification on the template DNA in different amplification systems by using the different primer pairs respectively. According to the method, short PCR products are connected to obtain a long library, and the connected library is used for sequencing on a machine, so that the data utilization rate can be greatly improved while the data quality is ensured.

Description

technical field [0001] The invention belongs to the field of biological sequencing, in particular to a method for designing and connecting amplification primers of DNA molecules, in particular to a method for building a library for increasing the amount of sequencing data of Pacbio amplicon. Background technique [0002] In some sequencing applications, library construction requires amplification, and the insert fragment is long, and long-read sequencing is required, such as full-length 16S rDNA sequencing, 18S rDNA sequencing, ITS sequencing, target gene sequencing, full-length transcription DNA sequencing and targeted full-length transcriptome sequencing, etc. In the Pacbio long-read sequencing platform, its sequencing reagents have been developed in several versions, and the enzymatic read length has been increased to more than 60k. A library molecule can be read, causing the library molecule formed by the amplification product to be read repeatedly in the sequencing well...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/10C40B50/06C12Q1/6869
CPCC12Q1/686C12Q1/6869C40B50/06C12Q1/6806
Inventor 陈智超唐冲阮凤英李娅宁郭梅
Owner BGI TECH SOLUTIONS
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