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Nucleic acid probe, and preparation method and application thereof

A technology of probes and DNA probes, applied in the fields of determining metagenomic sequence information, preparing probes, and preparing metagenomic sequencing libraries, which can solve the problems of large sequencing throughput, unavoidable host genome pollution, and cost

Inactive Publication Date: 2012-12-26
SHENZHEN HUADA GENE INST
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Problems solved by technology

[0003] However, it is difficult to avoid the contamination of the host genome during the extraction process of the samples studied by metagenomics. The proportion of host genome contamination in sample DNA reaches 80% or even more than 90%
The existence of a large number of host exogenous contamination in the sample makes the sequencing cost of effective data relatively greatly increased
The second generation of high-throughput sequencing makes it possible to study various components in environmental samples from the perspective of the whole genome, but it takes a huge amount of sequencing throughput to achieve the expected sequencing effect for the sample DNA with high contamination of the host sample
[0004] Therefore, current metagenomic research methods still need to be improved

Method used

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  • Nucleic acid probe, and preparation method and application thereof
  • Nucleic acid probe, and preparation method and application thereof
  • Nucleic acid probe, and preparation method and application thereof

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preparation example Construction

[0055] 2. Preparation of host genome-wide probe sequences

[0056] The preparation method of the whole genome probe sequence of the host mainly comprises the following steps:

[0057] (1) Using pure genomic DNA as a material, a biotin-labeled paired-end library was prepared by a high-throughput library construction method;

[0058] (2) Perform quality control and quantification of the prepared biotin-labeled paired-end library. The sequence obtained here can be directly used as a double-stranded probe for subsequent hybridization reactions, or a single-stranded probe can be prepared in the next step for subsequent hybridization reactions;

[0059] (3) Take a certain amount of biotin paired-end library, heat denature, ice bath, and combine with streptavidin-coated magnetic beads to prepare single-stranded probes;

[0060] More specifically:

[0061] (1) Take a certain amount of pure and high-quality exogenous host genomic DNA, and use Covaris S2 to interrupt and purify it. A...

Embodiment 1

[0089] Example 1: Analysis of Human Genome Sequence Contamination Removal from Metagenomic DNA of Human Oral Saliva Samples

[0090] Oral saliva samples were provided by Shenzhen Second People's Hospital, and metagenomic DNA was obtained by lysozyme digestion and phenol and chloroform extraction methods.

[0091] Take 1 μg of metagenomic DNA and use Covaris to break into the main band of 250bp. After purification, the DNA is repaired and purified by reagent I, mixed with reagent II and incubated to add "A" base at the 3' end, purified and then mixed with reagent III for incubation Add the Illumina adapter sequence, after purification, gel separation, cut out 300-400bp molecules and recover them, and take 5ng of the intermediate ligation products for subsequent hybridization operations.

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Abstract

The invention provides a probe, and a preparation method and an application thereof, wherein the method for preparing the probe comprises the steps of carrying out fragmentation on a DNA sample to obtain a first DNA fragment; blunting the first DNA fragment to obtain a blunted first DNA fragment; connecting the blunted first DNA fragment with a linker to obtain the first DNA fragment connected with the linker; carrying out amplification on the first DNA fragment connected with the linker by using PCR primers to obtain a first amplification product, wherein the PCR primers comprise a first PCR primer and a second PCR primer; the 5'-terminal bases of both the first PCR primer and the second PCR primer are marked by biotin; and the first amplification product forms a double-chain DNA probe. The probe obtained by the method can be effectively used for reducing pollutions of host genome DNA in a metagenome construction library.

Description

technical field [0001] The present invention relates to the field of biotechnology. Specifically, the present invention relates to nucleic acid probes and their preparation methods and applications. More specifically, the present invention relates to methods for preparing probes, nucleic acid probes, and the use of nucleic acid probes in preparing sequencing libraries. , A method for preparing a metagenomic sequencing library, a metagenomic sequencing library and a method for determining information about a metagenomic sequence. Background technique [0002] At present, metagenomics has become an emerging and important scientific research field in genomics. It is a discipline that studies the direct extraction of genomic genetic material from environmental samples such as human gastrointestinal tract and soil, and performs related analysis. With the rapid development of metagenomics research, large-scale metagenomics research has been carried out one after another. A large n...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12N15/11C12Q1/68C40B50/06C40B40/06
Inventor 耿春雨韩鸿雁卢志远章文蔚祝珍珍
Owner SHENZHEN HUADA GENE INST
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