CN113622033A - Preparation method and application of nucleic acid library for low host background interference - Google Patents

Preparation method and application of nucleic acid library for low host background interference Download PDF

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CN113622033A
CN113622033A CN202110901929.1A CN202110901929A CN113622033A CN 113622033 A CN113622033 A CN 113622033A CN 202110901929 A CN202110901929 A CN 202110901929A CN 113622033 A CN113622033 A CN 113622033A
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nucleic acid
probe
target
sequencing
cases
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涂波
丁泽琴
孙梓健
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Chengdu Baiwei Biological Technology Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing

Abstract

The invention relates to the technical field of amplification of target genes of animals, plants and microorganisms, in particular to a preparation method and application of a nucleic acid library for low host background interference; the technology of nucleic acid target sequencing analysis can be improved, and accurate target sequencing, multiple sequencing and high-throughput sequencing can be provided; the method can be used for sequencing low copy target genes of nucleic acid, and reducing the interference of host abundant nucleic acid; can be used for the target sequencing of nucleic acid target genes; can be used for removing the target gene of nucleic acid for sequencing and improving the detection accuracy.

Description

Preparation method and application of nucleic acid library for low host background interference
Technical Field
The invention relates to the technical field of amplification of target genes of animals, plants and microorganisms, in particular to a preparation method and application of a nucleic acid library for low host background interference.
Background
In the current technologies related to biological research, the nucleic acid sequencing technology is widely applied from Sanger sequencing to the third generation sequencing which mainly uses the second generation sequencing technology of Illumina sequencing platform and single molecule sequencing. Although the sequencing accuracy and precision of different sequencing platforms are improved with the continuous development of sequencing technologies, the problem that the contamination caused by mixing a large amount of nucleic acid sequences homologous or non-homologous to the target gene in a complex sample is difficult to eliminate is only not eliminated from the alternation of the sequencing platforms. This problem can be overcome by methods such as algorithm optimization in single species sequencing, but inevitable contamination is brought about for multi-species mixed sequencing (e.g., metagenomic sequencing).
In the current method for sequencing a target gene of a complex sample, strategies such as nucleic acid purification, polymerase chain reaction, sequencing data later removal and the like are improved in various experimental practices, but the defects that the deviation of sample nucleic acid extraction is caused in the use of a nucleic acid purification kit, a part of nucleic acid fragments cannot be amplified or the deviation of the amplification result and the actual abundance is caused by using a special primer in the polymerase chain reaction, the target gene is covered by a high-abundance homologous sequence and the like are inevitable. These disadvantages finally result in that the nucleic acid sequence with high homology with the target sequence cannot be completely removed, the sequence information and abundance of the target sequence cannot be completely and truly obtained, and the detection accuracy is reduced.
Disclosure of Invention
The invention aims to provide a preparation method and application of a nucleic acid library for low host background interference, which are used for removing high-abundance non-target sequences in a nucleic acid sample, simultaneously improving that low-copy target gene sequences cannot be interfered by the high-abundance non-homologous nucleic acid sequences in a sequencing process, obtaining a real and reliable result on any sequencing platform and improving the detection accuracy.
To achieve the above object, in a first aspect, the present invention provides a method for preparing a nucleic acid library with low host background interference, comprising:
designing a target sequence probe primer, and obtaining a nucleic acid template by using polymerase so as to obtain a probe;
the probe forms a DNA-RNA complex by hybridizing with the target sequence;
cleaving the hybrid using a non-pairing endonuclease and a single-strand specific nuclease;
the amplicon population obtained using the target sequence universal primers was used for sequencing library construction. .
In one embodiment, the target sequence probe primer is one of a single oligonucleotide, a series of oligonucleotides or a modified oligonucleotide.
In one embodiment, designing a target sequence probe primer, obtaining a nucleic acid template using a polymerase, and obtaining a probe comprises:
amplicon amplification of the targeted or non-targeted sequence using the amplification primers and the nucleic acid template produces one or more amplicons.
In one embodiment, designing a target sequence probe primer, obtaining a nucleic acid template using a polymerase, and further obtaining a probe, further comprises:
DNA templates are used to generate amplicons that target genes, which are DNA, RNA, or DNA/RNA hybridization probes.
In one embodiment, the probe forms a DNA-RNA complex by hybridizing to the target sequence by:
the probe and the target nucleic acid are hybridized through an annealing mode, the probe and the target nucleic acid are hybridized in a buffer solution, the probe and the target nucleic acid are hybridized under ultraviolet treatment, the probe is tethered on a solid or semisolid support and is hybridized with the nucleic acid, and the probe is hybridized with the nucleic acid tethered on the solid or semisolid support.
In one embodiment, the unpaired endonuclease is a T7 endonuclease.
In one embodiment, the single strand specific nuclease is S1 nuclease.
In a second aspect, the invention also provides the use of a library of nucleic acids for high throughput sequencing of the library of nucleic acids with low host background interference.
The preparation method and the application of the nucleic acid library for low host background interference can improve the nucleic acid target sequencing analysis technology and can provide accurate target sequencing, multiple sequencing and high-throughput sequencing; the method can be used for sequencing low copy target genes of nucleic acid, and reducing the interference of host abundant nucleic acid; can be used for the target sequencing of nucleic acid target genes; can be used for removing the target gene of nucleic acid for sequencing and improving the detection accuracy.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic flow diagram of a method for preparing a nucleic acid library with low host background interference according to the present invention.
FIG. 2 is a graph showing the results of example 4.
FIG. 3 is a diagram illustrating the diversity of community structures provided by the present invention.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
Referring to FIG. 1, FIG. 1 is a schematic flow chart of a method for preparing a nucleic acid library with low host background interference according to an embodiment of the present invention. Specifically, the method for preparing the nucleic acid library for low host background interference can comprise the following steps:
s101, designing a target sequence probe primer, and obtaining a nucleic acid template by using polymerase so as to obtain a probe;
in the present example, amplification primers were designed for the target sequence to be sequenced. The primers may be used for amplification of one or more amplicons. The primer comprises the following components: can be a single oligonucleotide, or a series of oligonucleotides, or a modified oligonucleotide. The use of nucleic acids as templates for probe origin for non-targeted or targeted gene elimination can be: linear DNA fragments, linear RNA fragments, nucleic acid fragments subcloned into circular vectors, or nucleic acid sequences in nucleic acid libraries. In some cases, the template is derived from the fragmentation product of DNA, RNA, cDNA, or genomic DNA. In some cases, the template DNA is fragmented by sonication, enzymatic digestion, heat treatment, repeated pipetting, mechanical shaking and nebulization. The target sequence probe primer is one of single oligonucleotide, a series of oligonucleotides or modified oligonucleotides. Performing amplicon amplification on the targeted or non-targeted sequence using the amplification primers and the nucleic acid template; generating one or more amplicons, wherein the amplicons are generated by: performing primer extension reaction by hybridizing a primer related to a target gene with a nucleic acid, and ligating an extension reaction product of the primer with a specific nucleic acid sequence to obtain an amplicon; one or more amplicons are ligated to the identifier sequence for generating a non-targeted gene binding probe. In some embodiments, the nucleic acid fragment serving as a probe template is generated to contain one or more targeted gene sequences. In some embodiments, the plurality of gene-targeting nucleic acid fragments is generated by a gene synthesizer. In some embodiments, the plurality of gene-targeting nucleic acid fragments are generated by restriction endonuclease cleavage followed by ligation using a nucleic acid ligase. In some embodiments, the plurality of gene-targeting nucleic acid fragments are produced by a homologous recombinase. In some embodiments, the sequence of the targeted gene comprises the nucleic acid sequence of the entire target gene. In some embodiments, the sequence of the targeted gene comprises a portion of the nucleic acid sequence of the target gene. In some embodiments, amplification of the amplicon can be accomplished by linear amplification, non-linear amplification, isothermal amplification, or rolling circle amplification. In some embodiments, at least one amplicon is ligated to at least one different amplicon. In some cases, the amplicon comprises a molecular barcode, a nucleic acid sequence, a nucleic acid other than A, T, G or C. In some cases, the identifier sequence is located at the 5' end of the amplicon. In some cases at the 3' end of the amplicon. In some embodiments, the amplicon used to generate the targeted gene generates a probe. In some cases, the probe is an oligonucleotide probe. In some cases, the probe is a DNA, RNA, or DNA/RNA hybridization probe. In some cases, the probe is one or more of a linear probe, a non-linear probe, or a stem-loop structure probe. In some cases, the probe comprises an oligonucleotide-like derivative. In some cases, the probe comprises a nucleotide modifying molecule. In some cases, the probe comprises a degenerate sequence. In some cases, the probe comprises a primer sequence. In some cases, the probe comprises a non-primer sequence. In some cases, the probe is generated by a DNA polymerase or an RNA polymerase, the probe comprising modified nucleotides or deoxynucleotides. In some cases, the probe is produced by an exonuclease. In some cases, the probe is generated by an endonuclease or a restriction endonuclease. In some cases, the probes are generated by a gene synthesizer.
S102, hybridizing the probe with a target sequence to form a DNA-RNA complex;
in embodiments of the invention, the target of the generated probe is one or more targeted genes. In some embodiments, the probe generated contains one or more nucleic acid fragments targeting a gene sequence. In some embodiments, the plurality of gene-targeting nucleic acid fragments is generated by a gene synthesizer. In some embodiments, the plurality of gene-targeting nucleic acid fragments are generated by restriction endonuclease cleavage followed by ligation using a nucleic acid ligase. In some embodiments, the target nucleic acid to which the probe binds is single-stranded DNA, double-stranded DNA, RNA, cDNA, and genomic DNA. In some cases, the nucleic acid is fragmented by: ultrasonic treatment, enzyme digestion, heating treatment, repeated liquid transfer treatment, ultraviolet treatment, mechanical oscillation treatment and atomization treatment.
In some embodiments, the probe hybridizes to a target nucleic acid. In some cases, the probe hybridizes to the target nucleic acid by annealing. In some cases, the probe hybridizes to the target nucleic acid in a buffer. In some cases, the probe hybridizes to the target nucleic acid under ultraviolet light treatment. In some cases, the probes are tethered to a solid or semi-solid support for hybridization to nucleic acids. In some cases, the probe hybridizes to a nucleic acid tethered to a solid or semi-solid support.
In some embodiments, the hybrid is removed after hybridization of the probe to the target nucleic acid. In some cases, the hybrids are spatially separated and removed by the modified magnetic beads. In some cases, enzymatic removal is performed by nuclease. In some cases, removal is by means of heat or mechanical agitation.
In some embodiments, nucleic acids not bound to the probe are isolated, enriched and purified. In some cases, the nucleic acid is isolated and purified by placing it in an agarose gel. In some cases, the nucleic acid adsorbent comprises: hydroxyl magnetic beads (magnetic microspheres), carboxyl magnetic beads (magnetic microspheres), aminated magnetic beads (magnetic microspheres), nucleic acid adsorption columns, glass powder, glass fibers and glass beads.
S103, cutting the hybrid by using unpaired endonuclease and single-strand specific nuclease;
in embodiments of the invention, the unpaired endonuclease can be a T7 endonuclease. The single strand specific nuclease may be S1 nuclease. Single-Strand-specific nuclease the primers or probes used in the present invention are specific for one or more regions of a nucleic acid target fragment. In some cases, the primer or probe has at least 50% complementary sequence to one or more regions of the nucleic acid target fragment. In some cases, the primer or probe has at least 75% complementary sequence to one or more regions of the nucleic acid target fragment. In some cases, the primer or probe has at least 90% complementary sequence to one or more regions of the nucleic acid target fragment. In some cases, a primer or probe has at least 100% complementary sequence to one or more regions of a nucleic acid target fragment.
In some embodiments, the primers or probes used in the invention are specific for one or more regions of the non-nucleic acid target fragment. In some cases, the primer or probe has at least 50% complementary sequence to one or more regions of the non-nucleic acid target fragment. In some cases, the primer or probe has at least 75% complementary sequence to one or more regions of the non-nucleic acid target fragment. In some cases, the primer or probe has at least 90% complementary sequence to one or more regions of the non-nucleic acid target fragment. In some cases, the primer or probe has at least 100% complementary sequence to one or more regions of the non-nucleic acid target fragment.
In some embodiments, the primers or probes used in the present invention will incorporate modified nucleotides, oligonucleotides or oligonucleotide-like derivatives. In some cases, the modification of the primer or probe may be at the 5' end of the sequence. In some cases, the modification of the primer or probe may be at the 3' end of the sequence. In some cases, the modification of the primer or probe may be anywhere in the middle segment of the sequence. In some cases, the primer or probe is modified by a fluorophore. In some cases, the primer or probe is modified by biotin. In some cases, the primer or probe is modified by methylation. In some cases, the primer or probe is modified by phosphorylation. In some cases, the primer or probe is modified by a locked nucleic acid. In some cases, the primer or probe is modified by C3-Spacer or C5-Spacer. In some cases, the primer or probe is modified by digoxigenin.
In some embodiments, the nucleic acid probe is hybridized to the sample nucleic acid, the nucleic acid probe binds to the target gene, and the non-target nucleic acid does not bind to the probe.
In some embodiments, the hybrid strand formed by the probe and the target gene is degraded away.
In some embodiments, the hybrid strand complex formed by the probe and the target gene is removed from the system by affinity adsorption. In some cases, the hybridized strand complexes are removed from the system by streptavidin magnetic beads (magnetic microspheres). The complex of the hybrid chain is removed from the system by polystyrene magnetic beads (magnetic microspheres).
In some embodiments, ribonuclease is used to degrade RNA that is not bound to the probe. In some cases, RNase a is used to degrade RNA in the system. In some cases, RNase H is used to degrade RNA in the system. In some cases, RNA in the S1 nucleose degradation system was used. In some cases, RNA in the system is degraded using one or more of RNase a, RNase H or S1 Nuclease.
S104, obtaining an amplicon population by using the target sequence universal primer for constructing a sequencing library.
In the present example, the nucleic acid fragments that do not hybridize to the probe are enriched prior to sequencing library construction. In some embodiments, the nucleic acid library is a high throughput sequencing library. In some cases, the nucleic acid library is a DNA sequencing library. In some cases, the nucleic acid library is an RNA sequencing library. In some cases, the nucleic acid library is a methylated DNA library. In some cases, the nucleic acid library is a methylated RNA library. In some cases, the nucleic acid library is a short fragment sequencing library of less than 550 bp. In some cases, the nucleic acid library is a long fragment sequencing library of 1kb or greater.
In some cases, the nucleic acid library comprises at least 100, at least 1000, at least 10000, at least 100000, at least 1000000, at least 10000000, at least 100000000, at least 1000000000, at least 10000000000 nucleic acid fragments.
In some cases, the nucleic acid is fragmented by a method selected from the group consisting of: ultrasonic treatment, enzyme digestion, heat treatment, repeated liquid transfer treatment and atomization treatment.
In some embodiments, the present invention provides methods comprising ligating and enriching sequencing linker (Adaptor) sequences, tag (Index) sequences and nucleic acid sequences. In some cases, the method of ligation and enrichment is accomplished by linear amplification. In some cases, the method of ligation and enrichment is accomplished by rolling circle amplification. In some cases, the method of ligation and enrichment is accomplished by isothermal amplification. In some cases, the method of ligation and enrichment is accomplished by a non-linear amplification method.
In some embodiments, the nucleic acid sequences of the sequencing library will comprise a nucleic acid molecule Barcode (Barcode). In some cases, the nucleic acid molecule barcode is located at the 5' end of the nucleic acid molecule of the library to be tested. In some cases, the nucleic acid molecule barcode is located at the 3' end of the nucleic acid molecule of the library to be tested. In some cases, the nucleic acid molecule barcode comprises a target sequence of an enzyme.
In some embodiments, the present invention provides a method comprising: the nucleic acid not bound to the probe is sequenced. In some cases, sequencing was performed using Sanger sequencing. In some cases, Sequencing was performed using massively parallel Sequencing with Sequencing by Synthesis (Sequencing by Synthesis). In some cases, sequencing is performed using single molecule sequencing methods.
In some embodiments, sample sequencing data containing identifier sequences is read and obtained from high throughput sequencing raw data using a high performance computer. In some cases, the high throughput sequencing raw data is sequencer photomicrograph results. In some cases, the high-throughput sequencing raw data is an electrical pulse recording result. In some cases, the high throughput sequencing raw data is sequencing data in fastq format. In some cases, the high-throughput sequencing raw data is sequencing data in fasta format. In some cases, the high throughput sequencing data is bulk sequencing data of Sanger sequencing.
In some embodiments, the product obtained by primer extension is an amplicon of at least 100 bases, at least 1000 bases, at least 10000 bases.
Prophetic examples:
example 1: purification and fragmentation of sample total DNA
Total DNA of the sample was purified by the CTAB method or a genomic DNA purification kit corresponding to the sample. Purified total DNA was assayed for nucleic acid concentration by a Qubit fluorometer or Nano Drop microspectrophotometer and total DNA integrity was detected using an Agilent 2100 bioanalyzer.
Example 2: screening of target gene and preparation of probe
Taking the detection of the endogenous prokaryotic microorganisms in arabidopsis leaves as an example, sequences of plant plastid and mitochondrial 16S genes (NC _000932.1) are searched in an NCBI Nucleotide nucleic acid database, corresponding DNA is chemically synthesized according to the inquired arabidopsis plastid and mitochondrial sequences, and the DNA is subcloned into a pUC18 cloning vector and named as pUC18-NC 321. Amplicons containing the T7 promoter were amplified separately using pUC18-NC321 as templates using primers AT 7-515F/U806R and U515F/AT 7-806R, and then the two amplicons were mixed in equimolar amounts to obtain a probe DNA template U4 AT a final concentration of 50 ng/. mu.l.
The RNA probe is synthesized by in vitro transcription by using T7 RNA polymerase and taking U4 as a template, and the reaction system is as follows:
components Volume of
10×Reaction Buffer 2μl
ATP(50mM) 2μl
CTP(50mM) 2μl
UTP(50mM) 2μl
GTP(50mM) 2μl
U4 DNA mix 5μl
T7 RNA polymerase Mix 2μl
ddH2O 3μl
The above components were mixed well and incubated at 37 ℃ for 2 hours. The U4 DNA template was then removed by incubation with 1. mu.l DNaseI for 10 min. The obtained RNA probe was recovered and purified by ZymoResearch RNA Clean & Concentrator-25, quantified by Qubit, and stored at-80 ℃ to obtain a probe named PU-4.
Example 3 binding of target genes to probes
PCR amplification is carried out by using 16S universal primer U27F/U1492R and pUC18-NC321 and Zymobiomics Microbial Community DNA Standard (D6306) as templates, amplicons AF1 and AZ1 are obtained after respectively carrying out DNA purification and recovery on amplification products, a target gene sample FZ1 to be treated is obtained after AF1 and AZ1 are mixed according to the proportion of 10:1, and the final concentration of the sample is 25 ng/. mu.l.
In this example, after the target gene sample FZ1 and the probe PU-4 obtained in example 2 were mixed in equal amounts and annealed above the thermal cycle, the obtained annealed product (MFZ1) would contain two components: component 1, AF1 and PU-4 form DNA-RNA hybrid in 515bp-806bp segment of AF1 fragment; component 2, containing AZ1 and PU-4, which are not reacted.
Example 4 removal of target Gene
To the annealed product MFZ1 obtained in example 3, 1. mu. l T7 EndonuclealseI (T7E1)/S1 nuclear mix was added, and incubated at 37 ℃ for 15 minutes, followed by addition of RNaseA and incubation at 37 ℃ for 10 minutes. The above reaction product was concentrated by purification using ZymoResearch DNA Clean & concentrate-100 to obtain CFZ1 as a product. The product CFZ1 was analyzed after agarose gel electrophoresis and showed that the 515-806bp region of the target gene AF1 had been cleaved and fragmented to form DNA fragments of 515bp and 756bp, respectively, while AZ1 in the product remained intact, as shown in FIG. 2, where M is the DNA molecular weight marker, FZ1 is the target gene DNA, and CFZ is the digested DNA product.
Example 5 verification of target Gene ablation
This example verifies the FA1 removal efficiency by sequencing the CFZ1 fragment using high throughput sequencing techniques. The embodiment is as follows:
PCR reactions were carried out using 16S rRNA gene U515F/U806R primers and FZ1 and CFZ1 as templates (3 technical repeats were carried out) to obtain amplification products SFZ1 and SCFZ1, respectively, followed by construction of a sequencing library using TruSeq DNA PCR-Free Sample Prep Kit (FC-121-3001/3003) from Illumina, and using Hiseq Rapid SBS Kit v2(FC-402 + 4023500 Cycle) from Illumina in a PE250 sequencing manner. And performing OTU clustering after performing data quality control on the obtained sequencing data, and finally performing colony structure diversity analysis after performing data conversion by using R language. The results of the analysis found that the relatively high fraction of AF1(Arabidopsis Chloroplast) was removed using the method of the invention, leaving only the non-target sequence CFZ 1. The results are shown in FIG. 3.
The preparation method of the nucleic acid library for low host background interference can improve the nucleic acid target sequencing analysis technology and can provide accurate target sequencing, multiple sequencing and high-throughput sequencing; the method can be used for sequencing low copy target genes of nucleic acid, and reducing the interference of host abundant nucleic acid; can be used for the target sequencing of nucleic acid target genes; can be used for removing target genes of nucleic acid for sequencing.
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (8)

1. A method for preparing a nucleic acid library for low host background interference, comprising:
designing a target sequence probe primer, and obtaining a nucleic acid template by using polymerase so as to obtain a probe;
the probe forms a DNA-RNA complex by hybridizing with the target sequence;
cleaving the hybrid using a non-pairing endonuclease and a single-strand specific nuclease;
the amplicon population obtained using the target sequence universal primers was used for sequencing library construction.
2. The method of claim 1 for low host background interference nucleic acid library preparation, which is characterized in that,
the target sequence probe primer is one of single oligonucleotide, a series of oligonucleotides or modified oligonucleotides.
3. The method of claim 1, wherein designing a target sequence probe primer, obtaining a nucleic acid template using a polymerase, and further obtaining a probe comprises:
amplicon amplification of the targeted or non-targeted sequence using the amplification primers and the nucleic acid template produces one or more amplicons.
4. The method of claim 1, wherein the target sequence probe primer is designed, and the polymerase is used to obtain the nucleic acid template and further obtain the probe, and further comprising:
DNA templates are used to generate amplicons that target genes, which are DNA, RNA, or DNA/RNA hybridization probes.
5. The method of claim 1, wherein the probe hybridizes to the target sequence to form a DNA-RNA complex by hybridization in the following manner:
the probe and the target nucleic acid are hybridized through an annealing mode, the probe and the target nucleic acid are hybridized in a buffer solution, the probe and the target nucleic acid are hybridized under ultraviolet treatment, the probe is tethered on a solid or semisolid support and is hybridized with the nucleic acid, and the probe is hybridized with the nucleic acid tethered on the solid or semisolid support.
6. The method of claim 1 for low host background interference nucleic acid library preparation, which is characterized in that,
the unpaired endonuclease is a T7 endonuclease.
7. The method of claim 1 for low host background interference nucleic acid library preparation, which is characterized in that,
the single-strand specific nuclease is S1 nuclease.
8. Use of a library of nucleic acids for low host background interference, wherein the library of nucleic acids is subjected to high throughput sequencing.
CN202110901929.1A 2021-08-06 2021-08-06 Preparation method and application of nucleic acid library for low host background interference Pending CN113622033A (en)

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Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1688718A (en) * 2002-09-19 2005-10-26 阿普里拉股份有限公司 Methods and compositions for detecting targets
CN104005090A (en) * 2014-05-28 2014-08-27 北京诺禾致源生物信息科技有限公司 Method for building high throughput sequencing library of low-quality sample deoxyribonucleic acid (DNA)
US20140242577A1 (en) * 2013-02-26 2014-08-28 Agilent Technologies, Inc. C-probe libraries for dna target enrichment
WO2014153408A1 (en) * 2013-03-19 2014-09-25 Directed Genomics, Llc Enrichment of target sequences
CN104178478A (en) * 2014-08-13 2014-12-03 邵华武 Liquid-phase hybrid capture enriching liquor for genome DNA sequencing library and hybrid method adopting liquid-phase hybrid capture enriching liquor
CN105671191A (en) * 2016-04-07 2016-06-15 南开大学 Nucleic acid isothermal detection technology and reagent relying on endonuclease activity
CN105734679A (en) * 2016-03-29 2016-07-06 重庆市肿瘤研究所 Preparation method of nucleic acid target sequence capture sequencing library
CN109750091A (en) * 2019-03-13 2019-05-14 江苏宏微特斯医药科技有限公司 Single tube detects the method and its kit of one or more object to be measured nucleic acid sequences
CN111051522A (en) * 2018-03-19 2020-04-21 伊鲁米纳公司 Methods and compositions for recombinase-mediated selective cleavage of nucleic acids

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1688718A (en) * 2002-09-19 2005-10-26 阿普里拉股份有限公司 Methods and compositions for detecting targets
US20140242577A1 (en) * 2013-02-26 2014-08-28 Agilent Technologies, Inc. C-probe libraries for dna target enrichment
WO2014153408A1 (en) * 2013-03-19 2014-09-25 Directed Genomics, Llc Enrichment of target sequences
CN104005090A (en) * 2014-05-28 2014-08-27 北京诺禾致源生物信息科技有限公司 Method for building high throughput sequencing library of low-quality sample deoxyribonucleic acid (DNA)
CN104178478A (en) * 2014-08-13 2014-12-03 邵华武 Liquid-phase hybrid capture enriching liquor for genome DNA sequencing library and hybrid method adopting liquid-phase hybrid capture enriching liquor
CN105734679A (en) * 2016-03-29 2016-07-06 重庆市肿瘤研究所 Preparation method of nucleic acid target sequence capture sequencing library
CN105671191A (en) * 2016-04-07 2016-06-15 南开大学 Nucleic acid isothermal detection technology and reagent relying on endonuclease activity
CN111051522A (en) * 2018-03-19 2020-04-21 伊鲁米纳公司 Methods and compositions for recombinase-mediated selective cleavage of nucleic acids
CN109750091A (en) * 2019-03-13 2019-05-14 江苏宏微特斯医药科技有限公司 Single tube detects the method and its kit of one or more object to be measured nucleic acid sequences

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