WO2011022970A1 - Amorces de séquençage destinées au séquençage direct des produits acides nucléiques de la pcr et procédé de séquençage utilisant celles-ci - Google Patents

Amorces de séquençage destinées au séquençage direct des produits acides nucléiques de la pcr et procédé de séquençage utilisant celles-ci Download PDF

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Publication number
WO2011022970A1
WO2011022970A1 PCT/CN2010/071385 CN2010071385W WO2011022970A1 WO 2011022970 A1 WO2011022970 A1 WO 2011022970A1 CN 2010071385 W CN2010071385 W CN 2010071385W WO 2011022970 A1 WO2011022970 A1 WO 2011022970A1
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WIPO (PCT)
Prior art keywords
sequencing
nucleic acid
primers
pcr
primer
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PCT/CN2010/071385
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English (en)
Chinese (zh)
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陆学东
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Lu Xuedong
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Publication of WO2011022970A1 publication Critical patent/WO2011022970A1/fr

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    • 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

Definitions

  • the present invention relates to the field of nucleic acid sequencing, and more particularly to a sequencing primer and sequencing method for direct sequencing of nucleic acid PCR products.
  • PCR product sequencing mostly uses traditional clonal sequencing technology, including: 1) PCR product is linked to plasmid carrier; 2) ligated product transforming bacteria; 3) bacterial culture; 4) cumbersome steps such as template preparation, which is cumbersome and time consuming. Book
  • the PCR product can also be sequenced by direct sequencing.
  • the most commonly used direct sequencing method for PCR products is still the Sanger dideoxynucleotide-mediated chain termination method, which is linearly amplified after common heterologous binding, thus sequencing Templates and primers are very strict.
  • the key factors affecting the direct sequencing of PCR products in this method include: 1) the quality of the sequencing template; 2) the specificity and amplification efficiency of the sequencing primers.
  • the quality of the template can be controlled by various methods such as PCR product purification, SAP-Exon I purification, and ethanol/sodium acetate purification to obtain a high-quality specific template.
  • the design and selection of sequencing primers has been a problem that plagues direct sequencing of PCR products.
  • primers with good PCR amplification can be used for sequencing reactions, such as cartridges and primers, random primers, long primers (greater than 24 bp), primers that are easy to form secondary structures, primers with low purity, etc. If a single PCR primer is used directly for sequencing, it often results in unsatisfactory sequencing results or failure of the sequencing reaction. It is also necessary to redesign the sequencing primers based on different PCR products, but this will result in an extension of the sequencing cycle and an increase in cost. Summary of the invention
  • the main object of the present invention is to provide a nucleic acid PCR product for direct
  • the sequencing primers are sequenced, and the sequencing primers have a wide range of applications.
  • Another object of the present invention is to provide an efficient, stable, and rapid sequencing method for direct sequencing of nucleic acid PCR products.
  • the present invention adopts the following technical solutions:
  • a sequencing primer for direct sequencing of a nucleic acid PCR product the base sequence of which is shown in SEQ ID NOS: 1 and 2.
  • a sequencing method for direct sequencing of a nucleic acid PCR product comprising the steps of:
  • the amplification primers comprising a specific primer sequence designed according to the target nucleic acid sequence and a sequencing primer sequence located at the 5' end of the specific primer, synthesizing a pair of sequencing Primer
  • the base sequence of the sequencing primer is shown in SEQ ID NOS: 1 and 2.
  • the step 3) is specifically: the PCR amplification product is detected by agarose gel electrophoresis, and the target nucleotide target amplified fragment is excised, and purified by a gel purification kit to obtain a purified sequencing reaction template.
  • the sequencing primers for direct sequencing of nucleic acid PCR products of the present invention show that the universal primer sequences are not related to the common pathogenic microorganisms, human and other mammalian common gene nucleic acid sequences known in the database. It has complete homology and does not cause non-specific sequencing reactions.
  • the sequencing results can specifically reflect the true base composition of the sequence, so rapid screening and identification of pathogenic microorganisms, molecular diagnosis of hereditary diseases and mononucleosides For acid polymorphism studies, the sequencing primers described herein can be utilized.
  • the sequencing method for direct sequencing of nucleic acid PCR products of the invention does not need to insert the PCR product to be tested into the plasmid vector, and directly performs nucleic acid sequence determination on the PCR product.
  • Traditional cloning Compared with sequencing technology, the cumbersome steps of molecular cloning, bacterial culture and template preparation repeated for sequencing are eliminated, which has the advantages of being fast, easy, economical, and better reflecting the real situation of the template, and thus can be widely used. It is used in the fields of gene mutation detection, genetic disease diagnosis, single nucleotide polymorphism analysis, rapid detection and identification of pathogenic microorganisms.
  • Figure 1 is an electrophoresis pattern of PCR amplification products of HBoV-positive specimens.
  • M is a DNA standard band
  • 3 and 6 are HBoV-positive specimens
  • 10 are negative controls
  • 1, 2, 4, 5, 7, 8, and 9 are HBoV-negative. specimen.
  • Figures 2A and 2B are sequencing diagrams of human Bocavirus PCR positive products.
  • Figure 3 is an electropherogram of PCR amplification products of four diarrhea virus-positive specimens.
  • M is the DNA standard band
  • 1 and 2 are RV-positive specimens
  • 3 and 4 are NV-positive specimens
  • 5 and 6 are EAV-positive specimens
  • 7 is As tV positive specimens
  • 8 is a positive control
  • 9 is a negative control.
  • Figure 4 is a sequence diagram of four viral PCR positive products. detailed description
  • Upstream primer SeqF (SEQ ID NO: 1): 5,- CGCCAGACGATATGCAGC-3,
  • downstream primer SeqR SEQ ID NO: 2): 5,- GTGGACAGCGGATAGGTAC-3, , synthetic boca virus VP gene fragment PCR amplification primer, Upstream primers and downstream primers 5,
  • the sequencing primers are ligated to the sequencing primers SeqF and SeqR (the underlined portion is the sequencing primer sequence):
  • the viral nucleic acid extraction kit was purchased from Axygen Corporation of the United States, and the nucleic acid extract was stored at -30 °C for use in strict accordance with the instructions.
  • the PCR kit was purchased from the Fermentas Company of Lithuania, 25 ⁇ l reaction system, and the upstream and downstream primers were at a concentration of HBoVF and HBoVR of 0. ⁇ , viral nucleic acid extract 2 ⁇ 1, 2 ⁇ PCR-
  • the amplified product was detected by Ig/dL agarose gel electrophoresis, and the VP gene target amplified fragment was cut at about 440 bp. Purified by Axygen's AxyPrepTM Gel Purification Kit to obtain a pure sequencing template.
  • Sequencing primers were sequenced with primers SeqF or SeqR, using Genome LabTM DTCS-Quick Start Kit 0 from Beckman, USA. Reaction conditions: 96 ° C for 20 s, 50 ° C for 20 s, 60 ° C for 4 min, 30 cycles.
  • the sequencing reaction products were purified according to the Genome LabTM DTCS-Quick Start Kit purification step.
  • the nucleic acid sequence was determined by the CEQTM 8800 Genetic Analysis System of Beckman Company, and the obtained product sequences were input into the gene pool, and BLAST was used for homology alignment.
  • the PCR amplification product of the VP gene fragment of 23 HBoV-positive samples was subjected to nucleic acid sequence determination.
  • the measured gene sequences were submitted to the gene bank, and the accession numbers were EU334499, EU375243, EU375244, FJ184310, FJ184311, EU358600-EU358603, EU363219 ⁇ EU36322L.
  • 15 were identical to the EU358602 nucleic acid sequence, and 2 were completely separated from EU363220.
  • the other four EU358601, EU358603, FJ184311, and FJ184310 have unique nucleic acid sequences.
  • the homology of the 6 strains with the Swedish strain ST1 (DQ000495) was 97.8%_98.8%, and the sequencing results are shown in Figure 2.
  • sequencing results demonstrate that the sequencing primers of the present invention have high specificity, and the results of BLAST alignment in GenBank indicate that the universal primer sequence is not related to the known nucleic acid sequences in the database (including humans, other mammals, eukaryotes, prokaryotes). ) has homology and therefore does not cause non-specific sequencing reactions, and the sequencing results can specifically reflect the true base composition of the sequence;
  • the sequencing primer has good amplification efficiency, the sequencing reaction extends well, the sequencing failure caused by rapid decay or interruption of the signal does not occur, the signal signal to noise ratio of the signal is high, the base peak quality is high, and the sequencing result is accurate and reliable.
  • Example 2 Detection and identification of rotavirus, Norovirus, Astrovirus, and intestinal adenovirus in fecal specimens of infants with viral diarrhea.
  • the primer sequences are shown in the table below: Target gene sequence (5'-3')
  • Viral nucleic acid extraction Virus DNA and RNA extraction kits were purchased from QIAGEN, and the extracted nucleic acids were stored at -80 °C for use.
  • RT-PCR kits for amplifying RV, NV and AstV were purchased from QIAGEN.
  • RT-PCR reaction conditions reverse transcription, 50 ° C 30 min; DNA denaturation, 95 ° C 15 min; 94 ° C 45s ⁇ 55 ° C 45s ⁇ 72 ° C lmin (35 cycles), 72 ° C extension lOmin;
  • the PCR kit for increasing EAV was purchased from Fermentas.
  • PCR reaction conditions denaturation at 94 ° C for 5 min, 94 ° C 45 s ⁇ 55 ° C 45 s ⁇ 72 ° C lmin (35 cycles), 72 ° C extension for 10 min.
  • the amplified product was analyzed by 1% agarose gel electrophoresis, and the results were observed using a gel imaging system.
  • the 6 ⁇ PCR product was added to 2 U SAP and 1 U Exon I at 37 °C for 1 h and 75 °C for 15 min.
  • the resulting purified PCR product was used as a template for the sequencing reaction.
  • Step 5) to step 7) Same as Embodiment 1.
  • the obtained sequence was imported into GenBank and aligned by BLAST.
  • the homology of RV with the known sequences EU679386, DQ870492, AF531912 was 95%-97%; the homology of NV with EU494692, EU794709, EU794707 was 95%_97%; EAV The homology with DQ315364, AB330122 is 98%-100%; the homology of AstV with AF361030, L13745, AY720891 is 93-95%, and the sequencing results are shown in Fig. 4.

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
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  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne des amorces destinées au séquençage direct des produits acides nucléiques de la PCR et un procédé de séquençage utilisant celles-ci. Les séquences nucléotidiques des amorces de séquençage sont présentées sous les noms de SEQ ID NO : 1 et 2. Les amorces de séquençage destinées au séquençage direct des produits acides nucléiques de la PCR n'induisent pas de réaction de séquençage non spécifique, et le résultat du séquençage à l’aide de celles-ci peut refléter la composition de base réelle de manière spécifique, ainsi les amorces peuvent être utilisées dans le criblage et l'identification d'un grand nombre de micro-organismes pathogènes communs, le diagnostic moléculaire de maladies héréditaires et la recherche d'un polymorphisme mononucléotidique. Le procédé de séquençage direct des produits acides nucléiques de la PCR utilisant les amorces de séquençage ci-dessus permet de réaliser un séquençage des acides nucléiques sur les produits de la PCR directement sans avoir à insérer les produits de la PCR à l'intérieur de plasmides. De ce fait, le procédé est rapide, facile, économique et plus précis.
PCT/CN2010/071385 2009-08-28 2010-03-29 Amorces de séquençage destinées au séquençage direct des produits acides nucléiques de la pcr et procédé de séquençage utilisant celles-ci WO2011022970A1 (fr)

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CN2009101897780A CN101638691B (zh) 2009-08-28 2009-08-28 用于核酸pcr产物直接测序的测序引物和测序方法
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CN101638691B (zh) * 2009-08-28 2011-12-21 陆学东 用于核酸pcr产物直接测序的测序引物和测序方法
CN107937503B (zh) * 2017-12-29 2018-10-26 北京睿博兴科生物技术有限公司广州分公司 一种对引物测序的方法
CN114703290A (zh) * 2022-01-25 2022-07-05 中国科学院南海海洋研究所 一种海参通用扩增引物、扩增方法及应用

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WO2001083696A2 (fr) * 2000-04-28 2001-11-08 Digital Gene Tech Inc PROCEDES D'ISOLEMENT ET DE SEQUENCAGE RAPIDES DE SEQUENCES GENETIQUES SPECIFIQUES
CN1483082A (zh) * 2000-10-30 2004-03-17 �����﹤����ʽ���� 测定核酸碱基序列的方法
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KR100442832B1 (ko) * 2002-07-10 2004-08-02 삼성전자주식회사 다중 중합효소 연쇄반응에 의한 ｍｏｄｙ２ 유전자의증폭을 위한 프라이머 세트

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WO2001083696A2 (fr) * 2000-04-28 2001-11-08 Digital Gene Tech Inc PROCEDES D'ISOLEMENT ET DE SEQUENCAGE RAPIDES DE SEQUENCES GENETIQUES SPECIFIQUES
CN1483082A (zh) * 2000-10-30 2004-03-17 �����﹤����ʽ���� 测定核酸碱基序列的方法
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