CN101638691B - sequencing primer and sequencing method for directly sequencing nucleic acid PCR product - Google Patents
sequencing primer and sequencing method for directly sequencing nucleic acid PCR product Download PDFInfo
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- CN101638691B CN101638691B CN2009101897780A CN200910189778A CN101638691B CN 101638691 B CN101638691 B CN 101638691B CN 2009101897780 A CN2009101897780 A CN 2009101897780A CN 200910189778 A CN200910189778 A CN 200910189778A CN 101638691 B CN101638691 B CN 101638691B
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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
Abstract
The invention relates to the field of nucleic acid sequencing, in particular to a sequencing primer and a sequencing method for directly sequencing a nucleic acid PCR product. The base sequences of the sequencing primer are shown in SEQ ID NO: 1 and 2. The sequencing primer for directly sequencing the nucleic acid PCR product can not cause a non-specific sequencing reaction, and a sequencing result can specifically reflect the true base composition of the sequences; The sequencing primer can be utilized for the rapid screening and the authentication of any pathogenic microorganism, the molecular diagnostics of hereditary diseases and the polymorphism research of mononucleotide. The sequencing method for directly sequencing the nucleic acid PCR product directly sequences the nucleic acid for the PCR product without inserting the PCR product to be tested into a plasmid carrier, has the advantages of rapidity, simplicity, convenience and economy and better reflects the true conditions of a template.
Description
Technical field
The present invention relates to the nucleic acid sequencing field, relate in particular to a kind of sequencing primer and sequence measurement that is used for directly sequencing nucleic acid PCR product.
Background technology
Traditional cloning and sequencing technology is adopted in the order-checking of PCR product mostly, and comprising: 1) the PCR product is connected with plasmid vector; 2) connect the product transform bacteria; 3) microbial culture; 4) loaded down with trivial details step, troublesome poeration and length consuming time such as template preparation.
The PCR product also can adopt the method for direct order-checking to check order, the most frequently used direct sequence measurement of PCR product is still the acid mediated chain termination method of Sanger dideoxyribonucleoside at present, with common PCR reacting phase ratio, because the sequencing reaction of chain termination method depends on the linear amplification that causes by after unidirectional primer and the template specific combination, thereby require very strict to sequencing template and primer.Influencing the PCR product key of success factor that directly checks order in this method comprises: the 1) quality of sequencing template; 2) specificity of sequencing primer and amplification efficiency.The quality of template can be cut glue by the PCR product and be reclaimed purifying, SAP-Exon I purifying, ethanol/several different methods such as sodium-acetate purifying and controlled, thereby obtain high-quality specificity template.Yet, because the diversity of PCR product makes that the design of sequencing primer and selection are directly problems of order-checking of puzzlement PCR product always.Be not that all good primers of pcr amplification can both be used for sequencing reaction; as degenerated primer, random primer, long primer (greater than 24bp), the primer that self easily forms secondary structure, primer that purity is not high or the like, usually can cause the undesirable or sequencing reaction failure of sequencing result if simply the PCR primer of routine is directly used in order-checking.Need redesign sequencing primer according to different PCR products in addition, but can cause the prolongation in order-checking cycle and the increase of cost.
Summary of the invention
For addressing the above problem, main purpose of the present invention is to be provided for the sequencing primer of directly sequencing nucleic acid PCR product, and described sequencing primer is applied widely.
Another object of the present invention is to provide efficiently, stablizes, is used for efficiently the sequence measurement of directly sequencing nucleic acid PCR product.
For achieving the above object, the present invention adopts following technical scheme:
The sequencing primer that is used for directly sequencing nucleic acid PCR product, the base sequence of described sequencing primer is shown in SEQ ID NO:1 and 2.
Be used for the sequence measurement of directly sequencing nucleic acid PCR product, may further comprise the steps:
1) according to target nucleic acid sequences Design to be detected and synthetic a pair of amplimer, described amplimer comprises according to the specific primer sequence of target nucleic acid sequences Design and is positioned at the sequencing primer sequence that Auele Specific Primer 5 ' is held, synthetic a pair of sequencing primer;
2) adopt described amplimer that testing sample is carried out pcr amplification, obtain pcr amplification product;
3) the described pcr amplification product of purifying obtains the sequencing reaction template of purifying;
4) with the sequencing reaction template of purifying, carry out sequencing reaction, obtain the sequencing reaction product with described sequencing primer;
5) the described sequencing reaction product of purifying;
6) the sequencing reaction product to purifying carries out sequencing.
The base sequence of described sequencing primer is shown in SEQ ID NO:1 and 2.
Described step 3) is specially: pcr amplification product downcuts target Nucleotide target amplification fragment to be measured after agarose gel electrophoresis detects, through gel-purified test kit purifying, obtain the sequencing reaction template of purifying.
The sequencing primer that is used for directly sequencing nucleic acid PCR product of the present invention, in GenBank this universal primer sequence of BLAST comparison result shows not with database in known common invasive organism, the biological common gene nucleic acid sequence of human and other mammalss has complete homology, can not cause non-specific sequencing reaction, sequencing result can reflect the true based composition of sequence specifically, so carry out pathogenic micro-organism rapid screening and evaluation, the molecular diagnosis of heredopathia and single nucleotide polymorphism research can utilize the described sequencing primer of the application.
The sequence measurement that is used for directly sequencing nucleic acid PCR product of the present invention does not need PCR product to be measured is inserted plasmid vector, and directly the PCR product is carried out determining nucleic acid sequence.Compare with traditional cloning and sequencing technology, save the loaded down with trivial details steps such as molecular cloning, microbial culture and template preparation of carrying out repeatedly for order-checking, have quick, easy, economical, and can reflect the advantage of the truth of template better, thereby can be widely used in fields such as detection in Gene Mutation, heredopathia diagnosis, single nucleotide polymorphism analysis, pathogenic micro-organism rapid detection and evaluation.
Description of drawings
Fig. 1 is a HBoV positive sample pcr amplification product electrophorogram, and M is the DNA index zone among the figure, and 3,6 is the HBoV positive sample, 10 negative contrasts, and 1,2,4,5,7,8,9 is the negative sample of HBoV.
Fig. 2 A and 2B are human bocavirus PCR positive products sequencer maps.
Fig. 3 is four kinds of diarrhea virus positive sample pcr amplification product electrophorograms, and M is the DNA index zone among the figure, and 1,2 is the RV positive sample, and 3,4 is the NV positive sample, and 5,6 is the EAV positive sample, and 7 is As tV positive sample, 8 positive contrasts, 9 negative contrasts.
Fig. 4 is four kinds of virus PCR positive products sequencer maps.
Embodiment
One, sample source and gathering: choose 28d-10 year respiratory tract infection infant, amount to 447 examples,, draw the about 0.5mL of nasopharyngeal secretions, be difficult to cooperate or secretory product lacks the person and then gets throat swab with disposal sputum collecting cannula in be admitted to hospital back the 2nd day of infant; Sample adds 2.0mL virus and transports liquid (containing 100U/mL penicillin, 100U/mL Streptomycin sulphate, 2000U/mL amphotericin B), puts in-30 ℃ of refrigerators to be checked.
Two, concrete operations step is as follows:
1) with software Primer5.0 design sequencing primer, it is synthetic to transfer to Invitrogen (Shanghai) biotech firm.
Upstream primer SeqF (SEQ ID NO:1): 5 '-CGCCAGACGATATGCAGC-3 ',
Downstream primer SeqR (SEQ ID NO:2): 5 '-GTGGACAGCGGATAGGTAC-3 ',
Synthetic bocavirus VP gene fragment pcr amplification primer, 5 ' end of upstream primer and downstream primer are connected sequencing primer SeqF, SeqR (underscore partly is the sequencing primer sequence) respectively:
Upstream primer HBoVF:
5’-CGCCAGACGATATGCAGCGCAAACCCATCACTCTCAATGC-3’
Downstream primer HBoVR:
5’-GTGGACAGCGGATAGGTACGCTCTCTCCTCCCAGTGACAT-3’，
2) human bocavirus nucleic acid extraction: viral nucleic acid extracts test kit and purchases the Axygen company in the U.S., and in strict accordance with the specification sheets operation, nucleic acid extractive-30 ℃ preservation is standby.
3) human bocavirus VP gene fragment amplification:
The PCR test kit is purchased the Fermentas company in Lithuania, and 25 μ l reaction systems, upstream and downstream primer HBoVF and HBoVR concentration are 0.1 μ M, viral nucleic acid extract 2 μ l, 2 * PCR-Mastermix, 12.5 μ l.PCR reaction conditions: 94 ℃ of 5min; 94 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 45s, 35 circulations; 72 ℃ of 8min.
4) PCR product purification:
Amplified production detects through the 1g/dL agarose gel electrophoresis, downcuts about 440bp VP of place gene target amplification fragment.Through the U.S. AxyPrepTM of Axygen company gel-purified test kit purifying, obtain purified sequencing template.
5) sequencing reaction:
Sequencing primer adopts sequencing primer SeqF or SeqR, adopts the Genome Lab TM DTCS-Quick Start Kit of U.S. Beckman company.Reaction conditions: 96 ℃ of 20s, 50 ℃ of 20s, 60 ℃ of 4min, 30 circulations.
6) order-checking purifying:
The sequencing reaction product carries out purifying according to Genome Lab TM DTCS-Quick Start Kit purification step.
7) sequencing:
CEQTM8800 genetic analysis systems through U.S. Beckman company carries out determining nucleic acid sequence, and products therefrom sequence input gene pool adopts BLAST to carry out the homology comparison.
Three, experimental result:
1) HBoV PCR detected result
In 447 parts of respiratory tract infection infant nasopharyngeal secretionses, have 23 examples to detect HBoV VP nucleic acid specificity fragment, positive rate is 5.1%, and positive amplified production is the single band of the about 440bp of length, conforms to expected results.Positive sample PCR product 1% agarose gel electrophoresis the results are shown in Figure 1.
2) positive pcr amplification product sequencing of HBoV and nucleotide sequence comparison
Pcr amplification product to the VP gene fragment of 23 parts of HBoV positive samples carries out determining nucleic acid sequence.The gene order that records is submitted gene pool to, and the acquisition accession number is EU334499, EU375243, EU375244, FJ184310, FJ184311, EU358600-EU358603, EU363219-EU363221.Have 15 strains and EU358602 nucleotide sequence identical in 23 parts of positive samples, 2 strains and EU363220 are identical, and other 4 strain EU358601, EU358603, FJ184311, FJ184310 have unique nucleotide sequence.This 6 strain and Sweden strain ST1 (DQ000495) nucleic acid sequence homology are 97.8%-98.8%, and sequencing result is seen Fig. 2.
Sequencing result proves that sequencing primer of the present invention has high specific, show in GenBank this universal primer sequence of BLAST comparison result shows not with database in known nucleotide sequence (comprising people, other mammalss, eucaryon, prokaryotic organism) have homology, therefore can not cause non-specific sequencing reaction, sequencing result can reflect the true based composition of sequence specifically;
Described sequencing primer has good amplification efficiency, and sequencing reaction extends good, the rapid decay of signal or interruption can not occur and the order-checking failure that causes, the signal to noise ratio height of signal, base peak quality is high, sequencing result accurately, reliable.
Rotavirus, norovirus, Astrovirus in embodiment 2 infant's viral diarrhea infant stool samples, the detecting and identifying of EAd.
One, sample source and gathering: gathers 192 parts of 0 to 3 years old diarrhoea infant stool samples, the stool proterties is rare water sample, magma sample and mucoid, makes 1% just suspension with physiological saline, and standby-80 ℃ refrigerator preservation.
Two, concrete operations step is as follows:
1) sequencing primer is synthetic with embodiment 1;
Synthetic rotavirus (RV), norovirus (NV), Astrovirus (AstV), the pcr amplification primer of EAd (EAV) target gene, 5 ' end of upstream primer and downstream primer are connected universal primer SeqF, SeqR (underscore partly is the universal primer sequence) respectively.Primer sequence sees the following form:
2) viral nucleic acid extracts: viral DNA, RNA extract test kit and purchase the company in QIAGEN, in strict accordance with the specification sheets operation, the nucleic acid of extraction put preserve in-80 ℃ of temperature standby.
3) viral nucleic acid detects: the RT-PCR test kit of amplification RV, NV and As tV is purchased the company in QIAGEN.RT-PCR reaction conditions: reverse transcription, 50 ℃ of 30min; The DNA sex change, 95 ℃ of 15min; 94 ℃ of 45s → 55 ℃ 45s → 72 ℃ of 1min (35 circulations), 72 ℃ are extended 10min; The PCR test kit of amplification EAV is purchased the company in Fermentas.The PCR reaction conditions: 94 ℃ of sex change 5min, 94 ℃ of 45s → 55 ℃ 45s → 72 ℃ of 1min (35 circulations), 72 ℃ are extended 10min.Adopt the analysing amplified product of 1% agarose gel electrophoresis, adopt the gel imaging system observations.
4) SAP-Exon I purified pcr product:
6 μ l PCR products add 2U SAP and 1U Exon I, 37 ℃ of 1h, 75 ℃ of 15min.The purified pcr product of gained is as the sequencing reaction template.
Step 5) to step 7) with embodiment 1.
Three, experimental result:
1) four kinds of acute gastroenteritis virus PCR detected results
The recall rate of four kinds of acute gastroenteritis virus PCR methods is in 192 parts of infant stool samples: RV50.5%, and NV 12.0%, and EAV 11.5%, and AstV 2.1%.Positive sample PCR product 1% agarose gel electrophoresis the results are shown in Figure 3.
2) positive pcr amplification product sequencing and nucleotide sequence comparison
With the calling sequence input GenBank of institute, through the BLAST comparison, the homology of RV and known array EU679386, DQ870492, AF531912 is 95%-97%; The homology of NV and EU494692, EU794709, EU794707 is 95%-97%; EAV and DQ315364, AB330122, homology be 98%-100%; The homology of AstV and AF361030, L13745, AY720891 is 93-95%, and sequencing result is seen Fig. 4.
SEQUENCE?LISTING
＜110〉east is learned in land
＜120〉be used for the sequencing primer and the sequence measurement of directly sequencing nucleic acid PCR product
<130>P11139
<160>2
<170>PatentIn?version?3.3
<210>1
<211>18
<212>DNA
＜213〉artificial sequence
<400>1
cgccagacga?tatgcagc 18
<210>2
<211>19
<212>DNA
＜213〉artificial sequence
<400>2
gtggacagcg?gataggtac 19
Claims (3)
1. a pair of sequencing primer that is used for directly sequencing nucleic acid PCR product, it is characterized in that: the base sequence of described sequencing primer is respectively shown in SEQ ID NO:1 and 2, described nucleic acid PCR product is a nucleic acid PCR amplification products therefrom, and the used amplimer of described nucleic acid PCR amplification comprises according to the specific primer sequence of target nucleic acid sequences Design respectively and is positioned at the described sequencing primer sequence that specific primer sequence 5 ' is held.
2. be used for the sequence measurement of the directly sequencing nucleic acid PCR product of non-medical diagnosis on disease purpose, it is characterized in that: may further comprise the steps:
1) according to target nucleic acid sequences Design to be detected and synthetic a pair of amplimer, described every amplimer comprises according to the specific primer sequence of target nucleic acid sequences Design and is positioned at the sequencing primer sequence that specific primer sequence 5 ' is held, the described sequencing primer sequence that is positioned at Auele Specific Primer 5 ' end respectively shown in SEQ ID NO:1 and 2, synthetic a pair of sequencing primer;
2) adopt described amplimer that testing sample is carried out pcr amplification, obtain pcr amplification product;
3) the described pcr amplification product of purifying obtains the sequencing reaction template of purifying;
4) with the sequencing reaction template of purifying, carry out sequencing reaction, obtain the sequencing reaction product with described sequencing primer;
5) the described sequencing reaction product of purifying;
6) the sequencing reaction product to purifying carries out sequencing.
3. the sequence measurement that is used for directly sequencing nucleic acid PCR product according to claim 2, it is characterized in that, described step 3) is specially: pcr amplification product downcuts target Nucleotide target amplification fragment to be measured after agarose gel electrophoresis detects, through gel-purified test kit purifying, obtain the sequencing reaction template of purifying.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009101897780A CN101638691B (en) | 2009-08-28 | 2009-08-28 | sequencing primer and sequencing method for directly sequencing nucleic acid PCR product |
| PCT/CN2010/071385 WO2011022970A1 (en) | 2009-08-28 | 2010-03-29 | Sequencing primers for direct sequencing nucleic acid pcr products and method for sequencing by using the same |
| HK10103365.9A HK1134940A1 (en) | 2009-08-28 | 2010-04-01 | Sequencing primer and sequencing method used in direct sequencing of nucleic acid pcr products |
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| CN2009101897780A CN101638691B (en) | 2009-08-28 | 2009-08-28 | sequencing primer and sequencing method for directly sequencing nucleic acid PCR product |
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| CN101638691A CN101638691A (en) | 2010-02-03 |
| CN101638691B true CN101638691B (en) | 2011-12-21 |
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| HK (1) | HK1134940A1 (en) |
| WO (1) | WO2011022970A1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101638691B (en) * | 2009-08-28 | 2011-12-21 | 陆学东 | sequencing primer and sequencing method for directly sequencing nucleic acid PCR product |
| CN107937503B (en) * | 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 | A method of primer is sequenced |
| CN114703290A (en) * | 2022-01-25 | 2022-07-05 | 中国科学院南海海洋研究所 | General amplification primer, amplification method and application of sea cucumber |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1302547A2 (en) * | 1992-06-17 | 2003-04-16 | City Of Hope | A method of detecting and discriminating between nucleic acid sequences |
| CN1467294A (en) * | 2002-07-10 | 2004-01-14 | 三星电子株式会社 | Multiplex pcr primer set for human glucokinase gene amplification |
| CN1483082A (en) * | 2000-10-30 | 2004-03-17 | �����﹤����ʽ���� | Method of determining nucleic acid base sequence |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU5741101A (en) * | 2000-04-28 | 2001-11-12 | Digital Gene Technologies, Inc. | Methods for rapid isolation and sequence determination of gene-specific sequences |
| CN101638691B (en) * | 2009-08-28 | 2011-12-21 | 陆学东 | sequencing primer and sequencing method for directly sequencing nucleic acid PCR product |
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2009
- 2009-08-28 CN CN2009101897780A patent/CN101638691B/en not_active Expired - Fee Related
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2010
- 2010-03-29 WO PCT/CN2010/071385 patent/WO2011022970A1/en active Application Filing
- 2010-04-01 HK HK10103365.9A patent/HK1134940A1/en not_active IP Right Cessation
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1302547A2 (en) * | 1992-06-17 | 2003-04-16 | City Of Hope | A method of detecting and discriminating between nucleic acid sequences |
| CN1483082A (en) * | 2000-10-30 | 2004-03-17 | �����﹤����ʽ���� | Method of determining nucleic acid base sequence |
| CN1467294A (en) * | 2002-07-10 | 2004-01-14 | 三星电子株式会社 | Multiplex pcr primer set for human glucokinase gene amplification |
Non-Patent Citations (4)
| Title |
|---|
| Jerome C.Regier,et al.Increased yield of PCR product from degenerate primers with nondegenerate, nonhomologous 5’tails.《Biotechniques》.2005,第38卷(第1期),34－38. * |
| Soowon Cho,et al.A highly conserved nuclear gene for low-level phylogenetics:elongation factor-1αrecovers morphology-based tree for Heliothine Moths.《Mol.Biol.Evol.》.1995,第12卷(第4期),650-656. * |
| 沈关心等.菌落PCR和质粒PCR对转化菌的筛选.《免疫学杂志》.2000,第16卷(第2期),149-151. * |
| 王虎等.DNA测序模板的制备和测序引物设计中的相关问题.《中国分子心脏病学杂志》.2004,第4卷(第1期),56-60. * |
Also Published As
| Publication number | Publication date |
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| CN101638691A (en) | 2010-02-03 |
| HK1134940A1 (en) | 2010-05-20 |
| WO2011022970A1 (en) | 2011-03-03 |
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