CN103667447A - Primer and kit for detecting NQO1 gene polymorphism, and application of primer and kit on pathological examination - Google Patents
Primer and kit for detecting NQO1 gene polymorphism, and application of primer and kit on pathological examination Download PDFInfo
- Publication number
- CN103667447A CN103667447A CN201310539817.1A CN201310539817A CN103667447A CN 103667447 A CN103667447 A CN 103667447A CN 201310539817 A CN201310539817 A CN 201310539817A CN 103667447 A CN103667447 A CN 103667447A
- Authority
- CN
- China
- Prior art keywords
- primer
- tetra
- nqo1
- exon
- gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- 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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- 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
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention discloses a primer and a kit for detecting NQO1 gene polymorphism, and an application of primer and kit on pathological examination. The primer comprises a specific primer for amplifying the fourth exon C465T site fragment and the sixth exon C609T site fragment of quinone oxidoreductase 1 gene and a primer for pyrosequencing the obtained nucleic acid fragment. The polymorphism of the fourth exon C465T site fragment and the sixth exon C609T site fragment of quinone oxidoreductase 1 gene related to tumor susceptibility can be detected by adopting the pyrosequencing technology through the primer, method and kit disclosed by the invention, the specificity is good and the accuracy is high.
Description
Technical field
The invention belongs to life science and biological technical field, be particularly related to the primer, test kit and the application in pathology detection that for quinone oxidoreductase 1 Gene NQO1 * 2 (C609T), NQO1*3 (C465T) loci polymorphism, detect, adopt tetra-sodium sequencing technologies, can detect quinone oxidoreductase 1 gene pleiomorphism, specificity is good, and accuracy is high.
Background technology
Quinone oxidoreductase 1 gene (NADPH:quinone oxidore-ductase1, NQO1) is first reported in 1958 by Ernster and Navazio, is called as at first DT-lipoamide dehydrogenase (DT-diaphorase).It is ubiquitous a kind of flavin protease in most eukaryotic cells, can remove the toxicity of many natural and synthetic compounds, can activate again some quinones antitumor drugs, thereby attract wide attention simultaneously.NQO1 is a kind of derivable reductase enzyme, and the external environment factors such as phenol antioxidant, polycyclic aromatic hydrocarbons, azoic dyestuff, anoxic all can induce NQO1 to express consumingly.This inducibility is expressed NQO1 may be played an important role in tumor prevention and oncotherapy.
Early 1990s, studies have found that on NQO1cDNA609 site and has C/T single nucleotide polymorphism (SNP), may change the secondary structure of enzyme, makes the activity decreased of enzyme, has increased the generation of oxyradical.At present in finder's NQO1 gene order, cause that the non-synonym coding single nucleotide polymorphism that protein coding aminoacid sequence changes has three, comprising being positioned at the C465T of the 4th exon and the C609T of the 6th exon, cause that respectively 139 coded amino acids become arginine (Arg) from tryptophane (Trp), 187 amino acids become Serine (Ser) from proline(Pro) (Pro).These aminoacid sequences change, may cause reduction or the disappearance of enzyme NQO1 activity in individuality, weakened NQO1 to the detoxification of quinones and changed the impact of tumour on specific proto-oncogene and cancer suppressor gene function, thereby the possibility that makes body suffer from tumour increases.
Research discovery, enzyme NQO1 has the individuality that 2 wild-type alleles are wild homozygote (C/C), and the vigor of NQO1 enzyme is normal; And the vigor with the individual enzyme of heterozygote (C/T) reduces, there is the completely dissolve of vigor of the individual NQO1 enzyme of no mutant homozygote was (T/T).
Still not a lot of for NQO1C465T research at present.On NQO1cDNA465 site, there is C/T single nucleotide polymorphism.Different crowd NQO1465T gene frequency lower (0～0.05).Much research also finds, NQO1C609T gene polymorphic resistant frequency has very big-difference on race distributes, and the homozygote individuality that genotype is (T/T) is 18%～20% in China and other Asia ethnic groups, is approximately Caucasian and Afro American 4 times.
In view of the generation of NQO1 and tumour, develop closely related, its the 6th exon (C609T) and the 4th exon (C465T) have non-synonym coding single nucleotide polymorphism, thereby the base sequence change that can cause protein coding causes the change of protein function, and then affects developing of tumour.To its detect will contribute to the further effect of clear and definite its genetic background in tumour occurs, and provide useful guidance for tumor individual therapy.
In clinical labororatory, the common method of inspection quinone oxidoreductase 1 Gene NQO1 * 2 (C609T), NQO1*3 (C465T) polymorphism is restriction fragment length polymorphism (restriction fragment length polymorphism, RELP) method at present.This technology is first-generation DNA molecular marker technology, utilize restriction enzyme can identify the distinguished sequence of DNA molecular, and at particular sequence place incision DNA molecular, produce distinctive restriction fragment, the defect of the method is that detection sensitivity is low and can only detect general type, can not be specific to mutant proportion.
The present invention carries out tetra-sodium order-checking and detects on original basis, the concrete sudden change situation of pleomorphism site can not only be detected, also can pick out mutant proportion according to collection of illustrative plates, makes detected result more specific.
Summary of the invention
Defect for prior art, the present invention is except utilizing two pairs of specific primers respectively the 4th exon C465T site fragment of quinone oxidoreductase 1 Gene NQO1 and the 6th exon C609T site fragment to be carried out conventional pcr amplification, also utilize a pair of tetra-sodium sequencing primer to carry out forward order-checking and backward sequencing to pcr amplified fragment, the concrete sudden change situation of pleomorphism site not only can be detected, also can pick out mutant proportion according to order-checking collection of illustrative plates, make detected result more specific, thus can be better for tumor individual therapy provides useful guidance.
In carrying out tetra-sodium order-checking process, four kinds of dNTP (dATP, dTTP, dCTP, dGTP) are sequentially added in sequencing reaction system.At each, take turns in sequencing reaction, only add wherein a kind of.As the pairing of the dNTP being added into and DNA profiling, archaeal dna polymerase can this dNTP of catalysis and the 3' end formation covalent linkage of tetra-sodium sequencing primer C465T-S or C609T-S, and the tetra-sodium group (PPi) of dNTP is just released.The mole number of the dNTP adding and the PPi discharging equates.Then, ATP sulfurylase (ATP sulfurylase) catalysis ammonium persulphate (ammonium persulfate, APS) and PPi form ATP, and the mole number of this ATP and PPi is also consistent.Utilize ATP as the energy, luciferase (luciferase) can be oxidized to fluorescein oxyluciferin (oxyluciferin), and then sends the visible light signal being directly proportional to ATP amount.Optical signals ccd video camera detects, and transfers waveform signal to by program pyrogramTM.The height of each crest (optical signal) with react in the Nucleotide number that adds be directly proportional.
Based on this tetra-sodium sequencing technologies, the invention provides for detection of the C609T of NQO1 gene and the primer of C465T loci polymorphism, it is characterized in that, described primer comprises the Auele Specific Primer of increase from sample nucleic acid NQO1 gene the 4th exon C465T site fragment and the 6th exon C609T site fragment, and its base sequence is:
C465T-F:5'-CTAGCTTTACTCGGACCCACTC-3'（SEQ ID No.1）
C465T-R:5'-GCAACAAGAGGGAAGCTCCATC-3'（SEQ ID No.2）
C609T-F:5'-TCTTACTGAGAAGCCCAGACCAACT-3'（SEQ ID No.3）
C609T-R:5'-CTCCAGGCGTTTCTTCCATCC-3'（SEQ ID No.4）
And the primer that obtained nucleic acid amplification product is carried out to tetra-sodium order-checking, its base sequence is:
C465T-S:5'-GCATTCAGAACCATCCACCTAC-3'（SEQ ID No.5）
C609T-S:5'-TTCTGTGGCTTCCAAGTCTTAG-3'（SEQ ID No.6）。
Further, the 5' of primer C465T-F and C609T-R end is by biotin labeling.
Further, the reaction conditions of described amplification be 94 ℃ 2 minutes; 98 ℃ 10 seconds, 60 ℃ 20 seconds, 68 ℃ 20 seconds, 35 circulations; 68 ℃ 5 minutes.
The present invention also provides and has detected the C609T of NQO1 gene and the application of the method for C465T loci polymorphism in pathology detection, comprising:
(1) extract the DNA in sample;
(2) DNA of usining in step (1), as template, is used pair of primers C465T-F and C465T-R amplification NQO1 gene the 4th exon C465T site fragment, obtains pcr amplification product 1; Use pair of primers C609T-F and C609T-R amplification NQO1 gene the 6th exon C609T site fragment, obtain pcr amplification product 2;
(3) whether the pcr amplification product 1 and 2 of getting in step (2) carries out agarose electrophoresis, detect and increase successfully;
(4) if increase successfully in step (3), utilize tetra-sodium sequencing primer C465T-S to check order to the pcr amplification product 1 in step (2), utilize tetra-sodium sequencing primer C609T-S to check order to the pcr amplification product 2 in step (2);
(5) according to the tetra-sodium sequencing result in step step (4), determine the C465T of NQO1 gene and the polymorphism in C609T site,
(6), according to the polymorphism result in step (5), determine the pathological state of sample;
It is characterized in that, described primer sequence is:
C465T-F:5'-CTAGCTTTACTCGGACCCACTC-3'
C465T-R:5'-GCAACAAGAGGGAAGCTCCATC-3'
C609T-F:5'-TCTTACTGAGAAGCCCAGACCAACT-3'
C609T-R:5'-CTCCAGGCGTTTCTTCCATCC-3'
C465T-S:5'-GCATTCAGAACCATCCACCTAC-3'
C609T-S:5'-TTCTGTGGCTTCCAAGTCTTAG-3'。
Further, the reaction conditions of described amplification be 94 ℃ 2 minutes; 98 ℃ 10 seconds, 60 ℃ 20 seconds, 68 ℃ 20 seconds, 35 circulations; 68 ℃ 5 minutes.
The present invention is also provided for detecting the C609T of NQO1 gene and the test kit of C465T loci polymorphism, comprise nucleic acid extracting reagent, pcr amplification system, tetra-sodium sequencing reagent, it is characterized in that, described pcr amplification system comprises that its base sequence is for the Auele Specific Primer of amplification gene NQO1 the 4th exon C465T site fragment and the 6th exon C609T site fragment:
C465T-F:5'-CTAGCTTTACTCGGACCCACTC-3'（SEQ ID No.1）
C465T-R:5'-GCAACAAGAGGGAAGCTCCATC-3'（SEQ ID No.2）
C609T-F:5'-TCTTACTGAGAAGCCCAGACCAACT-3'（SEQ ID No.3）
C609T-R:5'-CTCCAGGCGTTTCTTCCATCC-3'（SEQ ID No.4）
Described tetra-sodium sequencing reagent comprises the primer that obtained amplified nucleic acid fragment is carried out to tetra-sodium order-checking, and its base sequence is:
C465T-S:5'-GCATTCAGAACCATCCACCTAC-3'（SEQ ID No.5）
C609T-S:5'-TTCTGTGGCTTCCAAGTCTTAG-3'（SEQ ID No.6）。
Further, the 5' of primer C465T-F and C609T-R end is by biotin labeling.
Further, described pcr amplification system also comprises 2*Buffer, dNTP, KOD enzyme and pure water.
Further, described tetra-sodium sequencing reagent also comprises 75% ethanol and DMSO.
Further, described nucleic acid extracting reagent comprises erythrocyte cracked liquid.
The present invention is applied to sequencing technologies the detection of quinone oxidoreductase 1 gene polymorphism sites, design amplimer and tetra-sodium sequencing primer that NQO1*2 (C609T), NQO1*3 (C465T) pleomorphism site are included, carry out pcr amplification, expanding fragment length is respectively 464bp and 212bp, carries out subsequently tetra-sodium sequencing analysis.
Adopt tetra-sodium sequencing technologies, by specific amplimer of the present invention and tetra-sodium sequencing primer, can detect the quinone oxidoreductase relevant to tumor prevention and oncotherapy 1 Gene NQO1 * 2 (C609T), NQO1*3 (C465T) loci polymorphism, specificity is good, and accuracy is high.Can be for the clinical Subsidiary Index as tumorigenic early prevention, early diagnosis and screening.
Accompanying drawing explanation
Fig. 1 is C465T wild-type tetra-sodium sequencer map.
Fig. 2 is C609T wild-type tetra-sodium sequencer map.
Fig. 3 is C609T sudden change heterozygous tetra-sodium sequencer map.
Fig. 4 is the C609T homozygous tetra-sodium sequencer map that suddenlys change.
Embodiment
Below in conjunction with specific embodiments and the drawings, further set forth the present invention.Should be noted that, unaccounted normal condition and method in embodiment, conventionally according to the conventional employing method of affiliated field experimenter: such as, Ao Sibai and James Kingston chief editor's < < fine works molecular biology experiment guide > > the 4th edition, or the step of advising according to manufacturer and condition.
Embodiment 1: the primer and the test kit that detect Gene NQO1 loci polymorphism
The primer detecting for quinone oxidoreductase 1 Gene NQO1 * 2 (C609T), NQO1*3 (C465T) loci polymorphism, comprise the Auele Specific Primer of the quinone oxidoreductase 1 Gene NQO1 * 2 (C609T) that increases, NQO1*3 (C465T) site fragment and the primer that the nucleic acid fragment obtaining is carried out to tetra-sodium order-checking from sample nucleic acid, wherein
The specific primer sequence of quinone oxidoreductase 1 Gene NQO1 * 2 (C609T), NQO1*3 (C465T) the site fragment of increasing from sample nucleic acid is:
C465T-F:5'-CTAGCTTTACTCGGACCCACTC-3'（SEQ ID No.1）
C465T-R:5'-GCAACAAGAGGGAAGCTCCATC-3'（SEQ ID No.2）
C609T-F:5'-TCTTACTGAGAAGCCCAGACCAACT-3'（SEQ ID No.3）
C609T-R:5'-CTCCAGGCGTTTCTTCCATCC-3'（SEQ ID No.4）
Wherein C465T-F and C609T-R are 5' end biotin labeling primer;
The primer sequence that the nucleic acid fragment obtaining is carried out to tetra-sodium order-checking is:
C465T-S:5'-GCATTCAGAACCATCCACCTAC-3'（SEQ ID No.5）
C609T-S:5'-TTCTGTGGCTTCCAAGTCTTAG-3'（SEQ ID No.6）。
Adopt tetra-sodium sequencing technologies, by two couples of specific amplimer C465T-F, C465T-R and C609T-F, C609T-R and tetra-sodium sequencing primer, can detect the quinone oxidoreductase relevant to tumor disease 1 Gene NQO1 * 2 (C609T), NQO1*3 (C465T) loci polymorphism.
The test kit of the C465T loci polymorphism of a kind of C609T for detection of quinone oxidoreductase 1 Gene NQO1 * 2, NQO1*3, comprise nucleic acid extracting reagent, pcr amplification system, tetra-sodium sequencing reagent, wherein, described pcr amplification system comprises that its base sequence is for the Auele Specific Primer of amplification gene NQO1 the 4th exon C465T site fragment and the 6th exon C609T site fragment:
C465T-F:5'-CTAGCTTTACTCGGACCCACTC-3'（SEQ ID No.1）
C465T-R:5'-GCAACAAGAGGGAAGCTCCATC-3'（SEQ ID No.2）
C609T-F:5'-TCTTACTGAGAAGCCCAGACCAACT-3'（SEQ ID No.3）
C609T-R:5'-CTCCAGGCGTTTCTTCCATCC-3'（SEQ ID No.4）
Described tetra-sodium sequencing reagent comprises the primer that obtained amplified nucleic acid fragment is carried out to tetra-sodium order-checking, and its base sequence is:
C465T-S:5'-GCATTCAGAACCATCCACCTAC-3'（SEQ ID No.5）
C609T-S:5'-TTCTGTGGCTTCCAAGTCTTAG-3'（SEQ ID No.6）。
Preferably, the 5' of primer C465T-F and C609T-R end is by biotin labeling.
Preferably, described pcr amplification system also comprises 2*Buffer, dNTP, KOD enzyme and pure water.
Preferably, described tetra-sodium sequencing reagent also comprises 75% ethanol and DMSO.
Preferably, described nucleic acid extracting reagent comprises erythrocyte cracked liquid.
Embodiment 2:DNA extracts
DNA extraction reagent: erythrocyte cracked liquid, poba gene group DNA extraction agent box (TIANGEN company).
Operation steps: get 500ul whole blood, put in the centrifuge tube of 1.5ml, add 1ml erythrocyte cracked liquid.Spin upside down, make to mix completely, rotation shakes that after 15 seconds, to put into whizzer centrifugal, and centrifugation rate is 5000rpm, 10min..Outwell upper strata liquid, color precipitation is arranged at visible centrifuge tube bottom.Add 500ul erythrocyte cracked liquid, repeat this cleavage step once.Centrifugal 5000rpm, 5min, sucks supernatant, leaves white corpuscle precipitation, adds 200uL damping fluid GA, and vibration is to thoroughly mixing.Add 20 μ l Proteinase K solution, mix.Add 200 μ l damping fluid GB, fully put upside down and mix, place 10 minutes for 70 ℃, solution strain is limpid, brief centrifugal to remove the globule of cap wall.Add people's 200 μ l dehydrated alcohols, fully vibration mixes 15 seconds, now may occur flocks, brief centrifugal to remove the globule of cap wall.Previous step gained solution and flocks are all added in an adsorption column CB3 (adsorption column is put into collection tube), and centrifugal 30 seconds of 12,000rpm (～13,400 * g), outwells waste liquid, and adsorption column CB3 is put back in collection tube.Before adding in adsorption column CB3 500 μ l damping fluid GD(to use, please first check whether added dehydrated alcohol), centrifugal 30 seconds of 12,000rpm (～13,400 * g), outwells waste liquid, and adsorption column CB3 is put into collection tube.Before adding in adsorption column CB3 700 μ l rinsing liquid PW(to use, please first check whether added dehydrated alcohol), centrifugal 30 seconds of 12,000rpm (～13,400 * g), outwells waste liquid, and adsorption column CB3 is put into collection tube.In adsorption column CB3, add 500 μ l rinsing liquid PW, centrifugal 30 seconds of 12,000rpm (～13,400 * g), outwells waste liquid.Adsorption column CB3 is put back in collection tube, and centrifugal 2 minutes of 12,000rpm (～13,400 * g), outwells waste liquid.Adsorption column CB3 is placed in to room temperature and places several minutes, thoroughly to dry rinsing liquid remaining in sorbing material.Adsorption column CB3 is proceeded in a clean centrifuge tube, and to the unsettled dropping 100 μ l elution buffer TE in middle part of adsorption film, room temperature is placed 2-5 minute, and centrifugal 2 minutes of 12,000rpm (～13,400 * g), collects solution in centrifuge tube.Finally, obtain the DNA extracting.
Embodiment 3:PCR amplification
To the DNA extracting in embodiment 2, utilize pcr amplification system to carry out conventional pcr amplification, obtain amplification of DNA fragments amplified production.
The pcr amplification system that increases used to quinone oxidoreductase 1 Gene NQO1 * 2 (C609T) site fragment is 2*Buffer10 μ l, dNTP4 μ l, KOD enzyme 0.4 μ l, upstream primer C609T-F1 μ l, downstream primer C609T-R1 μ l, pure water 2.6 μ l, add 1 μ l template DNA in 19 μ l amplification systems, and cumulative volume is 20 μ l.
The pcr amplification system that increases used to quinone oxidoreductase 1 Gene NQO1 * 3 (C465T) site fragment is 2*Buffer10 μ l, dNTP4 μ l, KOD enzyme 0.4 μ l, upstream primer C465T-F1 μ l, downstream primer C465T-R1 μ l, pure water 2.6 μ l, add 1 μ l template DNA in 19 μ l amplification systems, and cumulative volume is 20 μ l.
Conventional pcr amplification reaction condition be 94 ℃ 2 minutes; 98 ℃ 10 seconds, 60 ℃ 20 seconds, 68 ℃ 20 seconds, 35 circulations; 68 ℃ 5 minutes.
Embodiment 4: agarose gel electrophoresis
Whether the DNA fragmentation amplified production 3 μ l that get in embodiment 3 carry out agarose gel electrophoresis, detect and increase successfully, and clear brighter object band is carried out to follow-up tetra-sodium sequencing reaction.
Embodiment 5: tetra-sodium order-checking
Tetra-sodium sequencing reagent: 75% ethanol, Denaturation Solution, Annealing Buffer, Binding Buffer, 1 * Wash Buffer, Streptavidin Sepharase High Performance(bead), PyroMark Gold Q96Reagents(E S dNTP), DMSO (dimethyl sulfoxide (DMSO)), tetra-sodium sequencing primer C465T-S and C609T-S, wherein, except tetra-sodium sequencing primer, all purchase the QIAGEN company from the U.S..
Tetra-sodium order-checking step: utilize tetra-sodium sequencing reagent, electrophoresis method through in embodiment 4 is confirmed to the successful pcr amplification product of amplification carries out tetra-sodium order-checking, and the step that specifically checks order is according to PyroMark Gold Q96(U.S. QIAGEN company) process specifications carries out.Wherein utilize tetra-sodium sequencing primer C465T-S to carry out backward sequencing to the 4th exon C465T site fragment of quinone oxidoreductase 1 Gene NQO1, utilize tetra-sodium sequencing primer C609T-S to carry out forward order-checking to the 6th exon C609T site fragment.
Amplified production for the 4th exon C465T site fragment and the 6th exon C609T site fragment of quinone oxidoreductase 1 Gene NQO1, utilize respectively tetra-sodium sequencing primer C465T-S and C609T-S to check order, according to the polymorphism in sequencer map spectrum analysis and definite these two sites.
Embodiment 6: clinical sample pathological state is analyzed
Get 4 increments of clinical censorship originally, number consecutively is No. 1 sample, No. 2 samples, No. 3 samples and No. 4 samples.According to the method for embodiment 2 to embodiment 5, utilize primer C465T-S and C609T-S respectively the amplified production of NQO1 gene the 4th exon C465T site fragment in No. 1 sample, No. 2 samples, No. 3 samples and No. 4 samples and the amplified production of the 6th exon C609T site fragment to be checked order.
The gene order of NQO1 gene the 4th exon C465T site wild-type is TTC
cgG.As shown in Figure 1, institute's calling sequence is the tetra-sodium sequencing result of the 4th exon C465T site fragment amplification product of the NQO1 gene in No. 1 sample: CC after measured
ggAA, the square frame in Fig. 1 is marked.Pleomorphism site is G, and owing to being backward sequencing, known No. 1 sample is C465T wild-type.The detected result homozygous with sudden change for the sudden change heterozygous of NQO1 gene the 4th exon C465T is identical with reality.
The gene order of NQO1 gene the 6th exon C609T site wild-type is AA
ccTC.As shown in Figure 2, institute's calling sequence is the tetra-sodium sequencing result of the 6th exon C609T site fragment amplification product of the NQO1 gene in No. 2 samples: AA after measured
ccTC, the square frame in Fig. 2 is marked.Pleomorphism site is C, and this site is forward order-checking, and known No. 2 spurious editions are C609T wild-type.
The gene order of NQO1 gene the 6th exon C609T site mutation heterozygous is AA
t/CcTC.After measured, as shown in Figure 3, institute's calling sequence is the tetra-sodium sequencing result of NQO1 gene the 6th exon C609T site fragment amplification product in No. 3 samples: AA
t/CcTC, the square frame in Fig. 3 is marked.Pleomorphism site is T/C, and this site is forward order-checking, and known 3 good samples are C609T sudden change heterozygous.
The homozygous gene order of NQO1 gene the 6th exon C609T site mutation is AA
tcTC.As shown in Figure 4, institute's calling sequence is the tetra-sodium sequencing result of the fragment amplification product in No. 4 samples: AA after measured
tcTC, the square frame in Fig. 4 is marked.Pleomorphism site is T, and this site is forward order-checking, and known No. 4 samples are that C609T sudden change is homozygous.
SEQUENCE LISTING
<110> Wuhan company limited of Ai Dikang medical test institute
<120> is for detection of primer, test kit and the application in pathology detection thereof of NQO1 gene pleiomorphism
<130>
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 22
<212> DNA
<213> artificial sequence
<400> 1
ctagctttac tcggacccac tc 22
<210> 2
<211> 22
<212> DNA
<213> artificial sequence
<400> 2
gcaacaagag ggaagctcca tc 22
<210> 3
<211> 25
<212> DNA
<213> artificial sequence
<400> 3
tcttactgag aagcccagac caact 25
<210> 4
<211> 21
<212> DNA
<213> artificial sequence
<400> 4
ctccaggcgt ttcttccatc c 21
<210> 5
<211> 22
<212> DNA
<213> artificial sequence
<400> 5
gcattcagaa ccatccacct ac 22
<210> 6
<211> 22
<212> DNA
<213> artificial sequence
<400> 6
ttctgtggct tccaagtctt ag 22
Claims (10)
1. for detection of the C609T of NQO1 gene and the primer of C465T loci polymorphism, it is characterized in that, described primer comprises the Auele Specific Primer of increase from sample nucleic acid NQO1 gene the 4th exon C465T site fragment and the 6th exon C609T site fragment, and its base sequence is:
C465T-F : 5'-CTAGCTTTACTCGGACCCACTC-3'
C465T-R: 5'-GCAACAAGAGGGAAGCTCCATC-3'
C609T-F: 5'-TCTTACTGAGAAGCCCAGACCAACT-3'
C609T-R: 5'- CTCCAGGCGTTTCTTCCATCC-3'
And the primer that obtained nucleic acid amplification product is carried out to tetra-sodium order-checking, its base sequence is:
C465T-S: 5'- GCATTCAGAACCATCCACCTAC -3'
C609T-S: 5'- TTCTGTGGCTTCCAAGTCTTAG -3'。
2. primer as claimed in claim 1, is characterized in that, the 5' end of primer C465T-F and C609T-R is by biotin labeling.
3. primer as claimed in claim 1, is characterized in that, the reaction conditions of described amplification be 94 ℃ 2 minutes; 98 ℃ 10 seconds, 60 ℃ 20 seconds, 68 ℃ 20 seconds, 35 circulations; 68 ℃ 5 minutes.
4. detect the C609T of NQO1 gene and the application of the method for C465T loci polymorphism in pathology detection, comprising:
(1) extract the DNA in sample;
(2) DNA of usining in step (1), as template, is used pair of primers C465T-F and C465T-R amplification NQO1 gene the 4th exon C465T site fragment, obtains pcr amplification product 1; Use pair of primers C609T-F and C609T-R amplification NQO1 gene the 6th exon C609T site fragment, obtain pcr amplification product 2;
(3) whether the PCR amplified production 1 and 2 of getting in step (2) carries out agarose electrophoresis, detect and increase successfully;
(4) if increase successfully in step (3), utilize tetra-sodium sequencing primer C465T-S to check order to the pcr amplification product 1 in step (2), utilize tetra-sodium sequencing primer C609T-S to check order to the pcr amplification product 2 in step (2);
(5), according to the tetra-sodium sequencing result in step step (4), determine the C465T of NQO1 gene and the polymorphism in C609T site;
(6), according to the polymorphism result in step (5), determine the pathological state of sample;
It is characterized in that, described primer sequence is:
C465T-F: 5'-CTAGCTTTACTCGGACCCACTC-3'
C465T-R: 5'-GCAACAAGAGGGAAGCTCCATC-3'
C609T-F: 5'-TCTTACTGAGAAGCCCAGACCAACT-3'
C609T-R: 5'- CTCCAGGCGTTTCTTCCATCC-3'
C465T-S: 5'- GCATTCAGAACCATCCACCTAC -3'
C609T-S: 5'- TTCTGTGGCTTCCAAGTCTTAG -3'。
5. method as claimed in claim 4, is characterized in that, the reaction conditions of described amplification be 94 ℃ 2 minutes; 98 ℃ 10 seconds, 60 ℃ 20 seconds, 68 ℃ 20 seconds, 35 circulations; 68 ℃ 5 minutes.
6. for detection of the C609T of NQO1 gene and the test kit of C465T loci polymorphism, comprise nucleic acid extracting reagent, pcr amplification system, tetra-sodium sequencing reagent, it is characterized in that, described pcr amplification system comprises that its base sequence is for the Auele Specific Primer of amplification gene NQO1 the 4th exon C465T site fragment and the 6th exon C609T site fragment:
C465T-F : 5'-CTAGCTTTACTCGGACCCACTC-3'
C465T-R: 5'-GCAACAAGAGGGAAGCTCCATC-3'
C609T-F: 5'-TCTTACTGAGAAGCCCAGACCAACT-3'
C609T-R: 5'- CTCCAGGCGTTTCTTCCATCC-3'
Described tetra-sodium sequencing reagent comprises the primer that obtained amplified nucleic acid fragment is carried out to tetra-sodium order-checking, and its base sequence is:
C465T-S: 5'- GCATTCAGAACCATCCACCTAC -3'
C609T-S: 5'- TTCTGTGGCTTCCAAGTCTTAG -3'。
7. test kit as claimed in claim 6, the 5' end of primer C465T-F and C609T-R is by biotin labeling.
8. test kit as claimed in claim 6, is characterized in that, described pcr amplification system also comprises 2* Buffer, dNTP, KOD enzyme and pure water.
9. test kit as claimed in claim 6, is characterized in that, described tetra-sodium sequencing reagent also comprises 75% ethanol and DMSO.
10. test kit as claimed in claim 6, is characterized in that, described nucleic acid extracting reagent comprises erythrocyte cracked liquid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310539817.1A CN103667447A (en) | 2013-11-04 | 2013-11-04 | Primer and kit for detecting NQO1 gene polymorphism, and application of primer and kit on pathological examination |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310539817.1A CN103667447A (en) | 2013-11-04 | 2013-11-04 | Primer and kit for detecting NQO1 gene polymorphism, and application of primer and kit on pathological examination |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103667447A true CN103667447A (en) | 2014-03-26 |
Family
ID=50306215
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310539817.1A Pending CN103667447A (en) | 2013-11-04 | 2013-11-04 | Primer and kit for detecting NQO1 gene polymorphism, and application of primer and kit on pathological examination |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103667447A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110946853A (en) * | 2019-11-26 | 2020-04-03 | 暨南大学 | Application of curcumenol in preparation of NQO2 protein inhibitor |
| CN112522376A (en) * | 2020-12-21 | 2021-03-19 | 济南和合医学检验有限公司 | Primer group, kit and method for detecting gene polymorphism |
| CN113755588A (en) * | 2021-09-08 | 2021-12-07 | 菲思特(上海)生物科技有限公司 | Genetic polymorphism detection kit for fluorouracil adverse reaction and curative effect prediction, detection method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012040492A1 (en) * | 2010-09-22 | 2012-03-29 | The Board Of Regents Of The University Of Texas System | Methods of treating cancer comprising targeting nqo1 |
-
2013
- 2013-11-04 CN CN201310539817.1A patent/CN103667447A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012040492A1 (en) * | 2010-09-22 | 2012-03-29 | The Board Of Regents Of The University Of Texas System | Methods of treating cancer comprising targeting nqo1 |
Non-Patent Citations (2)
| Title |
|---|
| FARHAD ZAKER等: "The Frequency and Association of C609T and C465T Polymorphisms of NAD(P)H:Quinone Oxidoreductase Gene With Adult Acute Myeloid Leukemia", 《LABMEDICINE》, vol. 42, no. 11, 30 November 2011 (2011-11-30) * |
| 李利等: "NQO1基因C609T、C465T多态性与子宫内膜癌易感性的关系", 《山东医药》, vol. 53, no. 3, 25 January 2013 (2013-01-25) * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110946853A (en) * | 2019-11-26 | 2020-04-03 | 暨南大学 | Application of curcumenol in preparation of NQO2 protein inhibitor |
| CN112522376A (en) * | 2020-12-21 | 2021-03-19 | 济南和合医学检验有限公司 | Primer group, kit and method for detecting gene polymorphism |
| CN112522376B (en) * | 2020-12-21 | 2022-07-19 | 济南和合医学检验有限公司 | Primer group, kit and method for detecting gene polymorphism |
| CN113755588A (en) * | 2021-09-08 | 2021-12-07 | 菲思特(上海)生物科技有限公司 | Genetic polymorphism detection kit for fluorouracil adverse reaction and curative effect prediction, detection method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102329876B (en) | Method for measuring nucleotide sequence of disease associated nucleic acid molecules in sample to be detected | |
| CN104031978B (en) | A kind of test kit based on the transgenation of ARMS fluorescence quantitative PCR detection and method | |
| Wapenaar et al. | The interferon gamma gene in celiac disease: augmented expression correlates with tissue damage but no evidence for genetic susceptibility | |
| CN104120132A (en) | FBN1 genetic mutant and application thereof | |
| CN106929591A (en) | A kind of human HLA B*5801 genetic polymorphism detection kits | |
| JP2017184642A (en) | Dementia marker, evaluation method of dementia using same, evaluation reagent, and evaluation kit | |
| CN106498035A (en) | A kind of construction method and its application for detecting chemotherapeutics gene SNP variation library for high-flux sequence | |
| CN104975100A (en) | Specific primer and kit for human ALDH2 gene polymorphism detection | |
| CN103667447A (en) | Primer and kit for detecting NQO1 gene polymorphism, and application of primer and kit on pathological examination | |
| CN108893532A (en) | A kind of gene detecting kit and detection method for SMA genetic screening | |
| CN106834434B (en) | Nucleic acid, kit and method for detecting COX-1, COX-2 and GPIIIa gene polymorphism | |
| CN105420392B (en) | One group of gene new mutation relevant to newborn's Tendon defection phenotype and detection kit | |
| KR102112951B1 (en) | Ngs method for the diagnosis of cancer | |
| CN110499368A (en) | One kind SNP marker relevant to carcinoma of mouth prognosis prediction and its application | |
| CN103131776A (en) | A719G single nucleotide polymorphism detection kit of thiopurine methyltransferase (TPMT) * 3C | |
| CN107267611B (en) | Primer group for detecting p.Pro189Ala locus genotype and detection kit and application thereof | |
| CN105838720A (en) | PTPRQ gene mutant and application thereof | |
| Fouad et al. | Serum level of growth arrest-specific 6 (Gas6) protein and genetic variations in the Gas6 gene in patients with type 2 diabetes mellitus | |
| CN107312833B (en) | LSP primer and kit for detecting human BRCA1 gene mutation | |
| CN108676852B (en) | Primer pair group and kit for detecting epigenetic modification difference of P53 gene in peripheral blood free DNA | |
| Avsar et al. | Association of MTHFR, MTRR and RAD54L Gene Variations with Meningioma and Correlation with Tumor’s Histopathological Characteristics on Turkish Cohort | |
| CN104152570A (en) | Basal cell carcinoma susceptibility gene SNP polymorphism detection kit | |
| RU2607031C1 (en) | Method of detecting candidate genes for population research of genetic polymorphism in children dwelling in strontium geochemical province environment | |
| Zhu et al. | Accurate identification of HLA-B* 15: 02 allele by two-dimensional polymerase chain reaction | |
| CN108085381B (en) | Application of gene mutation sites in preparation of reagent or kit for diagnosing chronic pancreatitis and evaluating prognosis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20140326 |