CN104120177A - Method and primer for detecting polymorphism of 5-HTTLPR fragment - Google Patents
Method and primer for detecting polymorphism of 5-HTTLPR fragment Download PDFInfo
- Publication number
- CN104120177A CN104120177A CN201410258375.8A CN201410258375A CN104120177A CN 104120177 A CN104120177 A CN 104120177A CN 201410258375 A CN201410258375 A CN 201410258375A CN 104120177 A CN104120177 A CN 104120177A
- Authority
- CN
- China
- Prior art keywords
- httlpr
- primer
- fragment
- polymorphism
- dna
- 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
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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method and primer for detecting the polymorphism of a 5-HTTLPR fragment. The primer comprises a forward primer and a reverse primer which are used for amplifying a 5-HTTLPR fragment, and the primer has a base sequence as follows: HTTLPR-F: 5'-CAGCACCTAACCCCTAATGT-3'; LPR-R: 5'-GCCGGTTGGGCTAGCGTCTA-3'. PCR (Polymerase Chain Reaction) amplification and agarose gel electrophoresis banding are combined, so that the polymorphism of the 5-HTTLPR fragment related to obsessive-compulsive disorder can be rapidly and simply detected at low cost.
Description
Technical field
The invention belongs to life science and biological technical field, particularly detect method and the primer of 5-HTTLPR segment polymorphism.
Background technology
Obsession is the mental disorder that a kind of heterogeneity is high, the cause of disease is complicated, both at home and abroad to its etiologic etiological research so far still without final conclusion, many scholars be devoted to genetics, spirit stress with the research of environment, neuroendocrine and synaptic plasticity aspect, wherein inherited genetic factors has played vital role.
Result of study about obsession genetics aspect is also not quite similar, and wherein most study is exactly 5-HTTLPR segment polymorphism and compulsive dependency.5-HTTLPR is the abbreviation of the chain polymorphic regions of serotonin transporter gene (serotonin transporter gene-linked polymorphic region).The polymorphism of 5-HTTLPR fragment is one of common polymorphisms of serotonin transporter (serotonin transporter, 5-HTT) gene.5-HTTLPR is positioned at the promoter region at about 1kb place, 5-HTT genetic transcription starting point upstream, for being rich in the tumor-necrosis factor glycoproteins of 20～30bp repeat element of GC.There is 44bp fragment insertion deletion in 5-HTTLPR fragment, affects the transcriptional activity of 5-HTT gene.The polymorphism of 5-HTTLPR fragment produces long segment (L) allelotrope and short-movie section (S) allelotrope.The allelic transcriptional activity of L is higher 2.5 times than S-allele.5-HTTLPR may affect by affecting the transcriptional activity of 5-HTT gene and the function of 5-HTT albumen the re-uptake speed of serotonin (serotonin, 5-HT), and the function of 5-HT is changed to some extent.
Up to now, inquire into the research of cognation between 5-HTTLPR segment polymorphism and obsession, there are many repugnances, Zhou Yun flies to wait and in Chinese Han Population, 61 routine patients with OCDs and 68 normal peoples that match is studied, result shows genotype and the control group no significant difference of 5-HTTLPR segment polymorphism, and L allelotrope and obsession are proportionate, make it suffer from compulsive danger and improved 1.929 times, be compulsive risk factor.5-HTILPR and obsession non-correlation are thought in the researchs such as Wang Zhen.Ramoz etc., by the large-scale cohort study to 352 families, think that 5-HTTLPR varient does not participate in obsession.And the research that Baca-Garcia etc. do thinks that L allelotrope is relevant with compulsive generation in the polymorphism of 5-HTTLPR, the researchs such as McDougle also show that L allelotrope and the obsession of 5-HTTLPR polymorphism are proportionate, and be negative correlation with the curative effect of selective serotonin reuptake inhibitor (selective serotonin reuptake inhibitors, SSRIs).Bengel etc. think, it is relevant that obsession and 5-HTTLPR genotype LL are significance.Separately there is result of study to show that patients with OCD genotype and gene frequency and control group are all variant, patients with OCD LL genotype and the genotypic odds ratio of non-LL are 1.086, illustrate that LL genotype makes compulsive ill risk increase by 1.086 times, this is consistent with the research of Bengel etc.In patients with OCD, the genotypic Yale Blang of LL forces scale (Yale-Brown Obsessive Compulsive Scale, YBOCS) score value apparently higher than LS type and SS type, illustrates that LL genotype and compulsive severity are proportionate.
Many patients with OCDs often have family history, and even their father and/or mother are patients with OCD equally, and well-known, family is the important component part of social support system, and this is consistent with many results of study.Studies have found that, parental separation is the Hazard Factor of patients with OCD, is negative correlation with obsession morbidity, however Kou Changgui think be whether the neurosal morbidity such as single-parent family and obsession without statistical correlations, may be different from selected objects relevant.
Because compulsive pathogenesis is more complicated, inherited genetic factors is occupied much ratios in compulsive pathogenesis, whether, with series of problems such as social environmental factor interactions, all needs further research.
What generally adopt for the detection of 5-HTTLPR polymorphism at present is polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method and sequencing.RFLP method pcr amplification target DNA, amplified production cuts into different big or small fragments with the digestion of specificity restriction endonuclease again, directly in gel electrophoresis, differentiates.Not homoallelic restriction enzyme site distributes different, produces the DNA fragmentation band of different lengths.The method is easy, result is easily judged, but also there is many drawbacks simultaneously, 1. the amplified production of target fragment is pure, under existing at nonspecific amplified production (particularly large fragment may contain restriction enzyme site), incomplete digestion may occur the amplified production of target fragment or enzyme is cut the assorted band of rear appearance; 2. the enzyme process of cutting is wanted fully (ratio that is substrate and enzyme is suitable, and digestion time will ensure), just can avoid false negative result to produce; 3. enzyme is cut positive findings and can be determined detected concrete sequence, and negative findings only can illustrate that the amplified production of target fragment is non-restriction endonuclease recognition sequence, but can not accurately judge concrete sequence; 4. enzyme recognition sequence, if any methylated base, will can not cut by restriction endonuclease so.Sequencing can judge the polymorphism of 5-HTTLPR more intuitively, but the polymorphism of 5-HTTLPR relates to disappearance or the insertion of large fragment base, thereby the possibility of error may appear detecting in order-checking.And the present invention adopts the directly method of aobvious band of pcr amplification reaction rear electrophoresis, can effectively overcome the defect that PCR-RFLP method and sequencing cause, thereby detect sample fast, accurately, simply and at low cost.
Summary of the invention
The object of the present invention is to provide the primer that detects 5-HTTLPR segment polymorphism, it is characterized in that, comprising: the forward and reverse primer of amplification 5-HTTLPR gene pleiomorphism; Its base sequence is:
HTTLPR-F：CAGCACCTAACCCCTAATGT
HTTLPR-R：GCCGGTTGGGCTAGCGTCTA
Further, the working concentration of described forward and reverse primer ratio is: HTTLPR-F:HTTLPR-R=1:1.
Further, described electrophoresis agarose working concentration is: 1%
Further, the amplification reaction condition of described forward and reverse amplimer is: first stage, 95 DEG C of denaturation 10min; Subordinate phase, 94 DEG C of 30sec of denaturation temperature, 64 DEG C of 90sec of annealing temperature, 72 DEG C of 30sec of elongating temperature, circulate 35 times; Phase III, 72 DEG C of 10min; Fourth stage, amplified reaction finishes, and amplified production is preserved at 4 DEG C.
A kind of method that the present invention also aims to provide the 5-HTTLPR of detection polymorphism, it comprises the steps:
(1) extract sample DNA;
(2) utilize pair for amplification primer HTTLPR-F and HTTLPR-R to increase to the DNA in (1), obtain amplified production;
(3) amplified production in (2) is carried out to agarose electrophoresis;
(4) band polymorphism sizes different from 5-HTTLPR in (3) are compared, determine whether polymorphism exists, and it is characterized in that,
HTTLPR-F：CAGCACCTAACCCCTAATGT
HTTLPR-R：GCCGGTTGGGCTAGCGTCTA
Further, the amplification reaction condition of step (2) is: first stage, 95 DEG C of denaturation 10min; Subordinate phase, 94 DEG C of 30sec of denaturation temperature, 64 DEG C of 90sec of annealing temperature, 72 DEG C of 30sec of elongating temperature, circulate 35 times; Phase III, 72 DEG C of 10min; Fourth stage, amplified reaction finishes, and amplified production is preserved at 4 DEG C.
The test kit that the present invention also aims to provide a kind of 5-HTTLPR of detection polymorphism, is characterized in that, described test kit comprises sample DNA extraction agent; Dehydrated alcohol; Detection system PCR reaction solution, order-checking system reaction solution, positive reference substance, negative control product and blank product, wherein detection system PCR reaction solution comprises pair for amplification product HTTLPR-F and HTTLPR-R, it is characterized in that:
HTTLPR-F：CAGCACCTAACCCCTAATGT
HTTLPR-R：GCCGGTTGGGCTAGCGTCTA。
Further, described test kit also comprises the reference sequences size of HTTLPR.
Beneficial effect of the present invention: the present invention has designed the forward and reverse primer of amplification 5-HTTLPR fragment, construct stable pcr amplification system, by adjusting the reaction conditions such as concentration, annealing temperature of forward and reverse primer, can make amplification efficiency reach best, simultaneously in the time of the increasing of 5-HTTLPR fragment, can the forward and reverse primer of enrichment and the correct pairing of sample DNA template after the specific amplification products that amplifies on a large scale, improve specific amplification.Moreover, in view of the S-allele of 5-HTTLPR is because the DNA sequence dna that allelic two the discontinuous regions of L lack respectively long 22bp produces, utilize the polymorphism of the traditional sequencing detection 5-HTTLPR such as Sanger order-checking can not accurately provide result, and the present invention adopts the directly method of aobvious band of pcr amplification reaction rear electrophoresis, under the same conditions, the positive reference substance for the treatment of inspection sample DNA and contain LS heterozygous genes type carries out pcr amplification reaction and agarose gel electrophoresis, thereby obtain the electrophorogram of sample amplification product and reference substance amplified production, distribute and just can determine the polymorphism of 5-HTTLPR fragment according to the band in electrophorogram, have directly perceived, simple to operate and low cost and other advantages.
Brief description of the drawings
Fig. 1 is the electrophorogram of the 5-HTTLPR fragment amplification product of 16 routine samples.
Fig. 2 is No. 83 sample Sanger sequencer maps.
Fig. 3 is No. 82 sample Sanger sequencer maps.
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: for example, 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
Detect the primer of 5-HTTLPR segment polymorphism, this primer comprises forward primer HTTLPR-F and the reverse primer HTTLPR-R of amplification 5-HTTLPR fragment, and its base sequence is:
HTTLPR-F：5’-CAGCACCTAACCCCTAATGT-3’
HTTLPR-R：5’-GCCGGTTGGGCTAGCGTCTA-3’。
In detection, utilize the 5-HTTLPR fragment in above-mentioned forward and reverse primer pair and positive reference substance and sample to be checked to increase, obtain amplified production, the then aobvious band of agarose gel electrophoresis, the specific band of acquisition amplified production.In the time that 5-HTTLPR fragment is LL homozygous genotype, only have the band of an amplified production, its size is 529bp; In the time that 5-HTTLPR fragment is SS homozygous genotype, only have the band of an amplified production, its size is 529bp; In the time that 5-HTTLPR fragment is LS heterozygous genes type, there is the band of two amplified productions, its size is respectively 529bp and 485bp.
Detect a test kit for 5-HTTLPR segment polymorphism, comprise sample DNA extraction agent; Detection system PCR reaction solution; Agarose gel electrophoresis system reagent; Positive reference substance, negative control product and blank product.
Sample DNA extraction agent: can select the extraction reagent that those skilled in the art know to carry out DNA extracting, also can use the test kit (as the DNA extraction test kit of TIANGEN Biotech (Beijing) Co., Ltd.) of biotech company's exploitation to carry out DNA extracting.
Detection system PCR reaction solution comprises: 2 × PCR Buffer; 2mM dNTPs; 1U/ μ l archaeal dna polymerase (as KOD FXDNA polysaccharase, Taq archaeal dna polymerase etc.); (10 μ m), (10 μ m) for HTTLPR-R for the forward and reverse primer HTTLPR-F of amplification 5-HTTLPR fragment.
Agarose gel electrophoresis system reagent comprises: 6 × sample-loading buffer, DNA molecular amount standard (DNA molecular weight marker), 50 × TAE electrophoretic buffer stock solution, 1 × TAE electrophoretic buffer working fluid, 1% agarose and 10mg/mL ethidium bromide (EB) solution.
6 × sample-loading buffer: 0.25% bromjophenol blue, 40% (W/V) aqueous sucrose solution, 4 DEG C of preservations.
DNA molecular amount standard: the DNA fragmentation by 100bp, 250bp, 500bp, 750bp, 1500bp and 2000bp size forms.
50 × TAE electrophoretic buffer stock solution: take 242g Tris, 37.2g Na
2eDTAH
2o, at ddH
2stirring and dissolving in O, then adds 57.1ml Glacial acetic acid, after fully stirring, uses ddH
2o is settled to 1000ml.
1 × TAE electrophoretic buffer working fluid: get 10ml50 × TAE electrophoretic buffer stock solution and join 490ml ddH
2in O.
1% agarose: 100ml1 for 1g agarose × TAE boiling water bath dissolves.
10mg/mL ethidium bromide solution: at 100ml ddH
2in O, add 1g ethidium bromide, magnetic agitation a few hours dissolve completely to guarantee it, are then transferred in brown bottle, are stored in room temperature.
Detection system PCR reaction solution reagent is formulated as follows:
Wherein, the base sequence of HTTLPR-F and HTTLPR-R is:
| Primer title | Base sequence |
| HTTLPR-F | 5’-CAGCACCTAACCCCTAATGT-3’ |
| HTTLPR-R | 5’ -GCCGGTTGGGCTAGCGTCTA -3’ |
Negative control product: the solution that does not contain 5-HTTLPR fragment.
Blank product: 2 μ l physiological saline or do not add any material.
Positive reference substance (positive control, PC): the solution that contains 5-HTTLPR fragment LS heterozygous genes type (simultaneously containing 5-HTTLPR reference sequences HTTLPR-L-ref and HTTLPR-S-ref).
HTTLPR-L-ref is 5-HTTLPR L allelotrope reference sequences, and length is 529bp, and the DNA sequence dna that in figure, first square frame frame is lived is the sequence of primer HTTLPR-F, and the DNA sequence dna that second square frame frame lived is the complementary sequence of primer HTTLPR-R.The base sequence of HTTLPR-L-ref is as follows:
ccctactgcagccctcccggcatcccccctgcaacctcccagcaactccctgtacccctcctaggatcgctcctgcatcccccattatcccccccttcaccctcgcggca
tcccccctgcaccccccagcatcccccctgcagcccccccagcatctcccctgcacccccagcatcccccctgcagcccttccagcatccccctgcacctctcccaggatctcccctgcaacccccattatcccccctgcacccctcgcagtatcccccctgcaccccccagca
tccccccatgcacccccggcatcccccctgcacccctccagcattctccttgcaccctaccagtattcccccgcatcccggcctccaagcctcccgcccaccttgcggtccccgccctggcgtctaggtggcaccagaatcccgcgcggactccacccgctgggagctgccctcgcttgcccgtggttgtccagctcagtccctc
HTTLPR-S-ref is 5-HTTLPR S-allele reference sequences, and length is 485bp, and the difference of its base sequence and HTTLPR-L-ref is the not DNA sequence dna containing underscore part in HTTLPR-L-ref.
Embodiment 2
Testing process:
(1) utilize blood DNA extraction agent box (TIANGEN Biotech (Beijing) Co., Ltd.) to extract the genomic dna in blood sample:
1) extract 500 μ l blood and add 1000 μ l erythrocyte cracked liquids, put upside down and mix, room temperature is placed 5 minutes, puts upside down and mixes several times more during this time.The centrifugal 5min of 3000rpm, sucks supernatant, leaves white corpuscle precipitation, adds 200 μ l damping fluid GA, and vibration is to thoroughly mixing.
2) add 20 μ l Proteinase K solution, mix.
3) add 200 μ l damping fluid GB, fully put upside down and mix, place 10 minutes for 70 DEG C, solution strain is limpid, brief centrifugal to remove the globule of cap wall.
4) add 200 μ l dehydrated alcohols, fully vibration mixes 15 seconds, now may occur flocks, brief centrifugal to remove the globule of cap wall.
5) previous step gained solution and flocks are all added in an adsorption column CB3 (adsorption column is put into collection tube), and 12,000rpm (13,400 × g) centrifugal 30 seconds, outwell waste liquid, adsorption column CB3 is put back in collection tube.
6) in adsorption column CB3, add 500 μ l damping fluid GD (please first check before use and whether added dehydrated alcohol), and 12,000rpm (13,400 × g) centrifugal 30 seconds, outwell waste liquid, adsorption column CB3 is put into collection tube.
7) in adsorption column CB3, add 700 μ l rinsing liquid PW (please first check before use and whether added dehydrated alcohol), and 12,000rpm (13,400 × g) centrifugal 30 seconds, outwell waste liquid, adsorption column CB3 is put into collection tube.
8) in adsorption column CB3, add 500 μ l rinsing liquid PW, and 12,000rpm (13,400 × g) centrifugal 30 seconds, outwell waste liquid.
9) adsorption column CB3 is put back in collection tube, and 12,000rpm (13,400 × g) centrifugal 2 minutes, outwell waste liquid.Adsorption column CB3 is placed in to room temperature and places several minutes, thoroughly to dry rinsing liquid remaining in sorbing material.
10) adsorption column CB3 is proceeded in a clean centrifuge tube, to the unsettled dropping 100 μ l elution buffer TE in middle part of adsorption film, room temperature is placed 2～5 minutes, 12,000rpm (13,400 × g) centrifugal 2 minutes, solution is collected in centrifuge tube, obtain poba gene group DNA solution.
(2) reagent configuration: configure the each X μ of the detection system PCR reaction solution l containing sample DNA template, every person-portion 18 μ l packing by detecting people's umber:
X=18 μ l reaction solution × (part negative control product+1, part positive reference substance+1, n part blood sample+1 part blank product), wherein n is for detecting sample number.
(3) application of sample: join 18 μ l using the poba gene group DNA solution obtaining in 2 μ l steps (1) as sample DNA template not containing in the detection system PCR reaction solution of sample DNA template; For positive control experiment, directly add 2 μ l positive reference substances and do not contain in the detection system PCR reaction solution of sample DNA template to 18 μ l; For negative control experiment, directly add 2 μ l negative control product and do not contain in the detection system PCR reaction solution of sample DNA template to 18 μ l; For blank experiment, add 2 μ l physiological saline and do not contain in the detection system PCR reaction solution of sample DNA template or do not add any material to 18 μ l.
(4) pcr amplification: detect and carry out on conventional PCR instrument, obtain pcr amplification product, available instrument comprises ABI veriti (Applied Biosystems company of the U.S.) etc.Reaction conditions is as follows:
(5) agarose gel electrophoresis
Operation steps is as follows:
A. gel shaped both mold ends is sealed with medical proof fabric, horizontal positioned, puts the comb of choosing well, between comb bottom and mould, stays 1mm space;
B. take the Erlenmeyer flask that DNA electrophoresis agarose 1g puts into 250ml, add 100ml1 × TAE damping fluid, after mixing, flask is placed on electric furnace, heated and boiled, until agarose dissolves completely;
C. close electric furnace, take off Erlenmeyer flask, put and under room temperature, be cooled to 70 DEG C of left and right (holding flask can tolerate), then add 10mg/ml ethidium bromide 5 μ l, after mixing, pour offset plate bed board into by gelating soln.This is tested glue plate used and approximately needs glue 100ml;
D. under room temperature, treat that gel solidifies completely, take approximately 30 minutes, throw off two end adhesive plasters, extract broach, offset plate is put into electrophoresis chamber;
E. add 1 × TAE damping fluid at electrophoresis chamber, be advisable to exceed gel surface 2mm;
F. by sample blending, of short duration centrifugal;
G. draw sample with sample injector, sequentially add respectively in point sample hole, notice that sample injector suction nozzle should be placed in gel point sample hole just, can not pierce through gel, also will prevent outside sample overfolw hole;
H. switch on power, regulating voltage to 80 volt, electrophoresis is after 30 minutes, gel slab taken out, the acquisition electrophorogram of taking pictures under ultraviolet lamp, and observe the pcr amplification product band of blood sample and positive reference substance in electrophorogram and distribute.
(6) result judgement: distribute according to the pcr amplification product band of blood sample and positive reference substance in the electrophorogram of observing in (5), determine the polymorphism of 5-HTTLPR fragment.
Embodiment 3 clinical samples detect
Fetch and deliver inspection patients with OCD lineal relative anticoagulation sample 16 examples, extract the genomic dna in anticoagulation sample by testing process described in embodiment 2, carry out pcr amplification reaction, obtain pcr amplification product, and pcr amplification product is carried out to agarose gel electrophoresis.
For every part of anticoagulation sample sample, the genomic dna solution that 2 μ l are extracted joins 18 μ l not containing in the detection system PCR reaction solution of sample DNA template, does the positive, feminine gender and blank experiment simultaneously.The regular-PCR instrument in 96 holes can detect 46 duplicate samples simultaneously, and each sample repeats for 2 times, a positive control, a negative control and a blank.Be 160 minutes detection time.
The amplified production of every duplicate samples genomic dna shows band through agarose gel electrophoresis, also same duplicate samples is carried out to traditional Sanger order-checking (while wherein order-checking, sequencing primer used is forward and reverse primer HTTLPR-F used in the present invention and HTTLPR-R) simultaneously, and the detected result of acquisition is compared.Detected result is as following table:
| Sample number into spectrum | Detected result of the present invention | The Sanger detected result that checks order | Compulsive ill risk assessment |
| 72 | LL | LL | Risk is high |
| 73 | LL | LL | Risk is high |
| 74 | SS | Fail to judge | Risk is low |
| 75 | LS | Fail to judge | There is risk, need periodic monitoring |
| 76 | SS | Fail to judge | Risk is low |
| 77 | SS | Fail to judge | Risk is low |
| 78 | SS | Fail to judge | Risk is low |
| 79 | LL | LL | Risk is high |
| 80 | SS | Fail to judge | Risk is low |
| 81 | SS | Fail to judge | Risk is low |
| 82 | LS | Fail to judge | There is risk, need periodic monitoring |
| 83 | SS | Fail to judge | Risk is low |
| 84 | LL | LL | Risk is high |
| 85 | LL | LL | Risk is high |
| 86 | SS | Fail to judge | Risk is low |
| 87 | LL | LL | Risk is high |
| PC | LS | Fail to judge | There is risk, need periodic monitoring |
From upper table and Fig. 1, the detected result of utilizing the present invention to detect positive reference substance (PC) is LS heterozygous genes type, still can not detect by Sanger method.Moreover, in positive reference substance (PC) and 16 routine samples, Sanger sequencing can only detect the sample of LL homozygous genotype, can not detect the sample of LS heterozygous genes type or SS homozygous genotype, can not detect positive reference substance (PC), this is the discontinuous disappearance that the S-allele of 5-HTTLPR exists large fragment, also may there is other base mutation simultaneously, and this can not reflect with order-checking well, (be LL homozygous genotype and detection method of the present invention can detect the 5-HTTLPR segment polymorphism of positive reference substance (PC) and 16 routine samples, LS heterozygous genes type or SS homozygous genotype), and its polymorphism can intuitively obtain result accurately by electrophorogram band, then provide risk assessment according to detected result, what is more important, the clinical experiment result of these detected results and risk assessment and patient's clinical symptom and hospital matches, this just suffices to show that detection method accuracy of the present invention is relatively good.Fig. 2 is the Sanger sequencer map of No. 83 samples, because of the peak of attaching most importance to, and can not judge its genotype, but according to detected result of the present invention, can judge that its genotype is SS homozygous genotype.Fig. 3 is the Sanger sequencer map of No. 82 samples, the sequence that order-checking is obtained and 5-HTTLPR reference sequences HTTLPR-L-ref and HTTLPR-S-ref contrast, discovery there is no identical place, thereby cannot differentiate its genotype, but according to detected result of the present invention, can judge that its genotype is LS homozygous genotype.Therefore,, with regard to detecting the polymorphism of 5-HTTLPR fragment, method provided by the invention can detect sample simply, fast, at low cost, thereby meets better clinical demand.
SEQUENCE LISTING
<110> ADICON Clinical Laboratories, Inc.
<120> detects method and the primer of 5-HTTLPR segment polymorphism
<130>
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213> artificial sequence
<400> 1
cagcacctaa cccctaatgt 20
<210> 2
<211> 20
<212> DNA
<213> artificial sequence
<400> 2
gccggttggg ctagcgtcta 20
<210> 3
<211> 529
<212> DNA
<213> HTTLPR-L-ref
<400> 3
cagcacctaa cccctaatgt ccctactgca gccctcccgg catcccccct gcaacctccc 60
agcaactccc tgtacccctc ctaggatcgc tcctgcatcc cccattatcc cccccttcac 120
cctcgcggca tcccccctgc accccccagc atcccccctg cagccccccc agcatctccc 180
ctgcaccccc agcatccccc ctgcagccct tccagcatcc ccctgcacct ctcccaggat 240
ctcccctgca acccccatta tcccccctgc acccctcgca gtatcccccc tgcacccccc 300
agcatccccc catgcacccc cggcatcccc cctgcacccc tccagcattc tccttgcacc 360
ctaccagtat tcccccgcat cccggcctcc aagcctcccg cccaccttgc ggtccccgcc 420
ctggcgtcta ggtggcacca gaatcccgcg cggactccac ccgctgggag ctgccctcgc 480
ttgcccgtgg ttgtccagct cagtccctct agacgctagc ccaaccggc 529
Claims (7)
1. the primer that detects 5-HTTLPR segment polymorphism, is characterized in that, described primer comprises the forward and reverse primer of amplification 5-HTTLPR fragment, and its base sequence is:
HTTLPR-F：5’-CAGCACCTAACCCCTAATGT-3’
HTTLPR-R：5’-GCCGGTTGGGCTAGCGTCTA-3’。
2. primer as claimed in claim 1, is characterized in that, the working concentration ratio of described forward and reverse primer is: HTTLPR-F: HTTLPR-R=1:1.
3. primer as claimed in claim 1, is characterized in that, 5-HTTLPR segment polymorphism is selected from LL homozygous genotype, LS heterozygous genes type and SS homozygous genotype.
4. primer as claimed in claim 1, is characterized in that, the amplification reaction condition of described forward and reverse amplimer is: first stage, 95 DEG C of denaturation 10min; Subordinate phase, 94 DEG C of 30sec of denaturation temperature, 58 DEG C of 90sec of annealing temperature, 72 DEG C of 30sec of elongating temperature, circulate 35 times; Phase III, 72 DEG C of 10min; Fourth stage, amplified reaction finishes, and amplified production is preserved at 4 DEG C.
5. a method that detects 5-HTTLPR segment polymorphism, it comprises the steps:
(1) extract sample DNA;
(2) utilize forward and reverse primer HTTLPR-F and HTTLPR-R to increase to the DNA in (1), obtain amplified production;
(3) amplified production in (2) is carried out to agarose gel electrophoresis, obtain electrophorogram;
(4) distribute according to the band in described electrophorogram, determine the polymorphism of 5-HTTLPR fragment, it is characterized in that,
HTTLPR-F：5’-CAGCACCTAACCCCTAATGT-3’
HTTLPR-R：5’-GCCGGTTGGGCTAGCGTCTA-3’。
6. method as claimed in claim 5, is characterized in that, the amplification reaction condition of step (2) is: first stage, 95 DEG C of denaturation 10min; Subordinate phase, 94 DEG C of 30sec of denaturation temperature, 64 DEG C of 90sec of annealing temperature, 72 DEG C of 30sec of elongating temperature, circulate 35 times; Phase III, 72 DEG C of 10min; Fourth stage, amplified reaction finishes, and amplified production is preserved at 4 DEG C.
7. method as claimed in claim 5, is characterized in that, in agarose gel electrophoresis, described agarose concentration is 1%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410258375.8A CN104120177A (en) | 2014-06-11 | 2014-06-11 | Method and primer for detecting polymorphism of 5-HTTLPR fragment |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410258375.8A CN104120177A (en) | 2014-06-11 | 2014-06-11 | Method and primer for detecting polymorphism of 5-HTTLPR fragment |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104120177A true CN104120177A (en) | 2014-10-29 |
Family
ID=51765807
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410258375.8A Pending CN104120177A (en) | 2014-06-11 | 2014-06-11 | Method and primer for detecting polymorphism of 5-HTTLPR fragment |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104120177A (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102202666A (en) * | 2007-12-14 | 2011-09-28 | H.隆德贝克有限公司 | Therapeutic uses of compounds having affinity to the serotonin transporter, serotonin receptors and noradrenalin transporter |
-
2014
- 2014-06-11 CN CN201410258375.8A patent/CN104120177A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102202666A (en) * | 2007-12-14 | 2011-09-28 | H.隆德贝克有限公司 | Therapeutic uses of compounds having affinity to the serotonin transporter, serotonin receptors and noradrenalin transporter |
Non-Patent Citations (3)
| Title |
|---|
| 冯冬梅: "《5_羟色胺转运体基因多态性与强迫症的相关性研究》", 《中国现代医生》, vol. 48, no. 2, 31 January 2010 (2010-01-31), pages 9 - 11 * |
| 刘恩益: "《五羟色胺转运体启动子区基因多态性与强迫症状和认知功能的关联研究》", 《中华临床医师杂志（电子版）》, vol. 7, no. 7, 30 April 2013 (2013-04-30), pages 2874 - 2876 * |
| 周云飞: "《5_羟色胺转运体启动子区基因多态性与强迫症的关联分析》", 《临床精神医学杂志》, vol. 17, no. 2, 30 April 2007 (2007-04-30), pages 76 - 78 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Kuo et al. | Identification of an association between genomic hypomethylation of FCGR2A and susceptibility to Kawasaki disease and intravenous immunoglobulin resistance by DNA methylation array | |
| CN105506106A (en) | Method and primer for detecting whole exons of FIX gene | |
| CN106399561A (en) | Prime, probe and kit for detecting polymorphism of CYP2C19 gene | |
| CN106544419A (en) | Detect primer, probe and the test kit of ApoE and SLCO1B1 gene pleiomorphisms | |
| O’Rielly et al. | Ankylosing spondylitis: beyond genome-wide association studies | |
| Arguello et al. | Origination of an X-linked testes chimeric gene by illegitimate recombination in Drosophila | |
| CN104745697B (en) | Detect the method and primer of NF1 the 31st No. 34 full extron of gene | |
| Kelly et al. | Next-generation sequencing and microarray-based interrogation of microRNAs from formalin-fixed, paraffin-embedded tissue: preliminary assessment of cross-platform concordance | |
| Kazantseva et al. | AVPR1A main effect and OXTR-by-environment interplay in individual differences in depression level | |
| CN105886606A (en) | Kit for rapid detection of polymorphism of Warfarin metabolic enzyme gene by virtue of pyrosequencing method and application of kit | |
| CN108531575A (en) | Detect primer, kit and the method for the full exon sequence mutation of TERC genes | |
| CN105441540A (en) | Non-syndromic deafness gene polymorphism detecting kit and application thereof | |
| CN107208314A (en) | A kind of construction method, classifying method and the reagent of haplotype parting sequencing library | |
| CN105969890A (en) | SNP (Single Nucleotide Polymorphism) molecular marker related to litter size of fragrant pig and application of SNP molecular marker | |
| CN104862407A (en) | Primer and method for detecting EZH2 genes | |
| CN101921852B (en) | Method for detecting single nucleotide polymorphism of cattle AdPLA gene | |
| Pirim et al. | Resequencing of LPL in African Blacks and associations with lipoprotein–lipid levels | |
| Gao et al. | Identification of key genes and underlying mechanisms in acute Kawasaki disease based on bioinformatics analysis | |
| Qian et al. | Genetic susceptibility to ulcerative colitis in the Chinese Han ethnic population: association with TNF polymorphisms | |
| CN106222287A (en) | The method of detection ELA2 gene and primer | |
| CN104120177A (en) | Method and primer for detecting polymorphism of 5-HTTLPR fragment | |
| CN103710438A (en) | Method and primers for detecting fifth exon mutation site of RUNX1 gene | |
| CN102796819A (en) | Kit for detecting microdeletion of Y chromosome | |
| Wang et al. | Variation analysis of PRIM1 gene in Chinese patients with primary ovarian insufficiency | |
| CN103540659A (en) | Method, primer and kit for detecting DNMT3A mutation site |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | 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: 20141029 |