CN104561285A - Amplification and sequencing primer and reagent kit for single nucleotide polymorphism detection of microRNA-146a (micro ribonucleic acid-146a) gene and in prozone - Google Patents
Amplification and sequencing primer and reagent kit for single nucleotide polymorphism detection of microRNA-146a (micro ribonucleic acid-146a) gene and in prozone Download PDFInfo
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Abstract
The invention discloses an amplification and sequencing primer and a reagent kit for single nucleotide polymorphism detection of a microRNA-146a (micro ribonucleic acid-146a) gene and in a prozone. The reagent kit comprises reaction liquid A for PCR (polymerase chain reaction) amplification, reaction tubes coating primers and primers S1, S2, S3 and S4 for sequencing. The primer can amplify segments where an SNP (single nucleotide polymorphism) in human miR-146a (micro ribonucleic acid-146a) and four SNPs in the gene prozone are located under the same reaction condition at one time, and then sequencing is carried out by the sequencing primers. The primers are directly coated in the reaction tubes to avoid difficulty in primer configuration. The kit can be used for detection of a clinical sample and provide more biological information of the polymorphism of miR-146a.
Description
Technical field
The present invention relates to the molecular biology method based on polymerase chain reaction (PCR) and DNA sequencing technology, particularly relate to a kind of amplification of detecting for 5 single nucleotide polymorphism (SNP) in microRNA (miR) 146a gene and gene proparea thereof and sequencing primer and test kit thereof.
Background technology
Microrna (microRNA, miR) is the strand non-coding RNA that endogenic length is about 19-21 Nucleotide, can regulate the expression of encoding gene.These RNA molecule are with the level of complicated mode Function protein instead of the expression suppressing target gene completely.Although these effects may be very slight, there is a large amount of genes to regulate and control by miR, show that they are very important Molecular regulators.Everybody admits miR and plays an important role in the biological action of many complexity at present, such as fetal development, tissue identification, propagation, differentiation, apoptosis, signal path, metabolism, tumour and virus infection etc.Recently also find that miR is relevant with autoimmune disorder.In numerous miR, miR-146a is study hotspot.
The biosynthetic process of this miRNA mainly comprises that miR-96 gene is transcribed, precursor RNA synthesis in core, core export, endochylema processing and assembling etc.MiR-146a rs2910164G/C allelotrope is positioned at the 60th Nucleotide of pre-miR-146a, namely on miR-146a.Allele C can cause the mispairing of pre-miR-146a hairpin structure, make △ G reduce to-40.3kcal/mol from-43.1kcal/mol, thus reduce the efficiency that the stability of pri-miR, the efficiency of pri-miR formation pre-miR or pre-miR form miR-146a.And rs2431697, rs57095329, rs6864584, rs2277920 are positioned at the intergenic region before miR-146a gene, the expression efficiency of miR-146a may be affected.At present, what these single nucleotide polymorphism researchs were more is and disease-related Journal of Sex Research comprise tumour, silver bits property sacroiliitis, multiple sclerosis, tuberculosis, systemic lupus erythematous and rheumatoid arthritis etc.
Summary of the invention
The object of the invention is to overcome in existing molecular diagnostic techniques detect the SNP type that miR-146a will comprise many, detect incomplete defect, there is provided a set of optimization primer for pcr amplification and order-checking, disposablely under the same conditions can carry out 4 PCR, smoothly the gene fragment comprising miR-146a be increased and checked order.
The present invention's second object is to provide a kind of this primer supporting and carries out the specific PCR response procedures that in miR-146a gene and front region sequence, 5 SNP detect, and reaches the object of rapid amplifying.
The present invention's the 3rd object is to provide a kind ofly carries out SNP in miR-146a gene and front region sequence thereof to clinical samples and carries out the fluorescent PCR amplification kit of sequential analysis.
Technical scheme of the present invention is summarized as follows:
The amplification detected for single nucleotide polymorphism in microRNA-146a gene and proparea and sequencing primer, comprising containing the amplification of rs2431697 nucleic acid fragment and sequencing primer is SEQ ID NO.1 and SEQ ID NO.2, be SEQ ID NO.3 and SEQ ID NO.4 containing the amplification of rs57095329 and rs6864584 nucleic acid fragment and sequencing primer, being SEQ ID NO.5 and SEQ ID NO.6 containing the amplification of rs2277920 nucleic acid fragment and sequencing primer, is SEQ ID NO.7 and SEQ ID NO.8 containing the amplification of rs2910164 nucleic acid fragment and sequencing primer.
Supporting amplification comprises the PCR reaction of rs2431697, rs57095329, rs6864584, rs2277920, rs2910164 nucleic acid fragment, is after 95 DEG C of denaturation 3min, 94 DEG C of sex change 15s, 56 DEG C of annealing 15s, 72 DEG C extend 35s, carry out 35 circulations, be then kept at 25 DEG C; Timely taking-up is checked order.
A kind of amplification for single nucleotide polymorphism detection in microRNA-146a gene and proparea and sequencing kit:
Reagent A: be made up of 2mL damping fluid, comprise
green I dyestuff, 0.2mM dNTPs (comprising dATP, dCTP, dGTP, dTTP), 80mM Tris-HCl, 80mM KCl, 40mM (NH4) 2SO4,6mM MgSO4,100U Taq archaeal dna polymerase;
Reagent S1: by for the sequencing primer 30 μ L aqueous solution containing rs2431697 nucleic acid fragment, comprise 4 μMs of SEQ ID NO.2;
Reagent S2: by for the sequencing primer 30 μ L aqueous solution containing rs57095329 and rs6864584 nucleic acid fragment, comprise 4 μMs of SEQ ID NO.4;
Reagent S3: by for the sequencing primer 30 μ L aqueous solution containing rs2277920 nucleic acid fragment, comprise 4 μMs of SEQ ID NO.6;
Reagent S4: by for the sequencing primer 30 μ L aqueous solution containing rs2910164 nucleic acid fragment, comprise 4 μMs of SEQ ID NO.8;
Bag is by the PCR reaction tubes of primer:
A reaction tubes: containing for the amplimer mixing composition containing rs2431697 nucleic acid fragment, comprise 1.5 × 10
-2nmol SEQ ID NO.1 and 1.5 × 10
-2nmol SEQ ID NO.2;
No. two reaction tubess: containing for the amplimer mixing composition containing rs57095329 and rs6864584 nucleic acid fragment, comprise 1.5 × 10
-2nmol SEQ ID NO.3 and 1.5 × 10
-2nmol SEQ ID NO.4;
No. three reaction tubess: containing for the amplimer mixing composition containing rs2277920 nucleic acid fragment, comprise 1.5 × 10
-2nmol SEQ ID NO.5 and 1.5 × 10
-2nmol SEQ ID NO.6;
No. four reaction tubess: containing for the amplimer mixing composition of A containing rs2910164 nucleic acid fragment, comprise 1.5 × 10
-2nmol SEQ ID NO.7 and 1.5 × 10
-2nmol SEQ ID NO.8;
No. five reaction tubess: same number pipe;
No. six reaction tubess: with No. two pipes;
No. seven reaction tubess: with No. three pipes;
No. eight reaction tubess: with No. four pipes.
Advantage of the present invention:
1) provide and a set ofly carry out the primer of fast PCR and the sequencing primer of optimization, the sequence of all 5 SNP of all miR-146a of disposable amplification is used for follow-up order-checking; 2) be with fluorescence dye in reaction system, directly can observe amplification by quantitative fluorescent PCR, the inconvenience avoiding electrophoresis to bring and pollution; 3) primer direct coated is in reaction tubes, avoids primer to configure difficulty.By optimization experiment reaction system and reaction conditions, form simple, quick, sensitive, special detection kit, can be used for the detection to clinical samples, can man power and material be reduced, more experiment information is provided.
Accompanying drawing explanation
Fig. 1 is the SNP distribution in miR-146a gene and proparea.
Fig. 2 behaves the sequencing result of SNP in miR-146a gene and proparea.
Embodiment
In following embodiment, method therefor is ordinary method if no special instructions.
Amplification of the present invention and sequencing primer nucleotides sequence are classified as:
SEQ ID NO.1 5’-attatgattggcaaatggaagg-3’
SEQ ID NO.2 5’-tttggctggatggaggat-3’
SEQ ID NO.3 5’-attgggcagccgataaag-3’
SEQ ID NO.4 5’-ctcctcgttgtgctactgtctc-3’
SEQ ID NO.5 5’-ggagacagtagcacaacgag-3’
SEQ ID NO.6 5’-tagccatagtcttccaaccc-3’
SEQ ID NO.7 5’-cccacatcagccttccag-3’
SEQ ID NO.8 5’-ccaggtcctcaagcccac-3’
Embodiment 1
A set of for the amplification of SNP detection in people miR-146a gene and proparea and the Auele Specific Primer of order-checking, comprising containing the amplification of rs2431697 nucleic acid fragment and sequencing primer is SEQ ID NO.1 and SEQ ID NO.2, be SEQ ID NO.3 and SEQ ID NO.4 containing the amplification of rs57095329 and rs6864584 nucleic acid fragment and sequencing primer, being SEQ ID NO.5 and SEQ ID NO.6 containing the amplification of rs2277920 nucleic acid fragment and sequencing primer, is SEQ ID NO.7 and SEQ ID NO.8 containing the amplification of rs2910164 nucleic acid fragment and sequencing primer.
Embodiment 2
For in miR-146a gene and front region sequence thereof SNP detect amplification and a sequencing kit, be made up of following reagent:
Reagent A: be made up of 2mL damping fluid, comprise
green I dyestuff, 0.2mM dNTPs (comprising dATP, dCTP, dGTP, dTTP), 80mM Tris-HCl, 80mM KCl, 40mM (NH4) 2SO4,6mM MgSO4,100U Taq archaeal dna polymerase;
Reagent S1: by for the sequencing primer 30 μ L aqueous solution containing rs2431697 nucleic acid fragment, comprise 4 μMs of SEQ ID NO.2;
Reagent S2: by for the sequencing primer 30 μ L aqueous solution containing rs57095329 and rs6864584 nucleic acid fragment, comprise 4 μMs of SEQ ID NO.4;
Reagent S3: by for the sequencing primer 30 μ L aqueous solution containing rs2277920 nucleic acid fragment, comprise 4 μMs of SEQ ID NO.6;
Reagent S4: by for the sequencing primer 30 μ L aqueous solution containing rs2910164 nucleic acid fragment, comprise 4 μMs of SEQ ID NO.8;
Bag is by the PCR reaction tubes of primer:
A reaction tubes: containing for the amplimer mixing composition containing rs2431697 nucleic acid fragment, comprise 1.5 × 10
-2nmol SEQ ID NO.1 and 1.5 × 10
-2nmol SEQ ID NO.2;
No. two reaction tubess: containing for the amplimer mixing composition containing rs57095329 and rs6864584 nucleic acid fragment, comprise 1.5 × 10
-2nmol SEQ ID NO.3 and 1.5 × 10
-2nmol SEQ ID NO.4;
No. three reaction tubess: containing for the amplimer mixing composition containing rs2277920 nucleic acid fragment, comprise 1.5 × 10
-2nmol SEQ ID NO.5 and 1.5 × 10
-2nmol SEQ ID NO.6;
No. four reaction tubess: containing for the amplimer mixing composition of A containing rs2910164 nucleic acid fragment, comprise 1.5 × 10
-2nmol SEQ ID NO.7 and 1.5 × 10
-2nmol SEQ ID NO.8;
No. five reaction tubess: same number pipe;
No. six reaction tubess: with No. two pipes;
No. seven reaction tubess: with No. three pipes;
No. eight reaction tubess: with No. four pipes.
Embodiment 3
All exons of real-time fluorescence quantitative PCR (the real-time FQ PCR) androgen receptor that increases are carried out with test kit of the present invention.First, sample number empirically calculates the requirement of each reagent, and experimental procedure is as follows:
1. amplification reaction solution configuration: configure reaction solution (1 is detected sample) in order, add water 120 μ L, reagent A 150 μ L, DNA sample 30 μ L, totally 300 μ L; Mixing.
2. each pipe is sequentially added into the amplification reaction solution of 25 μ L, builds lid.
3. the PCR program empirically designed increases: be after 95 DEG C of denaturation 3min, 94 DEG C of sex change 1min, 56 DEG C of annealing 1mins, and 72 DEG C extend 35s, and extended peroid obtains fluorescent value, carries out 35 circulations, is finally kept at 25 DEG C; Timely taking-up is checked order.
4. fluorescent value is 22-28 circulation take-off, and specific amplification is thought in the take-off simultaneously of each group, sees Fig. 1.
Embodiment 4
The primer supporting with test kit of the present invention checks order:
To detect 1 sample, 4 PCR check order as example is carried out.
1.PCR product purification (ExoI/SAP method):
1.1 preparation purification system: exonuclease 1 (ExoI) 3 μ L+ shrimp solution enzyme (SAP) 6 μ L+ water 3 μ L, totally 12 μ L, mixing.
The 1.2 purification system 2 μ L getting preparation mix (4) with each amplified production 8 μ L totally, add in another blank Sptting plate.
Sptting plate is placed on more than PCR instrument by 1.3,37 DEG C of 30min, 80 DEG C of 20min, can preserve about 1 week in 4 DEG C of refrigerators.
2. sequencing reaction:
In 2.110 μ L PCR purified products, every hole adds 20 μ L water, fully after mixing, centrifugal.
2.2 preparation sequencing reaction systems: get 4.8 μ L BDT Ready Reaction Premix (2.5 ×) and add 21.6 μ L BDT damping fluid (5 ×) and add 69.6 μ L Deionised water and mix.Get the above-mentioned mixed solution of 8 μ L each 1 μ L fully mixes with S1, S2, S3, S4 respectively at every turn, centrifugal fast.
2.3 every hole PCR primer diluents get 1 μ L to (4 hole) in reacting hole, and every hole adds 9 μ L sequencing reaction mixed solutions, mixing, centrifugal fast.
Sptting plate is placed in PCR instrument by 2.4, and PCR sealing load pad is put at Sptting plate top, to prevent liquid evaporation, shuts PCR instrument, runs sequencing reaction: after 96 DEG C of 10s, carrying out 25 circulations is 96 DEG C of 10s, 50 DEG C of 10s, 60 DEG C of 2min, finally preserves 15 DEG C and takes out.
3. sequencing reaction product purification:
1 μ LEDTA, 1 μ L NaAc and 25 μ L dehydrated alcohols are added, fully concussion mixing 30s in 3.1 sequencing reaction hole, every holes.Room temperature places the centrifugal 30min. of 15min, 3000rpm
3.2 are inverted Sptting plate, adjusting rotary speed, the centrifugal 1min of 500rpm
50 μ L 70% ethanol are added, the centrifugal 10min of 3000rpm in 3.3 every hole sequencing reaction products.
3.4 are inverted Sptting plate, the centrifugal 1min of 500rpm.
3.5 room temperature lucifuges leave standstill 30min, fully ethanol in volatilization plate.
Add methane amide 10 μ L, shrouding in 3.6 every holes, be placed on more than PCR 95 DEG C of 2min, put into rapidly-20 DEG C of refrigerator cooling 3-5min.
4. sequenator reads plate detection: use ABI3500 sequenator to detect sequencing result.The sequencing result software that makes to check order accordingly carries out data analysis, sees Fig. 2.
Embodiment 5
Sequencing result and miR-146a gene and proparea sequence criteria sequence thereof are compared:
1. sequencing result carry out observing whether have bimodal, prompting heterozygote;
2. do not find bimodal, standard order-checking and sequencing result Input Software being compared, is homozygote;
3. pair position different from standard sequence is analyzed, and is above-mentionedly decided to be new catastrophe point as non-.
Sequence table
<110> The First Affiliated Hospital of Wenzhou Medical University
<120> is used for amplification that in microRNA-146a gene and proparea, single nucleotide polymorphism detects and sequencing primer and test kit thereof
<160> 8
<170> PatentIn Version 2.1
<210> 1
<211> 22
<212> DNA
<213> artificial sequence
<220>
<221> prim_bind
<222> (1)...(22)
<400> 1
attatgattg gcaaatggaa gg 22
<210> 2
<211> 18
<212> DNA
<213> artificial sequence
<220>
<221> prim_bind
<222> (1)...(18)
<400> 2
tttggctgga tggaggat 18
<210> 3
<211> 18
<212> DNA
<213> artificial sequence
<220>
<221> prim_bind
<222> (1)...(18)
<400> 3
attgggcagc cgataaag 18
<210> 4
<211> 22
<212> DNA
<213> artificial sequence
<220>
<221> prim_bind
<222> (1)...(22)
<400> 4
ctcctcgttg tgctactgtc tc 22
<210> 5
<211> 20
<212> DNA
<213> artificial sequence
<220>
<221> prim_bind
<222> (1)...(20)
<400> 5
ggagacagta gcacaacgag 20
<210> 6
<211> 20
<212> DNA
<213> artificial sequence
<220>
<221> prim_bind
<222> (1)...(20)
<400> 6
tagccatagt cttccaaccc 20
<210> 7
<211> 18
<212> DNA
<213> artificial sequence
<220>
<221> prim_bind
<222> (1)...(18)
<400> 7
cccacatcag ccttccag 18
<210> 8
<211> 18
<212> DNA
<213> artificial sequence
<220>
<221> prim_bind
<222> (1)...(18)
<400> 8
ccaggtcctc aagcccac 18
Claims (2)
1. the amplification for single nucleotide polymorphism detection in microRNA-146a gene and proparea and sequencing primer, it is characterized in that on the basis of optimize PCR design of primers, disposablely under the same conditions can carry out 4 polymerase chain reactions, complete the amplification of the nucleic acid fragment containing all 5 SNP; And by sequencing primer, order-checking difference is carried out to 5 SNP, comprising containing the amplification of rs2431697 nucleic acid fragment and sequencing primer is SEQ ID NO.1 and SEQ ID NO.2, be SEQ ID NO.3 and SEQ ID NO.4 containing the amplification of rs57095329 and rs6864584 nucleic acid fragment and sequencing primer, being SEQ ID NO.5 and SEQ ID NO.6 containing the amplification of rs2277920 nucleic acid fragment and sequencing primer, is SEQ ID NO.7 and SEQ ID NO.8 containing the amplification of rs2910164 nucleic acid fragment and sequencing primer.
2., for amplification and the sequencing kit of single nucleotide polymorphism detection in microRNA-146a gene and proparea, it is characterized in that being made up of following reagent:
Reagent A: be made up of 2mL damping fluid, comprises 2 × SYBR Green I dyestuff, 0.2mM dNTPs, 80mM Tris-HCl, 80mM KCl, 40mM (NH4) 2SO4,6mM MgSO4,100U Taq archaeal dna polymerase;
Reagent S1: by for the sequencing primer 30 μ L aqueous solution containing rs2431697 nucleic acid fragment, comprise 4 μMs of SEQ ID NO.2;
Reagent S2: by for the sequencing primer 30 μ L aqueous solution containing rs57095329 and rs6864584 nucleic acid fragment, comprise 4 μMs of SEQ ID NO.4;
Reagent S3: by for the sequencing primer 30 μ L aqueous solution containing rs2277920 nucleic acid fragment, comprise 4 μMs of SEQ ID NO.6;
Reagent S4: by for the sequencing primer 30 μ L aqueous solution containing rs2910164 nucleic acid fragment, comprise 4 μMs of SEQ ID NO.8;
Bag is by the PCR reaction tubes of primer:
A reaction tubes: containing for the amplimer mixing composition containing rs2431697 nucleic acid fragment, comprise 1.5 × 10
-2nmol SEQ ID NO.1 and 1.5 × 10
-2nmol SEQ ID NO.2;
No. two reaction tubess: containing for the amplimer mixing composition containing rs57095329 and rs6864584 nucleic acid fragment, comprise 1.5 × 10
-2nmol SEQ ID NO.3 and 1.5 × 10
-2nmol SEQ ID NO.4;
No. three reaction tubess: containing for the amplimer mixing composition containing rs2277920 nucleic acid fragment, comprise 1.5 × 10
-2nmol SEQ ID NO.5 and 1.5 × 10
-2nmol SEQ ID NO.6;
No. four reaction tubess: containing for the amplimer mixing composition of A containing rs2910164 nucleic acid fragment, comprise 1.5 × 10
-2nmol SEQ ID NO.7 and 1.5 × 10
-2nmol SEQ ID NO.8;
No. five reaction tubess: same number pipe;
No. six reaction tubess: with No. two pipes;
No. seven reaction tubess: with No. three pipes;
No. eight reaction tubess: with No. four pipes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410828052.8A CN104561285A (en) | 2014-12-26 | 2014-12-26 | Amplification and sequencing primer and reagent kit for single nucleotide polymorphism detection of microRNA-146a (micro ribonucleic acid-146a) gene and in prozone |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410828052.8A CN104561285A (en) | 2014-12-26 | 2014-12-26 | Amplification and sequencing primer and reagent kit for single nucleotide polymorphism detection of microRNA-146a (micro ribonucleic acid-146a) gene and in prozone |
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| Publication Number | Publication Date |
|---|---|
| CN104561285A true CN104561285A (en) | 2015-04-29 |
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| CN201410828052.8A Pending CN104561285A (en) | 2014-12-26 | 2014-12-26 | Amplification and sequencing primer and reagent kit for single nucleotide polymorphism detection of microRNA-146a (micro ribonucleic acid-146a) gene and in prozone |
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| CN (1) | CN104561285A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106636307A (en) * | 2015-11-02 | 2017-05-10 | 广东医学院 | Application of miRNA-146a mutant in preparing test strip for early screening and rapid diagnosis of cervical cancer |
| CN109337960A (en) * | 2018-11-14 | 2019-02-15 | 昆明新开源暾秀生物科技有限公司 | A kind of diagnostic kit and its detection method of epilepsy in childhood |
-
2014
- 2014-12-26 CN CN201410828052.8A patent/CN104561285A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| LIN WEI ET AL.: "MicroRNA-146a and Ets-1 Gene Polymorphisms Are Associated with Pediatric Uveitis", 《PLOS ONE》 * |
| SE LOFGREN ET AL.: "Genetic association of miRNA-146a with systemic lupus erythematosus in Europeans through decreased expression of the gene", 《GENES AND IMMUNITY》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106636307A (en) * | 2015-11-02 | 2017-05-10 | 广东医学院 | Application of miRNA-146a mutant in preparing test strip for early screening and rapid diagnosis of cervical cancer |
| CN109337960A (en) * | 2018-11-14 | 2019-02-15 | 昆明新开源暾秀生物科技有限公司 | A kind of diagnostic kit and its detection method of epilepsy in childhood |
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