CN108396066A - Primer, probe and the kit of BIM genetic polymorphism detections - Google Patents
Primer, probe and the kit of BIM genetic polymorphism detections Download PDFInfo
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Abstract
The invention discloses the BIM gene pleiomorphism primers based on TaqMan fluorescence probes, probe and detection kit, the kit include the primer and probe for BIM_Exon2_3 and/or BIM_Exon2_4；The BIM_Exon2_3 primers that are directed to are as shown in SEQ ID NO.1 and SEQ ID NO.2, and the probe for BIM_Exon2_3 is as shown in SEQ ID NO.11；The primer for BIM_Exon2_4 is as shown in SEQ ID NO.3 and SEQ ID NO.4, and the probe for BIM_Exon2_4 is as shown in SEQ ID NO.12.The BIM gene specific Taqman probes that the present invention designs have sequence-specific, are bonded only to complementary region, and fluorescence signal and the copy number of amplification have one-to-one relationship, therefore high specificity, high sensitivity.Even if sample target gene content can detect if too low, sample B IM gene delection polymorphic detection susceptibilitys are improved.
Description
Technical field
The invention belongs to genetic test field, the BIM gene pleiomorphisms based on TaqMan fluorescence probes are particularly related to
Detection primer, probe and kit.
Background technology
Real-Time Fluorescent Quantitative PCR Technique efficiently solves traditional quantitative PCR and (does not set probe or mixed without real-time fluorescence dyestuff
Enter) limitation that PCR product is detached, carries out end point determination can only be taken out, it is primary glimmering to realize the detection of each round cycle
The intensity of optical signal, and be recorded among computer software, by each sample Ct values (recurring number for reaching threshold signal)
It calculates, quantitative result is obtained according to standard curve.Fluorescent material used in real-time fluorescence quantitative PCR can be divided into two kinds：Fluorescence
Probe and fluorescent dye.Most currently used real-time (real-time) round pcr is the fluorescent dye determinations such as SYBR Green.
Excess SYBR fluorescent dyes are added in PCR reaction systems in this method, after SYBR fluorescent dyes non-specifically mix DNA double chain,
Emit fluorescence signal, any fluorescence signal will not be emitted without mixing the SYBR dye molecules in chain, to ensure fluorescence signal
Increase and PCR product increase it is fully synchronized.SYBR is only combined with double-stranded DNA, by solubility curve, determines that PCR is anti-
Should whether special.Such method also has some limitations, requires high DNA or RNA quantitatively to detect for example, being not suitable for specificity；
The specific sequence that SYBR fluorescent dye determinations lack fluorescence probe combines, and only relies on the sequence-specific of primer, relatively poor.And
Do not have stringent correspondence because non-specific PCR reacts sometimes between fluorescence signal and the copy number of amplification, therefore total
Specificity, sensitivity are relatively poor.
With deeply developing for technical staff, TaqMan fluorescence probe methods are gradually widely used, and principle expands for PCR
A specific fluorescence probe is added when increasing while pair of primers is added, which is an oligonucleotides, both ends difference
One reporter fluorescence group of label and a quenching fluorescence group.When probe is complete, the fluorescence signal of reporter group transmitting is quenched
Go out group absorptions；When PCR amplification, the 5'-3' 5 prime excision enzyme activities of Taq enzyme degrade probe digestion, make reporter fluorescence group and quench
The fluorophor that goes out detaches, and to which fluorescence monitoring system can receive fluorescence signal, as soon as often expanding a DNA chain, there are one glimmering
Optical molecule is formed, realize the accumulation of fluorescence signal and PCR product formed it is fully synchronized.And novel TaqMan-MGB probes make this
Technology can not only carry out gene quantification analysis, but can analyzing gene mutation (such as SNP, Indel, splicing variation), be expected to become base
Because of the one preferred technique platform diagnosed and personalized medicine is analyzed.
BIM entitled BCL2-like 11 entirely are BCL-2 protein family members, are one of strongest pro apoptotic proteins of activity,
BIM contains BH3 structural domains, which contains important apoptotic function.BIM and other anti-apoptotic of BCL-2 protein families at
Member such as BCL2, BCL2L1/BCL-X (L) and MCL1 interact, and mutually adjust, adjust apoptosis process.BIM genes are through turning
A variety of transcripts are formed after record, and then translates and forms multiple protein isomers, and only 4 extras are aobvious in a usual isomer protein
Son or 3 exon coding informations, the two will not coexist.4 exons (exon 4), which encode structural domain containing BH3, has rush apoptosis
The isomers of function, and 3 exons (exon 3) coded product is free of BH3 functional domains without apoptotic function.East Asia
There are deletion polymorphisms for No. 2 intrones (intro 2) of No. 2 of BIM genes between 3 exons in crowd.Research is found
The excalation of this intron sequences can cause the BIM albumen for encoding no pro-apoptosis bioactivity to increase, thereby increases and it is possible to cause to lung cancer
In targeted drug EGFR TKI or BCR-ABL kinase inhibitors Imatinib initial drug-resistant or weaken TKI clinical efficacy.
According to studying before this, the BIM del polymorphism carrying rates of Chinese patients are 12.3%, and human genome monoploid schemes (Hap
Map) Chinese's BIM del carrying rates in the works are 20.5%.One related to TKI therapeutic responses to BIM del polymorphisms
The research (Ng study) of property points out that BIM deletion polymorphisms are one of drug resistant mechanism of primary TKI.BIM gene delections are polymorphic
Property presence, under antitumor drug effect in the apoptosis process that induces, do not contain the BIM γ eggs of BH3 homeodomains
White up-regulated expression, and the major protein Bim EL for playing apoptosis-promoting effect are reduced, and cause a large amount of non-functional BIM albumen cannot
It is combined with anti-apoptotic proteins by BH3 structural domains, neutralizes the effect of anti-apoptotic, be unable to activated b ax and Bak and inspire endogenous apoptosis
Approach, the phenomenon that generate endogenous drug resistance.
In conclusion BIM may be with the relevant small molecule of diseases such as lung cancer, chronic myelocytic leukemia (CML), breast cancer
Targeted drug primary drug resistance or unsatisfactory curative effect are related；The potential countermeasure for improving BIM del patient's curative effects may have BIM simulations
Object, BCL-2 inhibitor, hdac inhibitor.These hypothesis also need further prospective trial research verification.In confirmatory study or
In novel pro-apoptotic drug experimental study, the molecular detection technology of BIM is particularly important, and BIM molecular function state-detections contribute to
Clinic TKI or predication of chemotherapy effect ability are improved, or is provided with diagnosis for the use in conjunction of targeted drug and pro-apoptotic drug
Technology.
Invention content
An object of the present invention is to provide a kind of BIM genetic polymorphism detection reagents based on TaqMan fluorescence probes
Box, the kit have specificity and sensitivity well.
Realize that the technical solution of the above method is as follows.
BIM genetic polymorphism detection kits based on TaqMan fluorescence probes, include primer and probe, the primer
Sequence it is at least a pair of in following：For the SEQ ID NO.1 and SEQ ID NO.2 of BIM_Exon2_3；For BIM_
The SEQ ID NO.3 and SEQ ID NO.4 of Exon2_4；
Correspondingly, the probe in following at least one：For the SEQ ID NO.11 of BIM_Exon2_3；Needle
To the SEQ ID NO.12 of BIM_Exon2_4.
Further, further include the primer and probe for BIM E2 having as a contrast, the primer for BIM E2
As shown in SEQ ID NO.5 and SEQ ID NO.6, the probe for BIM E2 is as shown in SEQ ID NO.13.
Further, further include primer for ACTB_Exon4_5 and/or GAPD_Exon2_3 and the spy having as a contrast
Needle, the primer for ACTB_Exon4_5 is as shown in SEQ ID NO.7 and SEQ ID NO.8, for ACTB_Exon4_5
Probe be as shown in SEQ ID NO.14；The primer for GAPD_Exon2_3 such as SEQ ID NO.9 and SEQ ID
Shown in NO.10, the probe for GAPD_Exon2_3 is as shown in SEQ ID NO.15.
Preferably, the primer includes above five pairs, and the probe includes above five.
Another object of the present invention provides the above-mentioned primer and/or probe of BIM genetic polymorphism detections.
Primer and probe of the present invention designs more previously reported difference, and exon is used in Nature magazines
Inner primer PCR method does not carry out further improving specificity, as common PCR reaction methods using probe.The present invention not only increases
Except the design for having added probe, it is often more important that, the position of probe is creatively set in the of BIM genes by inventor
The joint (joint) of 2 exons and the 3rd exon, the 2nd exon and the 4th exon joint
(joint), the specificity of probe is greatly strengthened in this way so that PCR reaction products are not done by latent gene group DNA pollution
It disturbs.On the basis of optimizing PCR specific primers pair, the MGB for combining more special across exon joint (joint) is visited
Needle so that the PCR amplification in the present invention has the specificity of dual guarantee product.In the present invention, as long as occurring across outside the 2nd and the 3rd
The PCR product for showing sub- probe signals is the presence for showing to have deletion polymorphism, as long as occurring believing across the 2nd and the 4th exon probe
Number PCR product be show functional property BH3 structural domains BIM expression.
BIM genetic polymorphism detection kits of the present invention based on TaqMan fluorescence probes have the following advantages：
1, the BIM gene specific Taqman probes that the present invention designs have sequence-specific, are bonded only to complementary region, and
Fluorescence signal and the copy number of amplification have one-to-one relationship, therefore high specificity, high sensitivity.Even if target base in sample
It because content is too low, can also detect, improve sample B IM gene delection polymorphic detection susceptibilitys.
2, probe specificity is strong, is not easy non-specific amplification occur in PCR, successfully avoids the sequence of target gene homology,
Reduce false positive mutation rate.
3, since BIM missings are endogenous medicament-resistant mutation, BIM deletion mutant shapes can be detected using blood preparation
State, the limitation that knurl sample could detect need to be obtained in the past by changing, and kit of the present invention is easy to get with sample, feasible
The stronger advantage of property.
4, the Non-specific BIM gene pleiomorphisms missing probe that the present invention designs may be used TaqMan methods and detect a variety of samples
Product；It is easy to operate.
Description of the drawings
Fig. 1 is the result schematic diagram of qPCR methods detection BIM gene delection polymorphism ' negative ' specimens.
Fig. 2 is the result schematic diagram of Sanger direct sequencings detection BIM gene delection polymorphism ' negative ' specimens.
Fig. 3 is the result schematic diagram of qPCR methods detection BIM gene delection polymorphism positive samples.
Fig. 4 is the result schematic diagram of Sanger direct sequencings detection BIM gene delection polymorphism positive samples.
Fig. 5 is the result schematic diagram of the qPCR methods detection BIM gene delection polymorphisms of control group 1.
Specific implementation mode
It to facilitate the understanding of the present invention, below will be to invention is more fully described.The present invention can be with many not
With form realize, however it is not limited to embodiment described herein.Make to this on the contrary, purpose of providing these embodiments is
The understanding of the disclosure of invention is more thorough and comprehensive.
Unless otherwise defined, all technical and scientific terms used in the present invention and the technical field for belonging to the present invention
The normally understood meaning of technical staff it is identical.The term used in the description of the present invention is intended merely to describe specific reality
The purpose for applying example is not used in the limitation present invention.Term "and/or" used in the present invention includes one or more relevant listed
Any and all combinations of project.
The BIM genetic polymorphism detection kits based on TaqMan fluorescence probes described in the present embodiment, including primer and
Probe, moreover, selecting the 2nd exon expression using ACTB (beta actin), GAPD and BIM as reference and equal
One changes (normalization), and capable of more reliablely and stablely reacting BIM, whether there is or not the expression water of functional different transcripts
It is flat.
Sequence ttgcggcgtattggagacgagtttaac in the 4th exon of BIM genes corresponds to coded amino acid sequence
It is classified as LRRIGDEFN, here it is the BH3 structural domains of BIM apoptotic functions.The specific primer and probe sequence composition of the present embodiment is such as
Under.
1.BIM_Exon2_3：
Forward Primer SEQ ID NO.1GCCAGGCCTTCAACCACTATC
Reverse Primer SEQ ID NO.2AGCACAGTGAAAGATCACCTATATCCT
2.BIM_Exon2_4：
Forward Primer SEQ ID NO.3GTATTGGAGACGAGTTTAACGCTTACT
Reverse Primer SEQ ID NO.4TCGGCTGCTTGGTAATTATTCA
3.BIM Exon2:
Forward Primer SEQ ID NO.5TGCGAACCCTGCCACACT
Reverse Primer SEQ ID NO.6GAACGCAGCGAACCGAAT
4.ACTB_Exon4_5：
Forward Primer SEQ ID NO.7GGCACCCAGCACAATGAAG
Reverse Primer SEQ ID NO.8CCGATCCACACGGAGTACTTG
5.GAPD_Exon2_3：
Forward Primer SEQ ID NO.9TTCATTGACCTCAACTACATGGTTTAC
Reverse Primer SEQ ID NO.10GACGGTGCCATGGAATTTG
Probe sequence：
1.BIM_Exon2_3_PandP_1SEQ ID NO.11CAGTGCAATGGTAGTCAT
2.BIM_Exon2_4_PandP_1SEQ ID NO.12TGCAAGGAGGGTATTT
3.BIM Exon2PandP_1SEQ ID NO.13ATCGCATCATCGCGG
4.ACTB_Exon4_5_PandP_V2SEQ ID NO.14CAAGATCATTGCTCCTCCT
5.GAPD_Exon2_3_PandP_1SEQ ID NO.15TTCCAATATGATTCCACCCAT
(primed probe pays attention to being protected from light -20 degree preservations).
Inventor has found in research and development, different primers and between designing probe, testing result difference or bigger, example
Such as primer and probe of the control group high with above-mentioned primer and probe composition similarity-rough set.
The probe and primer of control group 1 are as follows：
Primer：
1.ACTB_Exon3_4_PandP_1
Forward Primer CGAGAAGATGACCCAGATCATG SEQ ID NO.19
Reverse Primer ACAGCCTGGATAGCAACGTACA SEQ ID NO.20
2.GAPD_Exon2_3_PandP_1
As hereinbefore.
3.BIM Exon2PandP_1
Forward Primer AAACGCAAGAAAAAAAGACCAAA SEQ ID NO.21
Reverse Primer CCTTCTCGGTCACACTCAGAACT SEQ ID NO.22
4.BIM_Exon2_3_PandP_1
As hereinbefore.
5.BIM_Exon2_4_PanP_1
Forward Primer CAGTGCAATGGCTTCCATGA SEQ ID NO.23
Reverse Primer GATCCATATCTCTGGGCGCATA SEQ ID NO.24
Probe：
1.BIM Exon2PandP_1CAAAGCAACCTTCTGATG SEQ ID NO.16
2.BIM_Exon2_3_PandP_1CAGTGCAATGGTAGTCAT(SEQ ID NO.11)
3.BIM_Exon2_4_PanP_1CAGGCTGAACCTGC SEQ ID NO.17
4.ACTB_Exon3_4_PandP_1AGACCTTCAACACCC SEQ ID NO.18
5.GAPD_Exon2_3_PandP_1SEQ ID NO.15
5 ' ends of every probe are marked with reporter group (Reporter, R) such as FAM, VIC；3 ' end marks of every probe
Note has fluorescent quenching group (Quencher, Q) such as TAMRA.
BIM real-time fluorescent quantitative RT-PCR methods：
QPCR materials and methods
One, total tissue RNA is extracted
1, using QIAsymphony RNA Kit (QIAGEN) and QIAsymphony SP automatic sample processing workstations
Extract the total serum IgE in tissue samples (with reference to product description).
2, using the concentration and quality of 2000 spectrophotometric determination RNA of Thermo NanoDrop (including OD260/
280, OD230/260), RNA freezes for use in -80 DEG C.
Two, RNA reverse transcriptions
The RNA extracted is reversed using PrimeScript RT reagent Kit (TaKaRa Bio Inc.)
It records (with reference to product description), RNA template quantities are that 1 μ g, cDNA products are placed in -20 DEG C of preservations in 20 μ L reverse transcription reaction systems.
Three, real-time fluorescence quantitative PCR (qPCR)
1, reaction system (per hole)：
Two multiple holes of each reaction setting.The corresponding probe beforehand dilution of each pair of primer and to be mixed into Mix spare (dense
Degree is 10 μm of ol/L).It is loaded in 96 orifice plates, gently 2000g centrifuges 3min after mixing, is placed in ABI 7500Fast type fluorescence
Quantitative PCR apparatus is reacted.
2, response parameter：1. 95 DEG C, 20sec；2. 95 DEG C, 3sec；3. 60 DEG C, 30sec；②to③40cycles.
Interpretation of result：Reverse transcription TaqMan probe real-time fluorescence quantitative PCR is carried out by internal reference of ACTB, GAPD gene,
The signal of ACTB or GAPD must define；BIM Exon2 signals are clear；BIM E3/E4 ratios, i.e. BIM E3 signals it is clear and with
The 2 of BIM E4(-deltaCt)Ratio>It is the positive when=0.3；<It is feminine gender when 0.3.
The sample genomic dna of BIM genetic polymorphism detections extracts：
1) 336 patient with breast cancer's primary tumor paraffin specimens for receiving radical mastectomy are collected, material is carried out respectively
After finishing, be cut into 4 μm of paraffin section with slicer, and place be no more than 30mg (<2mm3) be organized in 2ml centrifuge tubes；
And its blood preparation is through being collected by centrifugation blood cell layer；
2) under room temperature, 10,000 × g are centrifuged 10 minutes；Aspirate supernatant not encounter histocyte precipitation (paraffin
1ml dimethylbenzene need to be added in sample, and abundant vortex mixing is to remove paraffin)；
3) 1ml absolute ethyl alcohols washing removal dimethylbenzene is added, at room temperature, 10,000 × g is centrifuged 5 minutes；Supernatant is abandoned, no
Pour out histocyte precipitation；
4) it repeats to be washed with absolute ethyl alcohol；It 37 DEG C, is air-dried 15 minutes；
5) 200 μ l Buffer TL are added into organizing, 25 μ l OB protease are added, vortex mixing is placed on 55 DEG C of water
Oscillation incubation in shaking table is bathed to digest completely to tissue.If without shaking bath, vortex was taken out every 20-30 minutes during water-bath
Mixing is primary.Digestion time depends on sample usage amount and organization type, can obtain good lytic effect within general 3 hours；
6) under room temperature, 10,000 × g are centrifuged 5 minutes and are removed undissolved impurity, are carefully transferred to supernatant newly
Centrifuge tube in and leave undissolved impurity；
7) 220 μ l Buffer BL, vortex mixing is added.70 DEG C of water-baths 10 minutes.It, may after Buffer BL are added
Some precipitations are generated, but not influence the recycling of DNA.The volume of Buffer BL is adjusted according to the usage amount of sample；
8) 220 μ l absolute ethyl alcohols (room temperature, 96-100%) are added, maximum speed is vortexed to mix well.If sample
Dosage is more than 30mg, adjusts the dosage of absolute ethyl alcohol；If at this time seeing precipitation, 10 times are blown and beaten to dispel precipitation with pipette tips；
9) willDNA columns are sleeved in 2ml collecting pipes and (have been provided), and all lysates that the 6th step is obtained turn
EnterIn DNA columns (including all sediments), 8,000 × g centrifuges 1 minute to combine DNA, abandons filtrate and receipts
Collector；
10) willDNA columns are sleeved in new 2ml collecting pipes, and 500 μ l Buffer HB are added extremelyIn DNA columns, 8,000 × g is centrifuged 1 minute, abandons filtrate and collecting pipe；
11) willDNA columns are sleeved in new 2ml collecting pipes, and 700 μ l DNA Wash Buffer are added extremelyIn DNA columns, 8,000 × g is centrifuged 1 minute, abandons filtrate；
12)DNA columns are sleeved in the same 2ml collecting pipes, add 700 μ l DNA Wash
Buffer is extremelyIn DNA columns, 8,000 × g is centrifuged 1 minute, abandons filtrate；
13) willDNA columns are sleeved in the same 2ml collecting pipes, ＞ 12,000 × g centrifugation sky get rid of 2 minutes with
It is dryThe matrix of DNA columns；
14) willDNA columns are sleeved on 1.5ml sterile centrifugation tubes, and 50-200 μ l are added and are preheated to 70 DEG C
Elution Buffer are extremelyThe film center of DNA columns.It is stored at room temperature 3 minutes；
15) at room temperature, 10000 × g is centrifuged 1 minute, with eluted dna；
16) above-mentioned two step of step is repeated；
17) elution gained DNA sample is measured on Nanodrop2000 instruments total rna concentration and purity (- 80 degree preservations,
Avoid multigelation)；
18) DNA is verified for Sanger direct sequencings.
Sanger PCR sequencing PCRs detect breast cancer sample BIM deletion polymorphisms：
(1) dosage that the reaction system and dosage needed according to experiment calculates each component configures Mix liquid；(2) M is used
Millipore water dilutes synthetic primer (BIM_DEL_F:6-FAM GCTAACTCAACAAACCCATCAGAAC SEQ ID
NO.25)；BIM_DEL_R:AGCCAGTAAATATAAATCCAAAGCA SEQ ID NO.26)；(3) PCR reaction systems are prepared：
The PCR reactions of each 25 μ L include 1 μ L (20ng/ μ L) template DNA, 12.5 μ L Premix Ex Taq HS enzymes (Dalian TAKARA
Company), 1 μ L forward and reverse primer mixtures, 10.5 μ L ddH20.PCR reaction conditions are：94 DEG C of 5m of denaturation：Denaturation
94 DEG C of 30s, anneal 55 DEG C of 30s, 72 DEG C of extension 1m, 35 cycles；72 DEG C of extension 7m.By the PCR product of amplification through 2% agarose
After detected through gel electrophoresis target fragment is eligible, purified product.(4) PCR product is pressed using BigDyeTeminator V3.1
Operational manual is marked and purifies, and bidirectional sequencing is then carried out on ABI-3730 sequenators, and sequencing result is used
Chromas2.31 softwares are analyzed.
Above-mentioned two are representative sample, and Fig. 1 is BIM gene delection polymorphism ' negative ' specimens (the BIM E3/ of wherein an example
E4 ratios are 0.02,<0.3 interpretation is feminine gender), qPCR method results are feminine gender, with Sanger direct sequencings negative findings in Fig. 2
Unanimously (base is continuously distributed, non-overlapping in sequence results).Fig. 3 is BIM gene delection polymorphisms positive sample (BIM E3 signals
It defines and is 0.75 with BIM E3/E4 ratios,>=0.3 interpretation is the positive), qPCR method results are the positive, with Sanger in Fig. 4
The direct sequencing positive (base has overlapping in sequence results) result is consistent.
QPCR methods comparison Sanger PCR sequencing PCRs detect BIM deletion polymorphism results in 1.336 breast cancer samples of table
(note：336 breast cancer samples are had detected altogether, and common mulberry lattice PCR sequencing PCR (Sanger sequencing) detects 39
Example BIM deletion polymorphism samples, 55 positive samples are detected using this project approach.Sensitivity is specific up to 92.3%
Reach 94.6%.)
Be detected according to above-mentioned same detection method, as a result, it has been found that in Fig. 5 A to 5D, reference gene ACTB and
The Ct values such as the expression of GAPD and BIM Exon2, Exon2_4, Exon2_3 are not separated by or PCR is inefficient cause Ct values compared with
Greatly, it is seen that the testing result of control group 1 is all bad.Referring to Fig. 5 A-5D.
Several embodiments of the invention above described embodiment only expresses, the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the protection of the present invention
Range.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Sequence table
<110>Attached tumour hospital of Guangzhou medical university
<120>Primer, probe and the kit of BIM genetic polymorphism detections
<160> 25
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
gccaggcctt caaccactat c 21
<210> 2
<211> 27
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
agcacagtga aagatcacct atatcct 27
<210> 3
<211> 27
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
gtattggaga cgagtttaac gcttact 27
<210> 4
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
tcggctgctt ggtaattatt ca 22
<210> 5
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
tgcgaaccct gccacact 18
<210> 6
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
gaacgcagcg aaccgaat 18
<210> 7
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 7
ggcacccagc acaatgaag 19
<210> 8
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 8
ccgatccaca cggagtactt g 21
<210> 9
<211> 27
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 9
ttcattgacc tcaactacat ggtttac 27
<210> 10
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 10
gacggtgcca tggaatttg 19
<210> 11
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 11
cagtgcaatg gtagtcat 18
<210> 12
<211> 16
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 12
tgcaaggagg gtattt 16
<210> 13
<211> 15
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 13
atcgcatcat cgcgg 15
<210> 14
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 14
caagatcatt gctcctcct 19
<210> 15
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 15
ttccaatatg attccaccca t 21
<210> 16
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 16
caaagcaacc ttctgatg 18
<210> 17
<211> 14
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 17
caggctgaac ctgc 14
<210> 18
<211> 15
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 18
agaccttcaa caccc 15
<210> 19
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 19
cgagaagatg acccagatca tg 22
<210> 20
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 20
acagcctgga tagcaacgta ca 22
<210> 21
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 21
aaacgcaaga aaaaaagacc aaa 23
<210> 22
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 22
ccttctcggt cacactcaga act 23
<210> 23
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 23
cagtgcaatg gcttccatga 20
<210> 24
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 24
gatccatatc tctgggcgca ta 22
<210> 25
<211> 25
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 25
gctaactcaa caaacccatc agaac 25
Claims (9)
1. the BIM genetic polymorphism detection kits based on TaqMan fluorescence probes, which is characterized in that including being directed to BIM_
The primer and probe of Exon2_3 and/or BIM_Exon2_4；The primer for BIM_Exon2_3 such as SEQ ID NO.1 and
Shown in SEQ ID NO.2, the probe for BIM_Exon2_3 is as shown in SEQ ID NO.11；It is described to be directed to BIM_
The primer of Exon2_4 is as shown in SEQ ID NO.3 and SEQ ID NO.4, the probe for BIM_Exon2_4 such as SEQ ID
Shown in NO.12.
2. BIM genetic polymorphism detections kit according to claim 1, which is characterized in that further include having as a contrast
The primer and probe for BIM Exon2, the primer for BIM Exon2 such as SEQ ID NO.5 and SEQ ID NO.6
Shown, the probe for BIM Exon2 is as shown in SEQ ID NO.13.
3. BIM genetic polymorphism detections kit according to claim 1, which is characterized in that including being directed to BIM_
Exon2_3 and primer and probe for BIM_Exon2_4.
4. BIM genetic polymorphism detections kit according to claim 1 or 2 or 3, which is characterized in that further include having work
For the primer and probe for ACTB_Exon4_5 and/or GAPD_Exon2_3 of control, the drawing for ACTB_E4_E5
For object as shown in SEQ ID NO.7 and SEQ ID NO.8, the probe for ACTB_Exon4_5 is as shown in SEQ ID NO.14；
The primer for GAPD_Exon2_3 is as shown in SEQ ID NO.9 and SEQ ID NO.10, for GAPD_Exon2_3's
Probe is as shown in SEQ ID NO.15.
5. BIM genetic polymorphism detections kit according to claim 4, which is characterized in that including being directed to BIM_
The primer and probe of exon2_3, BIM_Exon2_4, BIM E2, ACTB_Exon4_5 and GAPD_Exon2_3.
6. the primer for BIM genetic polymorphism detections, which is characterized in that the primer is selected from following at least a pair of：For
SEQ ID NO.1 and SEQ the ID NO.2 of BIM_Exon2_3, for SEQ ID NO.3 and the SEQ ID of BIM_Exon2
NO.4。
7. the primer according to claim 6 for BIM genetic polymorphism detections, which is characterized in that further include with down toward
Few pair of primers：For SEQ ID NO.5 and SEQ the ID NO.6 of BIM Exon2, for the SEQ ID of ACTB_Exon4_5
NO.7 and SEQ ID NO.8, for the SEQ ID NO.9 and SEQ ID NO.10 of GAPD_Exon2_3.
8. the probe for BIM genetic polymorphism detections, which is characterized in that the probe is selected from following at least one：For
The SEQ ID NO.11 of BIM_Exon2_3, for the SEQ ID NO.12 of BIM_Exon2.
9. according to claim 8 be used for BIM genetic polymorphism detections probe, which is characterized in that further include it is following at least
One probe：For the SEQ ID NO.13 of BIM Exon2, for the SEQ ID NO.14 of ACTB_Exon4_5, for GAPD_
The SEQ ID NO.15 of Exon2_3_PandP_1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810456288.1A CN108396066A (en) | 2018-05-14 | 2018-05-14 | Primer, probe and the kit of BIM genetic polymorphism detections |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810456288.1A CN108396066A (en) | 2018-05-14 | 2018-05-14 | Primer, probe and the kit of BIM genetic polymorphism detections |
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| Publication Number | Publication Date |
|---|---|
| CN108396066A true CN108396066A (en) | 2018-08-14 |
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ID=63102242
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810456288.1A Pending CN108396066A (en) | 2018-05-14 | 2018-05-14 | Primer, probe and the kit of BIM genetic polymorphism detections |
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| CN (1) | CN108396066A (en) |
Citations (4)
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|---|---|---|---|---|
| CN103571923A (en) * | 2012-07-18 | 2014-02-12 | 益善生物技术股份有限公司 | Detection probes and detection liquid phase chip for BIM gene deletion mutation |
| CN104561251A (en) * | 2013-10-25 | 2015-04-29 | 上海市胸科医院 | Method and kit for detecting deletion mutation of cell apoptosis regulator gene (BIM) |
| CN105316393A (en) * | 2014-07-17 | 2016-02-10 | 上海市胸科医院 | Rapid detection method for detecting deletion mutation of cell apoptosis regulator gene (BIM) and detection kit thereof |
| CN105603069A (en) * | 2016-01-20 | 2016-05-25 | 安徽达健医学科技有限公司 | Bim (Bcl-2 interacting mediator of cell death) gene deletion fluorescent quantitative PCR (polymerase chain reaction) detection primer, probe and detection reagent kit |
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|---|---|---|---|---|
| CN103571923A (en) * | 2012-07-18 | 2014-02-12 | 益善生物技术股份有限公司 | Detection probes and detection liquid phase chip for BIM gene deletion mutation |
| CN104561251A (en) * | 2013-10-25 | 2015-04-29 | 上海市胸科医院 | Method and kit for detecting deletion mutation of cell apoptosis regulator gene (BIM) |
| CN105316393A (en) * | 2014-07-17 | 2016-02-10 | 上海市胸科医院 | Rapid detection method for detecting deletion mutation of cell apoptosis regulator gene (BIM) and detection kit thereof |
| CN105603069A (en) * | 2016-01-20 | 2016-05-25 | 安徽达健医学科技有限公司 | Bim (Bcl-2 interacting mediator of cell death) gene deletion fluorescent quantitative PCR (polymerase chain reaction) detection primer, probe and detection reagent kit |
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| 金田恩: "BIM缺失多态性与乳腺癌紫杉醇内源性耐药机制的关系", 《广州医科大学 硕士论文》 * |
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