CN103571925B - Specific detection primers and detection liquid phase chip for BIM gene mutation - Google Patents
Specific detection primers and detection liquid phase chip for BIM gene mutation Download PDFInfo
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Abstract
The present invention discloses a detection liquid phase chip and specific primers for BIM gene mutation. The liquid phase chip mainly comprises: ASPE primers comprising a 5' terminal tag sequence and a 3' terminal target gene mutational site-targeted specific primer sequence, wherein the specific primer sequence comprises deletion mutation-targeted SEQ ID NO.13, deletion mutation-targeted SEQ ID NO.14, A145G site-targeted SEQID NO.15, A145G site-targeted SEQ ID NO.16, C59T site-targeted SEQ ID NO.17, C59T site-targeted SEQ ID NO.18, T86C site-targeted SEQ ID NO.19, T86C site-targeted SEQ ID NO.20, G149C site-targeted SEQ ID NO.21, G149C site-targeted SEQ ID NO.22, and/or C104G site-targeted SEQ ID NO.23 and C104G site-targeted SEQ ID NO.24; anti-tag sequence coated microspheres; and amplification primers. According to the present invention, coincidence frequency of the detection results of the detection liquid phase chip and the sequencing method is up to 100%, and single and parallel detection on the wild-type with multiple mutational sites and the mutant-type with multiple mutational sites can be achieved.
Description
Technical field
The invention belongs to biology field, relate to medical science and biotechnology, particularly relate to a kind of BIM detection in Gene Mutation Auele Specific Primer and liquid-phase chip.
Background technology
BIM gene is present on people's No. 2 karyomit(e)s, is a kind of B cell leukemia/lymphoma 2(BCL2 gene) protein family urgees apoptosis member.BIM genetic expression one comprises BH3(BCL2-homology domain3) albumen of structural domain, by with Bcl2 family Bcl2-l1, BCL-XL, BCL2A1, some somatotrophic albumen such as MCL1 combine inhibition tumor cell growth, or and promote that protein B AX, the BAK1 of apoptosis combines promotion apoptosis of tumor cells.BH3 structural domain is the key function territory that BIM gene plays a role, and research finds that this functional domain and TKIs play a role closely related.In the nonsmall-cell lung cancer (NSCLC) of TKIs medicine treated with gefitinib, such as need the normal expression of BIM gene, and BH3-similar medicine can obviously strengthen its result for the treatment of.The abnormal human tumor cell caused of a lot of kinase expression of the mankind can by suppressing the expression of BIM gene or being kept the growth vigor of self by MAPK1 phosphorylation modification BIM albumen.In nearly all mankind's tumour, the activation expression of BIM gene plays a role for TKIs class medicine is necessary, suppresses the expression of BIM gene then can cause TKIs resistance.Show that BIM gene plays a significant role in TKIs medicine plays a role process.
At present, BIM detection method of gene mutation mainly contains: Illumina optical fiber superbead chip technology, Affymetrix6.0 chip technology.Although Illumina optical fiber superbead chip technology is the high throughput testing system of highly sensitive and accuracy, but level of automation is low, manual operations is many, be difficult to the needs meeting practical application, high-throughout Affymetrix suddenlys change 6.0 chip technology comparative maturities, but this chip technology in low-density clinical diagnosis cake core improper, be difficult to expansion in same reaction system and detect the relevant sudden change of numerous biological character or label sudden change.In addition, Affymetrix 6.0 chips that suddenly change are mainly stronger on chip of expression spectrum, and species are more, relatively weak on sudden change chip, and detect expensive, can not meet actual needs.
The BIM transgenation of target detect of the present invention, it is as shown in the table:
SEQ ID NO.58BIM deletion mutant
GTTGGTAGAGTTATCAATTAGGAAACCCAGTACAGAGTCTATTATAATTTAGATTGTACCTCATGATGAAGGCTAACTCAACAAACCCATCAGAACAGACACTGGAACAAAATGACATTTCTAAATACCATCCAGCTCTGTCTTCATAGGCTTCAGTGAGGTAAATCA
CTGTTCTCCATAGAGGCTGTGCCATTTTACATTCCCACCAACAGGGCACAAGGGTTCCAGTTTCTCCACATACTTACCAACACTTTTTTTTTTTTTTTTTTAACAGTAGTCATCCTAGAGGATATAGGTGATCTTTCACTGTGCTTTGGATTTATATTTACTGGCTTAGATTTGTATGGCCACCACCATAGTCAAGATACAGAACAACTCAACCACAAGGATTTCTCATGATACCTTTTTATAGCCACAGCCACCTCTCTCCCTCTTCCTTGAGCATTTTGTCATATGGTCATTGGTGATTAAA
(illustrate: this sequence is from the 169th bit base, there occurs the deletion mutantion of 2903 bit bases, in frame, sequence is absent region, those skilled in the art use routine techniques means, according to the based composition before and after absent region, in gene pool, just easily can obtain the based composition of this absent region, therefore, the content of the BIM deletion mutant of target detect of the present invention be fully disclosed in.）
SEQ ID NO.59 mutational site: A145G
GGGAGGTCATGCTTGGTTAATTGTTGCTAAGTAAATGGATTTTACCTTTGATTATTATTTTTCTGCCTTTATACAATGTGTTTTAGGCTTCTTTTAGACAAATAGACATTTTCCCATTACAAAGGACACTATCAGTTCTTCAGTATGGAATTTCTGTAAGAGTCAAGAAAACACACATCGGATCTTCCTACCTTTCTGTGGGGGTGTTTGAGGAGAGTGCTGTAGTAATGATTCTGTTGTAAAATGGGAAGTGTGACATTGATGGACTTAAGAATTTCTTAAAATACTGTCTTAAGCTGGCAAAACTCCTGGCATCCTCCACCTGACATAAACCAG
SEQ ID NO.60 mutational site: C59T
CTCTATGGTCTCCCTGCTTCCTGGTGTCAGCAGTGGCTGAGGGAGCCAAGCTTACACACCTCCCCCAACAACTGGACACAATAAAGCAGTGTGCACTGGGGCCCCTCTTTTGTTTGGAAAGTACCAGCAAAGCCAAGGAGAAATCTGAACTTCCAACTTACCTAGTAGAAACAGGCAGAAATCTTAGAATTGAAAAATATAATTAAACATAATTTTAAAAACAATCAGTGAATATGAGGCAAAATCAATAGAATTTACTCAATCTGAAGAGAGAGAAAATCTACTGGAAAAAAATGAACAGTCTCAGGAACTTACAGGAC
SEQ ID NO.61 mutational site: T86C
TGATTCGTGAGTAGAGTTGCCAAGACAGGTGTGGCAAGAAATAGCCCTGAGACAGGGCTGCTGGCACGCAGCTCTCCTCCTGTTCTGCCTTTCTGTGTGGACCACGAGGCAGCAGTGAGGGCTTCCCATGGAGCATGTCTCCCTTTCAGTCACACATCTTGCGCTCACAGTCACTGCCAGCCAGTTGACTGCAAGTGCTGTGTCTGTAAACCTTGCAGTGGGGACTTGACATTGCTTTCAGAG
SEQ ID NO.62 mutational site: G149C
AATCCAGGCACAGCCACAATCCAGGCACAGCCACAATCCAGGCACAGCTCTTAATTTTTTAACTTTTTATTTGAAATAATTTCAGACACACAAAAAGTTTGCAAAATAGTAGAGACACTTTTCTTATAGCCTTTACCCAGAGTACCCAGATGTTACCATTTTGCCACATTTTGCTTTATCCTTCTCCCTTCCAACTGCCACCATTCCATCCTGCCCCACATGCATGTAAGCACATCACATTTTTCTCCTGAACATTGAGAGTAAATTACCTGTTGCCCCTTGCCCCTAAATACTTCAGTGTGTATCTCCTAAAAATGAGGACATCCTCTTACCTAACCAGAGTTCAGCGA
SEQ ID NO.63 mutational site: C104G
TGAGGATGCTGAGTATTTGTGAATTTATCTCCTCTTTAAGATACAGGCCCCAGATTCTGGCCCCTGGCAGTTTCCCTGCATTGTCTTGAGTCTGGAGCTTTCTCCTTGGGTGGGTCTCAGAAATCTTACTGATTTCTGCTTTTTTGCCCACTAAAGGAGATAATTTCACTGCTAAACGGGTCAAATTCTCTTCTGAGGATGGGTCTAAATTCTTGCTAAATATACCTGAATTAACTCTGTCACTGTCTAAATATTTGTGTGTCCCCATAAATTCCTATGTTGAAATCCTAACCCC
Summary of the invention
An object of the present invention is to provide BIM gene mutation detection liquid-phase chip, and this liquid-phase chip can be used for separately or the deletion mutantion of parallel detection BIM gene six kinds of Common genes types, the wild-type of A145G, C59T, T86C, G149C and C104G and saltant type.
Realize above-mentioned purpose technical scheme as follows.
A kind of BIM gene mutation detection liquid-phase chip, includes
(A). the wild-type designed respectively for the different mutational site of BIM gene and the ASPE primer pair of saltant type: every bar ASPE primer holds the specific primer sequence for goal gene mutational site to form by 5 ' the tag sequence of holding and 3 ', described specific primer sequence is: for SEQ ID NO.13 and the SEQ ID NO.14 of deletion mutantion, for SEQ ID NO.15 and the SEQ ID NO.16 in A145G site, for SEQ ID NO.17 and the SEQ ID NO.18 in C59T site, for SEQ ID NO.19 and the SEQ ID NO.20 in T86C site, for SEQ ID NO.21 and the SEQ ID NO.22 in G149C site, and/or for the SEQ ID NO.23 in C104G site and SEQ ID NO.24, described tag sequence is selected from SEQ ID NO.1 ~ SEQ ID NO.12,
(B). microballoon that have anti-tag sequence bag quilt, that have different colours coding, is also provided with spacer sequence in the middle of described anti-tag sequence is connected with microballoon; Described anti-tag sequence is selected from SEQ ID NO.25 ~ SEQ ID NO.36, and described anti-tag sequence can correspondingly be matched with selected tag complementary in (A);
(C). for amplifying the primer needing that detect, that there is corresponding mutational site target sequence.
Wherein in an embodiment, described amplimer is: for SEQ ID NO.37, SEQ ID NO.38 and the SEQ ID NO.39 of deletion mutantion, for SEQ ID NO.40 and the SEQ ID NO.41 in A145G site, for SEQ ID NO.42 and the SEQ ID NO.43 in C59T site, for SEQ ID NO.44 and the SEQ ID NO.45 in T86C site, for SEQ ID NO.46 and the SEQ ID NO.47 in G149C site, and/or for the SEQ ID NO.48 in C104G site and SEQ ID NO.49.
Wherein in an embodiment, described ASPE primer is: the sequence that the sequence be made up of SEQ ID NO.1 and SEQ ID NO.13 and SEQ ID NO.2 and SEQ ID NO.14 for deletion mutantion form, for the sequence be made up of SEQ ID NO.3 and SEQ ID NO.15 in A145G site and the sequence that is made up of SEQ ID NO.4 and SEQ ID NO.16, for the sequence be made up of SEQ ID NO.5 and SEQ ID NO.17 in C59T site and the sequence that is made up of SEQ ID NO.6 and SEQ ID NO.18, for the sequence be made up of SEQ ID NO.7 and SEQ ID NO.19 in T86C site and the sequence that is made up of SEQ ID NO.8 and SEQ ID NO.20, for the sequence be made up of SEQ ID NO.9 and SEQ ID NO.21 in G149C site and the sequence that is made up of SEQ ID NO.10 and SEQ ID NO.22, and/or for the sequence be made up of SEQ ID NO.11 and SEQ ID NO.23 in C104G site and the sequence that is made up of SEQ ID NO.12 and SEQ ID NO.24.
Another object of the present invention is to provide the Auele Specific Primer for BIM detection in Gene Mutation.
For the Auele Specific Primer of BIM detection in Gene Mutation, described Auele Specific Primer is: for SEQ ID NO.13 and the SEQ ID NO.14 of deletion mutantion, for SEQ ID NO.15 and the SEQ ID NO.16 in A145G site, for SEQ ID NO.17 and the SEQ ID NO.18 in C59T site, for SEQ ID NO.19 and the SEQ ID NO.20 in T86C site, for SEQ ID NO.21 and the SEQ ID NO.22 in G149C site, and/or for the SEQ ID NO.23 in C104G site and SEQ ID NO.24.
Major advantage of the present invention is:
1. the detected result of BIM gene mutation detection liquid-phase chip provided by the present invention and the identical rate of sequencing are up to 100%.And the time required for detecting is well below conventional sequencing technologies, realistic especially application needs.Prepared BIM gene mutation detection liquid-phase chip has extraordinary signal-noise ratio, and substantially there is not cross reaction between designed probe and anti-tag sequence, choosing and the combination of tag sequence label and concrete ASPE primer of tag sequence label, anti-tag sequence label, can cross reaction be avoided, realize the parallel detection in multiple mutational site.
2. the present invention passes through the design experiences of contriver's long term accumulation and a large amount of experimental implementation, have chosen optimum combination from numerous Auele Specific Primers.The ASPE primer specificity primer of the present invention's design can the sensitive mutational site identifying target detect specifically, accurately distinguishes the genotype of various type; In same reaction system, substantially there is not cross reaction between different Auele Specific Primers, between the pcr amplification product that detects of Auele Specific Primer and non-targeted, detection specificity is good, and cross reacting rate is lower than 3%; Except Single locus catastrophe can be detected, also can the catastrophe in the simultaneously multiple mutational site of parallel detection, Detection results is consistent.
3. detection method step of the present invention is simple, the amplification that can complete 6 target sequences containing mutational site by One_step PCR is detected in 6 kinds of mutational sites, avoid the many uncertain factors existed in the complex operations processes such as repeated multiple times PCR, thus greatly can improve Detection accuracy, embody accurate qualitative and quantitative analysis feature simultaneously.
4. not only to overcome conventional solid chip susceptibility not high in the present invention, and the defect of the repeatability difference of detected result, improves existing liquid-phase chip technology simultaneously, makes prepared microballoon can be applicable to different test items, has very strong expansion.The fluorescent signal value detected improves greatly, thus the sensitivity detected is further enhanced, and signal to noise ratio strengthens, and detected result more accurately and reliably.
Embodiment
Embodiment 1BIM gene mutation detection liquid-phase chip, mainly includes:
One, ASPE primer
For wild-type and the saltant type of the deletion mutantion of BIM gene six kinds of Common genes types, A145G, C59T, T86C, G149C and C104G, design specific primer sequence respectively.ASPE primer is made up of " tag sequence+specific primer sequence ".ASPE primer sequence is as shown in the table:
The ASPE primer sequence (tag sequence+specific primer sequence) of table 1BIM gene
Every bar ASPE primer comprises two parts, and 5 ' end is for for the specificity tag sequence of anti-tag sequence on corresponding microballoon, and 3 ' end is saltant type or the special primer segments (as shown in Table 1 above) of wild-type.All ASPE primers are synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Every bar primer after synthesis is mixed with the stock solution of 100pmol/mL respectively with 10mmol/L Tris Buffer.
Two, the microballoon of anti-tag sequence bag quilt
According to designed ASPE Auele Specific Primer fragment, select tag sequence.Tag sequence of the present invention is when choosing, avoid various tag sequence as far as possible, anti-tag sequence, cross reaction between specific primer sequence, between the anti-tag sequence reducing each microballoon to greatest extent and the tag sequence secondary structure that may be formed from different ASPE primer sequence, simultaneously, the primer in the various mutations site in detection system and the specific requirements of PCR primer parallel detection can also be met, thus realize parallel detection various mutations type in same reaction system, cross reaction is avoided in whole parallel detection system, and make the specificity of liquid-phase chip testing product of the present invention, reach between sensitivity and repeatability and optimize and balance, the genotype of the various type of accurate differentiation.The anti-tag sequence that the 12 kinds of microballoon numberings selected are corresponding on microballoon is as shown in table 2:
The anti-tag sequence that table 2 microballoon numbering is corresponding on microballoon
The 12 kinds of microballoon purchased from American Luminex companies selected, are coated in anti-tag sequence on microballoon.Be connected with the spacer sequence of 5-10 T between anti-tag sequence and microballoon, before each anti-tag sequence, namely add the spacer sequence of the preceding paragraph 5-10 T, anti-tag sequence is synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.By the anti-tag sequence sterilizing ddH of synthesis
2o is made into the stock solution of 100nmol/ml.Described spacerarm is for by anti-tag and microsphere surface is spaced apart or anti-tag is placed in the sequence of hydrophilic environments.By arranging the spacer sequence of suitable length between anti-tag sequence and microballoon, can reduce sterically hindered, improving the efficiency of hybridization and the specificity of hybridization.Common spacer sequence comprises poly dT, i.e. poly(dT), oligomerization four polyoxyethylene glycol and (CH2) n spacerarm (n >=3), as (CH2) 12, (CH2) 18 etc.In addition, if there is poly(dA) interference, can also poly(TTG be used) as spacerarm.Spacerarm of the present invention is preferably 5-10 T, and the process of microballoon bag quilt is as follows:
Get 5 × 10 respectively
6the carboxylated microballoon (purchased from Luminex company) of individual above-mentioned numbering is suspended in (pH4.5) in the MES solution of 50ul0.1mol/L, adds the anti-tag molecule (100nmol/ml) of 10ul synthesis.The EDC(N-(3-Dimethylaminopropyl-N-ethylcarbodiimide of preparation 10ng/ml) (purchased from Pierce Chemical company) working fluid.The EDC working fluid of 2.5ul is added, constant-temperature incubation 30 minutes, then the EDC working fluid adding 2.5ul in microsphere suspensions, then constant-temperature incubation 30 minutes.After reaction terminates, the Tween-20 washing with 0.02% once, then is washed once with the SDS liquid of 0.1%.The microballoon being coated with anti-tag sequence after washing is resuspended in the Tris-EDTA solution [10mmol/L Tris(pH8.0)] of 100ul, in 1mmol/LEDTA, 2-8 DEG C keeps in Dark Place.
Three, the primer of the target sequence containing mutational site is amplified
For the deletion mutantion of BIM gene six kinds of Common genes types, A145G, C59T, T86C, G149C and C104G, design of amplification primers is to (be shown in table 3), wherein, for A145G, C59T, T86C, G149C and C104G site, amplify 5 corresponding target sequences respectively, detect for deletion mutantion, if measuring samples is wild-type homozygote, then amplify the product of 191bp, if measuring samples is saltant type homozygote, then amplify the product of 144bp, if measuring samples is heterozygote, then amplify two kinds of products simultaneously.
Table 3 amplifies the primer of the target sequence with mutational site
All primers are synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Every bar primer after synthesis is mixed with the stock solution of 100pmol/mL respectively with 10mmol/L Tris Buffer.
Embodiment 2 uses BIM gene mutation detection liquid-phase chip described in embodiment 2 to the detection of sample
The formula of described various solution is as follows:
The MES damping fluid (pH5.0) formula (250ml) of 50mM:
2 × Tm hybridization buffer
4 DEG C are stored in after filtration.
ExoSAP-IT test kit purchased from American USB company.
Biotin labeled dCTP is purchased from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
One, the DNA extraction of sample:
With reference to " molecular cloning " about the methods involving of DNA extraction, obtain DNA to be detected.
Two, the pcr amplification of testing sample
Design 6 pairs of primers, multiplex PCR one step amplifies 6 respectively containing the deletion mutantion of BIM gene six kinds of Common genes types, A145G, C59T, T86C, the target sequence of G149C and C104G, detect for deletion mutantion, if measuring samples is wild-type homozygote, then amplify the product of 191bp, if measuring samples is saltant type homozygote, then amplify the product of 144bp, if measuring samples is heterozygote, then amplify two kinds of products simultaneously, other five kinds of genotype product sizes are respectively 336bp, 320bp, 243bp, 350bp, 295bp, primer sequence (SEQ ID NO.37-49) is shown in shown in above-mentioned table 3.
First multiple PCR primer working fluid is prepared: the primer stock solution 100ul respectively getting SEQ ID NO.37-49 respectively, in 1.5ml Eppendorf tube, mixes and is multiple PCR primer working fluid.Multi-PRC reaction system is as follows:
Pcr amplification program is: 95 DEG C of 3min; 94 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 40s, 30 circulations; 72 DEG C of 10min; 4 DEG C save backup.
Three, the enzyme of PCR primer cuts process
1. get the reacted product of 7.5ul PCR, add 1ul10 × SAP damping fluid, 1ul SAP enzyme and 0.5ul Exo-I enzyme;
Hatch 15min for 2.37 DEG C, hatch 15min for 80 DEG C, the enzyme that deactivation is unnecessary.Product after enzyme cuts process is directly used in follow-up ASPE primer extension reaction.
Four, site-specific primer extension reaction (ASPE)
Utilize the ASPE primer of design in embodiment 1 to carry out primer extension reaction, in reaction process, mix biotin labeled dCTP, thus make biotin labelings multiple on reacted product band.
First the ASPE primer working fluid of mixing is prepared: get the corresponding wild-type of gene to be detected and saltant type ASPE primer stock solution 10ul respectively in 1.5ml Eppendorf tube, add 10mmol/L Tris Buffer to mend to 200ul, mix and be ASPE mix primer working fluid.The system of ASPE reaction is as follows:
Response procedures is: 96 DEG C of 2min; 94 DEG C of 30s, 54 DEG C of 1min, 72 DEG C of 2min, 30 circulations; 4 DEG C save backup.
Five, hybridization
1., according to the ASPE primer of design, the microballoon (as described in Example 1) of the corresponding 10 kinds of bag quilts of every group selection, often kind of microballoon concentration is 2.5 × 10
5individual/ml;
2. get 1ul respectively and often plant the microballoon of numbering in the Eppendorf tube of 1.5ml;
3. microballoon is in >=centrifugal the 1-2min of 10000g;
4. supernatant discarded, microballoon is resuspended in 2 × Tm hybridization buffer of 100ul, and vortex mixes;
5. get the above-mentioned microsphere suspensions of 25ul in the 96 corresponding holes of hole filter plate, control wells adds the ddH of 25ul
2o;
6. the ASPE reaction solution getting 5-25ul, in corresponding hole, uses ddH
2o complements to 50ul;
7. encase 96 orifice plates with lucifuge with masking foil, 95 DEG C of 60s, 37 DEG C of 15min hatch hybridization;
8. the microballoon after hybridization is in >=centrifugal the 2-5min of 3000g;
9. remove supernatant, microballoon is resuspended in 1 × Tm hybridization buffer of 75ul;
10. microballoon is in >=centrifugal the 2-5min of 3000g;
Microballoon is resuspended in 1 × Tm hybridization buffer of 75ul by 11., adds the SA-PE (SA-PE) that 15ul concentration is 10ug/ml;
Hatch 15min for 12.37 DEG C, detect on Luminex instrument.
Six, result detects and data analysis
Reaction after product is detected by Luminex serial analysis instrument.Detected result is as shown in table 4, table 5 and table 6.
Following requirement is had to fluorescent value (MFI) and data processing:
1. each site need have at least an allelotrope MFI to be greater than 300 and be greater than 10 × PCR negative control MFI;
2.NET MFI=sample MFI-PCR negative control MFI(NET MFI is less than 0 represent with 0);
3. meet the data of above two conditions, by following formulae discovery sudden change ratio:
Sudden change ratio=saltant type NET MFI ÷ (saltant type NET MFI+ wild-type NET MFI)
4. rule of thumb to the sudden change ratio definite threshold (cut-off value) of each detection site, to divide wild-type homozygote, heterozygote and saltant type homozygote.
Use present method to detect 20 increments BIM gene mutation site originally, experimental data meets above-mentioned requirements, therefore can calculate their sudden change ratio.Arranging of threshold value (cut-off value) is as follows: sudden change ratio range is considered as wild-type homozygote at 0%-20%; 30%-70% is considered as heterozygote; 80%-100% is considered as anomaly homozygote.Detect with sequencing and compare with liquid-phase chip result, calculate the identical rate of classifying method detected result provided by the present invention.Present method detect 20 increments this BIM genotype call results and the sequencing result rate of coincideing reach 100%.Visible BIM gene mutation detection liquid-phase chip provided by the present invention can detect the mutation type of BIM exactly, and result is reliable and stable.
Table 4 pattern detection result (MFI)
Table 5 sample B IM transgenation ratio (%)
Table 6 sample B IM gene mutation type analytical results
The liquid-phase chip of the ASPE primer that embodiment 3 is different is to the detection of BIM gene mutation site
One, the design (selection of Tag sequence and Anti-Tag sequence) prepared of liquid-phase chip
Liquid-phase chip is detected for BIM Gene A 145G, C59T, G149C and C104G site mutation, respectively for the specific primer sequence that wild-type and the saltant type design ASPE primer 3 ' of A145G, C59T, G149C and C104G are held, the Tag sequence that ASPE primer 5 ' is held then is selected from SEQ ID NO.1-SEQ ID NO.12, accordingly, be coated in and microballoon is selected from SEQ ID NO.25-SEQ ID NO.36 with the anti-tag sequence that corresponding tag complementary matches.Specific design is as shown in following table (table 7).The synthesis of ASPE primer, anti-tag sequence bag by microballoon, amplimer, detection method etc. as described in embodiment 1 and embodiment 2.
Design prepared by table 7 liquid-phase chip
One, sample detection
Adopt liquid-phase chip prepared by above-mentioned design, detect sample 61-80 by testing process described in embodiment 4 and method, detected result is as follows:
Table 8 sample B IM Gene A 145G detected result and Polymorphism Analysis
Table 9 sample B IM gene C 59T detected result and Polymorphism Analysis
Table 10 sample B IM gene G149C detected result and Polymorphism Analysis
Table 11 sample B IM gene C 104G detected result and Polymorphism Analysis
From the present embodiment, for the liquid-phase chip in different mutational site, ASPE primer uses tag sequences different in table 1, and its result is still reliable and stable.And ASPE primer is when selecting that in liquid-phase chip described in embodiment 1, tag sequence and specific primer sequence are arranged in pairs or groups, effect better (signal to noise ratio is better), see the present embodiment test group 2, test group 6, test group 9 and test group 12.Other different tag sequence and specific primer sequence are arranged in pairs or groups, and with coming to the same thing of embodiment 2 and the present embodiment, concrete data are omitted.The selection of embodiment 4BIM detection in Gene Mutation specific primer sequence
One, the design (selection of wild-type and saltant type specific primer sequence) prepared of liquid-phase chip
Liquid-phase chip is detected for the pleomorphism site of BIM gene T86C and G149C, with the complementary sequence forward or backwards of this place, mutational site target sequence for template, respectively for the specific primer sequence that wild-type and the saltant type design ASPE primer 3 ' of T86C and G149C are held, comprise preferred specific primer sequence in the embodiment of the present invention 1 and 2 alternative specific primer sequences, as shown in table 12.Wherein,
interior base is pleomorphism site.
Table 12 specific primer sequence
Liquid-phase chip is detected for the pleomorphism site of BIM gene T86C and G149C, different specific primer sequences is selected for T86C and G149C, the tag sequence that ASPE primer 5 ' is held then is fixed as the best effect sequence in embodiment 2, and select the anti-tag sequence corresponded, specific design is as shown in following table (table 13).The synthesis of ASPE primer, anti-tag sequence bag by microballoon, amplimer, detection method etc. as described in embodiment 1 and embodiment 2.
Design two prepared by table 13 liquid-phase chip
Two, sample detection
Adopt liquid-phase chip prepared by above-mentioned design, detect sample 81-100 by testing process described in embodiment 2 and method, detected result is as follows:
Table 14 sample B IM gene T86C detected result and Polymorphism Analysis
Table 15 sample B IM gene G149C detected result and Polymorphism Analysis
From the present embodiment, when ASPE primer selects that in embodiment 2, specific primer sequence and tag sequence are arranged in pairs or groups, Detection results is more than other Auele Specific Primer good (signal to noise ratio is better), and result more accurately and reliably.See the present embodiment test group 13 and test group 16.Other derives from the different specific primer sequence of the complementary sequence forward or backwards of place, target detect site sequence and tag sequence is arranged in pairs or groups, with coming to the same thing of embodiment 2 and the present embodiment, namely be still that the specific primer sequence described in embodiment 2 is better from different tag sequence arranging effects, concrete data are omitted.
Other multiple specific primer sequence for different mutational sites and tag sequence are arranged in pairs or groups, with coming to the same thing of embodiment 2 and the present embodiment, the Auele Specific Primer namely selected by embodiment 1, has better signal to noise ratio, Detection results is also better, and concrete data are omitted.
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (4)
1. a BIM gene mutation detection liquid-phase chip, is characterized in that, includes
(A). the wild-type designed respectively for the different mutational site of BIM gene and the ASPE primer pair of saltant type: every bar ASPE primer holds the specific primer sequence for goal gene mutational site to form by 5 ' the tag sequence of holding and 3 ', described specific primer sequence is: for SEQ ID NO.13 and the SEQ ID NO.14 of deletion mutantion, and the SEQ ID NO.15 be selected from for A145G site and SEQ ID NO.16, for SEQ ID NO.17 and the SEQ ID NO.18 in C59T site, for SEQ ID NO.19 and the SEQ ID NO.20 in T86C site, for SEQ ID NO.21 and the SEQ ID NO.22 in G149C site, with at least one group in the SEQ ID NO.23 in C104G site and SEQ ID NO.24, described tag sequence is selected from SEQ ID NO.1 ~ SEQ ID NO.12,
(B). microballoon that have anti-tag sequence bag quilt, that have different colours coding, is also provided with spacer sequence in the middle of described anti-tag sequence is connected with microballoon; Described anti-tag sequence is selected from SEQ ID NO.25 ~ SEQ ID NO.36, and described anti-tag sequence can correspondingly be matched with selected tag complementary in (A);
(C). for amplifying the primer needing that detect, that there is corresponding mutational site target sequence;
Described amplimer is: for SEQ ID NO.37, SEQ ID NO.38 and the SEQ ID NO.39 of deletion mutantion, and the SEQ ID NO.40 be selected from for A145G site and SEQ ID NO.41, for SEQ ID NO.42 and the SEQ ID NO.43 in C59T site, for SEQ ID NO.44 and the SEQ ID NO.45 in T86C site, for SEQ ID NO.46 and the SEQ ID NO.47 in G149C site, with at least one group in the SEQ ID NO.48 in C104G site and SEQ ID NO.49.
2. BIM gene mutation detection liquid-phase chip according to claim 1, it is characterized in that, described ASPE primer is: the sequence that the sequence be made up of SEQ ID NO.1 and SEQ ID NO.13 and SEQ ID NO.2 and SEQ ID NO.14 for deletion mutantion form, and be selected from for the sequence be made up of SEQ ID NO.3 and SEQ ID NO.15 in A145G site and the sequence that is made up of SEQ ID NO.4 and SEQ ID NO.16, for the sequence be made up of SEQ ID NO.5 and SEQ ID NO.17 in C59T site and the sequence that is made up of SEQ ID NO.6 and SEQ ID NO.18, for the sequence be made up of SEQ ID NO.7 and SEQ ID NO.19 in T86C site and the sequence that is made up of SEQ ID NO.8 and SEQ ID NO.20, for the sequence be made up of SEQ ID NO.9 and SEQ ID NO.21 in G149C site and the sequence that is made up of SEQ ID NO.10 and SEQ ID NO.22, with at least one group in the sequence be made up of SEQ ID NO.11 and SEQ ID NO.23 for C104G site and the sequence that is made up of SEQ ID NO.12 and SEQ ID NO.24.
3. BIM gene mutation detection liquid-phase chip according to claim 1 and 2, is characterized in that, described spacerarm is 5-10 T.
4. for the Auele Specific Primer of BIM detection in Gene Mutation, it is characterized in that, described Auele Specific Primer is, for SEQ ID NO.13 and the SEQ ID NO.14 of deletion mutantion, and the SEQ ID NO.15 be selected from for A145G site and SEQ ID NO.16, for SEQ ID NO.17 and the SEQ ID NO.18 in C59T site, for SEQ ID NO.19 and the SEQ ID NO.20 in T86C site, for SEQ ID NO.21 and the SEQ ID NO.22 in G149C site, with at least one group in the SEQ ID NO.23 in C104G site and SEQ ID NO.24.
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| 液相芯片技术的原理与应用进展;陈玮;《成都医学院学报》;20080930;第3卷(第3期);225-231 * |
| 贺韦东等.BIM在EGFR突变非小细胞肺癌中的表达及其意义.《山东大学学报(医学版)》.2011,第49卷(第12期), * |
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