Background technology
(Irinotecan was a kind of through suppressing the chemotherapeutics that the DNA topoisomerase I plays antitumous effect CPT-11) to irinotecan, obtained the drugs approved by FDA listing in 1998.In the dna replication dna process, topoisomerase I reversibility ground cuts the dna single chain, and re-assemblies formation dna double chain.The active metabolite SN-38 of irinotecan combines with topoisomerase I DNA mixture, stops re-assemblying of DNA chain, causes the dna double chain break, causes necrocytosis.
Yet the minimizing of the neutrophilic granulocyte that irinotecan causes and serious intestines toxicity problem become one of key factor that limits its dosage.The active metabolite SN-38 of irinotecan is through liver uridine diphosphate glucuronate transferring enzyme (UDP-GT) deactivation (mainly by UGT1A1, UGT1A7 and UGT1A9 metabolism), thereby the cell that protects the health is avoided Cytotoxic influence.Scientific research shows that the toxic side effect of irinotecan and the gene pleiomorphism of UGT1A1 are closely related.Three kinds of common allele type UGT1A1*28, UGT1A1*6 and UGT1A1*93 of UGT1A1 can cause the miopragia of this enzyme, thereby pharmaceutical activity metabolite residence time in blood is prolonged.
UGT1A1*28 is the sudden change of promotor TATA box.The heterozygote of UGT1A1*28 is similar with the enzymic activity of wild-type, but the corresponding enzymic activity of the sudden change homozygote of UGT1A1*28 only is 35% of a wild-type.The normal homozygote in this site is 6 multiple TA base pair A (TA)
6TAA (6/6), the sudden change homozygote is 7 multiple TA base pair A (TA)
7TAA (7/7), in the genotypic allelotrope of heterozygous mutation, one is wild-type, one is mutant (6/7).The UGT1A1 carrier of different genotype, the probability that when accepting irinotecan, produces toxic side effect is different.Wild-type UGT1A1 (6/6) can not produce toxic side effect when accepting irinotecan, and the probability of mutant heterozygote (6/7) generation toxic side effect is 12.5%, and mutant homozygote (7/7) then has 50% probability generation toxic side effect.In the Aisa people, each genotypic occurrence frequency of UGT1A1 gene is respectively that 6/6 type accounts for 70.2%, 6/7 type and accounts for 27.7%, 7/7 type and account for 2.1%.Therefore, drugs approved by FDA in 2005 are upgraded the product description of irinotecan, require to indicate that in information warning user's hereditary difference will influence its reaction to this medicine, should carry out UGT1A1*28 before the prompting medication and detect.
UGT1A1*93 is that (3156G>A), UGT1A1*6 are the point mutation (211G>A) on the exons 1 for point mutation on the promotor.Clinical study finds, has-the genotypic patient of 3156G has only 12.5% toxic side effects to occur, and-the genotypic patient of 3156A, have 50% spinoff after accepting irinotecan, to occur, and the time that toxic side effect occurs is also significantly early.The distribution frequency of UGT1A1*93 in the crowd of East Asia is about 9%.The ratio of the AUC of genotypic SN-38G of UGT1A1*6 and SN-38 (blood medicine-time lower curve area) significantly is lower than wild-type, and the survival rate that gets nowhere that UGT1A1*6 genotype patient accepts after the irinotecan all significantly descends with total survival rate.The distribution frequency of UGT1A1*6 in the crowd of East Asia is about 13.6%.
The launch of existing a plurality of both at home and abroad at present detection UGT1A1*28 allelotypes, like Third Wave, Genelex, Genzyme, LabCorp, Shen, Shanghai friend's biology and sky, Shanghai sky biology etc.But the product that these three gene locuss of joint-detection UGT1A1 are not arranged.It is the detection technique on basis that product in the market mainly is based upon with PCR; Like direct sequencing; PCR-single-strand conformation polymorphism analysis (SSCP) detects; These technology exist sensitivity low, the easy pollution of sample, the shortcoming that false positive rate is high, and regular-PCR method and quantitative fluorescent PCR can not satisfy clinical needs owing to detect the limitation of flux.And polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analytical technology and once can only carry out a kind of detection of sudden change, time and effort consuming based on the allelotrope discriminant analysis of TaqMan technology.
Based on the principle of chip, it is the suspension liquid-phase chip technology of carrier with the microballoon that U.S. Luminex company has developed.This techniques make use polystyrene microsphere as detection platform, carries out high-throughout many index parallel detection to nucleic acid and protein and other with fluorescence detector as the carrier of reaction.In the manufacturing processed of microballoon, mix the ruddiness and the infrared light staining agent of different ratios, thereby form the microballoon of 100 kinds of different colours codings of as many as.Different microballoon covalent attachment to the protein of different things to be detected or nucleic acid molecule as probe molecule, reporter molecules is with biotin labeling, and uses high-sensitive fluorescent dyeing.These microballoons and determinand, reporter molecules, fluorescent marker just form complete microballoon detection architecture and are used for reading of Luminex system.Excitated red respectively laser of Luminex reading system and green laser are used for the detection of microsphere system, and wherein red laser detects the red classification of microsphere surface intensity of fluorescence, and according to different color in the microballoon and number class, thereby confirm the type of reaction; Green laser detects the fluorescence intensity of fluorescent marker in the sample, detects microballoon kind, quantity through machine and computingmachine automatic statistical analysis laser again, thereby judges sample to be tested plurality of target tester concentration separately.Therefore, liquid-phase chip technology had both satisfied the requirement of high throughput testing, had possessed simultaneously quick and precisely, and was highly sensitive, and specificity is good, as a result advantage such as good reproducibility.We adopt the xTAG liquid-phase chip technology can detect multiple SNPs simultaneously, realize the operation of fast and convenientization of high-throughput, have improved detection efficiency greatly, in similar detection technique, maintain the leading position.
Summary of the invention
One of the object of the invention provides the UGT1A1 gene pleiomorphism and detects liquid-phase chip.This liquid-phase chip can be used for detecting the variation of normal genotype and three kinds of common allele type UGT1A1*28, UGT1A1*6 and UGT1A1*93 of UGT1A1 gene.
A kind of UGT1A1 gene pleiomorphism detects liquid-phase chip, mainly includes:
(1). be coated with the microballoon of the anti-tag sequence of special corresponding wild-type and anomaly respectively; Said anti-tag sequence is selected to UGT1A1*28 genotypic SEQ ID NO.13 and SEQ ID NO.14, is directed against genotypic SEQ IDNO.9 of UGT1A1*6 and SEQ ID NO.10 and/or is directed against UGT1A1*93 genotypic SEQ ID NO.11 and SEQ ID NO.12, and above-mentioned every kind of microballoon has the different colours coding;
(2). amplify have UGT1A1*28, the primer of UGT1A1*6 and/or the genotypic variation target sequence of UGT1A1*93; Said primer has biotin modification; So that corresponding PCR reaction product contains biotin labeling, and said PCR reaction product have can with the sequence of anti-tag complementary pairing.
Preferably; Said amplimer is for being directed against UGT1A1*28 and UGT1A1*6 genotypic SEQ ID NO.15 and SEQ IDNO.16; And/or to UGT1A1*93 genotypic SEQ ID NO.17 and SEQ ID NO.18, one end in above-mentioned every pair of primer has biotin modification.Preferably; The ASPE primer that said UGT1A1 gene pleiomorphism detection liquid-phase chip also includes corresponding wild-type and anomaly is right; Every kind of ASPE primer being made up of the specific primer sequence that is directed against the different genotype goal gene site of detecting of 3 ' end and the tag sequence of 5 ' end; Said specific primer sequence is for being directed against UGT1A1*6 genotypic SEQ ID NO.5 and SEQ ID NO.6 respectively; And/or to UGT1A1*93 genotypic SEQ ID NO.7 and SEQ ID NO.8, said tag sequence can with corresponding anti-tag sequence complementary pairing on the microballoon.
Another object of the present invention provides the method for using above-mentioned liquid-phase chip that the UGT1A1 gene pleiomorphism is detected.
A kind of method of using above-mentioned liquid-phase chip to the detection of UGT1A1 gene pleiomorphism mainly may further comprise the steps:
(1) use amplimer, pcr amplification testing sample DNA must contain biotin labeled PCR reaction product;
(2) the PCR reaction product is carried out hybridization with the microballoon that encapsulates special anti-tag sequence; Perhaps the PCR reaction product is carried out enzyme with the ExoSAP-IT test kit and is cut processing; Carry out primer extension reaction with said ASPE primer again; In reaction process, mix biotin labeled dCTP; Thereby make a plurality of biotin labeling on the reacted product band, the microballoon of the anti-tag sequence that being coated with of corresponding A SPE primer is special and the product behind the above-mentioned extension carry out hybridization;
(3) product behind the hybridization and Streptavidin-phycoerythrin react;
(4) detect through fluorescence detector.
Major advantage of the present invention is:
1. the identical rate of detection method provided by the present invention and PCR sequencing PCR is up to 100%.Prepared UGT1A1 gene pleiomorphism detects liquid-phase chip and has extraordinary signal-NR; And there is not cross reaction between institute's designed probe and the anti-tag sequence basically; Can also not need ASPE type specificity primer, detection method is easier.
2. gene pleiomorphism provided by the present invention detects liquid-phase chip and detection method thereof; Not only can be in one-time detection a plurality of pleomorphism sites of parallel detection; Realized simultaneously in one-time detection the parallel detection of different polymorphum types has been realized the important improvement to existing liquid-phase chip technology.
3. designed ASPE type specificity primer of the present invention has extraordinary specificity, can accurately distinguish the genotype of various types, and have better SNR, helps the parallel detection of many indexs.
4. detection method step of the present invention is simple; Three pleomorphism sites detect through a step multiplex PCR can accomplish two amplifications with target sequence of pleomorphism site; Many uncertain factors of existing in the complex operations processes such as repeated multiple times PCR have been avoided; Thereby can improve the detection accuracy rate greatly, embodied qualitative, quantitative analysis characteristic of accurate while.
5. the needed time of detection method provided by the present invention meets clinical needs especially well below sequencing technologies commonly used.
6. not only to have overcome traditional solid phase chip susceptibility not high in the present invention, and the defective of the repeatability difference of detected result is improved existing liquid-phase chip technology simultaneously, makes prepared microballoon can be applicable to the different detection project, has very strong expansion.The fluorescent signal value that detects improves greatly, thereby makes the sensitivity that detects be further enhanced, and SNR strengthens, and detected result more accurately and reliably.
Embodiment
Embodiment 1UGT1A1 gene pleiomorphism detects liquid-phase chip, mainly includes:
One, ASPE primer
To the variant sites of two kinds of common allele type UGT1A1*6 of UGT1A1 gene and UGT1A1*93 design specific primers sequence respectively.The ASPE primer is made up of " Tag+ specific primer sequence ".The ASPE primer sequence is as shown in the table:
Table 1 ASPE primer sequence
Every the ASPE primer comprises two parts, and 5 ' end is the specificity tag sequence to anti-tag sequence on the corresponding microballoon, and 3 ' end is mutant or wild-type specific primer sequence (shown in above-mentioned table 1).All ASPE primers are synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Every primer after synthetic is mixed with the stock solution of 100pmol/mL respectively with 10mmol/L Tris Buffer.
Two, the microballoon that encapsulates of anti-tag/ probe sequence
Target sequence according to required detection; Select the tag sequence; Reduce between the anti-tag sequence of each microballoon to greatest extent and secondary structure that tag and target fragment to be detected possibly form, corresponding anti-tag/ probe sequence is as shown in table 2 on six kinds of microballoons numberings of selection and the microballoon:
Corresponding anti-tag/ probe sequence on table 2 microballoon numbering and the microballoon
6 kinds of microballoons selecting are available from U.S. Luminex company, with the anti-tag/ probe sequence encapsulate with microballoon on.Be connected with the spacerarm preface of 5-10 T between anti-tag/ probe sequence and the microballoon; The spacerarm sequence that promptly before each anti-tag/ probe sequence, adds the preceding paragraph 5-10 T, the anti-tag/ probe sequence is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Synthetic anti-tag sequence is used sterilization ddH
2O is made into the stock solution of 100nmol/ml.Said spacerarm is to be used for specific probe and microsphere surface is spaced apart or specific probe placed the sequence of hydrophilic environments.Through the spacerarm sequence of suitable length is set between probe sequence and amino, can reduce sterically hinderedly, improve the efficient of hybridization and the specificity of hybridization.Common spacerarm sequence comprises poly dT, i.e. poly (dT), and oligomerization four polyoxyethylene glycol and (CH2) n spacerarm (n >=3) are like (CH2) 12, (CH2) 18 etc.In addition, if exist poly (dA) to disturb, can also use poly (TTG) as spacerarm.Spacerarm of the present invention is preferably 5-10 T, in China, and the synthetic technology comparative maturity of T, cost is relatively low.
The process that microballoon encapsulates is following:
Get 5 * 10 respectively
6The carboxylated microballoon of individual above-mentioned numbering (available from Luminex company) is suspended in the MES solution of 50ul 0.1mol/L (pH4.5), adds 10ul synthetic anti-tag molecule (100nmol/ml).EDC (N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide) (available from the Pierce Chemical company) working fluid of preparation 10ng/ml.Add the EDC working fluid of 2.5ul in the microballoon suspension, constant temperature was hatched 30 minutes, added the EDC working fluid of 2.5ul again, and constant temperature was hatched 30 minutes again.After reaction finished, the Tween-20 washing with 0.02% was once washed once with 0.1% SDS liquid again.The microballoon that is coated with the anti-tag sequence after the washing is resuspended in the Tris-EDTA solution of 100ul, and [10mmol/L Tris (pH8.0), among the 1mmol/L EDTA, 2-8 ℃ keeps in Dark Place.
Three. amplify the primer of target sequence with mutational site:
Three kinds of common allele type UGT1A1*28, UGT1A1*6 and UGT1A1*93 of UGT1A1 gene.UGT1A1*28 is the polymorphum of TA base pair multiplicity, and UGT1A1*93 is point mutation, all occurs in promoter region, but apart from each other; UGT1A1*6 occurs in exons 1.Utilize Primer5.0 design two pairs of primers (seeing table 3), amplify target sequence respectively.
Table 3 amplifies the primer of the target sequence with mutational site
Embodiment 2 utilization embodiment 1 described UGT1A1 gene pleiomorphism detects the detection of liquid-phase chip (not using the ASPE primer) to clinical sample
The prescription of said various solution is following:
MES damping fluid (pH5.0) prescription (250ml) of 50mM:
Reagent |
The source |
Final concentration |
The consumption of every 250ml |
MES(2[N-Morpholino] ethanesulfonic?acid) |
Sigma?M-2933 |
0.05M |
?2.44g |
5M?NaOH |
Fisher?SS256-500 |
--- |
5 |
2 * Tm hybridization buffer
Reagent |
The source |
Final concentration |
The consumption of every 250ml |
1MTris-HCl,pH8.0 |
SigmaT3038 |
0.2M |
50ml |
5M?NaCl |
Sigma?S5150 |
0.4M |
20ml |
Triton?X-100 |
Sigma?T8787 |
0.16% |
0.4ml |
Be stored in 4 ℃ after the filtration.
The primer of biotin modification is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
One, the DNA extraction of sample:
Extract the test kit explanation in a small amount with reference to AxyPrep whole blood genome, obtain DNA. to be detected
Two, the pcr amplification of testing sample
Utilize two pairs of primers of Primer5.0 design; Promoter fragment and the exons 1 that amplifies UGT1A1 with primer multiplex PCR one step of containing biotin modification totally two have biotin labeled target sequence of containing of pleomorphism site, the product size is respectively 586bp and 374bp.Primer sequence (SEQ NO.15-18) is seen shown in the above-mentioned table 4.
At first prepare PCR primer working fluid: the primer stock solution 100ul that respectively gets SEQ NO.15-16 respectively mixes and is PCR primer working fluid in the 1.5ml Eppendorf tube.The PCR reaction system is following:
10 * damping fluid (contains Mg
2+) 5ul
DNTP (each 2.5mmol/L) 4ul
Taq enzyme (5U/ul) 0.5ul
PCR primer working fluid (each 25pmol/mL) 4ul
Template DNA (10ng/ul) 1ul
ddH
2O 35.5ul
Be total to 50ul
The pcr amplification program is: 95 ℃ of 3min; 94 ℃ of 20s, 56 ℃ of 30s, 72 ℃ of 30s, 30 circulations; 72 ℃ of 10min; 4 ℃ of preservations are subsequent use.The base sequence of the amplified production that obtains: the sequence to UGT1A1*28, UGT1A1*6 variant sites is positioned on the 379-965bp of NG_009254, is positioned on the NG_0026011568-1942bp to the sequence of the variant sites of UGT1A1*93.
Three, hybridization
1. (microballoon concentration is 2.5 * 10 to choose the above-mentioned 6 kinds of microballoons of the UGT1A1*6, UGT1A1*93, the UGT1A1*28 that are coated with special anti-tag sequence
5Individual/ml).Every kind of microballoon has the different colours coding respectively, and every kind of microsphere surface is connected with one section specific oligonucleotide sequence (anti-tag) respectively simultaneously, and these anti-tag sequences can be respectively and the corresponding PCR product specific combination that contains biotin modification;
2. the microballoon of getting every kind of numbering of 1ul respectively is 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, the vortex mixing;
5. get the above-mentioned microballoon suspension of 25ul in the corresponding hole of 96 hole filter plates, control wells adds the ddH of 25ul
2O;
6. the PCR reaction solution of getting 5-25ul is used ddH in corresponding hole
2O complements to 50ul;
7. encase 96 orifice plates with lucifuge with masking foil, 95 ℃ of 60s, 60 ℃ of 15min are hatched hybridization;
8. the microballoon after the 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;
11 are resuspended in microballoon in 1 * Tm hybridization buffer of 75ul, and adding 15ul concentration is Streptavidin-phycoerythrin (SA-PE) of 10ug/ml;
12.60 ℃ hatch 15min, on the Luminex instrument, detect.
Four, the result detects and data analysis
The reaction after product is through Luminex serial analysis instrument detecting.With the carrier of polystyrene microsphere, as detection platform, nucleic acid molecule is carried out high-throughout many indexs parallel detection with fluorescence detector as reaction.In the manufacturing processed of microballoon, mix the ruddiness and the infrared light staining agent of different ratios, thereby form the microballoon of 100 kinds of different colours codings of as many as.Different microballoon covalent attachment to the nucleic acid molecule of different things to be detected as probe molecule, reporter molecules is with biotin labeling, and uses high-sensitive fluorescent dyeing.These microballoons and determinand, reporter molecules, fluorescent marker just form complete microballoon detection architecture and are used for reading of Luminex system.Excitated red respectively laser of Luminex reading system and green laser are used for the detection of microsphere system, and detected result is shown in table 4 and table 5.
Fluorescent value (MFI) and data processing there are following requirement:
1. each site need have an allelotrope MFI at least greater than 300 and greater than 10 * PCR negative control MFI;
2.NET MFI=sample MFI-PCR negative control MFI (NET MFI less than 0 with 0 the expression);
3. satisfy the data of above two conditions, calculate sudden change ratio by following formula:
Sudden change ratio=mutant NET MFI ÷ (mutant NET MFI+ wild-type NET MFI)
4. rule of thumb the sudden change ratio of each detection site is confirmed threshold value (cut-off value), to divide wild-type homozygote, heterozygote and mutant homozygote.
Use present method to detect the UGT1A1 gene pleiomorphism of great amount of samples, experimental data meets above-mentioned requirements, therefore can calculate their sudden change ratio.Being provided with as follows of threshold value (cut-off value): the sudden change ratio range is regarded as the wild-type homozygote at 0%-20%; 30%-70% is regarded as heterozygote; 80%-100% is regarded as the anomaly homozygote.Compare with the liquid-phase chip result with the PCR sequencing PCR detection, calculate the identical rate of classifying method detected result provided by the present invention.Present method detects 20 increments UGT1A1 genotype detection result and the sequencing result rate of coincideing originally and reaches 100%.Can detect the UGT1A1 genotype exactly it is thus clear that UGT1A1 gene pleiomorphism provided by the present invention detects liquid-phase chip, and the result is reliable and stable.
Table 4 pattern detection result (MFI)
Sequence number NO. |
*28-6
|
*28-7
|
?211G>A-w |
?211G>A-m |
-3156G>A-w |
-3156G>A-m |
Negative control |
17 |
5 |
?15 |
?3 |
16 |
3 |
1 |
668 |
675 |
?2015 |
?48 |
2098 |
32 |
2 |
3395 |
37 |
?2443 |
?38 |
661 |
682 |
3 |
2712 |
23 |
?2261 |
?37 |
2179 |
29 |
4 |
3340 |
13 |
?2783 |
?19 |
2441 |
40 |
5 |
2808 |
49 |
?2716 |
?35 |
2597 |
20 |
6 |
3097 |
49 |
?2883 |
?25 |
2130 |
16 |
7 |
652 |
586 |
?2492 |
?13 |
2104 |
49 |
8 |
665 |
643 |
?2697 |
?38 |
618 |
599 |
9 |
2654 |
25 |
?2515 |
?17 |
2168 |
38 |
10 |
3337 |
49 |
?602 |
?651 |
2630 |
13 |
11 |
3384 |
28 |
?2337 |
?41 |
2740 |
32 |
12 |
3161 |
24 |
?2558 |
?31 |
2113 |
16 |
13 |
2958 |
46 |
?2665 |
?34 |
2233 |
39 |
14 |
2817 |
21 |
?2913 |
?12 |
2601 |
21 |
15 |
2981 |
39 |
?2437 |
?40 |
35 |
2321 |
16 |
647 |
551 |
?2142 |
?15 |
2259 |
31 |
17 |
3243 |
14 |
?2097 |
?36 |
2955 |
27 |
18 |
687 |
568 |
?2902 |
?22 |
2891 |
39 |
19 |
2674 |
29 |
?2607 |
?32 |
2429 |
46 |
20 |
2581 |
33 |
?2752 |
?17 |
2004 |
12 |
Table 5 sample Polymorphism Analysis result
Embodiment 3 utilization embodiment 1 described UGT1A1 gene pleiomorphism detects the detection of liquid-phase chip (using the ASPE primer) to clinical sample
The prescription of said various solution such as embodiment 1 are said.
The ExoSAP-IT test kit is available from U.S. USB company.
Biotin labeled dCTP is available from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
One, the DNA extraction of sample:
Extract the test kit explanation in a small amount with reference to AxyPrep whole blood genome, obtain DNA. to be detected
Two, the pcr amplification of testing sample: of embodiment 1.
Three, the enzyme of PCR product is cut processing
With reference to the explanation of ExoSAP-IT test kit, detailed step is following:
1. get the reacted product of 7.5ul PCR, add 3ul ExoSAP-IT enzyme;
2.37 ℃ hatch 15min.Hatch 15min for 80 ℃, make unnecessary enzyme-deactivating.The product that enzyme is cut after the processing directly is used for follow-up ASPE primer extension reaction.
Four, site-specific primer extension reaction (ASPE)
Utilize the locus specificity primer of above-mentioned design to carry out primer extension reaction, in reaction process, mix biotin labeled dCTP, thereby make a plurality of biotin labeling on the reacted product band.
At first prepare blended ASPE primer working fluid: respectively get respectively 211G>A-w, 211G>A-m ,-3156G>A-w and the-corresponding ASPE primer of 3156G>A-m stock solution 10ul be in the 1.5ml Eppendorf tube; Add 10mmol/L Tris Buffer and mend, mix and be ASPE mix primer working fluid to 200ul.The system of ASPE reaction is following:
10 * damping fluid 2ul
MgCl
2(50mmol/L) 0.5ul
Biotin-dCTP(400umol/L) 0.25ul
DATP, dGTP, dTTP mixed solution (each 100umol/L) 1ul
Tsp enzyme (5U/ul) 0.25ul
Blended ASPE primer working fluid (each 500nmol/L) 1ul
Enzyme is cut the pcr amplification product 5ul of processing
ddH
2O 10.ul
Be total to 20ul
The PCR program is: 96 ℃ of 2min; 94 ℃ of 30s, 58 ℃ of 1min, 72 ℃ of 2min, 30 circulations; 4 ℃ of preservations are subsequent use.
Five, hybridization
1. according to designed ASPE primers, select UGT1A1*6, (microballoon concentration is 2.5 * 10 to the above-mentioned 4 kinds of microballoons of UGT1A1*93
5Individual/ml).Every kind of microballoon has the different colours coding respectively; Simultaneously every kind of microsphere surface is connected with one section specific oligonucleotide sequence (anti-tag) respectively, and these anti-tag sequences can be respectively and the tag sequence or the PCR product specific combination of corresponding ASPE primer 3 ' end;
2. 4 kinds of microballoons getting the above-mentioned UGT1A1*6 of 1ul, UGT1A1*93 respectively are 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, the vortex mixing;
5. get the above-mentioned microballoon suspension of 25ul respectively in the corresponding hole of 96 hole filter plates, control wells adds the ddH of 25ul
2O;
6. the ASPE reaction solution of getting 5-25ul UGT1A1*6 and * 93 is respectively used ddH in corresponding hole
2O complements to 50ul;
7. encase 96 orifice plates with lucifuge with masking foil, 95 ℃ of 60s, 37 ℃ of 15min are hatched hybridization;
8. the microballoon after the 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;
11 are resuspended in microballoon in 1 * Tm hybridization buffer of 75ul, and adding 15ul concentration is Streptavidin-phycoerythrin (SA-PE) of 10ug/ml;
12.37 ℃ hatch 15min, on the Luminex instrument, detect.
Seven, the result detects and data analysis
Data analysis such as embodiment 1 are said.
Use present method to detect the UGT1A1 gene pleiomorphism of great amount of samples, experimental data meets above-mentioned requirements, therefore can calculate their sudden change ratio.Being provided with as follows of threshold value (cut-off value): the sudden change ratio range is regarded as the wild-type homozygote at 0%-20%; 30%-70% is regarded as heterozygote; 80%-100% is regarded as the anomaly homozygote.Compare with the liquid-phase chip result with the PCR sequencing PCR detection, calculate the identical rate of classifying method detected result provided by the present invention.Present method detects 20 increments UGT1A1 genotype detection result and the sequencing result rate of coincideing originally and reaches 100%.Can detect the UGT1A1 genotype exactly it is thus clear that UGT1A1 gene pleiomorphism provided by the present invention detects liquid-phase chip, and the result is reliable and stable.
Table 6 pattern detection result (MFI)
Sequence number NO. |
?211G>A-w |
?211G>A-m |
-3156G>A-w |
-3156G>A-m |
Negative control |
?15 |
?0 |
15 |
5 |
1 |
?2179 |
?29 |
2097 |
36 |
2 |
?2783 |
?19 |
596 |
685 |
3 |
?2441 |
?40 |
2015 |
48 |
4 |
?2716 |
?35 |
2443 |
38 |
5 |
?2492 |
?13 |
2597 |
20 |
6 |
?2130 |
?16 |
2261 |
37 |
7 |
?2883 |
?25 |
2104 |
49 |
8 |
?2697 |
?38 |
608 |
599 |
9 |
?2515 |
?17 |
2558 |
31 |
10 |
?563 |
?681 |
2665 |
34 |
11 |
?2168 |
?38 |
2913 |
12 |
12 |
?2630 |
?13 |
2233 |
39 |
13 |
?2740 |
?32 |
2437 |
40 |
14 |
?2113 |
?16 |
2601 |
21 |
15 |
?2337 |
?41 |
44 |
2221 |
16 |
?2259 |
?31 |
2142 |
15 |
17 |
?2955 |
?27 |
2098 |
32 |
18 |
?2891 |
?39 |
2902 |
22 |
19 |
?2429 |
?46 |
2607 |
32 |
20 |
?2004 |
?12 |
2752 |
17 |
Table 7 sample UGT1A1 Polymorphism Analysis result
More than be to the specifying of possible embodiments of the present invention, but this embodiment is not in order to limiting claim of the present invention, does not allly break away from the equivalence that skill spirit of the present invention does and implement or change, all should be contained in the claim of the present invention.
Sequence table
< 110>Guangzhou Yishan Biotechnology Co., Ltd.
< 120>method and the liquid-phase chip of the detection of UGT1A1 gene pleiomorphism
<160>18
<170>PatentIn?version?3.1
<210>1
<211>44
<212>DNA
< 213>artificial sequence
<400>1
atcaaatctc?atcaattcaa?caatctcgtt?gtacatcaga?gacg 44
<210>2
<211>44
<212>DNA
< 213>artificial sequence
<400>2
tcatttcaat?caatcatcaa?caatctcgtt?gtacatcaga?gaca 44
<210>3
<211>40
<212>DNA
< 213>artificial sequence
<400>3
ctttttcaat?cactttcaat?tcatcccagc?ccacctgtcc 40
<210>4
<211>40
<212>DNA
< 213>artificial sequence
<400>4
ttcataacta?caatacatca?tcatcccagc?ccacctgtct 40
<210>5
<211>20
<212>DNA
< 213>artificial sequence
<400>5
ctcgttgtac?atcagagacg 20
<210>6
<211>20
<212>DNA
< 213>artificial sequence
<400>6
ctcgttgtac?atcagagaca 20
<210>7
<211>16
<212>DNA
< 213>artificial sequence
<400>7
cccagcccac?ctgtcc 16
<210>8
<211>16
<212>DNA
< 213>artificial sequence
<400>8
cccagcccac?ctgtct 16
<210>9
<211>24
<212>DNA
< 213>artificial sequence
<400>9
attgttgaat?tgatgagatt?tgat 24
<210>10
<211>24
<212>DNA
< 213>artificial sequence
<400>10
attgttgatg?attgattgaa?atga 24
<210>11
<211>24
<212>DNA
< 213>artificial sequence
<400>11
atgaattgaa?agtgattgaa?aaag 24
<210>12
<211>24
<212>DNA
< 213>artificial sequence
<400>12
atgatgatgt?attgtagtta?tgaa 24
<210>13
<211>26
<212>DNA
< 213>artificial sequence
<400>13
ttgccatata?tatatatata?agtagg 26
<210>14
<211>26
<212>DNA
< 213>artificial sequence
<400>14
gccatatata?tatatatata?agtagg 26
<210>15
<211>20
<212>DNA
< 213>artificial sequence
<400>15
ctgctacctt?tgtggactga 20
<210>16
<211>20
<212>DNA
< 213>artificial sequence
<400>16
cgtcagcatg?acatcaaagc 20
<210>17
<211>23
<212>DNA
< 213>artificial sequence
<400>17
gaacattcta?acggttcata?aag 23
<210>18
<211>18
<212>DNA
< 213>artificial sequence
<400>18
gcagcttcct?gggcacag 18