CN103849943B - MC1R detection in Gene Mutation specific primer and liquid-phase chip - Google Patents

Abstract

The invention discloses a kind of MC1R gene mutation detection liquid-phase chip and specific primer, this liquid-phase chip mainly includes: every kind of ASPE primer being held the specific primer sequence for genes of interest mutational site to form by 5 ' the tag sequences held and 3 ', described specific primer sequence is: for the SEQ ID NO.7 and SEQ ID NO.8 in C451T site, for the SEQ ID NO.9 and SEQ ID NO.10 in C478T site, and/or the SEQ ID NO.11 and SEQ ID NO.12 for G565T site；There is the anti coated microsphere of tag sequence；Amplimer.The testing result of detection liquid-phase chip provided by the present invention and the identical rate of sequencing are up to 100%, it is achieved the wild type in multiple mutational sites and saltant type parallel detection.

Description

MC1R detection in Gene Mutation specific primer and liquid-phase chip

Technical field

The invention belongs to biology field, relate to medical science and biotechnology, relate particularly to a kind of MC1R gene Mutation detection specific primer and liquid-phase chip.

Background technology

It is long that melanocortin 1 receptor (melanocortin 1receptor, MC1R) is positioned at No. 16 Chromosome 16qs 24.3 On arm, it is particularly located between No. 16 chromosome 89984286 to 89987384 base pairs.MC1R is melanocortin receptor One (297～317 aminoacid) minimum in (melanocortin receptor, MCR) family, this family is all G-protein Coupled receptor, has 7 transmembrane domain, and its native ligand is melanocortin Hormone Peptide.The clearest and the most definite melanocortin system (MC) the wide participation regulation and control of multiple physiologic pathway, including pigmentation, trophic behavior, body weight and energy metabolism balance, anti- The functions such as infection, sexual function and pain.MC1R mainly expresses in melanocyte, and MC1R genovariation can increase the black colour of skin Body suffers from the risk of tumor, additionally MC1R gene or a main freckle gene.

At present, MC1R detection method of gene mutation mainly has: Illumina optical fiber superbead chip technology, ground substance assistant laser Desorption ionization time-of-flight mass spectrometry technology (MALDI-TOF-MS) and fluorescent quantitative PCR technique, although Illumina optical fiber microballon Chip technology is the high throughput testing system of high sensitivity and accuracy, but automaticity is low, and manual operations is the most, difficult Needs with satisfied actual application.Matrix Assisted Laser Desorption ionization time-of-flight mass-spectrometric technique is a kind of Soft ionization techniques, The detection of protein and other has the powerful and function of maturation, but in field of nucleic acid detection, owing to nucleic acid divides The particularity of son itself, detection is subject to certain restrictions.Fluorescent quantitative PCR technique has highly sensitive, high specificity, automatization The feature that degree is high, but there is also the shortcoming that sample easily pollutes, false positive rate is high, and a kind of mutation type can only be detected every time.

Summary of the invention

An object of the present invention is to provide MC1R gene mutation detection liquid-phase chip, this liquid-phase chip can be used for individually or Three kinds of Common genes types C451T of parallel detection MC1R gene, the wild type of C478T and G565T and saltant type.

The technical scheme realizing above-mentioned purpose is as follows.

A kind of MC1R gene mutation detection liquid-phase chip, includes:

(A). the wild type separately designed for MC1R gene difference mutational site and the ASPE primer pair of saltant type: every ASPE primer is held the specific primer sequence for genes of interest mutational site to form by 5 ' the tag sequences held and 3 ', described spy Specific primer sequence is: for the SEQ ID NO.7 and SEQ ID NO.8 in C451T site, for the SEQ ID in C478T site NO.9 and SEQ ID NO.10, and/or the SEQ ID NO.11 and SEQ ID NO.12 for G565T site；Described tag sequence Selected from SEQ ID NO.1～SEQ ID NO.6；

(B). there are the microsphere that different anti-tag sequence is coated, have different colours coding, described anti-tag sequence It is additionally provided with spacer sequence in the middle of being connected with microsphere；Described anti-tag sequence is selected from SEQ ID NO.13～SEQ ID NO.18, and the tag complementary pairing that described anti-tag sequence can be correspondingly selected with (A)；

(C). for amplifying the primer of target sequence that need detection, that there is corresponding mutational site.

Wherein in an embodiment, described amplimer is: for C451T, C478T site SEQ ID NO.19 and SEQID NO.20, and/or the SEQ ID NO.21 and SEQ ID NO.22 for G565T site.

Wherein in an embodiment, described ASPE primer is: for C451T site by SEQ ID NO.1 and SEQ The sequence of ID NO.7 composition and the sequence that is made up of SEQ ID NO.2 and SEQ ID NO.8, for C478T site by SEQ The sequence of ID NO.3 and SEQ ID NO.9 composition and the sequence that is made up of SEQ ID NO.4 and SEQ ID NO.10, and/or pin To the sequence being made up of SEQ ID NO.5 and SEQ ID NO.11 in G565T site and by SEQ ID NO.6 and SEQ ID The sequence of NO.12 composition.

It is a further object of the present invention to provide the specific primer for MC1R detection in Gene Mutation.

Realize above-mentioned purpose technical scheme as follows.

For the specific primer of MC1R detection in Gene Mutation, described specific primer sequence is: for C451T site SEQID NO.7 and SEQ ID NO.8, for the SEQ ID NO.9 and SEQ ID NO.10 in C478T site, and/or for The SEQ ID NO.11 and SEQ ID NO.12 in G565T site.

Main advantages of the present invention are:

The testing result of MC1R gene mutation detection liquid-phase chip the most provided by the present invention is high with the identical rate of sequencing Reach 100%.And the time required for detection is well below conventional sequencing technologies, meet actual application needs especially.Prepared MC1R gene mutation detection liquid-phase chip there is extraordinary signal-noise ratio, and designed probe and anti- Cross reaction it is substantially absent from, tag sequence label, the choosing and tag label of anti-tag sequence label between tag sequence Sequence and the combination of concrete ASPE primer, it is possible to avoid cross reaction, it is achieved the parallel detection in multiple mutational sites.

2. the present invention passes through the design experiences of inventor's long term accumulation and substantial amounts of experimental implementation, draws from numerous specificitys Thing have chosen the combination of optimum.The ASPE primer specificity primer of present invention design sensitive can identify target detection specifically Mutational site, accurately distinguish the genotype of various type；In same reaction system, between different specific primers, Being substantially absent from cross reaction between the pcr amplification product of specific primer and non-targeted detection, detection specificity is good, intersects Response rate is less than 3%；Except Single locus catastrophe can be detected, it is also possible to dashing forward of the multiple mutational site of parallel detection simultaneously Change situation, Detection results is consistent.

3. the detection method step of the present invention is simple, and 3 kinds of mutational site detections can complete 2 by One_step PCR and contain There is an amplification of the target sequence in mutational site, it is to avoid many uncertain present in the complex operations processes such as repeated multiple times PCR Factor, thus it is greatly improved Detection accuracy, embody accurate qualitative and quantitative analysis feature simultaneously.

4. not only to overcome conventional solid chip sensitivity the highest for the present invention, the defect of the repeatable difference of testing result, Existing liquid-phase chip technology is improved so that prepared microsphere can be suitably used for different detection projects, has very simultaneously Strong expansion.The fluorescence signal value of detection is greatly improved, so that the sensitivity of detection is further enhanced, and signal to noise ratio Strengthening, testing result is more accurately and reliably.

Detailed description of the invention

Embodiment 1MC1R gene mutation detection liquid-phase chip, mainly includes:

One, ASPE primer

For three kinds of Common genes types C451T of MC1R gene, the wild type of C478T and G565T and saltant type, separately design Specific primer sequence.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 1MC1R gene

Every ASPE primer includes two parts, and 5 ' ends are for for the specificity tag of anti-tag sequence on corresponding microsphere Sequence, 3 ' ends are saltant type or the special primer segments (as shown in Table 1 above) of wild type.All ASPE primers are by the raw work in Shanghai Biotechnology Services Co., Ltd synthesizes.Every primer after synthesis is configured to 10mmol/LTris Buffer respectively The stock solution of 100pmol/mL.

Two, the coated microsphere of anti-tag sequence

According to designed ASPE specific primer fragment, select tag sequence, reduce each microsphere to greatest extent Between anti-tag sequence and the secondary structure that is likely to be formed of tag with ASPE specific primer fragment, 6 kinds of microspheres of selection are compiled Number anti-tag sequence corresponding with on microsphere is as shown in table 2:

Anti-tag sequence corresponding with on microsphere numbered by table 2 microsphere

Anti-tag sequence, purchased from Luminex company of the U.S., is coated on microsphere by the 6 kinds of microspheres selected.anti-tag It is connected the spacer sequence having 5-10 T between sequence with microsphere, before each anti-tag sequence, i.e. adds 5-10 T of the preceding paragraph Spacer sequence, anti-tag sequence is synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Anti-by synthesis Tag sequence sterilizing ddH2O is made into the stock solution of 100nmol/ml.Described spacerarm is for for by anti-tag and microsphere surface Spaced apart or that anti-tag is placed in hydrophilic environments sequence.By arranging between anti-tag sequence and microsphere The spacer sequence of suitable length, can reduce sterically hindered, improves efficiency and the specificity of hybridization of hybridization.Often The spacer sequence seen includes poly dT, i.e. poly(dT), oligomerization four Polyethylene Glycol and (CH2) n spacerarm (n >=3), as (CH2) 12, (CH2) 18 etc..It addition, if there is poly(dA) interference, it is also possible to poly(TTG) as spacerarm.This Bright spacerarm is preferably 5-10 T, and the coated process of microsphere is as follows:

Take 5 × 10 respectively⁶The carboxylated microsphere (purchased from Luminex company) of individual above-mentioned numbering is suspended in In the MES solution of 50ul0.1mol/L (pH4.5), add the anti-tag molecule (100nmol/ml) of 10ul synthesis.Preparation The EDC(N-(3-Dimethylaminopropyl-N-ethylcarbodiimide of 10ng/ml) (purchased from Pierce Chemical company) working solution.In microsphere suspensions, add the EDC working solution of 2.5ul, constant-temperature incubation 30 minutes, add The EDC working solution of 2.5ul, then constant-temperature incubation 30 minutes.After reaction terminates, washed once with the Tween-20 of 0.02%, then use The SDS liquid of 0.1% washed once.The microsphere being coated with anti-tag sequence after washing is resuspended in the Tris-EDTA of 100ul Solution [10mmol/L Tris(pH8.0)], in 1mmol/LEDTA, 2-8 DEG C keeps in Dark Place.

Three, the primer of the target sequence containing mutational site is amplified

For MC1R gene three kinds of Common genes types C451T, C478T and G565T, design amplimer, to (see Table 3), expands Increase and 2 target sequences containing 3 mutational sites.

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 primer after synthesis is used respectively 10mmol/L Tris Buffer is configured to the stock solution of 100pmol/mL.

Embodiment 2 uses the detection to sample of the MC1R gene mutation detection liquid-phase chip described in embodiment 1

The formula of described various solution is as follows:

MES buffer (pH5.0) formula (250ml) of 50mM:

2 × Tm hybridization buffer

4 DEG C it are stored in after filtration.

ExoSAP-IT test kit is purchased from USB company of the U.S..

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 correlation technique of DNA extraction, obtain DNA to be detected.

Two, the PCR amplification of testing sample

Design 2 to primer, multiplex PCR one step amplify 2 respectively containing three kinds of Common genes types C451T of MC1R gene and The target sequence of C478T, G565T, product size is respectively 363bp, 228bp, on primer sequence (SEQ IDNO.19-22) is shown in State shown in table 3.

First preparation multiple PCR primer working solution: respectively take the primer stock solution 100ul of SEQ ID NO.19-22 in In 1.5ml microcentrifugal tube, mix homogeneously is multiple PCR primer working solution.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℃10min；4℃ Save backup.

Three, the enzyme action of PCR primer processes

1. take the reacted product of 7.5ulPCR, add 1ul10 × SAP buffer, 1ul SAP enzyme and 0.5ul Exo-I Enzyme；

Hatch 15min for 2.37 DEG C, hatch 15min for 80 DEG C, inactivate unnecessary enzyme.After product after enzyme action process is directly used in Continuous 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, course of reaction mixes biotin mark The dCTP of note, so that biotin labelings multiple on reacted product band.

First the ASPE primer working solution of mixing is prepared: take the corresponding wild type of gene to be detected and saltant type ASPE respectively Primer stock solution 10ul, in 1.5ml microcentrifugal tube, adds 10mmol/L Tris Buffer and mends to 200ul, mix homogeneously It is ASPE mix primer working solution.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 corresponding 6 kinds of coated microspheres (as described in Example 1) of every group selection, every kind Microsphere concentration is 2.5 × 10⁵Individual/ml；

Take the microsphere of 1ul every kind numbering the most respectively in the microcentrifugal tube of 1.5ml；

3. microsphere is centrifuged 1-2min in >=10000g；

4. supernatant discarded, microsphere is resuspended in 2 × Tm hybridization buffer of 100ul, and vortex mixes；

5. taking the above-mentioned microsphere suspensions of 25ul in the 96 corresponding holes of hole filter plate, control wells adds the ddH of 25ul₂O；

6. take the ASPE reactant liquor of 5-25ul in corresponding hole, use ddH₂O complements to 50ul；

7. encasing 96 orifice plates with lucifuge with masking foil, 95 DEG C of 60s, 37 DEG C of 15min hatch hybridization；

8. the microsphere after hybridization is centrifuged 2-5min in >=3000g；

9. remove supernatant, microsphere is resuspended in 1 × Tm hybridization buffer of 75ul；

10. microsphere is centrifuged 2-5min in >=3000g；

Microsphere is resuspended in 1 × Tm hybridization buffer of 75ul by 11., adds the strepto-parent that 15ul concentration is 10ug/ml With element-phycoerythrin (SA-PE)；

Hatch 15min for 12.37 DEG C, detect on Luminex instrument.

Six, result detection and data analysis

Reaction afterproduct is detected by Luminex serial analysis instrument.Testing result is as shown in table 4, table 5 and table 6.

Fluorescent value (MFI) and data process are had claimed below:

The most each site needs an at least allele MFI more than 300 and to be more than 10 × PCR negative control MFI；

2.NET MFI=sample MFI-PCR negative control MFI(NET MFI representing with 0 less than 0)；

3. meet the data of two above condition, by following equation calculating sudden change ratio:

Sudden change ratio=saltant type NETMFI ÷ (saltant type NETMFI+ wild type NETMFI)

The most rule of thumb the sudden change ratio to each detection site determines threshold value (cut-off value), pure to divide wild type Zygote, heterozygote and saltant type homozygote.

Using the MC1R gene SNP site of this method 20 parts of samples of detection, experimental data meets above-mentioned requirements, therefore can count Calculate to obtain their sudden change ratio.To be considered as wild type at 0%-20% pure for the sudden change ratio range that is provided that of threshold value (cut-off value) Zygote；30%-70% is considered as heterozygote；80%-100% is considered as anomaly homozygote.Make with liquid-phase chip result with sequencing detection Comparison, calculates the identical rate of classifying method testing result provided by the present invention.The MC1R gene of this method 20 parts of samples of detection Type testing result and the sequencing result rate of coincideing reach 100%.Visible MC1R gene SNP detection liquid-phase chip provided by the present invention The SNP type of MC1R can be detected exactly, and result is reliable and stable.

One of table 4 pattern detection result (MFI)

Table 5 sample MC1R gene mutation ratio (%)

Sample number	C451T	C478T	G565T
				1	1%	2%	2%
2	2%	2%	2%
				3	2%	2%	2%
4	1%	2%	2%
				5	3%	1%	1%
6	2%	1%	2%
				7	2%	1%	2%
8	2%	2%	2%
				9	48%	3%	2%
10	1%	1%	2%
				11	2%	3%	2%
12	2%	2%	1%
				13	1%	2%	2%
14	1%	1%	97%
				15	1%	2%	2%
16	2%	2%	1%
				17	1%	99%	2%
18	3%	2%	1%
				19	2%	2%	1%
20	1%	1%	2%

Table 6 sample MC1R gene mutation type analysis result

The detection to MC1R gene SNP site of the liquid-phase chip of the ASPE primer that embodiment 3 is different

One, the design (Tag sequence and the selection of Anti-Tag sequence) that prepared by liquid-phase chip

By MC1R gene C 451T, C478T and G565T site mutation detection liquid-phase chip as a example by, be respectively directed to C451T, The specific primer sequence that the wild type of C478T and G565T and saltant type design ASPE primer 3 ' are held, and what ASPE primer 5 ' was held Tag sequence is then selected from SEQ ID NO.1-SEQ ID NO.6, and accordingly, be coated on microsphere joins with corresponding tag complementary To anti-tag sequence selected from SEQ ID NO.13-SEQ ID NO.18.Shown in specific design such as following table (table 7).ASPE draws The synthesis of thing, anti-tag sequence are coated microsphere, amplimer, detection method etc. as described in embodiment 1 and embodiment 2.

Design prepared by table 7 liquid-phase chip

One, sample detection

Use liquid-phase chip prepared by above-mentioned design, as described in embodiment 2, detect process and sample 21-40 is carried out by method Detection, testing result is as follows:

Table 8 sample MC1R gene C 451T testing result and Polymorphism Analysis

Table 9 sample MC1R gene C 478T testing result and Polymorphism Analysis

Table 10 sample MC1R gene G565T testing result and Polymorphism Analysis

From above-described embodiment, other uses different tag for the liquid-phase chip in different mutational sites, ASPE primer Sequence, its result is the most reliable and the most stable, and concrete data are omitted.And ASPE primer selects tag sequence in embodiment 1 to draw with specificity During the collocation of thing sequence, better (signal to noise ratio is more preferable), see the present embodiment test group 1, test group 5 and test group 9.Other is not Arranging in pairs or groups with specific primer sequence with tag sequence, identical with the result of embodiment 2 and the present embodiment, concrete data are omitted.

The selection of embodiment 4MC1R detection in Gene Mutation specific primer sequence

One, the design (wild type and the selection of saltant type specific primer sequence) that prepared by liquid-phase chip

As a example by the pleomorphism site of MC1R gene C 451T and G565T detects liquid-phase chip, with this place, mutational site mesh The complementary series forward or backwards of mark sequence is template, is respectively directed to wild type and the saltant type design ASPE of C451T and G565T The specific primer sequence that primer 3 ' is held, including specific primer sequence preferred in the embodiment of the present invention 1 and 2 alternative spies Specific primer sequence, as shown in table 11.Wherein, interior base is pleomorphism site.

Table 11 specific primer sequence

As a example by the pleomorphism site of MC1R gene C 451T and G565T detects liquid-phase chip, select for C451T and G565T With different specific primer sequences, the tag sequence that ASPE primer 5 ' is held then is fixed as the best effective infructescence in embodiment 1 Row, and select corresponding anti-tag sequence, shown in specific design such as following table (table 12).The synthesis of ASPE primer, Anti-tag sequence is coated microsphere, amplimer, detection method etc. as described in embodiment 1 and embodiment 2.

The two of design prepared by table 12 liquid-phase chip

Two, sample detection

Use liquid-phase chip prepared by above-mentioned design, as described in embodiment 2, detect process and sample 41-60 is carried out by method Detection, testing result is as follows:

Table 13 sample MC1R gene C 451T testing result and Polymorphism Analysis

Table 14 sample MC1R gene G565T testing result and Polymorphism Analysis

From the present embodiment, when ASPE primer selects specific primer sequence and the collocation of tag sequence in embodiment 1, effect Fruit more preferably (signal to noise ratio is more preferable), sees the present embodiment test group 10 and test group 13.Other derives from place, target detection site The different specific primer sequences of the complementary series forward or backwards of sequence are arranged in pairs or groups with tag sequence, with embodiment 2 and the present embodiment Result identical, be i.e. still that the specific primer sequence described in embodiment 2 from different tag sequence arranging effects more preferably, Concrete data are omitted.

Other is arranged in pairs or groups with tag sequence for the multiple specific primer sequence of different SNP site, with embodiment 2 and basis The result of embodiment is identical, i.e. specific primer selected by embodiment 1, has more preferable signal to noise ratio, and Detection results is also more preferable, Concrete data are omitted.

Embodiment described above only have expressed the several embodiments of the present invention, and it describes more concrete and detailed, but also Therefore the restriction to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that, for those of ordinary skill in the art For, without departing from the inventive concept of the premise, it is also possible to make some deformation and improvement, these broadly fall into the guarantor of the present invention Protect scope.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.

Claims (3)

1. a MC1R gene mutation detection liquid-phase chip, is characterized in that, includes:

(A). the wild type separately designed for MC1R gene difference mutational site and the ASPE primer pair of saltant type: every ASPE Primer is held the specific primer sequence for genes of interest mutational site to form by 5 ' the tag sequences held and 3 ', and described ASPE draws Thing is: for the sequence being made up of SEQ ID NO.1 and SEQ ID NO.7 in C451T site and by SEQ ID NO.2 and SEQ The sequence of ID NO.8 composition, and selected from the sequence being made up of SEQ ID NO.3 and SEQ ID NO.9 for C478T site And the sequence being made up of SEQ ID NO.4 and SEQ ID NO.10, and for G565T site by SEQ ID NO.5 and SEQ At least one pair of in the sequence of ID NO.11 composition and the sequence that is made up of SEQ ID NO.6 and SEQ ID NO.12；

(B). having the microsphere that different anti-tag sequence is coated, have different colours coding, described anti-tag sequence is with micro- Ball is additionally provided with spacer sequence in the middle of connecting；Described anti-tag sequence is selected from SEQ ID NO.13～SEQ ID NO.18, and Described anti-tag sequence can be correspondingly selected with (A) the pairing of tag complementary；

(C). for amplifying the primer of target sequence that need detection, that there is corresponding mutational site；Described amplimer is: For the SEQ ID NO.19 and SEQ ID NO.20 in C451T, C478T site, and the SEQ ID NO.21 for G565T site And SEQ ID NO.22.

MC1R gene mutation detection liquid-phase chip the most according to claim 1, is characterized in that, described ASPE primer is: pin To the sequence being made up of SEQ ID NO.1 and SEQ ID NO.7 in C451T site and by SEQ ID NO.2 and SEQ ID NO.8 The sequence of composition, for the sequence being made up of SEQ ID NO.3 and SEQ ID NO.9 in C478T site and by SEQ ID NO.4 The sequence formed with SEQ ID NO.10, and the sequence being made up of SEQ ID NO.5 and SEQ ID NO.11 for G565T site Row and the sequence being made up of SEQ ID NO.6 and SEQ ID NO.12.

MC1R gene mutation detection liquid-phase chip the most according to claim 1 and 2, it is characterised in that described spacerarm is 5-10 T.