CN104388555B - The method that DNA microarray chip detects the SNP related to type II diabetes - Google Patents
The method that DNA microarray chip detects the SNP related to type II diabetes Download PDFInfo
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Abstract
A kind of method that the SNP related to type II diabetes is detected the present invention relates to DNA microarray chip, belongs to DNA microarray chip technology field.The method that the present invention is provided is used as label by the use of 6 oligonucleotides-modified golden nanometer particles, it is used to the oligonucleotide probe that type II diabetes detects with reference to 6 filtered out and have developed a kind of to realize the detection to the DNA sample of simulation type II diabetes and the actual sample of type II diabetes while detect the micro-array chip detection method of 6 and type II diabetes relevant mutational site.
Description
Technical field
The side of the SNP related to type II diabetes is detected the present invention relates to a kind of DNA microarray chip
Method, belongs to DNA microarray chip technology field.
Background technology
Type II diabetes is a kind of multiple-factor inheritance different substantiality disease as caused by h and E factor collective effect.
From science of heredity angle study its pathogenesis to the examination of prediabetes, diagnose and intervene extremely important (A genome-wide
association study identified novel risk loci for type 2diabetes.Nature.2007,
445,881-885；Genome-Wide Association Analysis Identifies Loci for Type
2Diabetes and Triglyceride Levels.Science,2007,316,1331-1336.).In the something lost of complex disease
Pass in Mechanism Study, SNP (single nucleotide polymorphisms, SNPs) analysis can be complexity
The genetic mechanism research of character provides a convenient, effective approach.Screening can apply to relevant with type II diabetes morbidity
The oligonucleotide probe of SNPs detections, prevention and individualized treatment to type II diabetes etc. have great importance.
Micro-array biochip has amount of samples few and high-throughout characteristic, can to life science with it is various in medical science
Biochemical reaction process progress is integrated, so as to realize to the bioactive substances such as gene, part and antigen or even cell, tissue
Carry out the test and analysis of efficient quick.For example DNA microarray chip has been widely applied to genomics research, drug screening
With (Resolving Individuals Contributing Trace Amounts of DNA in clinical detection
Highly Complex Mixtures Using High-Density SNP Genotyping Microarrays.PLoS
Genet,2008,4,1-9；Technology Insight:microarrays—research and clinical
applications.Nature Rev.Endocrinol.,2007,3,594-605.)。
Golden nanometer particle has excellent chemical stability and unique optical property.By being repaiied on golden nanometer particle surface
The different biomolecule (such as DNA, antibody and agglutinin) of decorations is as the label probe of micro-array biochip, according to its resonance
Light scatter properties, can set up highly sensitive biology sample detection system.Resonance Light Scattering Method ratio based on micro-array chip
Traditional fluorescence detection has higher sensitivity and more preferable stability (Scanometric DNA Array
Detection with Nanoparticle Probes.Science,2000,289,1757-1760；Nanoparticle-
Based Bio–Bar Codes for the Ultrasensitive Detection of Proteins.Science,
2003,301,1884-1886.).At present, a kind of golden nanometer particle label is typically only capable to be examined for a kind of biomolecule
Survey, therefore, prepare a kind of golden nanometer particle label that can be detected simultaneously to a variety of determinands to simplifying microarray life
The detection method of thing chip has great importance.
The content of the invention
The present invention detects related to type II diabetes in order to solve the above technical problems, providing a kind of DNA microarray chip
The method of SNP, this method is by the use of 6 oligonucleotides-modified golden nanometer particles as label, and filters out
6 oligonucleotide probes for being used for type II diabetes detection have developed one kind can be while detect multiple related to type II diabetes
The micro-array chip detection method in mutational site, is realized to simulating the DNA sample of type II diabetes and the reality of type II diabetes
The detection of border sample.
In order to solve the above-mentioned technical problem, technical scheme is specific as follows：
A kind of method that DNA microarray chip detects the SNP related to type II diabetes, including it is following
Step：
(1) 6 oligonucleotides-modified golden nanometer particle labels are synthesized；
(2) 6 specific oligonucleotides probes for 6 mutational site detections of type II diabetes are filtered out；
(3) it will be prepared using standard preparation method on the specific oligonucleotides probe sampling liquid point sample to chip
DNA microarray chip；
(4) it is real to the target oligonucleotide and type II diabetes for simulating type II diabetes with described DNA microarray chip
Border sample is detected, then testing result is marked with 6 oligonucleotides-modified golden nanometer particle labels.
In the above-mentioned technical solutions, step (1) is comprised the following steps that：
The synthesis of 6 oligonucleotides-modified golden nanometer particle labels：By the oligonucleotides of 6 100 μM of sulfydryl modifications
With SP1:SP2:SP3:SP4:SP5:SP6=1.2:1.2:0.6:1:1.6:1.6 ratio mixing；Take 5 μ l oligonucleotides mixing
Solution is well mixed with 300 μ l 8nM golden nanometer particle, is stored at room temperature reaction 10h, adds isometric PBS bufferings molten
Liquid, reacts after being well mixed and stays overnight；Reacted solution traditional vacuum is concentrated into 100 μ l, by centrifuging purified reaction product,
Obtain 6 oligonucleotides-modified golden nanometer particle labels；
Wherein described oligonucleotide sequence is respectively：
SP1：(T10) CTCTCTTACATA, SP2：(T10) ATTTAAACCTTC,
SP3：(T10) TTCCTGGTTTCA, SP4：(T10) ACTTGATGCAAT,
SP5：(T10) ATAGACTTACTG, SP6：(T10)GGCAATTAAATT。
In the above-mentioned technical solutions, the average grain diameter of described 6 oligonucleotides-modified golden nanometer particle labels is
13nm。
In the above-mentioned technical solutions, 6 mutational sites described in step (2) are respectively：Rs11196205,
Rs11196218, rs6585205, rs10946398, rs10811661, rs7903146；
6 described specific oligonucleotides probe sequences are respectively：
P1：CTGCTCGTAGTTATAA(T10)-NH2,
P1A：CTGCTGGTAGTTATAA(T10)-NH2,
P2：TCGGGTGCTTATGAAA(T10)-NH2,
P2A：TCGGGTGTTTATGAAA(T10)-NH2,
P3：TCTAAGTCGTGGGGCA(T10)-NH2,
P3A：TCTAAGTAGTGGGGCA(T10)-NH2,
P4：GACAGCATAACGATAC(T10)-NH2,
P4A：GACAGCAGAACGATAC(T10)-NH2,
P5：TCATGAGAAAACTAAA(T10)-NH2,
P5A：TCATGGGAAAACTAAA(T10)-NH2,
P6：ATATAGTATCTAAAAA(T10)-NH2,
P6A：ATATAATATCTAAAAA(T10)-NH2。
In the above-mentioned technical solutions, the point sample concentration of specific oligonucleotides probe sampling liquid described in step (3) is 30 μM.
In the above-mentioned technical solutions, the concentration of the target oligonucleotide of the simulation type II diabetes described in step (4) is
500nM, the concentration of 6 described oligonucleotides-modified golden nanometer particle labels is 3nM.
In the above-mentioned technical solutions, with few core of the described DNA microarray chip to simulation type II diabetes in step (4)
Thuja acid target and type II diabetes actual sample are detected and again with 6 oligonucleotides-modified golden nanometer particle labels
It it is 30 DEG C to the reaction temperature that testing result is marked.
In the above-mentioned technical solutions, the target oligonucleotide sequence difference of the simulation type II diabetes described in step (4)
For：
T1：TTATAACTACGAGCAGTATGTAAGAGAG,
T1A:TTATAACTACCAGCAGTATGTAAGAGAG,
T2：TTTCATAAGCACCCGAGAAGGTTTAAAT,
T2A:TTTCATAAACACCCGAGAAGGTTTAAAT,
T3：TGCCCCACGACTTAGATGAAACCAGGAA,
T3A：TGCCCCACTACTTAGATGAAACCAGGAA,
T4：GTATCGTTCTGCTGTCATTGCATCAAGT,
T4A：GTATCGTTCTGCTGTCATTGCATCAAGT,
T5：TTTAGTTTTCCCATGACAGTAAGTCTAT,
T5A：TTTAGTTTTCTCATGACAGTAAGTCTAT,
T6：TTTTTAGATACTATATAATTTAATTGCC,
T6A：TTTTTAGATATTATATAATTTAATTGCC.
In the above-mentioned technical solutions, described in step (4)：
When being detected with described DNA microarray chip to the target oligonucleotide for simulating type II diabetes, the mould
Intend the target oligonucleotide T of type II diabetes1、T2、T3、T4、T5、T6、T1A、T2A、T3A、T4A、T5AAnd T6AConcentration be respectively：
0.005th, 0.01,0.05,0.1,0.5,1,5,10,50,100,500 and 1000nM, oligonucleotide probe P1、P2、P3、P4、P5、
P6、P1A、P2A、P3A、P4A、P5AAnd P6APoint sample concentration be 30 μM；
When being detected with the DNA microarray chip to type II diabetes actual sample, the concentration difference of actual sample
For：0.5th, 1,5 and 10nM, oligonucleotide probe P1、P2、P3、P4、P5、P6、P1A、P2A、P3A、P4A、P5AAnd P6APoint sample concentration it is equal
For 30 μM.
In the above-mentioned technical solutions, also comprise the following steps after step (4)：
The DNA microarray chip and 1mL silver enhancement solutions are reacted into 8min；
The silver enhancement solution is the mixed solution of silver nitrate solution and hydroquinone solution, silver nitrate solution and hydroquinone solution
Volume ratio is 1:1.
The invention has the advantages that：
(1) method that the present invention is provided is that screening is combined using 6 oligonucleotides-modified golden nanometer particles as label
Probe manufacturing DNA microarray chip out, is detected that (analog sample and actual sample are i.e. to analog sample and actual sample
For target oligonucleotide).A pair list for the simulation DNA sample related to type II diabetes and actual sample can be realized using this method
Nucleotide polymorphisms are detected.
(2) present invention is used for the method for synthesizing a plurality of oligonucleotides-modified golden nanometer particle, stable with simple, soon
Fast the characteristics of.The 6 oligonucleotides-modified golden nanometer particle labels synthesized using this method can be simultaneously to multiple with II
Detected in the related mutational site of patients with type Ⅰ DM.
(3) method of the invention filter out it is multiple can be used for the specific oligonucleotides probe of type II diabetes detection, pass through
To SNP and the analytical proof of gene frequency, these specific oligonucleotides probes are examined to target oligonucleotide
Measuring tool has high specific.
Brief description of the drawings
The present invention is described in further detail with reference to the accompanying drawings and detailed description.
The process schematic for the method that Fig. 1 provides for the present invention.
The 6 oligonucleotides-modified golden nanometer particle labels and unmodified gold of the method synthesis of Fig. 2 present invention
The comparison diagram of nano-particle；
Wherein Fig. 2 a are the electron microscope and solution thereon of unmodified golden nanometer particle；Fig. 2 b are to be closed with the method for the present invention
Into 6 oligonucleotides-modified golden nanometer particle labels electron microscope and solution thereon.
Testing result figure of the method for Fig. 3 present invention to 6 susceptibility gene mutation sites of simulation type II diabetes.
The method of Fig. 4 present invention is examined to the gene frequency in 6 susceptibility gene mutation sites of simulation type II diabetes
Survey result and corresponding scanning figure.
The method of Fig. 5 present invention is to the risk genes testing result of 15 type II diabetes actual samples and corresponding
Scanning figure.
Embodiment
A kind of method that DNA microarray chip detects the SNP related to type II diabetes, is specifically included
Following steps：
(1) 6 oligonucleotides-modified golden nanometer particle labels are synthesized
By the oligonucleotides of six 100 μM of sulfydryl modifications with SP1:SP2:SP3:SP4:SP5:SP6=1.2:1.2:0.6:1:
1.6:1.6 ratio mixing.5 μ l mixtures are taken to be well mixed with 300 μ l8nM golden nanometer particle (by molar absorption coefficient
For 4.2 × 108cm-1M-1Calculate), reaction 10h is stored at room temperature, isometric PBS cushioning liquid (10mM PB, 0.2M is added
NaCl, pH 7.5), react and stay overnight after being well mixed.Above-mentioned solution traditional vacuum is concentrated into 100 μ l, passes through centrifugation
(13000rpm, (9000rpm, 3 ×)) purified reaction product, obtains 6 oligonucleotides-modified golden nanometer particle labels.Will
Product after purification is dissolved in probe reaction cushioning liquid (0.67 × SSC, 0.1% (w/v) SDS), is preserved at 4 DEG C.Institute
The average grain diameter for stating 6 oligonucleotides-modified golden nanometer particle labels is 13nm.
Wherein described oligonucleotide sequence is respectively：
SP1：(T10) CTCTCTTACATA, SP2：(T10) ATTTAAACCTTC,
SP3：(T10) TTCCTGGTTTCA, SP4：(T10) ACTTGATGCAAT,
SP5：(T10) ATAGACTTACTG, SP6：(T10)GGCAATTAAATT。
Fig. 2 a are the electron microscope and solution thereon of unmodified golden nanometer particle；Fig. 2 b are to be synthesized with the method for the present invention
The electron microscope and solution thereon of 6 oligonucleotides-modified golden nanometer particle labels.By comparative illustration we by 6
It is oligonucleotides-modified to have arrived golden nanometer particle surface.
(2) 6 specific oligonucleotides probes for 6 mutational site detections of type II diabetes are filtered out；
6 described mutational sites are respectively：Rs11196205, rs11196218, rs6585205, rs10946398,
Rs10811661, rs7903146；
6 described specific oligonucleotides probe sequences are respectively：
P1：CTGCTCGTAGTTATAA(T10)-NH2,
P1A：CTGCTGGTAGTTATAA(T10)-NH2,
P2：TCGGGTGCTTATGAAA(T10)-NH2,
P2A：TCGGGTGTTTATGAAA(T10)-NH2,
P3：TCTAAGTCGTGGGGCA(T10)-NH2,
P3A：TCTAAGTAGTGGGGCA(T10)-NH2,
P4：GACAGCATAACGATAC(T10)-NH2,
P4A：GACAGCAGAACGATAC(T10)-NH2,
P5：TCATGAGAAAACTAAA(T10)-NH2,
P5A：TCATGGGAAAACTAAA(T10)-NH2,
P6：ATATAGTATCTAAAAA(T10)-NH2,
P6A：ATATAATATCTAAAAA(T10)-NH2。
(3) it will be prepared using standard preparation method on the specific oligonucleotides probe sampling liquid point sample to chip
DNA microarray chip；
Specific oligonucleotides probe is dissolved in 3 × SSC, 1.5M glycine betaines, 0.005% (w/v) SDS sampling liquid.Root
According to standard preparation method using the rich micro-array chip contact pin mark systems of Austria SmartArrayer 136 in three-dimensional macromolecule D bases
Point sample on piece, point sample amount is about 1nL/ points, and point sample concentration is 30 μM.DNA microarray chip will be obtained to be put into reaction box in 37
DEG C, it is incubated overnight in 75% relative humidity constant temperature and humidity incubator.
(4) (II type is also referred to as simulated to the target oligonucleotide for simulating type II diabetes with described DNA microarray chip
Diabetes DNA sample) and type II diabetes actual sample detected, reuse 6 oligonucleotides-modified golden nanometer particles
Testing result is marked label；
25 μ l testing samples reaction 1h is first added in each subarray for the DNA microarray chip made, is added
3nM 6 oligonucleotides-modified golden nanometer particle label reaction 1h, reaction temperature is 30 DEG C, is cleaned and centrifuge dripping.
It is described simulation type II diabetes target oligonucleotide sequence be respectively：
T1：TTATAACTACGAGCAGTATGTAAGAGAG,
T1A:TTATAACTACCAGCAGTATGTAAGAGAG,
T2：TTTCATAAGCACCCGAGAAGGTTTAAAT,
T2A:TTTCATAAACACCCGAGAAGGTTTAAAT,
T3：TGCCCCACGACTTAGATGAAACCAGGAA,
T3A：TGCCCCACTACTTAGATGAAACCAGGAA,
T4：GTATCGTTCTGCTGTCATTGCATCAAGT,
T4A：GTATCGTTCTGCTGTCATTGCATCAAGT,
T5：TTTAGTTTTCCCATGACAGTAAGTCTAT,
T5A：TTTAGTTTTCTCATGACAGTAAGTCTAT,
T6：TTTTTAGATACTATATAATTTAATTGCC,
T6A：TTTTTAGATATTATATAATTTAATTGCC.
When being detected with described DNA microarray chip to simulation type II diabetes DNA sample, the type of simulation II sugar
Urinate the target oligonucleotide T of disease1、T2、T3、T4、T5、T6、T1A、T2A、T3A、T4A、T5AAnd T6AConcentration be respectively：0.005、
0.01st, 0.05,0.1,0.5,1,5,10,50,100,500 and 1000nM, oligonucleotide probe P1、P2、P3、P4、P5、P6、P1A、
P2A、P3A、P4A、P5AAnd P6APoint sample concentration be 30 μM；
When being detected with the DNA microarray chip to type II diabetes actual sample, the concentration difference of actual sample
For：0.5th, 1,5 and 10nM, oligonucleotide probe P1、P2、P3、P4、P5、P6、P1A、P2A、P3A、P4A、P5AAnd P6APoint sample concentration it is equal
For 30 μM.
In order to further improve detection sensitivity, the DNA microarray chip described in step (4) and 1mL silver can also be increased
Strong solution reaction 8min；DNA microarray chip is detected according to the resonant light scattering principle of golden nanometer particle.
The silver enhancement solution is the mixed solution of silver nitrate solution and hydroquinone solution, silver nitrate solution and hydroquinone solution
Volume ratio is 1:1.
Embodiment 1：Simulate the detection of type II diabetes DNA sample
With reference to Fig. 1, Fig. 3, Fig. 4 and table 1 illustrate the present embodiment.
The standard method formulated using Stanford Univ USA Blang laboratory, by fixed concentration (30 μm of ol/L) widow
Nucleotide probe (P1-P6) point sample prepares DNA microarray chip on substrate.By using PBS (PH=7.4,50mmol/L phosphorus
Acid buffer+0.15mol/L sodium chloride) and 0.1mol/L monoethanolamines closing chip.And successively with target oligonucleotide T1、T2、
T3、T4、T5、T6、T1A、T2A、T3A、T4A、T5AAnd T6AConcentration be respectively：0.005nM, 0.01nM, 0.05nM, 0.1nM,
0.5nM, 1nM, 5nM, 10nM, 50nM, 100nM, 500nM and 1000nM mixture, T1A-T6AIn the mixture shared hundred
Point ratio is respectively：0,0.2,0.5,1,2,5,10,20,50,70,90 and 100%, and the oligonucleotides-modified Jenner's grain of rices of 3nM
Son (SPn@GNPs), 30 DEG C of each reaction 1h.It is micro- using resonant light scattering after further being reacted 8 minutes with 1mL silver enhancement solutions
Array bio-chip scanner is detected.
Testing result of the method for Fig. 3 the present embodiment to 6 susceptibility gene mutation sites of simulation type II diabetes.As schemed
Shown in 3, the susceptibility loci that the DNA microarray chip obtained using this method can be related to type II diabetes to 6
Detected.The oligonucleotide probe in 6 sites filtered out has high specific to target oligonucleotide detection.Fig. 4 is profit
With the method for the present embodiment to the gene frequency testing result in 6 susceptibility gene mutation sites of simulation type II diabetes and right
The scanning figure answered.(a)-(f) corresponds to 1-6 susceptibility gene mutation site respectively in figure.As shown in figure 4, can using this method
With the gene frequency of accurate detection as little as 0.2%.
Table 1 is quantitative testing result of the inventive method to type II diabetes susceptibility gene mutation site, as shown in table 1,
Can be to detecting that the detection limit in simulation type II diabetes susceptibility gene mutation site can as little as 0.005nM using this method.
Embodiment 2：The actual sample detection of type II diabetes
With reference to Fig. 1, Fig. 5 and table 1 illustrate the present embodiment.
The blood of 15 patients with NIDDM is collected, performing PCR amplification is entered for rs10811661 sites.Using the U.S. this
The standard method that Tan Fu universities Blang is formulated in laboratory, by fixed concentration (30 μm of ol/L) oligonucleotide probe (Pn) point sample
DNA microarray chip is prepared on substrate.By using PBS (PH=7.4,50mmol/L phosphate buffer+0.15mol/L chlorine
Change sodium) and 0.1mol/L monoethanolamines closing chip.And the II type glycosuria with various concentrations (0.5nM, 1nM, 5nM and 10nM) successively
The PCR primer of patient's blood, and the oligonucleotides-modified golden nanometer particle (SP of 3nMn@GNPs), 30 DEG C of each reaction 1h.Enter again
One step is detected after being reacted 8 minutes with 1mL silver enhancement solutions using resonant light scattering micro-array biochip scanner.
Fig. 5 is that the risk genes in 15 patients with NIDDM rs10811661 sites are carried out using the present embodiment method
The result of detection.As shown in figure 5, the rs10811661 sites for having 11 patients in 15 patients are T/C heterozygous genotypes, 2
Patient is C/C homozygous genotypes, and 2 patients are T/T homozygous genotypes.
Table 1 is the quantitative testing result to type II diabetes susceptibility gene mutation site using the inventive method, such as the institute of table 1
Show, can be with as little as 0.5nM to the detection limit of type II diabetes actual sample using this method.
The quantitative testing result in the patients with type Ⅰ DM susceptibility gene mutation site of table 1.11
Obviously, above-described embodiment is only intended to clearly illustrate example, and the not restriction to embodiment.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of change or
Change.There is no necessity and possibility to exhaust all the enbodiments.And the obvious change thus extended out or
Among changing still in the protection domain of the invention.
Claims (1)
1. a kind of DNA microarray chip detects the non-diseases diagnostic purpose of the SNP related to type II diabetes
Method, it is characterised in that comprise the following steps：
(1) 6 oligonucleotides-modified golden nanometer particle labels are synthesized；
Comprise the following steps that：By the oligonucleotides of 6 100 μM of sulfydryl modifications with SP1:SP2:SP3:SP4:SP5:SP6=1.2:
1.2:0.6:1:1.6:1.6 ratio mixing；The 5 μ l oligonucleotides mixed solutions and 300 μ l 8nM golden nanometer particle is taken to mix
Close uniform, be stored at room temperature reaction 10h, add isometric PBS cushioning liquid, react and stay overnight after being well mixed；After reacting
Solution traditional vacuum be concentrated into 100 μ l, by centrifuging purified reaction product, obtain 6 oligonucleotides-modified Jenner's grain of rices
Sub- label；
Wherein described oligonucleotide sequence is respectively：
SP1：(T10) CTCTCTTACATA, SP2：(T10) ATTTAAACCTTC,
SP3：(T10) TTCCTGGTTTCA, SP4：(T10) ACTTGATGCAAT,
SP5：(T10) ATAGACTTACTG, SP6：(T10)GGCAATTAAATT；
(2) 6 specific oligonucleotides probes for 6 mutational site detections of type II diabetes are filtered out；
6 described mutational sites are respectively：Rs11196205, rs11196218, rs6585205, rs10946398,
Rs10811661, rs7903146；
6 described specific oligonucleotides probe sequences are respectively：
P1：CTGCTCGTAGTTATAA(T10)-NH2,
P1A：CTGCTGGTAGTTATAA(T10)-NH2,
P2：TCGGGTGCTTATGAAA(T10)-NH2,
P2A：TCGGGTGTTTATGAAA(T10)-NH2,
P3：TCTAAGTCGTGGGGCA(T10)-NH2,
P3A：TCTAAGTAGTGGGGCA(T10)-NH2,
P4：GACAGCATAACGATAC(T10)-NH2,
P4A：GACAGCAGAACGATAC(T10)-NH2,
P5：TCATGAGAAAACTAAA(T10)-NH2,
P5A：TCATGGGAAAACTAAA(T10)-NH2,
P6：ATATAGTATCTAAAAA(T10)-NH2,
P6A：ATATAATATCTAAAAA(T10)-NH2；
(3) DNA microarray will be prepared on specific oligonucleotides probe sampling liquid point sample to chip using standard preparation method
Chip；
(4) with target oligonucleotide and type II diabetes actual sample of the described DNA microarray chip to simulation type II diabetes
Product are detected, then testing result is marked with 6 oligonucleotides-modified golden nanometer particle labels；
The average grain diameter of described 6 oligonucleotides-modified golden nanometer particle labels is 13nm；
The point sample concentration of the specific oligonucleotides probe sampling liquid is 30 μM；
The concentration of the target oligonucleotide of simulation type II diabetes described in step (4) is 500nM, 6 described few nucleosides
The concentration of the golden nanometer particle label of acid modification is 3nM；
With target oligonucleotide and type II diabetes of the described DNA microarray chip to simulation type II diabetes in step (4)
Actual sample is detected and again testing result is marked with 6 oligonucleotides-modified golden nanometer particle labels
Reaction temperature is 30 DEG C；
The target oligonucleotide sequence of simulation type II diabetes described in step (4) is respectively：
T1：TTATAACTACGAGCAGTATGTAAGAGAG,
T1A:TTATAACTACCAGCAGTATGTAAGAGAG,
T2：TTTCATAAGCACCCGAGAAGGTTTAAAT,
T2A:TTTCATAAACACCCGAGAAGGTTTAAAT,
T3：TGCCCCACGACTTAGATGAAACCAGGAA,
T3A：TGCCCCACTACTTAGATGAAACCAGGAA,
T4：GTATCGTTCTGCTGTCATTGCATCAAGT,
T4A：GTATCGTTCTGCTGTCATTGCATCAAGT,
T5：TTTAGTTTTCCCATGACAGTAAGTCTAT,
T5A：TTTAGTTTTCTCATGACAGTAAGTCTAT,
T6：TTTTTAGATACTATATAATTTAATTGCC,
T6A：TTTTTAGATATTATATAATTTAATTGCC；
Described in step (4)：
When being detected with described DNA microarray chip to the target oligonucleotide for simulating type II diabetes, the simulation II
The target oligonucleotide T of patients with type Ⅰ DM1、T2、T3、T4、T5、T6、T1A、T2A、T3A、T4A、T5AAnd T6AConcentration be respectively：0.005、
0.01st, 0.05,0.1,0.5,1,5,10,50,100,500 and 1000nM, oligonucleotide probe P1、P2、P3、P4、P5、P6、P1A、
P2A、P3A、P4A、P5AAnd P6APoint sample concentration be 30 μM；
When being detected with the DNA microarray chip to type II diabetes actual sample, the concentration of actual sample is respectively：
0.5th, 1,5 and 10nM, oligonucleotide probe P1、P2、P3、P4、P5、P6、P1A、P2A、P3A、P4A、P5AAnd P6APoint sample concentration be
30μM；
Also comprise the following steps after step (4)：
The DNA microarray chip and 1mL silver enhancement solutions are reacted into 8min；
The silver enhancement solution is the volume of silver nitrate solution and the mixed solution of hydroquinone solution, silver nitrate solution and hydroquinone solution
Than for 1:1.
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