CN102888446A - Gene chip for detecting stippled cartilage dysmorphism syndrome I type - Google Patents

Abstract

The invention belongs to the field of gene chips, and relates to a gene chip for detecting a stippled dysmorphism syndrome I type. The gene chip provided by the invention comprises a substrate and a probe, wherein the probe is immobilized on the substrate; and the probe is a nucleotide sequence represented by SEQ ID NO.1-50; and the substrate is one of a glass slide, a silicon wafer, a film and a high polymer material. The gene chip provided by the invention has the advantages of simple operation steps, high detection specificity, good stability, short time and low cost, and being capable of quickly and automatically detecting gene mutation of the stippled cartilage dysmorphism syndrome I type with low cost and high flux.

Description

Detect the gene chip of mottled cartilage abnormity syndrome i type

Technical field

The present invention relates to a kind of gene chip of biological technical field, more specifically, the present invention is a kind of gene chip that detects mottled cartilage abnormity syndrome i type (CDPX1).

Background technology

Mottled cartilage abnormity syndrome i type (CDPX1) is owing to be positioned ARSE gene on the Xp22.3 and disappearance or sudden change occur cause the shortage of arylsulfatase, peroxysome unusual, thereby causes the congenital disorders of dyschondroplasia.Main clinical manifestation is short and small for acra symmetry up and down, distinctive facies, neurodevelopment are slow, cataract, skin are fish scale-shaped change etc., and sickness rate is 1/500000 among the crowd.

Mottled cartilage abnormity syndrome i type belongs to the chain stealthy heredity of X.Key area is positioned Xp22.33, in the zone of about 3Mb, mainly is because sudden change or the disappearance of this zone ARSE gene cause between PABX and the DXS31.

But the father and mother that carry sudden change or disappearance are done the ill fetus of antenatal diagnosis early discovery, clinical diagnosis is not difficult to make according to its large joint characteristic x-ray performance, round pcr can effectively detect patient ARSE transgenation, and the FISH technology can effectively detect patient's ARSE genetically deficient.But there is no at present the method that a kind of quick, low-cost, large flux and automatization detect the transgenation of mottled cartilage abnormity syndrome i type.

Comparative genome hybridization (CGH) chip analysis technology is a kind of disruptive technology, can support the researchist to pass through the accurately research chromosomal variation relevant with disease of microarray.The principle of comparative genome hybridization (CGH) chip analysis is to hybridize simultaneously with the sample (test sample and control sample) of the different fluoresceins of two parts of marks at a chip, thus the difference of the number of copies of genomic dna between test experience sample and the control sample fast and intuitively.CGH is applied to the research that disease genetic is learned, a complete genomic scintigram can be provided, the prompting sample DNA exists amplification or disappearance at whole genomic which specific position, the zone of amplification occurs in those, very likely have Disease-causing gene, and the disappearance zone namely may comprise some suppressor genes.

Summary of the invention

The object of the invention is to overcome the deficiencies in the prior art, a kind of gene chip that detects mottled cartilage abnormity syndrome i type is provided.Diagnosing chip operation steps of the present invention is simple, and detection specificity is high, good stability, and the time is short, and cost is low.

The present invention is achieved by the following technical solutions, the present invention relates to a kind of gene chip that detects mottled cartilage abnormity syndrome i type, comprises the sheet base and be fixed on probe on the sheet base, and its middle probe is the nucleotide sequence shown in the SEQ ID NO.1-50.Described base is a kind of in slide glass, silicon chip, film, the macromolecular material.

Among the present invention, described probe numbering and sequence are as shown in table 1,

Table 1

Among the present invention, described gene chip claims again DNA chip, biochip, in the embodiments of the invention, after will a large amount of (common every square centimeter of reticular density is higher than 400) probe molecules being fixed on the upholder and the sample molecule of mark hybridize, by the hybridization signal intensity that detects each probe molecule and then quantity and the sequence information that obtains sample molecule; Particularly, its order-checking principle is the sequencing by hybridization method, namely by carrying out the method for determining nucleic acid sequence with the nucleic acid probe hybridization of one group of known array, has fixed the probe of eight known Nucleotide of sequence at a substrate surface; In solution, with fluorescently-labeled nucleotide sequence, when mating with the nucleic acid probe generation complementation of correspondence position on the gene chip, by determining the strongest probe location of fluorescence intensity, obtain the probe sequence of one group of sequence complete complementary; Can recombinate out the accordingly sequence of target nucleic acid;

Described biochip technology is owing to be fixed in a large amount of probes on the upholder simultaneously, can disposablely carry out determination and analysis to a large amount of sequences of sample, solve traditional mRNA blot (Southern Blotting and Northern Blotting etc.) deficiencies such as technological operation is numerous and diverse, level of automation is low, operating sequence quantity is few, detection efficiency is low; And, by designing different probe arrays, using specific analytical procedure can make this technology have multiple different using value, such as gene expression profile mensuration, consolidation detection, polymorphism analysis, genomic library mapping and sequencing by hybridization etc.

Among the present invention, the preparation method of described gene chip, mainly contain in-situ synthesis and point sample method: wherein, (1) in-situ synthesis is based on the composition principle of combinatorial chemistry, decide coupling site and the order of different chemical monomer on the substrate surface by one group of locating template, the Nucleotide of VITAMIN B4 (A), guanine (G), cytosine(Cyt) (C), four kinds of different bases of thymus pyrimidine (T) is pressed different order chemical couplings in corresponding site, the oligonucleotide probe that the original position composition sequence is different forms the DNA chip; (2) the standby gene chip of point sample legal system prepares cDNA (or oligonucleotide) probe library at first according to a conventional method, then by special mini sprinkler, respectively different probe solutions according to the order pointwise point that is ranked in advance at the sheet primary surface, and make probe be fixed in substrate surface by the physics and chemistry effect, probe fragment be except using oligonucleotide probe, also can use long gene fragment and nucleic acid analog probe (such as PNA etc.);

The preparation method of above-mentioned gene chip is known technology, and those skilled in the art can prepare the gene chip that detects mottled cartilage abnormity syndrome i type (CDPX1) according to existing technology.

The gene chip of the mottled cartilage abnormity syndrome i type of detection of the present invention (CDPX1) compared with prior art has following beneficial effect:

(1) described diagnosing chip operation steps is simple, and detection specificity is high, good stability;

(2) described diagnosing chip test of many times repeatability is high;

(3) detect required time weak point, can within a working days, finish to obtaining scanning result from the sample extracting.

The gene chip of the mottled cartilage abnormity syndrome i type of detection of the present invention (CDPX1), operation steps is simple, detection specificity is high, good stability, time short, scanning result can be finished within a working days to obtaining, cost is low from the sample extracting, can be fast, low-cost, large flux and automatization detect the transgenation of mottled cartilage abnormity syndrome i type (CDPX1).

Embodiment

Below in conjunction with specific embodiment, further set forth the present invention.Be interpreted as: these embodiment only are not used in for explanation the present invention and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, Sambrook equimolecular clone for example: laboratory manual is seen the condition described in the New York Cold Spring Harbor Laboratory press version in 1989, or the condition of advising according to manufacturer.

Step 1, preparation of samples

The water-bath temperature is transferred to 37 ℃ and 65 ℃, dissolves 10X Buffer C, the centrifugal several seconds after the of short duration vortex concussion.

Concerning each reaction, gDNA is joined in the PCR pipe of suitable nuclease free, and the water of adding nuclease free makes reaction volume reach 10.1 μ L.

Prepare enzyme on ice and cut mixing mother liquor (Digestion Master Mix), be sequentially added into following reagent:

2.9 μ L endonuclease reaction liquid are joined in the gDNA reaction tubes, make it volume and reach 13 μ L.Turn upside down and make it mixing.Carry out the PCR reaction, response procedures is: 37 ℃ are moved 2 hours, and 65 ℃ are moved 20 minutes.After the reaction reaction tubes is transferred on ice, got the gDNA that 2 μ L enzymes cut, dispose 0.8% agarose gel, with SYBR Gold dyeing, whether the leakage of electricity swimming detects enzyme and cuts complete.Most of enzyme is cut product length should be at 200bp-500bp.

Step 2, the fluorescent mark of DNA

In each reaction tubes, add 2.5 μ L random primers (Random Primers), make it volume and reach 13.5 μ L, turn upside down gently and carry out mixing.Carry out PCR reaction, response procedures is: 95 ℃ of sex change 3 minutes.Reaction tubes is transferred to 5min on ice.

Prepare mark mixing mother liquor (Labeling Master Mix), be sequentially added into following reagent:

11.5 μ L labeled reactant liquid are joined in the gDNA reaction tubes, make it volume and reach 25 μ L.Turn upside down gently and make it mixing.Carry out the PCR reaction, response procedures is: 37 ℃ are moved 2 hours, and 65 ℃ are moved 10 minutes.Reaction tubes is transferred on ice.

Step 3, the wash-out of mark gDNA

Add the 1X TE (pH 8.0) of 430 μ L in each reaction tubes.A Microcon YM-30 Filter column is put into a 1.5mL centrifuge tube, and the gDNA that mark is good is added on the filter membrane.The centrifugal 10m of 8000g under the room temperature.Abandon filtrate.Add the 1X TE (pH 8.0) of 480 μ L in each Filter column.The centrifugal 10m of 8000g under the room temperature.Abandon filtrate.

Be inverted Filter column, put into a new 1.5mL centrifuge tube.The centrifugal 1m of 8000g collects the purifying sample under the room temperature.Check and record the volume of every pipe elutant.Exceed 10 μ L such as fruit volume, elutant is added on the filter membrane again, the centrifugal 1m of 8000g under the room temperature.Abandon filtrate.

Repeat above-mentioned two the step until sample volume all less than or equal to 10 μ L.Adding 1X TE (pH 8.0) makes final volume reach 10 μ L.From each sample, get 1.5 μ L and detect output and specific activity (specific activity), use NanoDrop ND-1000UV-VIS spectrophotometer measurement.Two kinds of samples using respectively Cy5 and Cy3 mark of equivalent are mixed in the centrifuge tube of a new 1.5mL high temperature resistant (heat-resistant), reach final volume 158 μ L.

Step 4, chip is processed

The water that adds 1350 μ L rnase-frees in the bottle that 10X Blocking Agent lyophilized powder is housed.Before using or preserving, room temperature is placed 60m, vortex vibration mixing.

Water-bath is transferred to 95 ℃ and 37 ℃.

In the centrifuge tube of a rnase-free, be sequentially added into following reagent:

The biased sample that turns upside down, of short duration centrifugal subsequently.Sample hose is transferred to water-bath 3m in 95 ℃ of water-baths.Immediately sample hose is transferred to water-bath 30m in 37 ℃ of water-baths.Sample hose is taken out water-bath, centrifugal 1 minute of 17900g, collection tube original pattern product.

Step 5, chip hybridization

A clean pad is placed on Agilent SureHyb hybridization chamber bottom, makes the pad sign up, and align with the rectangular region of hybridization chamber bottom.Guarantee that pad and hybridization chamber bottom is with high! In the mode of " drag and dispense " 40 μ L hybridization sample mix liquid is added to packing ring inner compartment on the pad lentamente." the active side " of chip towards being placed down on the pad, faced up so that be printed on one of digitized bar code, and the barcode one that is printed on " Agilent " sign faces down.Guarantee that both align mutually.The hybridization chamber lid is covered on " pad-hybridization sample-chip " sandwich structure, and it is fixing that sliding clamp makes it.Anchor clamps on the manual fasten hybridization chamber.The hybridization chamber that vertical rotary assembles is with wetting chip, and whether observe bubble can unrestricted flow.If find static bubble, hybridization chamber can be put on the table and make it mobile by tapped chip or pad.The hybridization chamber that assembles is fixed on the turner, puts into 65 ℃ hybrid heater with the rotational speed of 20rpm.

Step 6, chip wash-out and scanning

At room temperature staining jar #1 is filled Wash Buffer 1.A chip carrier is put into staining jar #2.Put into a magnetic stir bar.The Wash Buffer 1 that packs into abundant in the staining jar #2 under the room temperature makes it to be enough to cover chip carrier.Staining jar is placed on the magnetic stirring apparatus.Staining jar #3 that will preheating in 37 ℃ of baking ovens is placed on the magnetic stirring apparatus with heating unit, the Wash Buffer 2 of the preheating of 3/4 volume of packing into.Put into a magnetic stir bar.Open heating unit, keep the temperature of Wash Buffer2 in 37 ℃; Control temperature with thermometer.A hybridization chamber is taken out from hybrid heater and timing.Record whether forms bubble in the crossover process and whether all bubbles rotate freedom.The dismounting hybridization chamber.Pry open sandwich structure from the end that barcode is arranged.By clean tweezers being stretched between two plectrums, separate gently two slides.Allow pad fall into the staining jar bottom.Chip is moved on the chip carrier of staining jar #2 (Wash Buffer 1).Allow as few as possible chip be exposed in the air.

Open magnetic stirrer 5min.Adjust rotating speed to reach good and within reason stirring.Chip carrier is transferred to the staining jar #3 that Wash Buffer 2 is housed, is preheating to 37 ℃, stirs 1min.In 5-10s, slowly chip carrier is shifted out.Reduce as far as possible the drop on the chip.The Wash Buffer 1 that abandoned and Wash Buffer 2.

Scan immediately chip to reduce in the environment oxygenant for the impact of strength of signal.

Embodiment 2

Adopt the gene chip of the mottled cartilage abnormity syndrome i type of detection of the present invention (CDPX1) to carry out correlated crowd detection screening, the result shows that described diagnosing chip operation steps is simple, and detection specificity is high, good stability; Test of many times repeatability is high; It is short to detect the required time, can finish within a working days to obtaining scanning result from the sample extracting.

The result of above-described embodiment shows, the gene chip of the mottled cartilage abnormity syndrome i type of detection of the present invention (CDPX1), operation steps is simple, detection specificity is high, good stability, the time is short, cost is low, can be fast, low-cost, large flux and automatization detect the transgenation of mottled cartilage abnormity syndrome i type (CDPX1).

SEQUENCE LISTING

＜110〉Gynaecology ﹠. Obstetrics Hospital Attached to Fudan Univ

＜120〉gene chip of the mottled cartilage abnormity of detection syndrome i type

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<170> PatentIn version 3.3

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Claims (3)

1. a gene chip that detects mottled cartilage abnormity syndrome i type is characterized in that comprise sheet base and probe, described probe is fixed on the sheet base, and described probe is the nucleotide sequence shown in the SEQ ID NO.1-50,

2. by the gene chip of the mottled cartilage abnormity of detection claimed in claim 1 syndrome i type, it is characterized in that described base is selected from slide glass, silicon chip, film or macromolecular material.

3. the purposes of the gene chip of claim 1 in the transgenation that detects mottled cartilage abnormity syndrome i type.