CN109280663A - A kind of full-automatic single nucleic acid strands preparation method - Google Patents
A kind of full-automatic single nucleic acid strands preparation method Download PDFInfo
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Abstract
The invention discloses a kind of full-automatic single nucleic acid strands preparation methods.This method prepares single nucleic acid strands using instrument for extracting nucleic acid, includes the following steps: that nucleic acid amplification product includes the double-strandednucleic acid of biotin labeling；Magnetic bead surfaces have coupled the functional group of recognizable biotin；The biotin can be combined with the functional group of magnetic bead surfaces；Make enrichment with magnetic bead by magnetic mode, while making the synchronous enrichment of the nucleic acid that biotin is marked, coating to magnetic bead surfaces；Denaturing soln makes enrichment coating become single-chain nucleic acid to the double-strandednucleic acid of magnetic bead surfaces；The magnetic bead for being coated with nucleic acid is drawn by magnetic mode, is added in subsequent reactions liquid.Single nucleic acid strands preparation method disclosed by the invention can fast and efficiently prepare single-chain nucleic acid, realize automation and high pass quantization.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of full-automatic single nucleic acid strands preparation method.
Background technique
In recent years, with the continuous development of economy and society, single-chain nucleic acid hybridization technique becomes modern medicine biology etc.
The important means of area diagnostic and detection, common single nucleic acid strands preparation method include: heating denaturalization, alkali process method, T7 reverse
Record method, denaturing high-performance liquid chromatography and asymmetric PCR.
Heating denaturalization: high temperature keeps nucleic acid single-stranded by double-strand solution, easy to operate, but what is be prepared single-stranded need to be stored in spy
Fixed condition, and this method is only applicable to high temperature resistant reagent, there is room temperature or cryogenic agent compared with big limitation.
T7 reverse transcription method: this method is at the end of PCR primer 5 ' plus T7 promoter, to purify pcr amplification product as template,
With the synthesizing single-stranded RNA of the external reverse transcription of t7 rna polymerase.Although single-stranded yield is higher for this method, but two steps is needed to complete, operation
Inconvenience, and need the pollution of strict control RNA enzyme.
Exonucleolytic enzyme process: since a PCR primer is phosphorylated, PCR product is when with exonuclease digestion, by phosphorus
The primer amplification chain of acidification is not cut, and finally obtained PCR product also needs repurity, inconvenient for operation, and enzyme after digestion
It is heated inactivation, and single-stranded yield relies on 5 prime excision enzyme activity.
Denaturing high-performance liquid chromatography: since a PCR primer is by biotin labeling, PCR amplification chain in DHPLC,
Can be separated with another common chain, this method can in 15 minutes directly from double stranded PCR products obtain needed for it is single-stranded, but needs are held high
Expensive instrument, it is difficult to universal.
Asymmetric PCR: being expanded with the primer of two inequalities, the method for obtaining a large amount of purpose single stranded DNAs.Compared to general
Logical PCR, this method is more demanding to nucleic acid amount, and sensitivity is low.
These methods require the operation that height relies on technical staff, and manual operation is although more flexible, but error is big, effect
Rate is low, can not achieve and fast and efficiently prepares single nucleic acid strands.
Summary of the invention
In view of the deficiencies of the prior art, the present invention intends to provide a kind of full-automatic single nucleic acid strands preparation side
Method.This method by the processes such as being enriched with, being denaturalized, utilizes instrument for extracting nucleic acid to realize the full-automatic of single nucleic acid strands preparation by magnetic bead
Change, it is accurate and efficient.
Above-mentioned purpose of the invention is achieved through the following technical solutions:
A kind of full-automatic single nucleic acid strands preparation method, by the nucleic acid amplification product and reagent that biotin is marked to be processed
It is added in sample-adding plate, then sample-adding plate is put into Full automatic instrument for extracting nucleic acid, start the program pre-set, by following (1)-
(4) the step of, completes full-automatic single nucleic acid strands preparation:
(1) instrument for extracting nucleic acid expands the nucleic acid that biotin is marked to be processed according to the mixing parameter pre-set
The mixture of volume increase object and combination liquid is mixed, and mixing liquid A is obtained；
(2) instrument for extracting nucleic acid draws magnetic bead according to the magnetic parameter pre-set from magnetic bead liquid, and is added to mixing
It in liquid A, is mixed according still further to the mixing parameter pre-set, obtains mixing liquid B, the magnetic bead surfaces coupling has recognizable
The functional group of biotin is coated with the nucleic acid enriching that biotin is marked to magnetic bead surfaces；
(3) instrument for extracting nucleic acid draws the magnetic bead for being coated with nucleic acid from mixing liquid B according to the magnetic parameter pre-set,
And be added in denaturing soln, it is mixed according still further to the mixing parameter pre-set, obtains mixing liquid C, be coated with enrichment
Double-strandednucleic acid to magnetic bead surfaces becomes single-chain nucleic acid；
(4) instrument for extracting nucleic acid draws the magnetic for being coated with single-chain nucleic acid from mixing liquid C according to the magnetic parameter pre-set
Pearl is added in subsequent reactions liquid.
The principle of the above method of the present invention is: magnetic bead surfaces have coupled the functional group of recognizable biotin, nucleic acid amplification
Product includes the double-strandednucleic acid of biotin labeling, and the biotin can be combined with the functional group of magnetic bead surfaces, passes through magnetic
Make the enrichment with magnetic bead for the double-strandednucleic acid for being coated with biotin labeling, while synchronizes the nucleic acid that biotin is marked also and being enriched to magnetic
Bead surface；Denaturing soln makes enrichment coating become single-chain nucleic acid to the double-strandednucleic acid of magnetic bead surfaces.
Instrument for extracting nucleic acid is the instrument that the extraction work of sample nucleic acid is automatically performed using mating nucleic acid extracting reagent, operation
After different parameters are arranged according to sample in personnel, which can be automatically performed the extraction and purification process of nucleic acid, lead in biotechnology
Domain is using relatively broad.But instrument for extracting nucleic acid to be used for the preparation of single nucleic acid strands currently without people.The present inventor's benefit
It with the achievable function of instrument for extracting nucleic acid, is acted on by enrichment with magnetic bead, obtains above-mentioned single nucleic acid strands full-automation preparation side
Method, it is quick, efficient, accurate to have the characteristics that.
Further, above-mentioned full-automatic single nucleic acid strands preparation method, mixing parameter described in step (1)-(4) include
Any combination that frequency and movement carry out includes continous way, intermittent or periodic in frequency；It include back and forth blowing in movement
Suction, up and down reciprocatingly knocking, horizontal oscillations formula rotate horizontally Patting type.
For step (1) when mixing, the Avidin of magnetic bead surfaces can excessively be destroyed by beaing mixing number, interfere specificity knot
It closes, beats very few and be not able to satisfy the requirement mixed well.Therefore, it is up and down reciprocatingly to beat to mix 30s that the present invention, which mixes parameter,
~2min, mixing frequency is 450 beats/min.
Success or failure of the procedure relation that step (3) mixes in denaturing soln to test.If it is manual operation, different operation
When personnel are tested using same procedure, as some tiny errors (such as the dynamics mixed and speed etc.) influence test
As a result.The present invention is verified by a large number of experiments, finally guarantees the suitable of the step using following blending manner with instrument for extracting nucleic acid
Benefit is completed: up and down reciprocatingly being beaten and is mixed 50s (mixing frequency is 460 beats/min), stands 250s；It up and down reciprocatingly beats again and mixes 30s
(mixing frequency is 460 beats/min), stands 250s.The blending manner will more fully be coated with the magnetic bead and denaturing liquid of nucleic acid
It mixes, during standing, so that the double-strandednucleic acid of magnetic bead surfaces fully becomes single-chain nucleic acid, and artificial with instrument substitution, has
Avoid influence of the human error to result to effect.
Further, above-mentioned full-automatic single nucleic acid strands preparation method, biotin described in step (1) are vitamin B7,
Also known as biotin, biotin；Step (2) functional group is Streptavidin, neutravidin, albumen avidin, chain
Avidin, yolk avidin or combinations thereof.
Further, above-mentioned full-automatic single nucleic acid strands preparation method, nucleic acid amplification product described in step (1) are PCR
Amplification, ApoE gene (ASPCR), strand displacement amplification (SDA), nucleic acid sequence based amplification (NASBA), rolling ring expand
Increase the product of the methods of (RCA), ring mediated isothermal amplification (LAMP) amplification.
Further, above-mentioned full-automatic single nucleic acid strands preparation method, step (2)-(4) described magnetic mode are permanent magnetism
Iron absorption enrichment magnetic bead or electromagnet absorption enrichment magnetic bead.
Further, above-mentioned full-automatic single nucleic acid strands preparation method, the magnetic bead are magnetizable molecule, this
The diameter of grain is 0.1 μm~10 μm.
Further, above-mentioned full-automatic single nucleic acid strands preparation method, denaturing soln urea, first described in step (3)
One or more of amide, methanol, ethyl alcohol and sodium hydroxide configure.
Denaturing soln described in preferred steps (3) is configured with sodium hydroxide, configures final concentration of 0.05M~0.5M.
The single nucleic acid strands of above method step (4) of the present invention preparation can be added in subsequent reactions liquid, reaction solution according to
The application purpose of single nucleic acid strands and selected, can be hybridization reaction solution, pcr amplification reaction liquid, ASPCR amplification reaction solution,
The reaction solutions such as SDA amplification reaction solution, NASBA amplification reaction solution, RCA amplification reaction solution, LAMP amplification reaction solution.
The present invention also provides a kind of method of genetic test, the preparation step including full-automatic single nucleic acid strands above-mentioned,
And following hybridization check step:
(5) instrument for extracting nucleic acid has been coated with the magnetic of single-chain nucleic acid according to the magnetic parameter pre-set, aspiration step (4)
Pearl is added in hybridization reaction solution, is then mixed with the mixing parameter pre-set；
(6) hybridization mixture prepared is added to micro-array chip surface, be incubated for；
(7) dry instrument is washed using chip and clean chip, then dried with centrifuge；
(8) signal-obtaining is carried out using micro-array chip scanner and GENE Assay analysis system and result judges.
Some embodiments of the present invention detect 15 hereditary hearing impairment genes using the above method, detection
As a result consistent with notional result height.
Full-automatic single nucleic acid strands preparation method of the invention has the advantage that
1, easy to automate and high-throughput by the processes such as being enriched with, being denaturalized by magnetic bead using instrument for extracting nucleic acid
Change.
2, it is realized because of all operating process by machine, manual operation bring can be avoided to make mistakes to greatest extent, greatly mentioned
Height prepares the efficiency and stability of single-chain nucleic acid.
Detailed description of the invention
Fig. 1 is the relevant micro-array chip dot matrix arrangement schematic diagram of 15 hereditary hearing impairment gene detecting kits；
Fig. 2 is wild type gene group using the analysis detection of 15 hereditary hearing impairment gene detecting kits as a result, its
In (a) be embodiment 1 theoretical hybridization survey as a result, (b) be embodiment 1 true hybridization survey result；
Fig. 3 is 235 heterozygous genes groups using the analysis detection of 15 hereditary hearing impairment gene detecting kits as a result, its
In (a) be embodiment 2 theoretical hybridization survey as a result, (b) be embodiment 2 true hybridization survey result；
Fig. 4 is the analysis detection knot that IVS7-2 heterozygous genes group uses 15 hereditary hearing impairment gene detecting kits
Fruit, wherein the theoretical hybridization that (a) is embodiment 3 survey as a result, (b) be embodiment 3 true hybridization survey result.
Specific embodiment
It is illustrated below by way of specific embodiment is further to summary of the invention of the invention, but should not be construed as the present invention
Range be only limitted to example below, invention thinking according to the present invention and entire contents, can will be each in following instance
Technical characteristic makes combination/replacement/adjustment/modification appropriate etc., this is will be obvious to those skilled in the art that still
Belong to the scope that the present invention protects.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Following embodiments specifically explain the present invention by taking the detection of the relevant 15 SNP/ mutation of hereditary hearing impairment as an example
It states.
Embodiment 1
One, the preparation of nucleic acid amplification product
With wild type gene group, (human genome DNA is extracted from blood cake, and relevant for hereditary hearing impairment
All it is wild type for 15 SNP/ mutation) it is template, the amplification examination of 15 hereditary hearing impairment gene detecting kits is added
Agent carries out multiple amplified allele according to the amplification program provided in the following kit specification, obtains nucleic acid amplification and produce
Object.
Amplification program see the table below:
Two, the preparation of single-chain nucleic acid
Before experiment, first by the nucleic acid amplification product that biotin is marked to be processed, in conjunction with liquid, hybridization solution, denaturing liquid, magnetic
Pearl suspension is added in sample-adding plate outside instrument for extracting nucleic acid, and sample-adding plate is put into instrument for extracting nucleic acid, starts the journey pre-set
Sequence, according to it is following 1. -4. step carry out the preparation of full-automatic single nucleic acid strands:
Instrument for extracting nucleic acid is ((mixed up and down reciprocatingly to beat blending manner mixing 2 minutes according to the mixing parameter pre-set
Even frequency is 450 beats/min)), by nucleic acid amplification product (tri- amplification systems of A, B, C each 20 that biotin is marked to be processed
μ L) and 30 μ L combination liquid mixture mix, obtain mixing liquid A, the nucleic acid amplification product include biotin labeling double-strand
Nucleic acid；
More efficiently nucleic acid amplification product can be mixed well in conjunction with liquid using the mixing of above-mentioned mixing parameter, and subtracted
Few error as brought by manual operation.
Instrument for extracting nucleic acid is according to the magnetic pre-set and mixes parameter, from magnetic bead combination liquid (+9 μ L magnetic of 6 μ L combination liquid
Pearl) in draw magnetic bead 60s, be added in 30 μ L combination liquid, up and down reciprocatingly beat and mix 30s (mix frequency be 460 beats/min), obtain
To the suspension containing magnetic beads handled well；Magnetic is carried out using instrument for extracting nucleic acid, can sufficiently draw magnetic bead in a relatively short period of time, reduces magnetic
Pearl loss, such mixing parameter can more efficiently mix well magnetic bead in conjunction with liquid, so that magnetic bead and nucleic acid expand
The combination for increasing production object is more abundant.
Instrument for extracting nucleic acid is according to the magnetic parameter (draw magnetic bead 60s) that pre-sets by magnetic mode from handling well
It draws magnetic bead in 30 μ L suspension containing magnetic beads to be added in mixing liquid A, magnetic bead upper surface has coupled the functional group of recognizable biotin
Streptavidin, then according to mix parameter mix (with up and down reciprocatingly beat blending manner mix 30s (mix frequency be 460
Beat/min), stand 270s；Then 30s (mixing frequency as 460 beats/min) is mixed up and down reciprocatingly to beat blending manner again, stood
270s), mixing liquid B is obtained, while the nucleic acid enriching that biotin is marked is coated with to magnetic bead surfaces；
Magnetic is carried out using instrument for extracting nucleic acid, can sufficiently draw magnetic bead in a relatively short period of time, reduces magnetic bead loss, it is above-mentioned
Mixing parameter more fully magnetic bead and mixing liquid A can be mixed so that magnetic bead surfaces functional group and nucleic acid amplification produce
Biotin on object sufficiently combines, and increases the success rate of subsequent experimental.
Instrument for extracting nucleic acid is according to the magnetic parameter (draw magnetic bead 60s) pre-set by magnetic mode from mixing liquid B
Draw and be coated with the magnetic bead 60s of nucleic acid, and be added in 120 μ L denaturing solns (concentration is 0.1M NaOH solution), then according to
Parameter mixing is mixed (up and down reciprocatingly to beat and mix 50s (mixing frequency is 460 beats/min), stand 250s；It up and down reciprocatingly beats again
30s (mixing frequency is 460 beats/min) is mixed, 250s is stood), mixing liquid C is obtained, while enrichment coating being made to arrive magnetic bead surfaces
Double-strandednucleic acid becomes single-chain nucleic acid；
Such blending manner can more fully mix the magnetic bead for being coated with nucleic acid and denaturing liquid, during standing, make
The double-strandednucleic acid for obtaining magnetic bead surfaces more fully becomes single-chain nucleic acid, to improve the success rate of subsequent experimental.
Three, subsequent hybridization check
4. instrument for extracting nucleic acid is according to the magnetic parameter (draw magnetic bead 60s) pre-set by magnetic mode from mixing liquid
C draws the magnetic bead 60s for being coated with nucleic acid, is added in 50 μ L hybridization reaction solutions, up and down reciprocatingly beats and mixes 30s (mixing frequency
It is 460 beats/min)；
Magnetic is carried out using instrument for extracting nucleic acid, can sufficiently be drawn from mixing liquid C in a relatively short period of time and be coated with nucleic acid
Magnetic bead, reduce magnetic bead loss, the magnetic bead and 50 μ of single-chain nucleic acid will can be more fully coated with by up and down reciprocatingly beaing to mix
L hybridization solution mixes, and the remaining denaturing liquid of magnetic bead surfaces is neutralized, to improve the success rate of subsequent experimental.
5. instrument for extracting nucleic acid is according to the magnetic pre-set and mixes parameter, nucleic acid has been coated with from mixing liquid C absorption
Magnetic bead 60s is added in 20 μ L hybridization reaction solutions, is up and down reciprocatingly beaten and is mixed 10s (mixing frequency is 450 beats/min), after progress
Continuous hybridization reaction.
6. the hybridization mixture prepared is added to micro-array chip surface, 50 DEG C are incubated for 1 hour；
7. dry instrument SlideWasherTM24 (Bo Ao, Beijing) is washed using chip, successively at 42 DEG C and at room temperature respectively with two
Kind of solution respectively clean once (cleaning solution I:SSC final concentration of 0.3 ×, SDS final concentration of 0.1%；Cleaning solution II: SSC final concentration
It is 0.06 ×), then chip is dried with centrifuge；
8. using micro-array chip scanner LuxScanTM10K-A/B (Bo Ao, Beijing) and 15 hereditary hearing impairment phases
Correlation gene testing and analysis system (Bo Ao, Beijing) carries out signal-obtaining and result judgement.
15 hereditary hearing impairment gene detecting kits relevant micro-array chip dot matrix arrangement schematic diagram such as Fig. 1 institute
Show.Use the wild type gene group as template, tested and analyzed using 15 hereditary hearing impairment gene detecting kits,
Shown in actual results of hybridization such as Fig. 2 (b).
Four, result judges
When using wild type gene group as template, 15 deaf amplifing reagents are all during entire PCR amplification
The specific primer of wild type will do it massive amplification, and the specific primer of all saltant types not will do it amplification,
Then passing through can only be enriched in the coated magnetic bead surfaces of Streptavidin and can carry out with the wild-type probe on micro-array chip
The single-chain nucleic acid product of hybridization, single-chain nucleic acid products of these last enrichment coatings to magnetic bead surfaces are by hybridization by magnetic bead and glimmering
It is optically coupled in the different wild-type probes of micro-array chip, shown in theoretical results of hybridization such as Fig. 2 (a).
When using wild type gene group to be detected as template using 15 hereditary hearing impairment gene detecting kits
Analysis, it is completely the same with the theoretical results of hybridization in Fig. 2 (a) shown in actual results of hybridization such as Fig. 2 (b).With further it is mating
15 hereditary hearing impairment GENE Assay analysis network analyses confirm in the sample that relevant 15 SNP/ of hereditary hearing impairment are prominent
Change is all wild type.
Embodiment 2
One, the preparation of nucleic acid amplification product
With 235 heterozygous genes groups (human genome DNA be extracted from blood cake, and for hereditary hearing impairment correlation
15 SNP/ mutation for, except the 235del C on GIB2 gene be heterozygous mutant in addition to, remaining 14 loci gene type is
Wild type) it is template, the amplifing reagent of 15 hereditary hearing impairment gene detecting kits is added, is said according to the following kit
The amplification program provided in bright book carries out multiple amplified allele, obtains nucleic acid amplification product.
Amplification program see the table below:
Two, the preparation of single-chain nucleic acid；And
Three, subsequent hybridization check
According to the operation of step 2 and step 3 in embodiment 1.Use 15 hereditary hearing impairment gene detecting kits
The 235 heterozygous genes group is tested and analyzed, shown in actual results of hybridization such as Fig. 3 (b).
Four, result judges
When with 235 heterozygous genes groups (human genome DNA 15 SNP/ relevant for hereditary hearing impairment mutation and
Speech, in addition to the 235del C on GIB2 gene is heterozygous mutant, remaining 14 loci gene type is the human gene of wild type
Group DNA) it is template, 235del C heterozygosis of 15 deaf amplifing reagents during entire PCR amplification, on GIB2 gene
The specific primer of saltant type and remaining 14 wild type site will do it massive amplification, then pass through Streptavidin coating
Magnetic bead surfaces on can only be enriched to can be with 14 kinds of wild-type probes on micro-array chip and the 235del C on GIB2 gene
The single-chain nucleic acid product of the single-chain nucleic acid product that saltant type probe is hybridized, last these enrichment coating to magnetic bead surfaces passes through
Hybridize 235del C saltant type magnetic bead and fluorescence being connected in the different wild-type probes and GIB2 gene of micro-array chip
On probe, in theoretical results of hybridization such as Fig. 3 (a).
Divide when detect using 15 hereditary hearing impairment gene detecting kits using 235 heterozygous genes groups as template
Analysis, it is completely the same with the theoretical results of hybridization in Fig. 3 (a) shown in true results of hybridization such as Fig. 3 (b).Further with matched
15 hereditary hearing impairment GENE Assay analysis network analyses confirm the relevant 15 SNP/ mutation of hereditary hearing impairment in the sample
In, in addition to the 235del C on GIB2 gene is heterozygous mutant, remaining 14 loci gene type is wild type.
Embodiment 3
One, the preparation of nucleic acid amplification product
With IVS7-2 heterozygous genes group (human genome DNA is extracted from peripheral blood, concentration be 3ng/ μ L, and
For 15 SNP/ mutation relevant for hereditary hearing impairment, in addition to IVS7-2A > G on SLC26A4 gene is heterozygous mutant,
Remaining 14 loci gene type is wild type) it is template, the amplification of 15 hereditary hearing impairment gene detecting kits is added
Reagent carries out multiple amplified allele according to the amplification program provided in the following kit specification, obtains nucleic acid amplification
Product.
Amplification program see the table below:
Two, the preparation of single-chain nucleic acid
Before experiment, first by the nucleic acid amplification product that biotin is marked to be processed, in conjunction with liquid, hybridization solution, denaturing liquid, magnetic
Pearl suspension is added in sample-adding plate outside instrument for extracting nucleic acid, and sample-adding plate is put into instrument for extracting nucleic acid, starts the journey pre-set
Sequence, according to it is following 1. -4. step carry out the preparation of full-automatic single nucleic acid strands:
With embodiment 1, but first mixes parameter are as follows: mixes 30s up and down reciprocatingly to beat blending manner, mixing frequency is
450 beats/min.
The blending manner of the present embodiment can also avoid in addition to mixing well by nucleic acid amplification product and in conjunction with liquid because beaing
Mix the Avidin that number excessively destroys magnetic bead surfaces, interference specific binding.
3. 2. being carried out according to the operation in step 2 in embodiment 1 2. and 3..
Three, subsequent hybridization check
It is carried out according to the operation of step 3 in embodiment 1.Using 15 hereditary hearing impairment gene detecting kits to this
IVS7-2 heterozygous genes group is tested and analyzed, shown in actual results of hybridization such as Fig. 4 (b).
Three, result judges.
When with (the human genome DNA 15 SNP/ mutation relevant for hereditary hearing impairment of IVS7-2 heterozygous genes group
For, in addition to IVS7-2A > G on SLC26A4 gene is heterozygous mutant, remaining 14 loci gene type is the people of wild type
Type genomic dna) it is template, 15 deaf amplifing reagents are during entire PCR amplification, on SLC26A4 gene
The specific primer of IVS7-2A > G heterozygous mutant and remaining 14 wild type site will do it massive amplification, then pass through
Can only be enriched in the coated magnetic bead surfaces of Streptavidin can be with the 14 kinds of wild-type probes and SLC26A4 on micro-array chip
The single-chain nucleic acid product that IVS7-2A > G saltant type probe on gene is hybridized, these last enrichment coatings arrive magnetic bead surfaces
Single-chain nucleic acid product magnetic bead and fluorescence are connected to the different wild-type probes and SLC26A4 base of micro-array chip by hybridizing
Because on upper IVS7-2A > G saltant type probe, in theoretical results of hybridization such as Fig. 4 (a).
When being detected using 15 hereditary hearing impairment gene detecting kits using IVS7-2 heterozygous genes group as template
Analysis, it is completely the same with the theoretical results of hybridization in Fig. 4 (a) shown in true results of hybridization such as Fig. 4 (b).Further with mating
15 hereditary hearing impairment GENE Assay analysis network analyses confirm in the sample that relevant 15 SNP/ of hereditary hearing impairment are prominent
In change, in addition to IVS7-2A > G on SLC26A4 gene is heterozygous mutant, remaining 14 loci gene type is wild type.
Claims (10)
1. a kind of full-automatic single nucleic acid strands preparation method, the nucleic acid amplification product that biotin is marked to be processed and reagent are added
Enter to be loaded in plate, which is characterized in that sample-adding plate is put into instrument for extracting nucleic acid, starts the program pre-set, by following
(1)-(4) the step of, completes full-automatic single nucleic acid strands preparation:
(1) instrument for extracting nucleic acid produces the nucleic acid amplification that biotin is marked to be processed according to the mixing parameter pre-set
The mixture of object and combination liquid is mixed, and mixing liquid A is obtained；
(2) instrument for extracting nucleic acid draws magnetic bead according to the magnetic parameter pre-set from magnetic bead liquid, and is added to mixing liquid A
In, it is mixed according still further to the mixing parameter pre-set, obtains mixing liquid B, the magnetic bead surfaces coupling has recognizable life
The functional group of object element is coated with the nucleic acid enriching that biotin is marked to magnetic bead surfaces；
(3) instrument for extracting nucleic acid draws the magnetic bead for being coated with nucleic acid from mixing liquid B according to the magnetic parameter pre-set, and adds
Enter into denaturing soln, mixed according still further to the mixing parameter pre-set, obtain mixing liquid C, makes enrichment coating to magnetic
The double-strandednucleic acid of bead surface becomes single-chain nucleic acid；
(4) instrument for extracting nucleic acid draws the magnetic bead for being coated with single-chain nucleic acid from mixing liquid C according to the magnetic parameter pre-set,
It is added in subsequent reactions liquid.
2. full-automatic single nucleic acid strands preparation method according to claim 1, which is characterized in that described in step (1)-(4)
Mixing parameter include frequency and movement carry out any combination, in frequency include continous way, intermittent or periodic；Dynamic
It include reciprocal blowing and drawing type, up and down reciprocatingly knocking, horizontal oscillations formula or horizontal rotation Patting type on work.
3. full-automatic single nucleic acid strands preparation method according to claim 1, which is characterized in that biology described in step (1)
Element is vitamin B7；Step (2) functional group is Streptavidin, neutravidin, albumen avidin, chain affinity
Element, yolk avidin or combinations thereof.
4. full-automatic single nucleic acid strands preparation method according to claim 1, which is characterized in that nucleic acid described in step (1)
Amplified production is PCR amplification, ApoE gene, strand displacement amplification, nucleic acid sequence based amplification, rolling circle amplification or ring
The product of mediated isothermal amplification method amplification.
5. full-automatic single nucleic acid strands preparation method according to claim 1, which is characterized in that step (2)-(4) described magnetic
The parameter of suction is permanent magnet absorption enrichment magnetic bead or electromagnet absorption enrichment magnetic bead.
6. full-automatic single nucleic acid strands preparation method according to claim 1, which is characterized in that the magnetic bead is magnetizable
Molecule, the diameter of particle are 0.1 μm~10 μm.
7. full-automatic single nucleic acid strands preparation method according to claim 1, which is characterized in that denaturation described in step (3)
One or more of solution urea, formamide, methanol, ethyl alcohol and sodium hydroxide configure.
8. full-automatic single nucleic acid strands preparation method according to claim 7, which is characterized in that denaturation described in step (3)
Solution is configured with sodium hydroxide, configures final concentration of 0.05M~0.5M.
9. full-automatic single nucleic acid strands preparation method according to claim 1, which is characterized in that step (4) is described subsequent anti-
Answer liquid be hybridization buffer, PCR amplification buffer, ASPCR amplification buffer, SDA amplification buffer, NASBA amplification buffer,
RCA amplification buffer or LAMP amplification buffer.
10. a kind of method of genetic test, which is characterized in that including full-automatic nucleic acid described in any one of claim 1 to 9
Single-stranded preparation method and hybridization check step below:
(5) instrument for extracting nucleic acid has been coated with the magnetic bead of single-chain nucleic acid, has added according to the magnetic parameter pre-set, aspiration step (4)
Enter into hybridization reaction solution, is then mixed with the mixing parameter pre-set；
(6) hybridization mixture prepared is added to micro-array chip surface, be incubated for；
(7) dry instrument is washed using chip and clean chip, then dried with centrifuge；
(8) signal-obtaining is carried out using micro-array chip scanner and GENE Assay analysis system and result judges.
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