CN102634507A - Multi-gene multi-zone specific capture method - Google Patents
Multi-gene multi-zone specific capture method Download PDFInfo
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Abstract
The invention discloses a kit for capturing a specific nucleic acid target with multiple genes in multiple zones. The kit is characterized in that the kit comprises a first primer, a capture component and a second primer, wherein the first primer is used for performing first unidirectional PCR (polymerase chain reaction) amplification on a target gene region sequence to be captured, and one end of the first primer is connected with a first linker DNA (deoxyribonucleic acid) fragmentation marked with biotin and is in complementary combination with the target gene region sequence to be captured to form an oligonucleotide single stranded sequence; the capture component comprises an avidin and a solid-phase carrier combined with the avidin; the avidin is used for capturing a target gene region sequence after the first PCR amplification and combining with the avidin on the target gene region sequence after the first PCR amplification; and the second primer is used for performing second unidirectional PCR amplification on the target gene region sequence after the combination with the avidin, and one end of the second primer is connected with a second linker DNA fragmentation and is in complementary combination with the target gene region, and is opposite to the direction of the PCR amplification performed by the first primer. The kit has good specificity, high homogeneity and high coverage rate and is simple to operate.
Description
Technical field
The invention belongs to the gene sequencing technical field, be specifically related to a kind of specificity catching method of polygene multizone.
Background technology
The world today, the nucleic acid sequencing technology has obtained the progress of advancing by leaps and bounds, the genome sequence of the Human Genome Project and many animals, plant and mikrobe the completion of checking order in succession.Sequencing technologies is by original first-generation Sanger PCR sequencing PCR, progressively develops high throughput sequencing technologies.High throughput sequencing technologies can check order to an identical or different DNA short segments (60-500bp) up to a million even up to ten million simultaneously simultaneously, and produces huge sequence data.Yet the processing of these huge data has proposed stern challenge to scientist.Concerning a gene order-checking, possibly check order and accomplish the time that only needs couple of days, but will be an intact week or the more times that need of data processing.This has just postponed the speed of gene order-checking greatly.The new-generation sequencing technology has expedited the emergence of colourful Genome Atlas.Yet for many researchs, especially those need check order when confirming the dna sequence dna heritable variation relevant with various diseases to more than one hundred million people in batches, and complete human genome preface of resurveying still costs an arm and a leg.Variation let alone with disease-related mostly betides protein-coding region, and this only accounts for 2% of human genome.Therefore, the target area sequence measurement supreme with cost benefit is one of emphasis of Future Development, relies on these method research high information quantity zones to reduce cost greatly.Some research is unnecessaryly known whole genome sequences, but special genes group zone is studied, such as exon, SNP zone or with the zone of disease-related, therefore improved the speed of data processing greatly.And to reach the sequence research of specific region, how will at first to solve efficient capture to them.
Gene trap has three kinds of methods at present: based on the hybrid capture method of chip; Hybrid capture method based on in-solution; The method of separating specific gene group zone with molecular inversion probes.
Based on the hybrid capture method of chip, genomic dna is at first interrupted, and then with the sequence capturing chip hybridization that customizes, does not have the quilt in the hybridization to wash off.The target group of enrichment by wash-out and amplification, check order with the high-flux sequence appearance subsequently then.Several big advantage of this method: orientation is caught genome target district (the present length of the chip piece appointment genome area that can catch 5MB; Data are reliable; As long as you select the sequence capturing chip that the zone of wanting to catch will design and synthesize a customization, and are easy to use laborsaving.But the requirement of genomic dna is many, and is inapplicable for rare precious genome, and its specificity is not as good as the in-solution method.
Flow process based on the hybrid capture method of in-solution is that genomic dna interrupts, and---selected sizeable library and RNA bait are hatched altogether and were formed---wash-out magnetic bead---pcr amplification---order-checking of RNA-DNA heterozygote in 24 hours.
1) splendid specificity: the sequence of reading up to 90% derives from RNA " bait " and catches, and wherein surpasses 50% with the target sequence overlaps fully;
2) outstanding homogeneity: to the genome larger sequence fragment, overlap RNA " bait " over half with acquisition sequence at least and surpass 80%, even to the exon sequence of small segment, this numeral also surpasses 60%;
3) remarkable circulation ratio: the variance rate between 2 groups of technology revision tests is less than 10-5;
4) can accurately detect SNP.In sum, the specificity based on the parallel target sequencing technologies of the high-throughput of liquid phase sequence capturing has been verified in the experimental result perfection, accuracy, circulation ratio and application prospects.But because used bait is RNA, so need very high operational requirement, reagent or utensil all need be used no RNA enzyme.And operation steps is more numerous and diverse.
Molecular inversion probes is separated the method in specific gene group zone, and (in-solution) catching method can carry out the orientation preface of resurveying to the genomic locus of big component in this solution.This flux can reach 1Mb, even higher.Use the strand 80 aggressiveness storehouses of height MULTIPLE COMPOSITE and come cyclisation cyclisation simultaneously target genome area.This capture technique can be used one group of all target of universal primer amplification.
As shown in Figure 1, a capture oligo comprises two strands and catches the arm sequence, and these two arm sequences can be complementary respectively with the two ends flanking sequence of target sequence, thereby make the target sequence cyclisation.The oligonucleotide sequence that connects two arm sequences is a fixed sequence program.The sequence that includes a 40bp in each cyclization, it can be complementary with the capture oligonucleotide sequence.This sequence provides the universal primer sequence of downstream amplification usefulness.This technological capture region can reach 800bp, satisfies the requirement of susceptibility and high degree of specificity simultaneously.
Uridylic glycosylase (uracil glycosylase) is to be present in the intravital a kind of important DNA repair enzyme of multiple biology; Remove effect for the uridylic of mispairing in the dna replication dna and the uridylic that causes because of the cytosine(Cyt) deaminizating, keeping bringing into play crucial effects aspect the genomic integrity.
Weak point is to need a 40bp carrier oligonucleotide, and 3 ' end of this oligonucleotide must dock with 3 ' end of genomic fragment accurately.Nuclease can be with the sequence excision of 5 ' unnecessary end.These molecular mechanisms make the complex designization of catching arm and make this method very thorny with the standard primer-design software.Will be with this technology popularization to the investigator, the arm of catching that makes its design can cover any given human genome target sequence is very big challenge.
Summary of the invention
The object of the invention is to provide a kind of test kit that the polygene multizone is caught the specific nucleic acid target that carries out, and has solved all weak points in the gene trapping in the prior art.
In order to solve these problems of the prior art, technical scheme provided by the invention is:
A kind ofly carry out the test kit that the polygene multizone is caught the specific nucleic acid target, it is characterized in that said test kit comprises first primer, catches the component and second primer:
Said first primer is used to goal gene regional sequence to be caught unidirectional pcr amplification for the first time; Said first primer, one end links to each other with the first linker DNA fragment that is marked with vitamin H, and with the complementary bonded oligonucleotide of goal gene regional sequence to be caught single stranded sequence;
Said catch component comprise avidin and with avidin bonded solid phase carrier; Said avidin is used to catch the goal gene regional sequence behind the unidirectional pcr amplification for the first time, and with the said first time unidirectional pcr amplification after the goal gene regional sequence on vitamin H combine;
Said second primer is used for the goal gene regional sequence after the avidin combination is carried out the unidirectional pcr amplification second time; Said second primer, one end links to each other with the second linker DNA fragment; And combine, and carry out the in the opposite direction of pcr amplification with first primer with said goal gene regional sequence is complementary.
Preferably, said solid phase carrier is a microballoon.
Preferably, said solid phase carrier is a magnetic microsphere.
Preferably, said avidin is selected from white of an egg affinity element, streptavidin, the yolk affinity is plain and type affinity is plain.
Preferably, the said first linker DNA fragment has the continuous nucleotide sequence of SEQ No:1, and 5 ' end carries out mark through vitamin H; The second linker DNA fragment has the continuous nucleotide sequence of SEQ No:2.
Preferably, said first primer is by the first linker DNA fragment, ACTG base group and be used for specific amplification goal gene target sequence, and the Auele Specific Primer that contains at least 15 successive nucleotide sequences of goal gene is formed by connecting; Said second primer is by the second linker DNA fragment, ACTG base group and be used for specific amplification goal gene target sequence, and the Auele Specific Primer that contains at least 15 successive nucleotide sequences of goal gene is formed by connecting.
Preferably, said first primer carries out for the first time that a PCR reaction system of pcr amplification comprises PCR damping fluid, dNTPs, MgCl
2, TaqDNA polysaccharase, goal gene regional sequence to be caught be as template DNA, a said PCR reaction system final concentration consists of:
PCR damping fluid final concentration 1～10 *;
dNTPs 0.01～1.5mM；
The primer sequence final concentration is 0.01～2 μ M;
Template DNA 0.01～10ng/ μ L;
TaqDNA polysaccharase 0.01～1.0U/ μ L;
MgCl
2Final concentration is 0.5～5mM.
Preferably, said second primer carries out for the second time that the 2nd PCR reaction system of pcr amplification comprises PCR damping fluid, dNTPs, MgCl
2, TaqDNA polysaccharase, goal gene regional sequence to be caught be as template DNA, said the 2nd PCR reaction system final concentration consists of:
PCR damping fluid final concentration 1～10 *;
dNTPs 0.01～1.5mM；
The primer sequence final concentration is 0.01～2 μ M;
Template DNA 0.01～10ng/ μ L;
TaqDNA polysaccharase 0.01～1.0U/ μ L;
MgCl
2Final concentration is 0.5～5mM.
Another object of the present invention is to provide a kind of method that the polygene multizone is caught the specific nucleic acid target of carrying out, it is characterized in that said method comprising the steps of:
(1) genome that will be to be caught interrupts the back at random and adopts first primer to carry out the pcr amplification first time; Said first primer, one end links to each other with the first linker DNA fragment that is marked with vitamin H, and with the complementary bonded oligonucleotide of goal gene regional sequence to be caught single stranded sequence;
(2) use and to catch component the goal gene regional sequence that carries out behind the unidirectional pcr amplification is for the first time caught; Said catch component comprise avidin and with avidin bonded solid phase carrier; Vitamin H on the goal gene regional sequence behind said avidin and the said first time unidirectional pcr amplification combines;
(3) use the goal gene regional sequence after second primer combines avidin to carry out the unidirectional pcr amplification second time; Said second primer, one end links to each other with the second linker DNA fragment, and combines with said goal gene regional sequence is complementary, and carries out the in the opposite direction of pcr amplification with first primer;
(4) the goal gene regional sequence behind unidirectional pcr amplification general's second time is removed the avidin biotin composite, obtains the single-stranded DNA banks that two ends have identical or different DNA joint.
Another purpose of the present invention is to provide a kind of application of test kit aspect gene sequencing that the polygene multizone is caught the specific nucleic acid target of carrying out.
As used herein, term " avidin " comprise white of an egg affinity plain (Avidin, A), the strepto-affinity plain (Streptavidin, SA), the yolk affinity is plain and type affinity element etc.This term also comprises the avidin of wild-type, mutant and derivative type.
As used herein, (biontin B) is distributed widely in the animal and plant tissue term " vitamin H ", often from higher yolk of content and hepatic tissue, extracts molecular weight 244.31Kd.Biotin molecule has two ring texturees, and wherein the I ring for the imidazolone ring is and the main position of avidin bonded; II ring is thiphene ring, and C2 is last to have a valeric acid side chain, and its terminal carboxyl(group) is the macromolecular only structure of binding antibody and other biological, and after chemically modified, vitamin H can become the verivate that has the various active group---the activation vitamin H.
As used herein, the general selection of term " solid phase carrier " can combine like macro-molecular proteins such as avidin or Streptavidins; Must keep active behind the biomacromolecule immobilization, and carry out, preferably its reactive group orientating reaction solution for helping sufficient reacting.
The linker DNA fragments sequence is following:
Joint A:5 '-/BioTEG/GAGGATCCAGAATTCTCGAGTT-3 ';
Joint B:5 '-CTCGAGAATTCTGGATCCTC-3 ';
Catch primer and comprise first primer and second primer, form by the Auele Specific Primer (specific primer) of linker DNA fragment+ACTG base group+cooperate with genomic DNA fragment to be amplified.Promptly the structure of first primer is joint A+ACTG+specific primer; The structure of second primer is joint B+ACTG+specific primer; In two primers Auele Specific Primer according to the different genes that will catch and difference.
To catch design of primers and become three parts: shank, middle portion and end part; Its each several part effect is different.Wherein middle portion is an ACTG base group, and middle portion adds ACTG, its objective is in order when order-checking begins, to correct with these four known bases; 4 base sequences of ACTG are arranged arbitrarily.Auele Specific Primer requires to be designed to identical or close annealing temperature, so length is unfixing.Auele Specific Primer according to the heterogeneic difference that will catch design.
Joint and Auele Specific Primer are according to the general primer principle of design:
(1) length: 15-30bp generally is not more than 38, otherwise the righttest elongating temperature of PCR can surpass the best use of temperature (74 degree) of Taq enzyme, thereby reduces the specificity of product.
(2) G ten C content: should be between 45% one 55%, the renaturation temperature in the pcr amplification generally is that the Tm value of low Tm value primer deducts the 5-10 degree.
(3) randomness of base distribution: should avoid occurring continuously the single base more than 4.Especially be not taken in it and 3 ' bring out at present and to surpass 3 continuous G or C, otherwise primer is caused in G ten C enrichment sequence area mistakes.
(4) primer self: can not contain self complementary sequence, otherwise can form hair clip appearance secondary structure.
(5) between the primer: do not have between two primers more than 4 complementation or homology base, not so can form primer dimer, should avoid the complementary overlapping of 3 ' end especially.
(6) specificity: should be with the homology of non-specific amplification sequence less than 70%, or be less than continuous 8 complementary base.
(7) 3 ' of primer end: 3 ' end of primer influences the effect of extension of Taq enzyme to a great extent, and 3 ' end mispairing should not take place.3 ' end base of primer is preferably selected A, G, C for use, and avoids selecting for use T as much as possible, should avoid occurring continuously the T more than 2 especially.
(8) avoid the secondary structure district of primer: the reason that some primer is invalid is the influence of primer iteron secondary structure.
The present invention proposes the two unidirectional amplifications of step of polygene multizone and catches specific nucleic acid target method, and this method had both been gathered the advantage of the high specific of in-solution, by having simple to operate, time saving and energy saving advantage, had again with the few advantage of sample size.
Preferred polygene multizone two unidirectional amplifications of step are caught specific nucleic acid target method and can be adopted following step to carry out:
(1) at first genome is interrupted with Ultrasonic Cell Disruptor at random;
(2) carry out unidirectional pcr amplification in the genome with single primer adding fragmentation.Single primer is held with the linker DNA that is connected to vitamin H 5 ' by the DNA special primer and is linked to each other;
The strand purpose sheet segment DNA that (3) will carry vitamin H (Bio) with the Streptavidin magnetic bead is caught;
(4) with Auele Specific Primer the DNA that catches is carried out the unidirectional pcr amplification second time;
(5) remove the strand that has vitamin H, obtain the single-stranded DNA banks (library DNA identical sequence molecule also can be the molecule of different series) that two ends have identical or different DNA joint;
(6) library DNA is used for order-checking or other any application.
With respect to scheme of the prior art, advantage of the present invention is:
(1) specificity of the inventive method is with better based on the specificity of in-solution catching method.
(2) design of primers is easy, and entire operation is simpler.
(3) homogeneity and fraction of coverage are higher.
Description of drawings
Below in conjunction with accompanying drawing and embodiment the present invention is further described:
Fig. 1 is the schematic flow sheet that molecular inversion probes is separated specific gene group region method in the prior art;
Fig. 2 carries out the method flow synoptic diagram that the polygene multizone is caught the specific nucleic acid target for the present invention;
Fig. 3 carries out the method coverage result that the polygene multizone is caught the specific nucleic acid target for the present invention;
The structural representation of catching primer that Fig. 4 adopts for the present invention; On sequential structure was formed, the said primer of catching was made up of the Auele Specific Primer (specific primer) of linker DNA fragment+ACTG base group+cooperate with genomic DNA fragment to be amplified.
Embodiment
Below in conjunction with specific embodiment such scheme is further specified.Should be understood that these embodiment are used to the present invention is described and are not limited to limit scope of the present invention.The implementation condition that adopts among the embodiment can be done further adjustment according to the condition of concrete producer, and not marked implementation condition is generally the condition in the normal experiment.
Embodiment
One, the extraction of genomic dna and purifying
1, in 200 μ l whole bloods, add 400 μ l Lysis Solution and 20 μ l Proteinase K solution, mixing obtains the homogeneous suspension.
2, in 56 ℃ of water-baths, hatch 10min.
3, add 200 μ l absolute ethyl alcohol and mixings.
4, the cleavage mixture of preparation is transferred to Gene JET Genomic DNA Purification Column.The centrifugal 1min of 6000g discards waste liquid.Gene JET Genomic DNA Purification Column is transferred to the new collection tube of 2ml.
5, add 500 μ l Wash buffer I, after the centrifugal 1min of 8000g discards waste liquid purification column is put back to collection tube.
6, add 500 μ l Wash buffer II to Gene JET Genomic DNA Purification Column, the centrifugal 3min of 12000g.
7, with the centrifugal 1min of the sub-12000g of void column.
8, Gene JET Genomic DNA Purification Column transfers to the EP pipe of new 1.5ml.
9, the distilled water of interpolation 100 μ l or Elution buffer are to the center of Gene JET Genomic DNA Purification Column, incubated at room 2min, the centrifugal 1min of 8000g.
10, discard purification column, the DNA of purifying perhaps is used for the downstream experiment immediately or is stored in-20 ℃.
Two, genomic dna purity detecting (Nanodrop)
1, the Nanodrop icon on the double-click computer screen starts software.
2, select required measurement pattern, can eject the prompting of initialize instrument on the screen. add the zero(ppm) water of 2 microlitres in the well of instrument, the loam cake that closes makes the formation fluid column, clicks then and confirms can hear the sound of SV folding to begin initialize.
3, the information on the screen disappears after five or six seconds, and the expression initialize is accomplished. and with lens wiping paper zero(ppm) water is wiped clean, added the Buffer of 2-3 microlitre, loam cake and click Blank. closes
4, after Blank accomplishes, with lens wiping paper Buffer is wiped clean and promptly can begin to go up appearance, click Measure and begin to measure.
Three, the fragmentation of genomic dna (fragmentization)
Genome DNA sample obtains from different sources, from the bacterium to the Mammals.When the nanodrop of OD260/280 detected value at 1.8-2.0, the about 0.3 μ g/ μ l of concentration.The power setting of Ultrasonic Cell Disruptor is become 200W, broken 5 seconds, stopped broken number of times 200 times 5 seconds.
Four, design of primers
Joint A:5 '-/BioTEG/GAGGATCCAGAATTCTCGAGTT-3 ';
Joint B:5 '-CTCGAGAATTCTGGATCCTC-3 ';
Described Auele Specific Primer (specific primer) for being used for specific amplification goal gene target sequence, contains at least 15 successive nucleotide sequences of goal gene; As the Auele Specific Primer that carries out 1-5 exon of pcr amplification von Willebrand disease gene has the continuous nucleotide sequence of SEQ No:5～14.
Catch primer and comprise first primer and second primer, form by the Auele Specific Primer (specific primer) of linker DNA fragment+ACTG base group+cooperate with genomic DNA fragment to be amplified.Promptly the structure of first primer is joint A+ACTG+specific primer; The structure of second primer is joint B+ACTG+specific primer; In two primers Auele Specific Primer according to the different genes that will catch and difference.
To catch design of primers and become three parts: shank, middle portion and end part; Its each several part effect is different.Wherein middle portion is an ACTG base group, and middle portion adds ACTG, its objective is in order when order-checking begins, to correct with these four known bases; 4 base sequences of ACTG are arranged arbitrarily.Auele Specific Primer requires to be designed to identical or close annealing temperature, so length is unfixing.Auele Specific Primer according to the heterogeneic difference that will catch design.
Five, for the first time unidirectional PCR reaction
Reaction system is following:
Temperature of reaction:
Six, product purification
Product is with PCR purification kit purifying
The binding buffer that test kit is provided directly adds reaction tubes to stop 72 ℃ extension, takes place in order to prevent the nonspecific reaction after solution cools off.Product is eluted in the elutriant of 30 μ l.
Seven, magnetic capture
(1) gets 25 μ l M-270 Streptavidin magnetic beads,, use 25 μ l, 2 * Binding and wash buffer resuspended then with 500 μ l, 2 * Binding and wash buffer washed twice.
(2) primer extension product of getting 25 μ l purifying joins in the Streptavidin magnetic bead.
(3) mixture shakes and mixes 30min at ambient temperature, so that magnetic bead can be good at combining the heterozygosis of biotinylated primer extension thing and dna profiling double-stranded.
(4) product is transferred to the centrifuge tube of new 1.5ml, with 500 μ l, 1 * Binding and wash buffer washing 5 times.
Eight, primer extension reaction for the second time
Reaction system is following:
Temperature of reaction:
Nine, the acquisition of strand target dna
(1) magnetic bead is resuspended in 500 μ l hot wash buffer (1 * PCR buffer, 2.5mM MgCl2,0.1%Tween-20 (v/v)), and transfers to new 1.5ml EP pipe to above-mentioned solution.
(2) mixture is bathed (perhaps be higher than capture primer Tm value 5 ℃) concussion and is hatched 2min at 65 ℃ of thermostat metals, and the EP pipe is on the magnet scoop time, rapidly supernatant is removed clean, in case cooling.(this step is to remove those that do not extend and background fragments capture primer non-specific binding)
(3) deposition is resuspended in the EB buffer of 30 μ l, it is transferred to EP pipe (this step help to reduce non-specific library is segmental carry) of new 1.5ml.On the PCR appearance, hatch 3min for 95 ℃, and on MPC (Magnetic particle collector), remove supernatant, carefully stay magnetic bead.
Ten, order-checking
11, interpretation of result
1, repeatability
Made 6 samples, each sample has up to a hundred exons, its repeated result such as table 1.
The repeated result of table 1 exon
2, coverage
Coverage result of the present invention is as shown in Figure 3.
3, specificity
The specificity comparative result of the present invention and prior art catching method commonly used is as shown in table 2.
The specificity comparative result of table 2 the present invention and prior art catching method commonly used
Above-mentioned instance only is explanation technical conceive of the present invention and characteristics, and its purpose is to let the people who is familiar with this technology can understand content of the present invention and enforcement according to this, can not limit protection scope of the present invention with this.All equivalent transformations that spirit is done according to the present invention or modification all should be encompassed within protection scope of the present invention.
Claims (10)
1. one kind is carried out the test kit that the polygene multizone is caught the specific nucleic acid target, it is characterized in that said test kit comprises first primer, catches the component and second primer:
Said first primer is used to goal gene regional sequence to be caught unidirectional pcr amplification for the first time; Said first primer, one end links to each other with the first linker DNA fragment that is marked with vitamin H, and with the complementary bonded oligonucleotide of goal gene regional sequence to be caught single stranded sequence;
Said catch component comprise avidin and with avidin bonded solid phase carrier; Said avidin is used to catch the goal gene regional sequence behind the unidirectional pcr amplification for the first time, and with the said first time unidirectional pcr amplification after the goal gene regional sequence on vitamin H combine;
Said second primer is used for the goal gene regional sequence after the avidin combination is carried out the unidirectional pcr amplification second time; Said second primer, one end links to each other with the second linker DNA fragment; And combine, and carry out the in the opposite direction of pcr amplification with first primer with said goal gene regional sequence is complementary.
2. test kit according to claim 1 is characterized in that said solid phase carrier is a microballoon.
3. test kit according to claim 1 is characterized in that said solid phase carrier is a magnetic microsphere.
4. test kit according to claim 1 is characterized in that said avidin is selected from white of an egg affinity element, streptavidin, the yolk affinity is plain and type affinity is plain.
5. test kit according to claim 1 is characterized in that the said first linker DNA fragment has the continuous nucleotide sequence of SEQ No:1, and 5 ' end carries out mark through vitamin H; The second linker DNA fragment has the continuous nucleotide sequence of SEQ No:2.
6. test kit according to claim 1; It is characterized in that said first primer by the first linker DNA fragment, ACTG base group be used for specific amplification goal gene target sequence, the Auele Specific Primer that contains at least 15 successive nucleotide sequences of goal gene is formed by connecting; Said second primer is by the second linker DNA fragment, ACTG base group and be used for specific amplification goal gene target sequence, and the Auele Specific Primer that contains at least 15 successive nucleotide sequences of goal gene is formed by connecting.
7. test kit according to claim 1, a PCR reaction system of pcr amplification comprises PCR damping fluid, dNTPs, MgCl to it is characterized in that carrying out for the first time by said first primer
2, TaqDNA polysaccharase, goal gene regional sequence to be caught be as template DNA, a said PCR reaction system final concentration consists of:
PCR damping fluid final concentration 1～10 *;
dNTPs 0.01～1.5mM；
The primer sequence final concentration is 0.01～2 μ M;
Template DNA 0.01～10ng/ μ L;
TaqDNA polysaccharase 0.01～1.0U/ μ L;
MgCl
2Final concentration is 0.5～5mM.
8. test kit according to claim 1, the 2nd PCR reaction system of pcr amplification comprises PCR damping fluid, dNTPs, MgCl to it is characterized in that carrying out for the second time by said second primer
2, TaqDNA polysaccharase, goal gene regional sequence to be caught be as template DNA, said the 2nd PCR reaction system final concentration consists of:
PCR damping fluid final concentration 1～10 *;
dNTPs 0.01～1.5mM；
The primer sequence final concentration is 0.01～2 μ M;
Template DNA 0.01～10ng/ μ L;
TaqDNA polysaccharase 0.01～1.0U/ μ L;
MgCl
2Final concentration is 0.5～5mM.
9. one kind is carried out the method that the polygene multizone is caught the specific nucleic acid target, it is characterized in that said method comprising the steps of:
(1) genome that will be to be caught interrupts the back at random and adopts first primer to carry out the pcr amplification first time; Said first primer, one end links to each other with the first linker DNA fragment that is marked with vitamin H, and with the complementary bonded oligonucleotide of goal gene regional sequence to be caught single stranded sequence;
(2) use and to catch component the goal gene regional sequence that carries out behind the unidirectional pcr amplification is for the first time caught; Said catch component comprise avidin and with avidin bonded solid phase carrier; Vitamin H on the goal gene regional sequence behind said avidin and the said first time unidirectional pcr amplification combines;
(3) use the goal gene regional sequence after second primer combines avidin to carry out the unidirectional pcr amplification second time; Said second primer, one end links to each other with the second linker DNA fragment, and combines with said goal gene regional sequence is complementary, and carries out the in the opposite direction of pcr amplification with first primer;
(4) the goal gene regional sequence behind unidirectional pcr amplification general's second time is removed the avidin biotin composite, obtains the single-stranded DNA banks that two ends have identical or different DNA joint.
10. one kind is carried out the application of test kit aspect gene sequencing that the polygene multizone is caught the specific nucleic acid target.
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| CN104963000A (en) * | 2014-12-15 | 2015-10-07 | 北京贝瑞和康生物技术有限公司 | Method and kit for rapid construction of single-cell DNA sequencing library |
| CN110491448A (en) * | 2019-07-15 | 2019-11-22 | 广州奇辉生物科技有限公司 | A kind of method, system, platform and storage medium handling PCR primer |
| CN116083529A (en) * | 2022-12-16 | 2023-05-09 | 上海亿康医学检验所有限公司 | Method for targeted enrichment of DNA of genome target region and application thereof |
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| CN101460633A (en) * | 2006-03-14 | 2009-06-17 | 基尼宗生物科学公司 | Methods and means for nucleic acid sequencing |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104963000A (en) * | 2014-12-15 | 2015-10-07 | 北京贝瑞和康生物技术有限公司 | Method and kit for rapid construction of single-cell DNA sequencing library |
| CN110491448A (en) * | 2019-07-15 | 2019-11-22 | 广州奇辉生物科技有限公司 | A kind of method, system, platform and storage medium handling PCR primer |
| CN110491448B (en) * | 2019-07-15 | 2023-02-07 | 广州奇辉生物科技有限公司 | Method, system, platform and storage medium for processing PCR primers |
| CN116083529A (en) * | 2022-12-16 | 2023-05-09 | 上海亿康医学检验所有限公司 | Method for targeted enrichment of DNA of genome target region and application thereof |
| CN116083529B (en) * | 2022-12-16 | 2024-03-12 | 上海亿康医学检验所有限公司 | Method for targeted enrichment of DNA of genome target region and application thereof |
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