CN106047871A - Primer set, DNA in-vitro expansion method, kit and use of primer set - Google Patents
Primer set, DNA in-vitro expansion method, kit and use of primer set Download PDFInfo
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Abstract
The invention relates to the field of molecular biology and especially relates to a primer set, a DNA in-vitro expansion method, a kit and a use of the primer set. The specific primer set has an amplification area basically covering all areas of a TYR gene so that in amplification, the primer set can comprehensively acquire TYR gene nucleotide sequence cases and ensure abrupt change sites. The primer set is suitable for long fragment amplification, can amplify a single band under PCR conditions and does not produce a nonspecific band. Compared with the existing short fragment, a long fragment acquired by primer set-based amplification has an uniform sequencing result and can be subjected to data analysis through a simple process.
Description
Technical field
The present invention relates to biology field, especially relate to a kind of primer sets, DNA amplification in vitro method, test kit
And application.
Background technology
Albinism (albinism) is a kind of skin of being caused by melanic biosynthesis defect of a class and appendages
The autosomal recessive hereditary diseases of official's short of melanin.Albinism is divided into 3 types: oculocutaneous albinism (all get involved by skin and eye
And), (there is the albinic performance of general and other be in ocular albinism (only eye is involved) and albinism related syndromes
System is abnormal).In each albinoidism, it is oculocutaneous albinism more than 90%, oculocutaneous albinism morbidity worldwide
Rate is 1/20000, and the sickness rate of Chinese population is slightly higher, about 1/18000.
According to relating to the difference of gene, oculocutaneous albinism (oculocutaneous albinism, OCA) is broadly divided into 4
Planting hypotype, one of which hypotype is caused by tyrosinase cdna (tyrosinase, TYR) sudden change, the tyrosine of TYR gene code
Enzyme is responsible for the first two steps rate-limiting step of B16 cell.Owing to oculocutaneous albinism various patient clinical phenotype is the most overlapping, only
With the albefaction degree of eye, skin and hair, it is difficult to accurately distinguish various non-syndrome oculocutaneous albinism, therefore, gene test
Become unique reliable method of oculocutaneous albinism typing.
The secondary sequencing technologies of high flux (next-generation sequencing, NGS) fast-developing in recent years is
Diagnosis and the carrier screening of heredopathia it is widely used on the basis of based on first generation sequencing technologies.Secondary order-checking is by mould
Plate preparation and Sequence Detection process and data analysis process two parts form.Wherein, with the order-checking of synthesis limit, limit it is technically
Core, by the means of extensive parallel order-checking, simultaneously to millions of short dna sequencing fragments, it is thus achieved that the sequence of magnanimity
Information, then utilizes bioinformatics tools to be analyzed.Nature Methods magazine commented on 10 years in the past in 2014
The ten big technology the deepest on biological study impact, secondary order-checking ranks first.The strategy of secondary order-checking includes full-length genome, outer aobvious
Subgroup, target area, express spectra, methylate, chromosome co-immunoprecipitation order-checking etc..Wherein, target area sequence of resurveying is pointer
To target area interested or gene, the method being captured by target region or expanding is enriched with, then carries out large scale sequencing.With
Full-length genome, full exon group sequence measurement are compared, target area resurvey sequence can extensive, low cost examination specified disease
Know pathogenic sites, in terms of clinical diagnosis, have huge application prospect.
Summary of the invention
The application mainly solving the technical problems that provide a kind of primer sets, DNA amplification in vitro method, test kit and
Application, it is possible to detect TYR gene mutation accurately, delicately.
For solving above-mentioned technical problem, first technical scheme that the embodiment of the present invention uses is: provide a kind of primer
Group, compared with prior art, its difference is, this primer sets includes for expanding the multiple long segment of TYR gene in PCR
Detection primer pair, it is at least 1 right that this primer sets includes selected from following primer centering:
Sequence primer pair as shown in SEQ ID NO:1 and SEQ ID NO:2；
Sequence primer pair as shown in SEQ ID NO:3 and SEQ ID NO:4；
Sequence primer pair as shown in SEQ ID NO:5 and SEQ ID NO:6；
Sequence primer pair as shown in SEQ ID NO:7 and SEQ ID NO:8；
Sequence primer pair as shown in SEQ ID NO:9 and SEQ ID NO:10；
Sequence primer pair as shown in SEQ ID NO:11 and SEQ ID NO:12；
Sequence primer pair as shown in SEQ ID NO:13 and SEQ ID NO:14；
Sequence primer pair as shown in SEQ ID NO:15 and SEQ ID NO:16；
Sequence primer pair as shown in SEQ ID NO:17 and SEQ ID NO:18；
Sequence primer pair as shown in SEQ ID NO:19 and SEQ ID NO:20；
Sequence primer pair as shown in SEQ ID NO:21 and SEQ ID NO:22；
Sequence primer pair as shown in SEQ ID NO:23 and SEQ ID NO:24；
Sequence primer pair as shown in SEQ ID NO:25 and SEQ ID NO:26；
Sequence primer pair as shown in SEQ ID NO:27 and SEQ ID NO:28；
Sequence primer pair as shown in SEQ ID NO:29 and SEQ ID NO:30；
Sequence primer pair as shown in SEQ ID NO:31 and SEQ ID NO:32.
Wherein, primer sets includes described 16 pairs of primers pair.
For solving above-mentioned technical problem, second technical scheme that the embodiment of the present invention uses is: provide a kind of DNA external
Amplification method, reacts amplification of DNA fragments by PCR, compared with prior art, its difference is, uses above-mentioned primer
Group is as primer.
Wherein, reaction system is 20 μ l, expands enzyme 0.5U including long segment；Template 30ng；Forward primer 0.4 μ l；Downstream
Primer 0.4 μ l；
Described polymerase chain reaction includes:
The amplification stage: denaturation 1min at 94 DEG C；Degeneration 10s at 98 DEG C；Anneal at 65 DEG C 30s, downward at 68 DEG C
Stretch 10min；
Described amplification step cycle is performed 30 times；
After circulation performs 30 times, at 68 DEG C, hatch 30min.
For solving above-mentioned technical problem, the 3rd technical scheme that the embodiment of the present invention uses is: provide a kind of test kit,
Compared with prior art, its difference is, this test kit includes above-mentioned primer sets.
Wherein, this test kit also includes one or more of reagent:
For the reagent from sample extraction genomic DNA；
Utilize the reagent to carrying out PCR reaction of the primer in described primer sets；
For processing pcr amplification product so that amplified production can carry out the reagent of high-flux sequence；
And
For the pcr amplification product after processing being carried out the reagent of high-flux sequence.
Wherein, described high-flux sequence is Ion Torrent order-checking.
Wherein, the described primer utilized in described primer sets uses the above-mentioned external expansion of DNA to the reagent carrying out PCR reaction
Increasing method.
For solving above-mentioned technical problem, the 4th technical scheme that the embodiment of the present invention uses is: provide one to utilize
Any one primer sets stated, or any one above-mentioned DNA amplification in vitro method, or any one above-mentioned test kit is at TYR gene
Application in abrupt climatic change.
For solving above-mentioned technical problem, the 5th technical scheme that the embodiment of the present invention uses is: provide one to utilize
Any one primer sets stated, or any one above-mentioned DNA amplification in vitro method, or any one above-mentioned test kit is in melanin conjunction
Become the application in abnormal examination.
The beneficial effects of the present invention is: the primer sets of the present invention relates to most of region of TYR gene, including outer aobvious
Son and intron, can the nucleotide sequence situation of the more TYR gene of Overall Acquisition patient, specify mutational site；And this primer
Group expands for long segment, and the band amplified under suitable conditions is single, nothing but specific band, and due to amplified fragments
Long, sequencing result is more uniformly distributed compared with short fragment amplification method, and data analysis is the simplest；The test kit of the present invention uses long
Fragment DNA cloning method combines the mode of Ion Torrent sequencing technologies, has efficient, high specificity, the high and low one-tenth of sensitivity
This advantage.
Accompanying drawing explanation
In order to be illustrated more clearly that the technical scheme of the embodiment of the present application, will make required in the embodiment of the present application below
Accompanying drawing be briefly described.It should be evident that drawings described below is only some embodiments of the application, for
From the point of view of those of ordinary skill in the art, on the premise of not paying creative work, it is also possible to obtain other according to these accompanying drawings
Accompanying drawing.
Fig. 1 is the order-checking of the mutational site on family member TYR gene in the embodiment of the present invention 1.
Fig. 2 is genealogical chart on family member TYR gene in the embodiment of the present invention 1.
Detailed description of the invention
In order to make the purpose of the application, technical scheme and advantage clearer, below in conjunction with drawings and Examples, right
The application is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the application, not
For limiting the application.
Embodiments provide a kind of primer sets, DNA amplification in vitro method, test kit and primer sets, test kit and
The application in TYR detection in Gene Mutation and B16 cell exception examination of the DNA amplification in vitro method.Described in this specification
" to suddenly change " can be the insertion of one or more base, replace and/or lack, or gene amplification sudden change.Described " amplification sudden change "
Refer to phenomenon and process that the copy number in the region of the exon of gene or the exon of gene or gene optionally increases,
Belonging to the one of gene copy number variation (Copy number variations, CNV), amplification sudden change is likely to result in accordingly
The increase of protein expression.
The primer sets of the embodiment of the present invention is respectively used for amplifying the long segment of the respective regions of TYR gene, then will amplification
Target fragment carry out gene type assay to determine gene mutation situation, such as, the target fragment after amplification is carried out high flux
Secondary order-checking, to obtain the sequence information of the target fragment of amplification, and is derived from abrupt climatic change result.Wherein, high flux two
Be prior art for sequencing technologies, be widely used on the basis of based on the first generation sequencing technologies diagnosis of heredopathia with
The examination of carrier, details are introduced the most in the introduction, the most no longer state.
The primer sets of the embodiment of the present invention relates to most of region of TYR gene, including exon and intron, and its bag
Include selected from shown in table 1 at least the 1 of primer centering to or whole 16 pairs of primers: concrete primer is specific as follows to corresponding amplification region
Shown in table 1.
Table 1 detection primer pair
For designing specific primer, it is necessary first to obtain the information of oculocutaneous albinism Disease-causing gene TYR: pass through
It is white that Online Mendelian Inheritance in Man (OMIM, http://omim.org/) data base obtains Oculocutaneous
Change sick Disease-causing gene information, particularly obtain positional information and the mutational site information of TYR gene, be shown in Table 2.Omim database is
A kind of continuous updating about human gene and the data base of genetic disorder, be mainly focused on heritable or genetic gene
Disease, including text message and coherent reference information, sequence record, collection of illustrative plates and other data bases relevant.
The positional information of table 2 oculocutaneous albinism Disease-causing gene TYR
| Sequence number | Clinical typing | Disease-causing gene | Between the gene whole district | Full length gene |
| 1 | OCA I | TYR | chr11:88,911,040-89,028,927 | 117,888bp |
Secondly, according to TYR gene design specific primer:
With genomic DNA as template, the standard sequence provided according to UCSC Genome Bioinformatics data base
(GRCh37/hg19), and TYR sequence location, design 16 total lengths to long segment primer amplification Disease-causing gene TYR, comprise altogether
Intron and exon, the fragment length of every couple of primer amplification about 10kb, concrete primer sequence and amplification target area such as table 1
Shown in.Then mutational site is screened through the secondary order-checking of high flux (NGS).
Again, by round pcr, the target gene (i.e. TYR gene) in template is carried out selective amplification.Wherein, use
The primer that above-mentioned primer sets is reacted as PCR.
Extract peripheral blood genomic DNA as template.Extracting method can use multiple commercially available test kit, such as
The DNA extraction kit of QIAGEN company, or the genome DNA extracting reagent kit of OMEGA.
In embodiments of the present invention, it is 20 μ l that PCR reacts total system, expands enzyme 0.5U including long segment；Template 30ng；On
Trip primer 0.4 μ l；Downstream primer 0.4 μ l.PCR amplification condition is: denaturation 1min at 94 DEG C；Degeneration 10s at 98 DEG C；65
Anneal at DEG C 30s, extends 10min at 68 DEG C, and amplification cycles performs 30 times；Then, at 68 DEG C, 30min is hatched.
Every pair of primer carries out PCR amplification with above-mentioned PCR amplification condition, can amplify the DNA fragmentation of about 10kb length, this
16 pairs of primers designed by inventive embodiments, the DNA fragmentation of 16 sections of about 10kb length of coamplification, detailed sequence is shown in SEQ ID
NO.33-SEQ ID NO.48, all fragment overall length sequences almost contain all exons and the intron sequences of TYR gene.
The test kit that the embodiment of the present invention provides, including above-mentioned primer sets, also includes one or more of reagent:
For the reagent from sample extraction genomic DNA, such as DNA extraction liquid, lyases, buffer etc.；Utilize described primer sets
In primer to carrying out the reagent of PCR reaction, such as DNA cloning enzyme；For processing pcr amplification product so that amplified production can
Carry out the reagent of high-flux sequence, and for the pcr amplification product after processing being carried out the reagent of high-flux sequence.
The high-flux sequence method that the embodiment of the present invention is used is Ion Torrent sequencing.Life is preferably used
The Ion PGM sequenator of Technologies company is carried out.Ion Torrent sequencing technologies is also generally referred to by those skilled in the art as
Secondary sequencing technologies, it is based on chemical change produced by DNA building-up process.Archaeal dna polymerase, with single stranded DNA as template, is pressed
Base complementrity principle, the DNA that synthesis is complementary.When DNA often extends a base, a proton will be discharged, cause local
PH changes.Microsphere surface in each micropore of Ion Torrent quasiconductor sequence testing chip contains about 1,000,000 DNA moleculars to be copied
Shellfish.During order-checking, nucleic acid molecule continues to flow through chip micropore.If a nucleotide is complementary with the DNA molecular in certain micropore,
Then this nucleotide is synthesized in DNA molecular, and discharges proton, and in this micropore, the pH of solution changes.Ion transducer
After pH change being detected, this chemical information is changed into digital electronic information.If DNA contains two identical bases, then
Recording voltage signal is double.If base is not mated, then without proton release, the most just there is no the change of voltage signal.Ion
Torrent sequencing technologies belongs to the synthesis directly detecting DNA, i.e. limit synthesis frontier inspection is surveyed.It addition, Ion Torrent sequencing technologies
Need not the links such as CCD scanning, fluorescence excitation, within several seconds, just can detect the base that synthesis is inserted, substantially reduce the operation time,
Thus improve detection efficiency.
Test kit needs the function realized, and such as extracting genomic DNA etc. from peripheral blood is the ordinary skill in the art,
And there is a lot of ripe perfect commercial reagents available on market.
Test kit comprise for processing amplified production so that amplified production can be used for the examination in high throughput sequencing technologies
Agent.Pcr amplification product typically cannot be used directly for high-flux sequence, in addition it is also necessary to processes, and such as, end reparation, connection connects
Head and label, purification, reparation breach etc..For grasp high-flux sequence those of ordinary skill for, above-mentioned process step and
Required reagent will be appreciated that.Embodiments herein also provides exemplary processing method.
The reagent for the amplified production after processing being carried out high-flux sequence that test kit comprises.High-flux sequence is usual
Microwell chips is carried out.At present, business-like chip and reaction reagent are easily buied, such as, be purchased from Life
Technologies Inc.。
Specifically, the extraction of sample genomic dna: take peripheric venous blood 2ml, sodium citrate anticoagulant.Use QIAamp
DNA MiniKit (Qiagen) extracts Whole Blood Genomic DNA, and places-20 DEG C of preservations.
PCR expands, and reaction system is 20 μ l, long segment amplification enzyme 0.5U, DNA profiling 30ng and upstream and downstream primer (10 μMs)
Each 0.4 μ l.Reaction condition: 94 DEG C of denaturations 1min；98 DEG C of 10s, 65 DEG C of annealing 30s, 68 DEG C extend 10min；After 30 circulations
Hatch 30min for 68 DEG C.Take amplified production 4 μ l to identify through 0.8% agarose gel electrophoresis.
Ion Torrent checks order: after PCR primer mixing, by Ion Shear Plus Reagent Kit (Life
Technologies) endonuclease bamhi, and use Ion Plus Fragment Library Kit (Life
Technologies) and Ion Xpress Barcode Adapter 1-16 (Life technology) connect label joint
(Barcode Adapter), different family members uses different label joints (Barcode Adapter).Connection completes
The library template of mean size about 250bp is prepared afterwards, again with Ion Plus Fragment after purification process by E-gel electrophoresis
Library Kit (Life technologies) carries out PCR amplification preparation library, and the DNA library prepared needs to pass through
Qubit 2.0 carries out quantitatively, being then diluted to corresponding concentration and carrying out emulsion-based PCR by Ion One Touch again, and pass through magnetic
Positive template is enriched with on Ion OneTouch ES by pearl granule, finally uses Ion PGM 200 Sequencing
Kit (Life technologies) and Ion Torrent318 chip carry out sequencing reaction at Ion Torrent PGM platform,
Totally 125 order-checking circulation.
Sequencing result analyze: use Ion Torrent Suite v3.0 software carry out Ion Torrent data extract and
Sequence alignment and filtration.Hepatolenticular degeneration data base, after dbSNP filtering based on database, is retrieved in the mutational site obtained
(http://www.wilsondisease.med.ualberta.ca/database.asp), screens possible pathogenic mutation position
Point.
Core families genetic analysis and sudden change checking: the follow-up sudden change position that secondary order-checking finds and obtains through data base's comparison
Point, in patient and family member thereof, corresponding positions point carries out Sanger sequencing analysis, is determined for compliance with mendelian recessive genetic development
Site be that this first demonstrate,proves this Disease-causing gene mutational site.
In embodiments of the present invention, devising 16 pairs of primers, the PCR amplification method using DNA long fragment is white to Oculocutaneous
Change sick Disease-causing gene TYR to be enriched with, then carry out entirely at Ion Torrent PGM/ion Proton secondary order-checking platform
Gene is resurveyed sequence.The amplified fragments applying the test kit of this primer can cover all exons of TYR gene, intron comprehensively
And other regions, thus this test kit and primer molecular diagnosis in there is the highest practical clinical be worth.
Embodiment
1, family situation:
Proband's clinical manifestation is that skin, hair and eye iris melanin lack the most completely, the horizontal chatter of eyeball, fear
Light is obvious.Without other patients in father and mother both sides family, the equal phenotype of proband father and mother is normal, non-consanguineous mating, and the pregnancy period is without medication
History or poor environment contact history.This research is ratified through Inst of Population & Family Planning Sciences, Shenzhen City's Medical Ethics Committee,
All gene diagnosises all obtain the agreement of family numbers of patients and sign Informed Consent Form.
2, detection method:
(1) specimen collection and DNA extraction: take each 2mL of peripheric venous blood of proband and father and mother thereof, sodium citrate anticoagulant.
The DNA extraction kit (QIAamp DNA MiniKit, Qiagen Products) using market to sell extracts whole blood genome
DNA, and place-20 DEG C of preservations.
(2) Disease-causing gene full-length gene order is analyzed: examination Disease-causing gene tyrosinase cdna (tyrosinase, TYR)
Positional information as shown in table 2.
With genomic DNA as template, the standard sequence provided according to UCSC Genome Bioinformatics data base
(GRCh37/hg19), design 16 is to long segment primer amplification said gene total length (including whole intron and exon) altogether, often
Fragment length to primer amplification about 10kb.The PCR primer of the embodiment of the present invention is reasonable in design, efficient, high specificity, sensitivity
Height, the band amplified under suitable conditions by this primer is single, nothing but specific band, and owing to amplified fragments is long, with
Short fragment amplification method is compared sequencing result and is more uniformly distributed, and data analysis is the simplest, and concrete primer sequence is as shown in table 1.
(3) PCR amplification: reaction system is 20 μ L, long segment amplification enzyme 0.5U, DNA profiling 30ng and upstream and downstream primer (10
μM) each 0.4 μ L.Reaction condition: 94 DEG C of denaturations 1min；98 DEG C of 10s, 65 DEG C of annealing 30s, 68 DEG C extend 10min；30 circulations
Hatch 30min for latter 68 DEG C.Take amplified production 4 μ L to identify through 0.8% agarose gel electrophoresis.
Every pair of primer amplifies the DNA fragmentation of about 10kb length according to above-mentioned PCR amplification condition, set by the embodiment of the present invention
Primer, the DNA fragmentation of 16 sections of about 10kb length of coamplification, detailed sequence are shown in SEQ ID NO.33-SEQ ID NO.48 by meter 16.
(4) Ion Torrent order-checking: after PCR primer mixing, by Ion Shear Plus Reagent Kit (Life
Technologies company produces) endonuclease bamhi, and use Ion Plus Fragment Library Kit (Life
Technologies company produces) and Ion Xpress Barcode Adapters 1-16 (Life technologies company
Produce) connect Barcode Adapter, different family members uses different Barcode Adapter.Lead to after having connected
Cross the library template that E-gel electrophoresis prepares mean size about 250bp, again with Ion Plus Fragment after purification process
Library Kit (Life technologies company produce) carries out PCR amplification preparation library.The DNA library needs prepared
Carry out quantitatively, being then diluted to corresponding concentration and carrying out emulsion-based PCR by Ion OneTouch again, and lead to by Qubit 2.0
Cross magnetic bead particles on Ion OneTouch ES, positive template to be enriched with, finally use Ion PGM 200
Sequencing Kit (Life technologies company produce) and Ion Torrent 318 chip are at Ion Torrent
PGM platform carries out sequencing reaction, totally 125 order-checking circulations.
(5) sequencing result analysis: use Ion Torrent Suite v3.0 software to carry out Ion Torrent data and extract
And sequence alignment and filtration.The mutational site obtained, after dbSNP filtering based on database, is retrieved the data bases such as HGMD, LOVD and looks into
Inquiry pertinent literature, and query and search albinism data base (http://www.ifpcs.org/albinism/) filter out possible
Mutational site.
(6) core families genetic analysis and sudden change checking: secondary order-checking discovery the candidate obtained through data base's comparison dash forward
Displacement point, in patient and family member thereof, corresponding positions point carries out Sanger sequencing analysis, is determined for compliance with mendelian recessive heredity
The site of rule is the Disease-causing gene mutational site of this proband.
3, interpretation of result:
(1) gene sequencing quality: from table 3 it can be seen that the sample order-checking reading of father, mother and patient is respectively
663752,579868 and 416271, order-checking mean depth is respectively 363.8,319.2 and 241.7, and average length of reading is respectively
184bp, 185bp and 189bp.
The sequencing quality of table 3 Ion Torrent
(2) gene sequencing diagnosis: proband: the TYR gene of proband detects 2 kinds of heterozygous mutants altogether, is respectively
C.929insC and c.1199G > T.Father: the TYR gene test of father to a kind heterozygous mutant, c.1199G > T.Mother: mother
TYR gene test to a kind heterozygous mutant, c.929insC, refer to table 4.
Albinism gene mutation situation in table 4 family member
* mutational site is with reference to human genome GRCh37/hg19 version；aAllele father originates,bAllele mother
Source；Disease-causing gene runic shows.
(3) Sanger the result: as it is shown in figure 1, proband is OCA I type patient, Disease-causing gene is TYR gene, causes
C.1199G c.929insC disease sport and > T constitutes double heterozygote sudden change, and the most c.1199G > T sudden change heredity is from father,
C.929insC heredity is from mother.Result above meets Mendel's autosomal recessive inheritance, AR rule, as shown in Figure 2.Suggestion should
Prenatal gene diagnosis does in two tire During Pregnancies in family, is similar to the infant of the state of an illness to avoid being born in proband again.
Above-mentioned case illustrates, the follow-up mutational site that the secondary order-checking of high flux finds and obtains through data base's comparison, is suffering from
In person and family member thereof, corresponding positions point carries out Sanger sequencing analysis, and the site being determined for compliance with mendelian recessive genetic development is
The Disease-causing gene mutational site of this proband.
In embodiments of the present invention, first, DNA long fragment amplification method is used to combine secondary sequencing technologies examination Oculocutaneous
The sudden change of albinism TYR Disease-causing gene.Compared with traditional method, this sequence measurement covers gene Zone Full, complete including TYR
Portion's exon and intron, can Overall Acquisition patient in Disease-causing gene TYR nucleotide sequence situation, specify mutational site, for
Detection gene mutation provides reliable method, has the strongest sensitivity and accuracy, and the clinic for oculocutaneous albinism is examined
The disconnected diagnosis basis that molecular biology is provided.
Secondly, the PCR primer of the embodiment of the present invention is reasonable in design, efficient, high specificity, highly sensitive, utilizes this primer
By detection peripheral blood extract DNA carry out amplification in vitro, obtain required PCR primer and check order again, with genome sequencing,
Exon group sequence measurement is compared, and has efficient, the advantage of low cost.
Again, the band that the primer of embodiment of the present invention design amplifies under suitable conditions is single, without non-specific bar
Band, and owing to amplified fragments is long, sequencing result is more uniformly distributed compared with short fragment amplification method, and data analysis is the simplest.
4th, the embodiment of the present invention carries out PCR amplification check order for the whole exon of TYR gene, intron, Ke Yiwei
Find that oculocutaneous albinism new mutation scientific research in Disease-causing gene provides technological means.
5th, the embodiment of the present invention can be applied in other diseases relevant to TYR sudden change (such as cutaneous malignant melanocyte
Tumor, vitiligo etc.) detection or examination.
Obviously, above-described embodiment is only for clearly demonstrating example, and not restriction to embodiment.Right
For those of ordinary skill in the field, can also make on the basis of the above description other multi-form change or
Variation.Here without also cannot all of embodiment be given exhaustive.And the obvious change thus extended out or
Change among still in the protection domain of the invention.
Claims (10)
1. a primer sets, it is characterised in that this primer sets includes the inspection for expanding the multiple long segment of TYR gene in PCR
Surveying primer pair, it is at least 1 right that this primer sets includes selected from following primer centering:
Sequence primer pair as shown in SEQ ID NO:1 and SEQ ID NO:2；
Sequence primer pair as shown in SEQ ID NO:3 and SEQ ID NO:4；
Sequence primer pair as shown in SEQ ID NO:5 and SEQ ID NO:6；
Sequence primer pair as shown in SEQ ID NO:7 and SEQ ID NO:8；
Sequence primer pair as shown in SEQ ID NO:9 and SEQ ID NO:10；
Sequence primer pair as shown in SEQ ID NO:11 and SEQ ID NO:12；
Sequence primer pair as shown in SEQ ID NO:13 and SEQ ID NO:14；
Sequence primer pair as shown in SEQ ID NO:15 and SEQ ID NO:16；
Sequence primer pair as shown in SEQ ID NO:17 and SEQ ID NO:18；
Sequence primer pair as shown in SEQ ID NO:19 and SEQ ID NO:20；
Sequence primer pair as shown in SEQ ID NO:21 and SEQ ID NO:22；
Sequence primer pair as shown in SEQ ID NO:23 and SEQ ID NO:24；
Sequence primer pair as shown in SEQ ID NO:25 and SEQ ID NO:26；
Sequence primer pair as shown in SEQ ID NO:27 and SEQ ID NO:28；
Sequence primer pair as shown in SEQ ID NO:29 and SEQ ID NO:30；
Sequence primer pair as shown in SEQ ID NO:31 and SEQ ID NO:32.
Primer sets the most according to claim 1, it is characterised in that described primer sets comprises described 16 pairs of primers pair.
3. a DNA amplification in vitro method, reacts amplification of DNA fragments by PCR, it is characterised in that use such as claim 1 or
Primer sets described in 2 is as PCR primer.
DNA amplification in vitro method the most according to claim 3, it is characterised in that the reaction system of described PCR is 20 μ l,
Enzyme 0.5U is expanded including long segment；Template 30ng；Forward primer 0.4 μ l；Downstream primer 0.4 μ l；
The reaction condition of described PCR is:
The amplification stage: denaturation 1min at 94 DEG C；Degeneration 10s at 98 DEG C；Anneal at 65 DEG C 30s, extends at 68 DEG C
10min；
Described amplification step cycle is performed 30 times；
After circulation performs 30 times, at 68 DEG C, hatch 30min.
5. a test kit, it is characterised in that this test kit includes the primer sets described in claim 1 or 2.
Test kit the most according to claim 5, it is characterised in that this test kit also includes one or more of reagent:
For the reagent from sample extraction genomic DNA；
Utilize the reagent to carrying out PCR reaction of the primer in described primer sets；
For processing pcr amplification product so that amplified production can carry out the reagent of high-flux sequence；
And
For the pcr amplification product after processing being carried out the reagent of high-flux sequence.
Test kit the most according to claim 6, it is characterised in that described high-flux sequence is Ion Torrent order-checking.
Test kit the most according to claim 6, it is characterised in that the described primer utilized in described primer sets is to carrying out
The reagent of PCR reaction uses the DNA amplification in vitro method described in claim 4.
9. the DNA amplification in vitro method of any one of the primer sets of any one of claim 1 to 2, or claim 3 to 4, or right
Require the test kit of 5 to 8 any one, the application in TYR detection in Gene Mutation.
10. the DNA amplification in vitro method of any one of the primer sets of any one of claim 1 to 2, or claim 3 to 4, or power
Profit requires the test kit of 5 to 8 any one, the application in B16 cell exception examination.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109972209A (en) * | 2019-01-18 | 2019-07-05 | 南开大学 | The method that single-chain antibody library is rearranged to full length antibody library based on the one-step method of emulsion-based PCR |
-
2016
- 2016-08-05 CN CN201610639373.2A patent/CN106047871A/en active Pending
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| Title |
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| LIN LIU ET AL.: "Comparison of Next-Generation Sequencing Systems", 《JOURNAL OF BIOMEDICINE AND BIOTECHNOLOGY》 * |
| MICHAEL A QUAIL ET AL.: "A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers", 《BME GENOMICS》 * |
| 廖沛熙等: "眼皮肤白化病研究的新途径及TYR基因2种新突变分析", 《中国病理生理杂志》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109972209A (en) * | 2019-01-18 | 2019-07-05 | 南开大学 | The method that single-chain antibody library is rearranged to full length antibody library based on the one-step method of emulsion-based PCR |
| CN109972209B (en) * | 2019-01-18 | 2022-05-03 | 南开大学 | Method for rearranging single-chain antibody library into full-length antibody library by one-step method based on emulsion PCR |
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