CN105506106A - Method and primer for detecting whole exons of FIX gene - Google Patents
Method and primer for detecting whole exons of FIX gene Download PDFInfo
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- CN105506106A CN105506106A CN201511023610.4A CN201511023610A CN105506106A CN 105506106 A CN105506106 A CN 105506106A CN 201511023610 A CN201511023610 A CN 201511023610A CN 105506106 A CN105506106 A CN 105506106A
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Abstract
The invention discloses a method, primer and a kit for detecting whole exons of an FIX gene. Each of the primer and the kit includes seven pairs of forward and reverse primers which are expanded to cover the whole exon sequence of the FIX gene; a pair of universal primers is utilized as sequencing primers for detecting mutation conditions of the whole exon sequence of the FIX gene by virtue of a Sanger sequencing method. The primer and the method can be applied to the aspects of gene diagnosis, family female carrier detection, prenatal gene diagnosis and the like of patients with hemophilia B.
Description
Technical field
The invention belongs to life science and biological technical field, particularly detect primer and the method for the full exons mutation of FIX gene
Background technology
Hemophilia B is also referred to as plasma thromboplastin component (FIX) deficiency disease, it is a kind of X-linkage recessive hereditary disease, pathogenesis is that FIX (F9) transgenation causes FIX content in blood plasma to reduce or functional defect, and the sickness rate in the male sex is 1/30000, rare in women.Hemophilia B symptom main manifestations is that each position of entire patient is spontaneous or excessively hemorrhage after damaging, and there is no effective cure method at present, can only be alleviated by blood transfusion or FIX concentrate formulation or be delayed symptom, bring white elephant thus to patient home and society.
FIX gene is positioned at Xq26.3-27.2, total length 34kb, is made up of the tune region in 8 exons, 7 introns and flanking sequence.8 exon length from 25bp to 1935bp not etc., exons 1 coded signal peptide, the protein excretion of bootable synthesis is in blood; Exon 2 and 3 coding propetide and GLA structural domains; Exon 4 is encoded skins somatomedin structural domain I, this structural domain can with Ca
2+combine closely; Exon 5 is encoded skins somatomedin domain II; The encoding activating structural domain of exon 6, in coagulation process, under the acting in conjunction of XIa, VII a and tissue factor, FIX will be cut off at the 221st hyte propylhomoserin, the 269th arginine and the 365th Serine place, form light chain and heavy chain, now FIX is just transformed to activated FIXa; Exon 7 and 8 coding catalyst structure domains, play a very important role in the conversion of FXa at catalysis FX.FIX genetic flaw type is very various, the sudden change FIX transgenation of collecting in up-to-date human mutation database is more than 1000, comprise point mutation, the insertion of small segment and disappearance, large fragment copy the various mutations types such as rearrangement, wherein modal be caused by single base mutation missense mutation, nonsense mutation and splice mutation.
Before gene sequencing technology is not yet universal, family linkage analysis is the method being most commonly used to hemophilia family female carrier detection and prenatal gene diagnosis.Because linkage analysis need provide the blood sample of several member in family, make the genetic information of family complete as far as possible, and could adopt when the significant site with polymorphism meaning must be detected, thus may face many restrictions when reality uses.When the pleomorphism site adopted is positioned at outside gene, also may linkage analysis be made due to homologous recombination phnomena to occur mistake, cause final result to judge to produce larger error.Direct mutagenesis detection to F9 gene direct Sequencing by DNA sequencer, is found out the accurate location of sudden change, can be provided information more accurately.Owing to achieving automated operation, the PCR that we adopt, in conjunction with DNA direct Sequencing method, thus substantially reduces Diagnostic Time, the man power and material saved, and order-checking efficiency is high, and can determine position and the form of transgenation, result is also more clear and definite.
Several genes mutation detection techniques comprises the methods such as protein truncation test (PTT), single strand conformation polymorphism (SSCP), heteroduple analysis (HA), denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel elec-trophoresis (TGGE) (TGGE), but all these methods all have certain limitation, thus limit the recall rate of transgenation.The method of detection in Gene Mutation conventional is at present gene sequencing and quantitative fluorescent PCR etc.Because FIX capacity is large, mutation type is many, mutation rate is high, mutational site is dispersed in distribution, so prevent the further investigation to FIX transgenation, fluorescence quantitative PCR method is also inapplicable.
Summary of the invention
The invention provides the primer detecting the full exon of FIX gene, adopt Sanger sequencing, can be used for the situation of the full exons mutation of rapid detection FIX gene, for the gene diagnosis of HB.
The object of the present invention is to provide the primer detecting the full exon of FIX gene, comprising: 7 of the full exons mutation of amplification covering detection FIX gene aligns, reverse primer; Its base sequence is:
FIX-1-F：TGTAAAACGACGGCCAGTGTACAACTAATCGACCTTACCACTT
FIX-1-R：AACAGCTATGACCATGCGTGCTGGCTGTTAGACTCT
FIX-2+3-F：TGTAAAACGACGGCCAGTCCCTAAAGAGAAATTGGCTTTCAG
FIX-2+3-R：AACAGCTATGACCATGGAATTGCTTACCAACATACTGCTTC
FIX-4F：TGTAAAACGACGGCCAGTTGGCTTCCAGGTCAGTAGTT
FIX-4R：AACAGCTATGACCATGAGGTGAGTCGGAACATCATTATTAC
FIX-5F：TGTAAAACGACGGCCAGTCACTCTTATTTCAAGGCTCCAA
FIX-5R：AACAGCTATGACCATGAAGGAAGCAGATTCAAGTAGGA
FIX-6F：TGTAAAACGACGGCCAGTGCTTGAGACTCTATTCACTGATTAG
FIX-6R：AACAGCTATGACCATGCATCCCAATAGGTCTGTCTAGTA
FIX-7F：TGTAAAACGACGGCCAGTGCCAGCACCTAGAAGCCAATA
FIX-7R：AACAGCTATGACCATGCCCTTCTGCCTTTAGCCCAAT
FIX-8F：TGTAAAACGACGGCCAGTGGTGAACATAATATTGAGGAGACAG
FIX-8R：AACAGCTATGACCATGTTAGTTAGTGAGAGGCCCTGTTAA
Further, described 7 to align, the working concentration ratio of reverse primer is: FIX-1F:FIX-1R=1:1; FIX-2+3F:FIX-2+3R=1:1; FIX-4F:FIX-4R=1:1; FIX-5F:FIX-5R=1:1; FIX-6F:FIX-6R=1:1; FIX-7F:FIX-7R=1:1; FIX-8F:FIX-8R=1:1.
The present invention also aims to provide a kind of method detecting the full exon of FIX gene, it comprises the steps:
(1) sample DNA is extracted;
(2) 7 couples of amplimer FIX-1F and FIX-1R are utilized; FIX-2+3F and FIX-2+3R; FIX-4F and FIX-4R; FIX-5F and FIX-5R; FIX-6F and FIX-6R; FIX-7F and FIX-7R; FIX-8F and FIX-8R increases to the DNA in (1), obtains the amplified production covering and detect the full exon sequence of FIX gene;
(3) utilize 1 couple of sequencing primer M13-F and M13-R to carry out forward and backward sequencing to the amplified production in (2), obtain the gene order of described amplified production;
(4) gene order in (3) and wild-type FIX gene order are compared, determine whether mutational site exists; Wherein said primer sequence is:
FIX-1-F：TGTAAAACGACGGCCAGTGTACAACTAATCGACCTTACCACTT
FIX-1-R：AACAGCTATGACCATGCGTGCTGGCTGTTAGACTCT
FIX-2+3-F：TGTAAAACGACGGCCAGTCCCTAAAGAGAAATTGGCTTTCAG
FIX-2+3-R：AACAGCTATGACCATGGAATTGCTTACCAACATACTGCTTC
FIX-4F：TGTAAAACGACGGCCAGTTGGCTTCCAGGTCAGTAGTT
FIX-4R：AACAGCTATGACCATGAGGTGAGTCGGAACATCATTATTAC
FIX-5F：TGTAAAACGACGGCCAGTCACTCTTATTTCAAGGCTCCAA
FIX-5R：AACAGCTATGACCATGAAGGAAGCAGATTCAAGTAGGA
FIX-6F：TGTAAAACGACGGCCAGTGCTTGAGACTCTATTCACTGATTAG
FIX-6R：AACAGCTATGACCATGCATCCCAATAGGTCTGTCTAGTA
FIX-7F：TGTAAAACGACGGCCAGTGCCAGCACCTAGAAGCCAATA
FIX-7R：AACAGCTATGACCATGCCCTTCTGCCTTTAGCCCAAT
FIX-8F：TGTAAAACGACGGCCAGTGGTGAACATAATATTGAGGAGACAG
FIX-8R：AACAGCTATGACCATGTTAGTTAGTGAGAGGCCCTGTTAA
M13-F：TGTAAAACGACGGCCAGT
M13-R：AACAGCTATGACCATG
The present invention also aims to provide a kind of test kit detecting the full exon of FIX gene, described test kit comprises sample DNA extraction agent; Dehydrated alcohol; Detection system PCR reaction solution, order-checking system reaction solution, positive reference substance, negative controls and blank product, wherein detection system PCR reaction solution comprises 7 couples of amplimer FIX-1F and FIX-1R; FIX-2+3F and FIX-2+3R; FIX-4F and FIX-4R; FIX-5F and FIX-5R; FIX-6F and FIX-6R; FIX-7F and FIX-7R; FIX-8F and FIX-8R, order-checking system comprises 1 couple of sequencing primer M13-F and M13-R, comprising:
FIX-1-F：TGTAAAACGACGGCCAGTGTACAACTAATCGACCTTACCACTT
FIX-1-R：AACAGCTATGACCATGCGTGCTGGCTGTTAGACTCT
FIX-2+3-F：TGTAAAACGACGGCCAGTCCCTAAAGAGAAATTGGCTTTCAG
FIX-2+3-R：AACAGCTATGACCATGGAATTGCTTACCAACATACTGCTTC
FIX-4F：TGTAAAACGACGGCCAGTTGGCTTCCAGGTCAGTAGTT
FIX-4R：AACAGCTATGACCATGAGGTGAGTCGGAACATCATTATTAC
FIX-5F：TGTAAAACGACGGCCAGTCACTCTTATTTCAAGGCTCCAA
FIX-5R：AACAGCTATGACCATGAAGGAAGCAGATTCAAGTAGGA
FIX-6F：TGTAAAACGACGGCCAGTGCTTGAGACTCTATTCACTGATTAG
FIX-6R：AACAGCTATGACCATGCATCCCAATAGGTCTGTCTAGTA
FIX-7F：TGTAAAACGACGGCCAGTGCCAGCACCTAGAAGCCAATA
FIX-7R：AACAGCTATGACCATGCCCTTCTGCCTTTAGCCCAAT
FIX-8F：TGTAAAACGACGGCCAGTGGTGAACATAATATTGAGGAGACAG
FIX-8R：AACAGCTATGACCATGTTAGTTAGTGAGAGGCCCTGTTAA
M13-F：TGTAAAACGACGGCCAGT
M13-R：AACAGCTATGACCATG
Further, described detection system PCR reaction solution also comprises 2 × PCRBuffer; DNTPs; KODFXDNAPolymerase.
Further, described order-checking system also comprises order-checking refined solution, EDTA, dehydrated alcohol, 75% ethanol, HIDI and BigdyeTerminatorV3.1.
Further, described order-checking refined solution comprises shrimp alkaline phosphotase and exonuclease I.
The present invention devises amplification and covers and detect that 7 of FIX gene full exon sequence aligns, reverse primer, and the upstream primer 5 ' at pcr amplification of novelty end and downstream primer 5 ' are held and added the M13-F primer sequence of a segment length 18bp and the M13-R primer sequence of long 16bp respectively.Introduced M13-F and M13-R primer sequence all can be brought in such amplified production two ends, and when carrying out sequencing reaction subsequently, all amplified productions can use unified M13-F and M13-R primer to carry out forward and reverse order-checking.Because FIX gene has 8 exons, each pattern detection needs amplification 7 PCR primer, if each product uses respective sequencing primer to check order, operation will be very loaded down with trivial details, also very easily causes pollution.And the such design of this test kit effectively avoids this problem, enormously simplify the operation steps of sequencing reaction, makes whole process very convenient.Simultaneously by the reaction conditions such as concentration, annealing temperature of the forward and reverse primer of adjustment, amplification efficiency can be made to reach best.
Utilize expansion primer of the present invention and sequencing primer, Sanger sequencing is adopted to detect the sudden change of the full exon sequence of FIX gene, whole full exon sequence can be expanded, by the analysis of sequencing result, the transgenation situation of the full exon of FIX gene can be got information about very much, not by FIX sudden change variation and the impact not having mutantional hotspot, all mutational sites to be detected can be covered, and save testing cost significantly; There is very high specificity and accuracy; Adopt PCR method amplifying target genes and its gene pleiomorphism of order-checking detection, have highly sensitive, simple to operate, low cost and other advantages.
Accompanying drawing explanation
The detected result figure of Fig. 1 sample 1.
The detected result figure of Fig. 2 sample 2.
The detected result figure of Fig. 3 sample 3.
The detected result figure of Fig. 4 sample 4.
The detected result figure of Fig. 5 sample 5.
The detected result figure of Fig. 6 sample 6.
The detected result figure of Fig. 7 sample 7.
Embodiment
Below in conjunction with specific embodiments and the drawings, set forth the present invention further.Should be noted that, unaccounted normal condition and method in embodiment, usually according to the conventional employing method of affiliated field experimenter: such as, " the fine works molecular biology experiment guide " the 4th edition of Ao Sibai and James Kingston chief editor, or the step of advising according to manufacturer and condition.
Embodiment 1
Detect the primer of the full exon of FIX gene, comprising: 7 of the full exon of amplification covering detection FIX gene aligns, reverse primer; The base sequence of described expansion primer is:
FIX-1-F：TGTAAAACGACGGCCAGTGTACAACTAATCGACCTTACCACTT
FIX-1-R：AACAGCTATGACCATGCGTGCTGGCTGTTAGACTCT
FIX-2+3-F：TGTAAAACGACGGCCAGTCCCTAAAGAGAAATTGGCTTTCAG
FIX-2+3-R：AACAGCTATGACCATGGAATTGCTTACCAACATACTGCTTC
FIX-4F：TGTAAAACGACGGCCAGTTGGCTTCCAGGTCAGTAGTT
FIX-4R：AACAGCTATGACCATGAGGTGAGTCGGAACATCATTATTAC
FIX-5F：TGTAAAACGACGGCCAGTCACTCTTATTTCAAGGCTCCAA
FIX-5R：AACAGCTATGACCATGAAGGAAGCAGATTCAAGTAGGA
FIX-6F：TGTAAAACGACGGCCAGTGCTTGAGACTCTATTCACTGATTAG
FIX-6R：AACAGCTATGACCATGCATCCCAATAGGTCTGTCTAGTA
FIX-7F：TGTAAAACGACGGCCAGTGCCAGCACCTAGAAGCCAATA
FIX-7R：AACAGCTATGACCATGCCCTTCTGCCTTTAGCCCAAT
FIX-8F：TGTAAAACGACGGCCAGTGGTGAACATAATATTGAGGAGACAG
FIX-8R：AACAGCTATGACCATGTTAGTTAGTGAGAGGCCCTGTTAA
Described primer also comprises 1 pair of sequencing primer, and its base sequence is
M13-F：TGTAAAACGACGGCCAGT
M13-R：AACAGCTATGACCATG
In the detection, above-mentioned 7 are first utilized to increase to covering the DNA fragmentation detecting the full exon of FIX gene to forward and reverse amplimer, obtain amplified production, then utilize above-mentioned 1 pair of sequencing primer to check order to amplified production, obtain the gene order of amplified production.
In design of primers, often pair of designed primer is all positioned at the exon sequence both sides that will increase, and namely amplification region includes the full sequence of this exon.Because the mutational site of FIX is a lot, mutation type is indefinite.Therefore described whole exon sequence can all increase out by primer of the present invention, also ensures to undergo mutation in the position, where of no matter exon, all there will not be undetected situation.Because the 2nd and 3 exon sequences are all shorter, the position in gene order is 9291-9454,9643-9667 respectively, and middle intron sequences also only has 189bp, and therefore the present invention is when detection the 2nd and 3 exon, adopts with a pair amplimer.
Detect the test kit of the full exon of FIX gene, comprising: tissue DNA extraction agent box (such as using the extraction test kit of sky root biology); Dehydrated alcohol; Detection system PCR reaction solution, order-checking system reaction solution, positive reference substance, negative controls and blank product, wherein
Detection system PCR reaction solution comprises: 2 × PCRBuffer; 2mMdNTPs; KODFXDNAPolymerase (1U/ μ l); 7 couples of upstream and downstream primers F IX-1F (10 μm) and the FIX-1R (10 μm) of testing goal gene; FIX-2+3F (10 μm) and FIX-2+3R (10 μm); FIX-4F (10 μm) and FIX-4R (10 μm); FIX-5F (10 μm) and FIX-5R (10 μm); FIX-6F (10 μm) and FIX-6R (10 μm); FIX-7F (10 μm) and FIX-7R (10 μm); FIX-8F (10 μm) and FIX-8R (10 μm).
Order-checking system comprises: order-checking refined solution, EDTA (125mmol), dehydrated alcohol, 75% ethanol, HIDI (height deionized formamide), sequencing primer: M13-F (3.2 μm) and M13-R (3.2 μm), and BigdyeTerminatorV3.1 (buying from AppliedBiosystems company of the U.S.), the refined solution that wherein checks order comprises shrimp alkaline phosphotase (SAP) 0.6U and exonuclease I (EXONI) 1.2U.
Detection system PCR reaction solution is formulated as follows:
Wherein, FIX-1F and FIX-1R; FIX-2+3F and FIX-2+3R; FIX-4F and FIX-4R; FIX-5F and FIX-5R; FIX-6F and FIX-6R; FIX-7F and FIX-7R; The base sequence of FIX-8F and FIX-8R is:
FIX-1-F：TGTAAAACGACGGCCAGTGTACAACTAATCGACCTTACCACTT
FIX-1-R：AACAGCTATGACCATGCGTGCTGGCTGTTAGACTCT
FIX-2+3-F：TGTAAAACGACGGCCAGTCCCTAAAGAGAAATTGGCTTTCAG
FIX-2+3-R：AACAGCTATGACCATGGAATTGCTTACCAACATACTGCTTC
FIX-4F：TGTAAAACGACGGCCAGTTGGCTTCCAGGTCAGTAGTT
FIX-4R：AACAGCTATGACCATGAGGTGAGTCGGAACATCATTATTAC
FIX-5F：TGTAAAACGACGGCCAGTCACTCTTATTTCAAGGCTCCAA
FIX-5R：AACAGCTATGACCATGAAGGAAGCAGATTCAAGTAGGA
FIX-6F：TGTAAAACGACGGCCAGTGCTTGAGACTCTATTCACTGATTAG
FIX-6R：AACAGCTATGACCATGCATCCCAATAGGTCTGTCTAGTA
FIX-7F：TGTAAAACGACGGCCAGTGCCAGCACCTAGAAGCCAATA
FIX-7R：AACAGCTATGACCATGCCCTTCTGCCTTTAGCCCAAT
FIX-8F：TGTAAAACGACGGCCAGTGGTGAACATAATATTGAGGAGACAG
FIX-8R：AACAGCTATGACCATGTTAGTTAGTGAGAGGCCCTGTTAA
Positive reference substance: the solution containing FIX sequence.
Negative controls: without the solution of FIX sequence.
Blank product: 2 μ l physiological saline or do not add any material.
Embodiment 2
The operating process of blood DNA extraction agent box (sky root is biological):
(1) genomic dna in extracting blood:
1) extract 500uL blood and add 1000uL erythrocyte cracked liquid, put upside down mixing, room temperature places 5 minutes, and period puts upside down mixing several times again.The centrifugal 5min of 3000rpm, sucks supernatant, leaves leukocyte cell pellet, adds 200uL damping fluid GA, and vibration is to thoroughly mixing.
2) 20 μ l Proteinase K Solution are added, mixing.
3) add 200 μ l damping fluid GB, fully put upside down mixing, place 10 minutes for 70 DEG C, solution strain is limpid, and brief centrifugation is to remove the globule of cap wall.
4) add 200 μ l dehydrated alcohols, fully vibration mixing 15 seconds, now may occur flocks, brief centrifugation is to remove the globule of cap wall.
5) all add in an adsorption column CB3 (adsorption column puts into collection tube) by previous step gained solution and flocks, centrifugal 30 seconds of 12,000rpm (13,400 × g), outwells waste liquid, is put back in collection tube by adsorption column CB3.
6) in adsorption column CB3, add 500 μ l damping fluid GD (please first check whether before use and added dehydrated alcohol), centrifugal 30 seconds of 12,000rpm (13,400 × g), outwells waste liquid, adsorption column CB3 is put into collection tube.
7) in adsorption column CB3, add 700 μ l rinsing liquid PW (please first check whether before use and added dehydrated alcohol), centrifugal 30 seconds of 12,000rpm (13,400 × g), outwells waste liquid, adsorption column CB3 is put into collection tube.
8) in adsorption column CB3, add 500 μ l rinsing liquid PW, centrifugal 30 seconds of 12,000rpm (13,400 × g), outwells waste liquid.
9) put back in collection tube by adsorption column CB3, centrifugal 2 minutes of 12,000rpm (13,400 × g), outwells waste liquid.Adsorption column CB3 is placed in room temperature and places several minutes, thoroughly to dry rinsing liquid remaining in sorbing material.
10) proceeded to by adsorption column CB3 in a clean centrifuge tube, the unsettled dropping 100 in the middle part to adsorption film μ l elution buffer TE, room temperature places 2-5 minute, 12, centrifugal 2 minutes of 000rpm (13,400 × g), by solution collection in centrifuge tube.
(2) reagent configuration: by detecting people's number configuration detection system PCR reaction solution each X μ l, every person-portion 18 μ l packing:
X=18 μ l reaction solution × (n part sample+1 part of positive control+1 part of negative control+1 part of blank)
N is for detecting number of samples.
(3) application of sample: add each 2 μ lDNA in 3 detection system PCR reaction solutions; Positive control and negative control directly add 2 μ l positive reference substance and negative controls; Blank adds 2 μ l physiological saline or does not add any material.
(4) increase: detect and carry out on Standard PCR instrument, available instrumentation comprises ABIveriti (AppliedBiosystems company of the U.S.) etc.Reaction conditions is as follows:
(5) sanger order-checking:
Get 9 μ lPCR products and 2 μ l purification system.Purifying is carried out according to following program:
By 1 μ l purified product respectively with sequencing primer: M13-F (3.2 μm) and M13-R (3.2 μm), mix according to following system:
Sequencing reaction program:
Precipitation link:
In the product completing sequencing reaction, add the EDTA of 2 μ l125mmol, leave standstill 5min; Add 15ml dehydrated alcohol, whirlpool mixes; The centrifugal 30min of 3700rpm; Be inverted centrifugal 15sec, add 50ml70% ethanol, whirlpool mixes; The centrifugal 15min of 3700rpm; Be inverted centrifugal 15sec, be placed on 95 DEG C of metal baths; Denatured test is carried out after adding 10 μ lHIDI.Denaturation program:
After denaturation program terminates, upper sequenator (ABI3730) checks order.
(7) result judges: sequencing result and wild-type reference sequence (GeneBankNo.:K02402.1) are compared, report according to actual catastrophe to result.
Embodiment 3
Get 7 parts, clinical patients sample, whether 7 increments are originally all uncertain suffers from HB, detects this 7 increment and originally whether there is FIX sudden change.Genome, reagent preparation detecting is extracted by method described in embodiment 2.Every increment product add 2 μ l in detection system PCR reaction solution.Do the positive simultaneously, negative, each portion of blank.Detect with regular-PCR instrument, the time is 160 minutes.
The part forward sequencing result of No. 1 full exon sequence of sample 1 as shown in Figure 1, is wild-type, does not detect that FIX suddenlys change.
No. 2 of sample 2 and the part forward sequencing result of No. 3 full exon sequences as shown in Figure 1, are wild-type, do not detect that FIX suddenlys change.
The part forward sequencing result of No. 4 full exon sequence of sample 3 as shown in Figure 1, is wild-type, does not detect that FIX suddenlys change.
The part forward sequencing result of No. 5 full exon sequence of sample 4 as shown in Figure 1, is wild-type, does not detect that FIX suddenlys change.
The part forward sequencing result of No. 6 full exon sequence of sample 5 as shown in Figure 1, is wild-type, does not detect that FIX suddenlys change.
The part forward sequencing result of No. 7 full exon sequence of sample 6 as shown in Figure 1, is wild-type, does not detect that FIX suddenlys change.
The part forward sequencing result of No. 8 full exon sequence of sample 7 as shown in Figure 1, is wild-type, does not detect that FIX suddenlys change.
As can be seen from detected result, primer of the present invention has been included exon sequence, can expand the full exon of FIX gene, and sequencing result entirely accurate.Primer of the present invention can expand the full exon sequence of FIX gene accurately, no matter is wild-type or saltant type.
Claims (3)
1. detect the primer of the full exon of FIX gene, it is characterized in that, comprising: amplification covers the forward and reverse primer detecting the full exon of FIX gene; Its base sequence is:
FIX-1-F：TGTAAAACGACGGCCAGTGTACAACTAATCGACCTTACCACTT
FIX-1-R：AACAGCTATGACCATGCGTGCTGGCTGTTAGACTCT
FIX-2+3-F：TGTAAAACGACGGCCAGTCCCTAAAGAGAAATTGGCTTTCAG
FIX-2+3-R：AACAGCTATGACCATGGAATTGCTTACCAACATACTGCTTC
FIX-4F：TGTAAAACGACGGCCAGTTGGCTTCCAGGTCAGTAGTT
FIX-4R：AACAGCTATGACCATGAGGTGAGTCGGAACATCATTATTAC
FIX-5F：TGTAAAACGACGGCCAGTCACTCTTATTTCAAGGCTCCAA
FIX-5R：AACAGCTATGACCATGAAGGAAGCAGATTCAAGTAGGA
FIX-6F：TGTAAAACGACGGCCAGTGCTTGAGACTCTATTCACTGATTAG
FIX-6R：AACAGCTATGACCATGCATCCCAATAGGTCTGTCTAGTA
FIX-7F：TGTAAAACGACGGCCAGTGCCAGCACCTAGAAGCCAATA
FIX-7R：AACAGCTATGACCATGCCCTTCTGCCTTTAGCCCAAT
FIX-8F：TGTAAAACGACGGCCAGTGGTGAACATAATATTGAGGAGACAG
FIX-8R：AACAGCTATGACCATGTTAGTTAGTGAGAGGCCCTGTTAA。
2. the primer as described in one of claim 1 to 2, is characterized in that, described 7 to align, the working concentration ratio of reverse primer is: FIX-1F:FIX-1R=1:1; FIX-2+3F:FIX-2+3R=1:1; FIX-4F:FIX-4R=1:1; FIX-5F:FIX-5R=1:1; FIX-6F:FIX-6R=1:1; FIX-7F:FIX-7R=1:1; FIX-8F:FIX-8R=1:1.
3. detect a method for the full exon sequence of FIX gene, it comprises the steps:
(1) sample DNA is extracted;
(2) 7 couples of amplimer FIX-1F and FIX-1R are utilized; FIX-2+3F and FIX-2+3R; FIX-4F and FIX-4R; FIX-5F and FIX-5R; FIX-6F and FIX-6R; FIX-7F and FIX-7R; FIX-8F and FIX-8R.DNA in (1) is increased respectively, obtains the amplified production covering and detect the full exon of FIX gene;
(3) utilize 1 couple of sequencing primer M13-F and M13-R to carry out forward and backward sequencing respectively to the amplified production in (2), obtain the gene order of described amplified production;
(4) gene order in (3) and wild-type FIX sequence are compared, determine whether mutational site exists;
Wherein said primer sequence is:
FIX-1-F：TGTAAAACGACGGCCAGTGTACAACTAATCGACCTTACCACTT
FIX-1-R：AACAGCTATGACCATGCGTGCTGGCTGTTAGACTCT
FIX-2+3-F：TGTAAAACGACGGCCAGTCCCTAAAGAGAAATTGGCTTTCAG
FIX-2+3-R：AACAGCTATGACCATGGAATTGCTTACCAACATACTGCTTC
FIX-4F：TGTAAAACGACGGCCAGTTGGCTTCCAGGTCAGTAGTT
FIX-4R：AACAGCTATGACCATGAGGTGAGTCGGAACATCATTATTAC
FIX-5F：TGTAAAACGACGGCCAGTCACTCTTATTTCAAGGCTCCAA
FIX-5R：AACAGCTATGACCATGAAGGAAGCAGATTCAAGTAGGA
FIX-6F：TGTAAAACGACGGCCAGTGCTTGAGACTCTATTCACTGATTAG
FIX-6R：AACAGCTATGACCATGCATCCCAATAGGTCTGTCTAGTA
FIX-7F：TGTAAAACGACGGCCAGTGCCAGCACCTAGAAGCCAATA
FIX-7R：AACAGCTATGACCATGCCCTTCTGCCTTTAGCCCAAT
FIX-8F：TGTAAAACGACGGCCAGTGGTGAACATAATATTGAGGAGACAG
FIX-8R：AACAGCTATGACCATGTTAGTTAGTGAGAGGCCCTGTTAA
M13-F：TGTAAAACGACGGCCAGT
M13-R：AACAGCTATGACCATG。
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