CN108660210A - Detect the primer and kit and method that apc gene is mutated - Google Patents
Detect the primer and kit and method that apc gene is mutated Download PDFInfo
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- CN108660210A CN108660210A CN201810510677.8A CN201810510677A CN108660210A CN 108660210 A CN108660210 A CN 108660210A CN 201810510677 A CN201810510677 A CN 201810510677A CN 108660210 A CN108660210 A CN 108660210A
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Abstract
The present invention relates to mutator detection fields, the primer and kit in particular to detection detection apc gene mutation and method.Detect the primer of apc gene mutation, including any pair or multipair in following primer pair；The nucleotide sequence of primer pair 1 31 is successively as shown in SEQ ID NO.1 62.The primer that detection apc gene provided by the invention is mutated is to consider to design by various aspects, and obtain by screening, and when the product of the primer amplification is sequenced, peak shape is neat, and peak figure is complete, and quality is high.By carrying out apc gene detection to people (especially newborn), can be early discovery with Disease-causing gene crowd, diagnosing and treating for later stage disease provides scientific basis, early stage is contributed to take measures to prevent the growth of polyp, the generation of canceration is avoided, the canceration rate of familial adenomatous polyposis is effectively reduced.
Description
Technical field
The present invention relates to mutator detection field, primer in particular to detection detection apc gene mutation and
Kit and method.
Background technology
Familial adenomatous polyposis (fam ilial adenom ato μ s polypo-sis, FAP) be polyposis
Disease, is a kind of autosomal dominant disease, which betides adenomatous polyposis colii gene (adenomato μ s
Polyposis coli, APC) mutation it is closely related, be more than 80% FAP patient can detect apc gene be mutated.The whole world FAP
Incidence is the 1/7000~1/10000 of natus.
FAP is equal to Effect of gender, male to female ratio 1：1, all show that the autosome of height is aobvious in most of case
Property heredity, meet mendel's law, genepenetrance is 70% to 95%, and Sporadic cases accounts for about 1/3, and the FAP patient of adult is
Can have the family history of parent and their children has half that can suffer from FAP, and is related to several generations.It is generally acknowledged that 40
When year or having arrived in family the maximum illness age and add 10 years old age, do not occur adenoma person, it, will not even if there is family history
Occurs adenoma again.Area is not present in the disease and ethnic difference is anisotropic, and the average adenoma generation age is about 16 years old, and the colorectal cancer age occurs
About 40 years old.
Apc gene, code area are made of 16 exons, encode a kind of larger mRNA of about 10.5kb, and expression is a kind of
The albumen of 2843 amino acid, about 300000 dalton of molecular weight, APC albumen have tumor inhibition effect, inhibit Polypus
The generation of canceration.The genepenetrance of apc gene is up to 100%, in filial generation heredity than about 50%, in recent years, with detection technique
Raising, the incidence of FAP is in rising trend.There is very high kainogenesis mutation rate in 10%-30% is without the patient of family history.
Apc gene mutation type mainly has point mutation and frame frameshift mutation, the former includes that nonsense mutation, dislocation mutation and splicing are wrong
Accidentally, the latter includes missing and is inserted into.Apc gene mutation has more than 720 kinds, these mutation spread whole gene, 60% or more it is prominent
Become positioned at 5 ' ends of the 16th exon.It concentrates 10% or so code area wherein between codon the 1286th~1513
About 65% somatic mutation is referred to as mutation cluster region (MCR).Most of mutation belongs to dislocation mutation, by missing or 1~8
The insertion of a base-pair causes.About 95% mutation makes the APC albumen be in the result is that form terminator codon in advance in downstream
It truncates and changes, this may weaken the intrinsic inhibition cell Proliferation function of APC albumen, so as to cause the obstacle of APC protein functions.
Include to the diagnostic method of FAP both at home and abroad at present：1. digital rectal examination：Since the polyp of FAP is most in rectum performance,
Therefore simple digital rectal examination can provide preliminary diagnosis for the inspection of next step.2. electronics colonoscopy and clysis with barium：It will be seen that
The range of lesion, and biopsy can be got, specify tumour property.3. gastrofiberscope and duodenoscopy：It will be seen that stomach and
Duodenum whether there is polyp.4. funduscopy：Due to more than FAP and depositing congenital retinal pigment epithelium hyperplasia
(CHRPE), there are the diagnostic sensitivity and specificity of height, therefore funduscopy can be classified as in family and adenoma in large intestine not yet occurs
The screening of member or diagnostic means.X-ray, CT or MRI：It will be seen that being showed outside the colons such as osteoma, central nerve neuroma.This
Time-consuming and cumbersome for the method for a little clinical detections, and is only applicable to the baby being born, helpless to pre-natal diagnosis.
In view of this, special propose the present invention.
Invention content
The first object of the present invention is to provide the primer of detection apc gene mutation, when the product of the primer amplification is sequenced,
Peak shape is neat, and peak figure is complete, and quality is high.
The second object of the present invention is to provide the kit of detection apc gene mutation, is that the detection of apc gene mutation carries
For facility.
The third object of the present invention is the method for providing detection apc gene mutation, is familial adenomatous polyposis
Diagnosis provides technical support.
In order to realize that the above-mentioned purpose of the present invention, spy use following technical scheme：
Detect the primer of apc gene mutation, including any pair or multipair in following primer pair；
The nucleotide sequence of primer pair 1-31 is successively as shown in SEQ ID NO.1-62.
The primer provided by the invention for detecting apc gene mutation is to consider to design by various aspects, and pass through and screen
It arrives, when the product of the primer amplification is sequenced, peak shape is neat, and peak figure is complete, and quality is high, and other primer amplifications, such as SEQ ID
Primer pair nucleotide sequence shown in NO.63-78, sequencing result have miscellaneous peak, peak figure imperfect, of poor quality.
The present invention also provides the kits of detection apc gene mutation, draw containing what above-mentioned detection apc gene was mutated
Object, the detection for apc gene mutation provide facility.
Further, the kit further includes dNTPs, buffer solution, enzyme, distilled water any one or more of.
Since segment to be amplified is longer, enzyme generally selects LA Taq enzymes, can also select other that can expand compared with long segment
Enzyme.
Further, the kit further includes DNA extracts reagents.DNA extracts reagents are used for the extraction of sample genome,
General sample is blood.
Further, the kit further includes nucleic acid purification reagent.After PCR amplification, amplified production is purified, with
Convenient for sequencing.
Further, the nucleic acid purification reagent is centrifugal column type QIAquick Gel Extraction Kit.
Further, the kit further includes sequencing reagent.
Further, the sequencing reagent includes Bigdye, Hi-Di any one or more of.
The present invention also provides a kind of methods of detection apc gene mutation, and PCR amplification is carried out using above-mentioned primer pair,
It expands obtained product to be purified, gene sequencing, with unmutated sequence alignment.
Further, the program of the PCR amplification is as follows：
94-95 DEG C of pre-degeneration 10-15 minutes；94-95 DEG C is denaturalized 30-35 seconds, and 62 DEG C are annealed 30 seconds, 72 DEG C of extension 45-50
Second, 13-15 cycle；94-95 DEG C is denaturalized 30-35 seconds, and 55 DEG C are annealed 30 seconds, and 72 DEG C extend 45-50 seconds, 30-40 cycle；72
DEG C extend 10-15 minutes.
As in various embodiments, PCR response procedures can be 94 DEG C of pre-degenerations 15 minutes；94 DEG C be denaturalized 30 seconds, 62
DEG C annealing 30 seconds, 72 DEG C extend 45 seconds, 14 cycle；94 DEG C are denaturalized 30 seconds, and 55 DEG C are annealed 30 seconds, and 72 DEG C extend 45 seconds, 35
Cycle；Last 72 DEG C extend 10 minutes.
Or 95 DEG C of pre-degenerations 10 minutes；95 DEG C are denaturalized 35 seconds, and 62 DEG C are annealed 30 seconds, and 72 DEG C extend 50 seconds, 15
Cycle；95 DEG C are denaturalized 35 seconds, and 55 DEG C are annealed 30 seconds, and 72 DEG C extend 50 seconds, 40 cycles；Last 72 DEG C extend 15 minutes.Etc..
The reaction system of PCR amplification can be 10-50 μ L, and such as 15 μ L PCR reaction systems include：10×PCR buffer
1.5 μ L, 2.4 dNTP μ L, 0.15 μ L of Takara LA Taq enzymes, 0.6 μ L of primer (l0 μM), genomic DNA (100ng/ μ L) 0.5
μ L, use ddH2O supplies reaction system.
After the completion of PCR amplification, amplified production is purified, is generally recycled using DNA QIAquick Gel Extraction Kits.
10 μ L of DNA sequencing reaction system, including：1 μ L of PCR purified products, 0.5 Bigdye μ L, 5 × seq, 1.7 μ L and survey
0.5 μ L of sequence primer, add ddH2O supplies 10 μ L.Reaction condition is 96 DEG C of pre-degeneration 1min, carry out (96 DEG C of 10sec, 55 DEG C
10sec, 60 DEG C of 2.5min) 33 cycles.
2.5 μ L EDTA often are added in pipe after reaction, 30 μ L100% ethyl alcohol cover, and shake 4 times, are protected from light standing 15
Minute, supernatant liquid is abandoned in 3860rpm, 25 DEG C of centrifugation 40min, suction；100 μ L70% pre-cooled ethanols are added, cover, 3860rpm, 25
DEG C centrifugation 15 minutes, suction abandon supernatant liquid；It makes alcohol clean in room temperature volatilization, 8 μ LHi-Di dissolving DNAs is added；In PCR instrument
Denaturation：95 DEG C 4 minutes, on ice stand 4 minutes.It is put into sequenator and is sequenced.
Compared with prior art, beneficial effects of the present invention are：
(1) primer of detection apc gene mutation provided by the invention, when the product of the primer amplification is sequenced, peak shape is neat,
Peak figure is complete, and quality is high.
(2) primer provided by the invention and kit can be used for normal person's apc gene screening, pregnancy period mother and newborn
Screening, FAP patient's periodic review etc..
(3) for the present invention by carrying out apc gene detection to people (especially newborn), discovery that can be early has cause
The crowd of ospc gene, the diagnosing and treating for later stage disease provide scientific basis, and early stage is contributed to take measures to prevent polyp
Increase, avoid the generation of canceration, effectively reduces the canceration rate of familial adenomatous polyposis.
(4) the present invention is based on the method for PCR reactions and generation sequencing, SNP detections are carried out, accuracy is significantly improved,
And it is traumatic to human body small, it is convenient to carry out.
Description of the drawings
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technology description to be briefly described.
Fig. 1 is sequencing result figure of the embodiment of the present invention 2 using SEQ ID NO.1-2 amplified productions；
Fig. 2 is sequencing result figure of the embodiment of the present invention 2 using SEQ ID NO.3-4 amplified productions；
Fig. 3 is sequencing result figure of the embodiment of the present invention 2 using SEQ ID NO.5-6 amplified productions；
Fig. 4 is sequencing result figure of the embodiment of the present invention 2 using SEQ ID NO.7-8 amplified productions；
Fig. 5 is sequencing result figure of the embodiment of the present invention 2 using SEQ ID NO.9-10 amplified productions；
Fig. 6 is sequencing result figure of the embodiment of the present invention 2 using SEQ ID NO.11-12 amplified productions；
Fig. 7 is sequencing result figure of the embodiment of the present invention 2 using SEQ ID NO.13-14 amplified productions；
Fig. 8 is sequencing result figure of the embodiment of the present invention 2 using SEQ ID NO.15-16 amplified productions；
Fig. 9 is sequencing result figure of the embodiment of the present invention 2 using SEQ ID NO.17-18 amplified productions；
Figure 10 is sequencing result figure of the embodiment of the present invention 2 using SEQ ID NO.19-20 amplified productions；
Figure 11 is sequencing result figure of the embodiment of the present invention 2 using SEQ ID NO.21-22 amplified productions；
Figure 12 is sequencing result figure of the embodiment of the present invention 2 using SEQ ID NO.23-24 amplified productions；
Figure 13 is sequencing result figure of the embodiment of the present invention 2 using SEQ ID NO.25-26 amplified productions；
Figure 14 is sequencing result figure of the embodiment of the present invention 2 using SEQ ID NO.27-28 amplified productions；
Figure 15 is sequencing result figure of the embodiment of the present invention 2 using SEQ ID NO.29-30 amplified productions；
Figure 16 is sequencing result figure of the embodiment of the present invention 2 using SEQ ID NO.31-32 amplified productions；
Figure 17 is sequencing result figure of the embodiment of the present invention 2 using SEQ ID NO.33-34 amplified productions；
Figure 18 is sequencing result figure of the embodiment of the present invention 2 using SEQ ID NO.35-36 amplified productions；
Figure 19 is sequencing result figure of the embodiment of the present invention 2 using SEQ ID NO.37-38 amplified productions；
Figure 20 is sequencing result figure of the embodiment of the present invention 2 using SEQ ID NO.39-40 amplified productions；
Figure 21 is sequencing result figure of the embodiment of the present invention 2 using SEQ ID NO.41-42 amplified productions；
Figure 22 is sequencing result figure of the embodiment of the present invention 2 using SEQ ID NO.43-44 amplified productions；
Figure 23 is sequencing result figure of the embodiment of the present invention 2 using SEQ ID NO.45-46 amplified productions；
Figure 24 is sequencing result figure of the embodiment of the present invention 2 using SEQ ID NO.47-48 amplified productions；
Figure 25 is sequencing result figure of the embodiment of the present invention 2 using SEQ ID NO.49-50 amplified productions；
Figure 26 is sequencing result figure of the embodiment of the present invention 2 using SEQ ID NO.51-52 amplified productions；
Figure 27 is sequencing result figure of the embodiment of the present invention 2 using SEQ ID NO.53-54 amplified productions；
Figure 28 is sequencing result figure of the embodiment of the present invention 2 using SEQ ID NO.55-56 amplified productions；
Figure 29 is sequencing result figure of the embodiment of the present invention 2 using SEQ ID NO.57-58 amplified productions；
Figure 30 is sequencing result figure of the embodiment of the present invention 2 using SEQ ID NO.59-60 amplified productions；
Figure 31 is sequencing result figure of the embodiment of the present invention 2 using SEQ ID NO.61-62 amplified productions；
Figure 32 is sequencing result figure of the embodiment of the present invention 3 using SEQ ID NO.63-64 amplified productions；
Figure 33 is sequencing result figure of the embodiment of the present invention 3 using SEQ ID NO.65-66 amplified productions；
Figure 34 is sequencing result figure of the embodiment of the present invention 3 using SEQ ID NO.67-68 amplified productions；
Figure 35 is sequencing result figure of the embodiment of the present invention 3 using SEQ ID NO.69-70 amplified productions；
Figure 36 is sequencing result figure of the embodiment of the present invention 3 using SEQ ID NO.71-72 amplified productions；
Figure 37 is sequencing result figure of the embodiment of the present invention 3 using SEQ ID NO.73-74 amplified productions；
Figure 38 is sequencing result figure of the embodiment of the present invention 3 using SEQ ID NO.75-76 amplified productions；
Figure 39 is sequencing result figure of the embodiment of the present invention 3 using SEQ ID NO.77-78 amplified productions.
Specific implementation mode
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
Embodiment 1
A kind of kit of detection familial adenomatous polyposis apc gene mutation, including following component：
(1) PCR amplification is carried out to 16 exons coding districts of apc gene and devises 31 since the 16th exon is larger
Primer (nucleotide sequence is successively as shown in SEQ ID NO.1-62) is expanded.
(2) PCR amplification reagent：10 × PCR b μ ffer, dNTP, Takara LA Taq enzymes；
(3) PCR product purified reagent：Plain agar sugar gel DNA QIAquick Gel Extraction Kits (centrifugal column type) (Tiangeng, DP209)；
(4) sequencing reagent：2.2 μ L BigDyemix (Bigdye, 5 × seq), 2.5 μ L EDTA, 30 μ L ethanol solutions
(100%), 100 μ L ethanol solutions (70%), 8 μ L Hi-Di.
This kit is stored in -20 DEG C, reduces multigelation to the greatest extent.
Above-mentioned primer pair can be amplified such as NCBI Reference Sequence:APC bases shown in NM_000038.5
Because of sequence.
Embodiment 2
A kind of kit of detection familial adenomatous polyposis apc gene mutation, is included the following steps using step：
(1) sample genomic dna extracts
Sample genomic dna is extracted using poba gene group DNA extraction kit (0.1-1ml) (Tiangeng, DP318).
(2) using different primer pairs to apc gene PCR amplification
PCR amplification is carried out for apc gene design primer (as shown in SEQ ID NO.1-62).15 μ L PCR reaction systems
Including：10 × PCR b μ ffer, 1.5 μ L, 2.4 dNTPs μ L, 0.15 μ L of Takara LA Taq enzymes, upper and lower primer (l0 μM) are each
0.3 μ L, genomic DNA (100ng/ μ L) 0.5 μ L, use ddH2O supplies reaction system.
PCR response procedures are：94 DEG C of pre-degenerations 15 minutes；94 DEG C are denaturalized 30 seconds, and 62 DEG C are annealed 30 seconds, and 72 DEG C extend 45
Second, 14 cycles；94 DEG C are denaturalized 30 seconds, and 55 DEG C are annealed 30 seconds, and 72 DEG C extend 45 seconds, 35 cycles；Last 72 DEG C extend 10 points
Clock.
(3) PCR product purifies
Rubber tapping purifying PCR is carried out using plain agar sugar gel DNA QIAquick Gel Extraction Kits (centrifugal column type) (Tiangeng, DP209)
Product obtains corresponding target DNA fragment；
(4) DNA sequencing reaction
10 μ L of DNA sequencing reaction system, including：1 μ L of PCR purified products, 0.5 Bigdye μ L, 5 × seq, 1.7 μ L and survey
0.5 μ L of sequence primer (one of corresponding amplimer), add ddH2O supplies 10 μ L.Reaction condition is 96 DEG C of pre-degeneration 1min,
Carry out (96 DEG C of 10sec, 55 DEG C of 10sec, 60 DEG C of 2.5min) 33 cycles；
2.5 μ L EDTA often are added in pipe after reaction, 30 μ L100% ethyl alcohol cover, and shake 4 times, are protected from light standing 15
Minute, supernatant liquid is abandoned in 3860rpm, 25 DEG C of centrifugation 40min, suction；100 μ L70% pre-cooled ethanols are added, cover, 3860rpm, 25
DEG C centrifugation 15 minutes, suction abandon supernatant liquid；It makes alcohol clean in room temperature volatilization, 8 μ LHi-Di dissolving DNAs is added；In PCR instrument
Denaturation：95 DEG C 4 minutes, on ice stand 4 minutes.It is put into sequenator and is sequenced.
(5) interpretation of result
To apc gene direct Sequencing, result reading is carried out on sequenator, and sequencing detection knot is consulted with software Chromas
Fruit, sequencing result schematic diagram such as Fig. 1 to Figure 31.Fig. 1 to Figure 31 is a part for sequencing result, to illustrate provided by the invention draw
When object is sequenced after being expanded, peak shape is neat, and peak figure is complete, and quality is high.
The sequence of measurement is compared with GenBank database apc gene sequences, apc gene sequence such as NCBI
Reference Sequence:Shown in NM_000038.5, the amino acid sequence such as NCBI Reference of corresponding coding
Sequence:Shown in NP_000029.2.
It by the gene mutation analysis to familial adenomatous polyposis, is then compared with measurement mutation, is family
The diagnosis of Adenomatous Polyposis provides infrastructural support.
Therefore, by being detected to apc gene, can be early discovery with Disease-causing gene crowd, be later stage disease
The diagnosing and treating of disease provides scientific basis, and early stage is contributed to take measures to prevent the growth of polyp, avoids the hair of canceration
It is raw, effectively reduce the canceration rate of familial adenomatous polyposis.
Embodiment 3
Apc gene amplimer screening experiment, including following primer sequence and reagent：
(1) totally 8 pairs of apc gene the 5th, the 8th and the 16th exon code area primer, nucleotide sequence is successively such as SEQ
Shown in ID NO.63-78.
(2) PCR amplification reagent：10 × PCR b μ ffer, dNTP, Takara LA Taq enzymes；
(3) PCR product purified reagent：Plain agar sugar gel DNA QIAquick Gel Extraction Kits (centrifugal column type) (Tiangeng, DP209)；
(4) sequencing reagent：2.2 μ L BigDyemix (Bigdye, 5 × seq), 2.5 μ L EDTA, 30 μ L ethanol solutions
(100%), 100 μ L ethanol solutions (70%), 8 μ L Hi-Di.
Apc gene amplimer screening experiment operating procedure includes：
(1) sample genomic dna extracts, with embodiment 2.
(2) apc gene PCR amplification, with embodiment 2.
(3) PCR product purifies, with embodiment 2.
(4) DNA sequencing reaction, with embodiment 2.
(5) interpretation of result
Direct Sequencing is carried out to apc gene the 5th, the 8th and the 16th exon, result reading is carried out on sequenator, is used
Software Chromas consults sequencing assay result, sequencing result schematic diagram such as Figure 32 to Figure 39.
Figure 32 to Figure 39 is a part for sequencing result, the results showed that, apc gene the 5th, the 8th and the 16th exon are surveyed
Sequence result has miscellaneous peak, peak figure imperfect, of poor quality.
The above results illustrate that the primer of detection apc gene mutation provided by the invention, sequencing result are complete without miscellaneous peak, peak figure
It is whole, quality, it has a clear superiority.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from the present invention's
Many other change and modification can be made in the case of spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
SEQUENCE LISTING
<110>Pudong, Shanghai decodes life science institute
<120>Detect the primer and kit and method that apc gene is mutated
<130> 2010
<160> 78
<170> PatentIn version 3.3
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<210> 30
<211> 21
<212> DNA
<213>Artificial sequence
<400> 30
attttggctt aaaactttca t 21
<210> 31
<211> 20
<212> DNA
<213>Artificial sequence
<400> 31
gtggaatact tggaatttat 20
<210> 32
<211> 17
<212> DNA
<213>Artificial sequence
<400> 32
agatgccttg ggactta 17
<210> 33
<211> 20
<212> DNA
<213>Artificial sequence
<400> 33
aaatgattgc tatgggaagt 20
<210> 34
<211> 19
<212> DNA
<213>Artificial sequence
<400> 34
ctgttgctgg atggtagtt 19
<210> 35
<211> 22
<212> DNA
<213>Artificial sequence
<400> 35
tttgaatact acagtgttac cc 22
<210> 36
<211> 18
<212> DNA
<213>Artificial sequence
<400> 36
atttggcata aggcatag 18
<210> 37
<211> 18
<212> DNA
<213>Artificial sequence
<400> 37
gaagaagctc tgctgccc 18
<210> 38
<211> 20
<212> DNA
<213>Artificial sequence
<400> 38
cacattcctg ctgtccaaaa 20
<210> 39
<211> 24
<212> DNA
<213>Artificial sequence
<400> 39
aaaacaaagt gagcaaagac aatc 24
<210> 40
<211> 21
<212> DNA
<213>Artificial sequence
<400> 40
ctctgtgcag aacttggatg g 21
<210> 41
<211> 20
<212> DNA
<213>Artificial sequence
<400> 41
gagaaacgtc atgtggatca 20
<210> 42
<211> 19
<212> DNA
<213>Artificial sequence
<400> 42
aattcaacag ctttgtgcc 19
<210> 43
<211> 18
<212> DNA
<213>Artificial sequence
<400> 43
aagattggaa ctaggtca 18
<210> 44
<211> 19
<212> DNA
<213>Artificial sequence
<400> 44
ctttctgtat aaatggctc 19
<210> 45
<211> 21
<212> DNA
<213>Artificial sequence
<400> 45
cagagggtcc aggttcttcc a 21
<210> 46
<211> 24
<212> DNA
<213>Artificial sequence
<400> 46
ctccttctcc agcagctaac tcat 24
<210> 47
<211> 23
<212> DNA
<213>Artificial sequence
<400> 47
taattccaag gtcttcaatg ata 23
<210> 48
<211> 21
<212> DNA
<213>Artificial sequence
<400> 48
tgcttgaggt ttacttggtt c 21
<210> 49
<211> 20
<212> DNA
<213>Artificial sequence
<400> 49
ccaaagacat accagacaga 20
<210> 50
<211> 18
<212> DNA
<213>Artificial sequence
<400> 50
tcacctaata tgccaccc 18
<210> 51
<211> 19
<212> DNA
<213>Artificial sequence
<400> 51
atgacctgtt gcaggaatg 19
<210> 52
<211> 20
<212> DNA
<213>Artificial sequence
<400> 52
ctctaggaga agtggtggct 20
<210> 53
<211> 21
<212> DNA
<213>Artificial sequence
<400> 53
aagcaaacat gccttcaatc t 21
<210> 54
<211> 21
<212> DNA
<213>Artificial sequence
<400> 54
ggttctgttg gctcatctgt c 21
<210> 55
<211> 18
<212> DNA
<213>Artificial sequence
<400> 55
gacctgccca gcaaccat 18
<210> 56
<211> 20
<212> DNA
<213>Artificial sequence
<400> 56
caccagcctg aacagacgaa 20
<210> 57
<211> 18
<212> DNA
<213>Artificial sequence
<400> 57
aagctccaag cccaacct 18
<210> 58
<211> 21
<212> DNA
<213>Artificial sequence
<400> 58
ttctcaccca aacatcctct g 21
<210> 59
<211> 23
<212> DNA
<213>Artificial sequence
<400> 59
ttcctcaggt gctacaaatg gtg 23
<210> 60
<211> 24
<212> DNA
<213>Artificial sequence
<400> 60
gcttgagctg ctagaactga atgg 24
<210> 61
<211> 22
<212> DNA
<213>Artificial sequence
<400> 61
aactgagata aaaccaggac aa 22
<210> 62
<211> 22
<212> DNA
<213>Artificial sequence
<400> 62
aaccctctaa caagaatcaa ac 22
<210> 63
<211> 23
<212> DNA
<213>Artificial sequence
<400> 63
cttttaagga tgattaccag ttt 23
<210> 64
<211> 23
<212> DNA
<213>Artificial sequence
<400> 64
agtttcaaat aagttgtact gcc 23
<210> 65
<211> 19
<212> DNA
<213>Artificial sequence
<400> 65
ctctaatgct caagggaca 19
<210> 66
<211> 20
<212> DNA
<213>Artificial sequence
<400> 66
aaccatcttg cttcatactt 20
<210> 67
<211> 22
<212> DNA
<213>Artificial sequence
<400> 67
aatagagtaa atgtatgtgc cc 22
<210> 68
<211> 17
<212> DNA
<213>Artificial sequence
<400> 68
tgcctagacc aattccg 17
<210> 69
<211> 20
<212> DNA
<213>Artificial sequence
<400> 69
tgaatactac agtgttaccc 20
<210> 70
<211> 18
<212> DNA
<213>Artificial sequence
<400> 70
aggctgacca cttctact 18
<210> 71
<211> 19
<212> DNA
<213>Artificial sequence
<400> 71
gtcatgtgga tcagcctat 19
<210> 72
<211> 18
<212> DNA
<213>Artificial sequence
<400> 72
gactgtgccc ctcctcta 18
<210> 73
<211> 20
<212> DNA
<213>Artificial sequence
<400> 73
ttacctccac ctgtggcaag 20
<210> 74
<211> 21
<212> DNA
<213>Artificial sequence
<400> 74
gcccctctgt ctggtatgtc t 21
<210> 75
<211> 20
<212> DNA
<213>Artificial sequence
<400> 75
agaactaacc tccaaccaac 20
<210> 76
<211> 19
<212> DNA
<213>Artificial sequence
<400> 76
tggctccatt accattatt 19
<210> 77
<211> 19
<212> DNA
<213>Artificial sequence
<400> 77
gtagacagat gagccaaca 19
<210> 78
<211> 18
<212> DNA
<213>Artificial sequence
<400> 78
cacaaggtaa gacccaga 18
Claims (10)
1. detecting the primer of apc gene mutation, which is characterized in that including any pair or multipair in following primer pair；
The nucleotide sequence of primer pair 1-31 is successively as shown in SEQ ID NO.1-62.
2. detecting the kit of apc gene mutation, which is characterized in that contain detection apc gene mutation described in claim 1
Primer.
3. the kit of detection apc gene mutation according to claim 2, which is characterized in that the kit further includes
DNTPs, buffer solution, enzyme, distilled water any one or more of.
4. the kit of detection apc gene mutation according to claim 2, which is characterized in that the kit further includes
DNA extracts reagents.
5. the kit of detection apc gene mutation according to claim 2, which is characterized in that the kit further includes
Nucleic acid purification reagent.
6. the kit of detection apc gene mutation according to claim 5, which is characterized in that the nucleic acid purification reagent
For centrifugal column type QIAquick Gel Extraction Kit.
7. the kit of detection apc gene mutation according to claim 2, which is characterized in that the kit further includes
Sequencing reagent.
8. the kit of detection apc gene mutation according to claim 7, which is characterized in that the sequencing reagent includes
Bigdye, Hi-Di any one or more of.
9. a kind of method of detection apc gene mutation, which is characterized in that carry out PCR expansions using primer pair described in claim 1
Increase, the product expanded is purified, gene sequencing, with unmutated sequence alignment.
10. the method for detection apc gene mutation according to claim 9, which is characterized in that the program of the PCR amplification
It is as follows：
94-95 DEG C of pre-degeneration 10-15 minutes；94-95 DEG C is denaturalized 30-35 seconds, and 62 DEG C are annealed 30 seconds, and 72 DEG C extend 45-50 seconds,
13-15 cycle；94-95 DEG C is denaturalized 30-35 seconds, and 55 DEG C are annealed 30 seconds, and 72 DEG C extend 45-50 seconds, 30-40 cycle；72℃
Extend 10-15 minutes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810510677.8A CN108660210A (en) | 2018-05-24 | 2018-05-24 | Detect the primer and kit and method that apc gene is mutated |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810510677.8A CN108660210A (en) | 2018-05-24 | 2018-05-24 | Detect the primer and kit and method that apc gene is mutated |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108660210A true CN108660210A (en) | 2018-10-16 |
Family
ID=63777684
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810510677.8A Pending CN108660210A (en) | 2018-05-24 | 2018-05-24 | Detect the primer and kit and method that apc gene is mutated |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108660210A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115216531A (en) * | 2022-06-30 | 2022-10-21 | 湖南家辉生物技术有限公司 | Novel APC gene mutation c.794_795insG and diagnostic reagent thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103555837A (en) * | 2013-11-01 | 2014-02-05 | 武汉大学 | Human ROR2 gene mutation and application thereof |
| CN107523608A (en) * | 2016-06-22 | 2017-12-29 | 海门中科基因生物科技有限公司 | A kind of kit for detecting the mutation of PKU Disease-causing gene |
| CN108018353A (en) * | 2017-11-08 | 2018-05-11 | 泰州达安达瑞医学检验有限公司 | A kind of colorectal cancer early screening primer sets and kit based on 4 genes |
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2018
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| CN103555837A (en) * | 2013-11-01 | 2014-02-05 | 武汉大学 | Human ROR2 gene mutation and application thereof |
| CN107523608A (en) * | 2016-06-22 | 2017-12-29 | 海门中科基因生物科技有限公司 | A kind of kit for detecting the mutation of PKU Disease-causing gene |
| CN108018353A (en) * | 2017-11-08 | 2018-05-11 | 泰州达安达瑞医学检验有限公司 | A kind of colorectal cancer early screening primer sets and kit based on 4 genes |
Non-Patent Citations (2)
| Title |
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| GUIBO GAO等: "Novel insertion mutation p.Asp610GlyfsX23 in APC gene causes familial adenomatous polyposis in Chinese families", 《GENE》 * |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115216531A (en) * | 2022-06-30 | 2022-10-21 | 湖南家辉生物技术有限公司 | Novel APC gene mutation c.794_795insG and diagnostic reagent thereof |
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