CN101974643A - Epithelial type cadherin expressed gene CDH1 mutation detection kit and application thereof - Google Patents
Epithelial type cadherin expressed gene CDH1 mutation detection kit and application thereof Download PDFInfo
- Publication number
- CN101974643A CN101974643A CN201010550700.XA CN201010550700A CN101974643A CN 101974643 A CN101974643 A CN 101974643A CN 201010550700 A CN201010550700 A CN 201010550700A CN 101974643 A CN101974643 A CN 101974643A
- Authority
- CN
- China
- Prior art keywords
- cdh1
- pair
- gene
- mutation detection
- sudden change
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 230000035772 mutation Effects 0.000 title claims abstract description 22
- 238000001514 detection method Methods 0.000 title claims abstract description 15
- 108091007854 Cdh1/Fizzy-related Proteins 0.000 title abstract description 30
- 108050007957 Cadherin Proteins 0.000 title description 2
- 102000000905 Cadherin Human genes 0.000 title 1
- 238000012408 PCR amplification Methods 0.000 claims abstract description 12
- 206010064571 Gene mutation Diseases 0.000 claims abstract description 6
- 238000012163 sequencing technique Methods 0.000 claims abstract description 4
- 238000012360 testing method Methods 0.000 claims description 16
- 108091008146 restriction endonucleases Proteins 0.000 claims description 13
- 239000000203 mixture Substances 0.000 claims description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims description 4
- 229920002684 Sepharose Polymers 0.000 claims description 2
- 239000007850 fluorescent dye Substances 0.000 claims description 2
- 101150083915 cdh1 gene Proteins 0.000 abstract description 26
- 108090000623 proteins and genes Proteins 0.000 abstract description 14
- 238000004458 analytical method Methods 0.000 abstract description 12
- 238000000034 method Methods 0.000 abstract description 7
- 102000038594 Cdh1/Fizzy-related Human genes 0.000 abstract description 3
- 230000003321 amplification Effects 0.000 abstract description 2
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 2
- 239000012807 PCR reagent Substances 0.000 abstract 1
- 238000011156 evaluation Methods 0.000 abstract 1
- 239000002075 main ingredient Substances 0.000 abstract 1
- 102100025805 Cadherin-1 Human genes 0.000 description 26
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 18
- 108020004414 DNA Proteins 0.000 description 15
- 208000005718 Stomach Neoplasms Diseases 0.000 description 12
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 11
- 230000000694 effects Effects 0.000 description 10
- 239000012634 fragment Substances 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 9
- 239000013612 plasmid Substances 0.000 description 9
- 102200144876 rs2276331 Human genes 0.000 description 9
- 102220040094 rs546716073 Human genes 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 7
- 108060001084 Luciferase Proteins 0.000 description 6
- 239000005089 Luciferase Substances 0.000 description 6
- 206010017758 gastric cancer Diseases 0.000 description 6
- 201000011549 stomach cancer Diseases 0.000 description 6
- 201000000498 stomach carcinoma Diseases 0.000 description 6
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 5
- 229910052791 calcium Inorganic materials 0.000 description 5
- 239000011575 calcium Substances 0.000 description 5
- 238000002844 melting Methods 0.000 description 5
- 230000008018 melting Effects 0.000 description 5
- 238000007894 restriction fragment length polymorphism technique Methods 0.000 description 5
- 238000011144 upstream manufacturing Methods 0.000 description 5
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 4
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 4
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 108700024394 Exon Proteins 0.000 description 4
- 108010093825 Mucoproteins Proteins 0.000 description 4
- 102000001621 Mucoproteins Human genes 0.000 description 4
- 108700008625 Reporter Genes Proteins 0.000 description 4
- VCULZBXVVZYUSW-QVUOKDEMSA-N [(2r)-3-[2-[5-[(3as,4s,6ar)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethoxy-hydroxyphosphoryl]oxy-2-hexadecanoyloxypropyl] hexadecanoate Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)NCCOP(O)(=O)OC[C@@H](COC(=O)CCCCCCCCCCCCCCC)OC(=O)CCCCCCCCCCCCCCC)SC[C@@H]21 VCULZBXVVZYUSW-QVUOKDEMSA-N 0.000 description 4
- 238000000246 agarose gel electrophoresis Methods 0.000 description 4
- 102220363899 c.7A>G Human genes 0.000 description 4
- 230000004087 circulation Effects 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- 230000014509 gene expression Effects 0.000 description 4
- 238000004321 preservation Methods 0.000 description 4
- 238000004153 renaturation Methods 0.000 description 4
- 238000000137 annealing Methods 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 210000005259 peripheral blood Anatomy 0.000 description 3
- 239000011886 peripheral blood Substances 0.000 description 3
- 230000004853 protein function Effects 0.000 description 3
- 102220061264 rs139110184 Human genes 0.000 description 3
- 102220041858 rs200161607 Human genes 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- 230000004304 visual acuity Effects 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 101000587430 Homo sapiens Serine/arginine-rich splicing factor 2 Proteins 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 241000254064 Photinus pyralis Species 0.000 description 2
- 102100029666 Serine/arginine-rich splicing factor 2 Human genes 0.000 description 2
- 230000008034 disappearance Effects 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 102220047420 rs74550754 Human genes 0.000 description 2
- 238000010079 rubber tapping Methods 0.000 description 2
- 102100033886 Arylsulfatase F Human genes 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108700019961 Neoplasm Genes Proteins 0.000 description 1
- 102000048850 Neoplasm Genes Human genes 0.000 description 1
- 102100023935 Transmembrane glycoprotein NMB Human genes 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000002146 bilateral effect Effects 0.000 description 1
- 238000000546 chi-square test Methods 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000009786 epithelial differentiation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000012252 genetic analysis Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000011880 melting curve analysis Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 230000000869 mutational effect Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000010363 phase shift Effects 0.000 description 1
- 238000000197 pyrolysis Methods 0.000 description 1
- 238000003030 reporter gene method Methods 0.000 description 1
- 102220046174 rs1801552 Human genes 0.000 description 1
- 102220042314 rs3743674 Human genes 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 230000009182 swimming Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 108091007466 transmembrane glycoproteins Proteins 0.000 description 1
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a CDH1 mutation detection kit and an application thereof. The kit mainly comprises the following main ingredients: (1) 19 pairs of CDH1 gene PCR amplifying and sequencing primers; and (2) PCR reagent for amplification. The PCR amplification product obtained by the kit can screen gene micromutation by a high-resolution dissolved curve process or carries out polymorphism analysis on restrictive segment length. The kit has high-efficiency mutation detection and functional evaluation actions. The invention is used for CDH1 gene mutation detection.
Description
Technical field
The present invention relates to a kind of CDH1 detection in Gene Mutation test kit, particularly it is in East Asia cancer of the stomach group of people at high risk's gene diagnosis and consequence assessment.
Background technology
The CDH1 gene is positioned at human chromosomal 16q22.1, comprises 16 exons, and the about 100kb of total length, its transcription product are the 4.5kb messenger RNA(mRNA), by the epitheliated type calcium of translating synthetic 120kD mucoprotein (E-cadherin, E-cad).E-cad is one of mucoprotein family member of calcium, and the mucoprotein family molecule of calcium is transmembrane glycoprotein, mediates calcium dependent iuntercellular and sticks.E-cad is in epithelial cell differentiation and keep in the cell polarity and play a crucial role, because the E-cad abnormal expression that the CDH1 transgenation causes has vital role in kinds of tumors such as cancer of the stomach form.Therefore the CDH1 gene is an important tumor susceptibility gene of cancer of the stomach.But, about system's report of the sudden change of CDH1 gene seldom, mainly be confined to several specific catastrophe points in the heredity diffuse type cancer of the stomach in the past because the limitation of detection method.In Chinese patients with gastric cancer, yet there are no report about the system's examination and the case-control analysis of this transgenation.For missense mutation that detects and promoter region mutation functional consequence do not appear in the newspapers yet.Therefore be necessary to develop the appraisal procedure of a kind of conveniently sudden change detection and sudden change consequence, to be used for the genetic diagnosis of cancer of the stomach.
Summary of the invention
The purpose of this invention is to provide a kind of CDH1 gene mutation body detection kit and application thereof.
This detection kit mainly comprises following composition:
(1) CDH1 gene promoter area and the 1st to the 16th exon district pcr amplification and sequencing primer are 19 pairs, and each sees Table 1 to primer sequence;
(2) conventional pcr amplification reagent is as dNTP, Taq archaeal dna polymerase etc.
The pcr amplification and the sequencing primer sequence of table 1CDH1 gene 1-16 exon and promoter region
The PCR product that this test kit amplifies can adopt the detection that suddenlys change of following three kinds of modes: a kind of mode is by high resolving power solubility curve method examination gene micromutation; Another kind of mode is that PCR product enzyme is cut laggard row agarose gel electrophoresis, analyzes according to electrophoretic band.Therefore, can also comprise in the mentioned reagent box: restriction enzyme or LC Green Plus
+Fluorescence dye.
Said restriction enzyme be detect CDH1 gene extron 13 c.2076T＞the Nae I enzyme of C sudden change etc.
In having the test kit of restriction enzyme, sepharose can also be arranged.
The said test kit of the present invention is used to detect the CDH1 gene mutation body.
The sudden change detection and the functional assessment step of this test kit comprise:
(1) extracts the sample peripheral blood DNA.
(2) each exon of CDH1 gene and promoter region fragment PCR thereof amplification.：
Carry out pcr amplification for CDH1 gene 1-16 exon and promoter region according to table 1 design primer.Reaction system is genomic dna (100ng/ μ l) 1.0 μ l, dNTP Mixture (2.5mM each) 0.8 μ l, 10 * PCR buffer (Mg
2+Free) 1.0 μ l, MgCl
2(25mM) 1.0 μ l, upstream primer (10 μ M) 0.2 μ l, downstream primer (10 μ M) 0.2 μ l, dimethyl sulfoxide (DMSO) (DMSO) 0.4 μ l, ddH
2O 5.32 μ l, Taq archaeal dna polymerase (5Unit/ μ l) 0.08 μ l.Reaction conditions: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 30sec, 60 ℃ of (corresponding annealing temperature) renaturation 30sec, 72 ℃ are extended 40sec, 40 circulations; 72 ℃ are extended 7min, 4 ℃ of preservations again.
(3) high resolving power solubility curve method examination gene micromutation: sneak into LC Green dyestuff in the pcr amplification, on LightScanner detector (American I daho company), carry out melting curve scanning, utilize the morphological differences of Lightscanner software analysis curve, distinguish homozygote and heterozygote.
Or, restriction fragment length polymorphism is analyzed: seek special restriction enzyme at the mutational site, the PCR product is mixed with the restriction endonuclease reaction system, react for some time at a certain temperature with reference to the restriction endonuclease working instructions, behind agarose gel electrophoresis, divide wild-type, homozygous mutation or heterozygous mutant by the band difference section.
This test kit has efficiently, and sudden change detects and consequence assessment effect.Report in the embodiment that the case-control analysis is carried out in transgenation at CDH1, relate to the comprehensive examination of peripheral blood DNA CDH1 transgenation of 236 routine Chinese patients with gastric cancer and 240 routine normal controls.Detect 15 CDH1 of place transgenations altogether, wherein 7 places are new mutant.And report the gene frequency of some polymorphic sites in Chinese population.Adopting the function evaluating method of test kit to propose wherein 4 kinds of new mutants forms for cancer of the stomach and may have the nosetiology meaning.This test kit has vital role in the nosetiology meaning assessment of patients with gastric cancer gene diagnosis and sudden change.
Description of drawings
Fig. 1 is the HRM analysis chart of CDH1 gene Exon 14: heterozygote and homozygote set of curves are as shown.
Fig. 2 is CDH1 gene Exon 13RFLP rear electrophoresis figure, and the right the 1st swimming lane is Marker among the figure, wild-type 304bp, heterozygous 304+183+121bp, pure and mild mutant 183+121bp.
Fig. 3 is the CDH1 gene new mutant sequencer map that detects with this test kit.(A)c.44_46del?TGC.(B)+76G＞T.(C)c.604G＞A(V202I).(D)c.894C＞T(A298A).(E)c.1224G＞A(A408A).(F)c.1320+7A＞G.(G)c.1888C＞G(L630V).
Fig. 4 carries the sequence dna fragment that c.44_46del_TGC CDH1 suddenlys change.The underscore place shows that CDH1 c.44_46delTGC among the figure.
Fig. 5 is the sequence dna fragment that carries CDH1+76G＞T sudden change.The underscore place shows CDH1+76G＞T sudden change among the figure.
Fig. 6 be carry CDH1 c.604G＞sequence dna fragment of A (V202I) sudden change.Among the figure underscore place show CDH1 c.604G＞A (V202I) sudden change.
Fig. 7 be carry CDH1 c.894C＞sequence dna fragment of T (A298A) sudden change.Among the figure underscore place show CDH1 c.894C＞T (A298A) sudden change.
Fig. 8 be carry CDH1 c.1224G＞sequence dna fragment of A (A408A) sudden change.The underscore place shows CDH1c.1224G＞A (A408A) sudden change among the figure.
Fig. 9 be carry CDH1 c.1320+7A＞sequence dna fragment of G sudden change.Among the figure underscore place show CDH1 c.1320+7A＞G sudden change.
Figure 10 be carry CDH1 c.1888C＞sequence dna fragment of G (L630V) sudden change.The underscore place shows CDH1c.1888C＞G (L630V) sudden change among the figure.
Figure 11 is two luciferase reporter gene detected results of CDH1 gene promoter region mutation.
Embodiment
Embodiment 1: system's examination of Chinese patients with gastric cancer CDH1 transgenation and case-control analysis
Select 238 routine Chinese Jiangsu, Anhui and Zhejiang area patients with gastric cancer, and 240 routine normal controls, extract peripheral blood, extract DNA.Carry out pcr amplification and high resolving power melting curve (HRM) analysis for CDH1 gene 2-12,14-16 exon region according to table 1 design primer.Reaction system is: genomic dna (100ng/ μ l) 1.0 μ l, dNTP Mixture (2.5mM each) 0.8 μ l, 10 * PCR buffer (Mg
2+Free) 1.0 μ l, MgCl
2(25mM) 1.0 μ l, upstream primer (10 μ M) 0.2 μ l, downstream primer (10 μ M) 0.2 μ l, dimethyl sulfoxide (DMSO) (DMSO) 0.4 μ l, ddH
2O 4.32 μ l, LC Green dyestuff 1.0 μ l, Taq archaeal dna polymerase (5Unit/ μ l) 0.08 μ l.Reaction conditions: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 30sec, 60 ℃ of (corresponding annealing temperature) renaturation 30sec, 72 ℃ are extended 40sec, 40 circulations; 72 ℃ are extended 7min, 4 ℃ of preservations again.HRM instrument LightScanner 96 (American I daho company) detects: open Lightscanner software, time shutter 80ms is set, the scanning temperature range scans for 70-95 ℃.After finishing, scanning use the Scanning program to carry out the melting curve analysis, the automatic gene somatotype.The melting temperature(Tm) of heterozygote is low than homozygote, and the downcomer of its melting curve also has difference than homozygous melting curve in advance between dissimilar homozygotes.Fig. 1 is the HRM analysis chart of CDH1 gene extron 14, and heterozygote and homozygote set of curves are as shown.For CDH1 gene the 13rd exon, carry out pcr amplification and restriction fragment length polymorphism (RFLP) analysis according to table 1 design primer.Reaction system is genomic dna (100ng/ μ l) 1.0 μ l, dNTP Mixture (2.5mM each) 0.8 μ l, 10 * PCR buffer (Mg
2+Free) 1.0 μ l, MgCl
2(25mM) 1.0 μ l, upstream primer (10 μ M) 0.2 μ l, downstream primer (10 μ M) 0.2 μ l, dimethyl sulfoxide (DMSO) (DMSO) 0.4 μ l, ddH
2O 5.32 μ l, TaqDNA polysaccharase (5Unit/ μ l) 0.08 μ l.Reaction conditions: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 30sec, 60 ℃ of (corresponding annealing temperature) renaturation 30sec, 72 ℃ are extended 40sec, 40 circulations; 72 ℃ are extended 7min, 4 ℃ of preservations again.Rflp analysis: at CDH1 gene the 13rd exons mutation site c.2076T＞C seeks special restriction enzyme Nae I.The recognition site of this enzyme is GC
CGGC, when take place c.2076T＞during the C sudden change (underscore shows), this restriction endonuclease plays a role, and the CDH1 gene of wild-type can not be discerned by this restriction endonuclease because this site is T.Concrete operations: PCR product 5 μ l, Nae I restriction endonuclease 0.125 μ l, 10 * NEBuffer, 0.5 μ l, 37 ℃ are reacted after 1 hour 2% agarose gel electrophoresis, observation experiment result under ultraviolet.The possible several situations of band appear in pcr amplified fragment total length 304bp on the electrophorogram: 1. wild-type (TT): 304bp; 2. heterozygous mutant type (TC): 304+183+121b; 3. homozygous mutation type (CC): 183+121bp (Fig. 2).For CDH1 gene promoter and exons 1 zone, because the possible catastrophe point of this section is more, taking behind the pcr amplification directly, order-checking detects.With HRM or the preliminary examination of RFLP method is sudden change male sample, adopts BigDye Terminator V3.1 test kit, goes up order-checking at ABI 3130 genetic analysis instrument (American AB I company) and confirms sudden change.The result detects 15 CDH1 of place transgenations altogether, contains point mutation and small insertion/disappearance.Wherein 7 places are newfound catastrophe point.Fig. 3 is the sequencer map of 7 kinds of newly discovered sudden changes of this research.These 7 kinds of sudden changes are followed successively by (A) c.44_46del TGC; (B)+76G＞T; (C) c.604G＞A (V202I); (D) c.894C＞T (A298A); (E) c.1224G＞A (A408A); (F) c.1320+7A＞G.; (G) c.1888C＞G (L630V).Fig. 4-the 10th carries the sequence dna fragment of these 7 kinds of CDH1 new mutants, respectively corresponding SEQ ID NO:41-47.Further, adopt relatively suddenly change carrying rate in patient and normal people of Mantel-Haenszel χ 2 statistical analysis techniques such as grade, reported that these suddenly change at the gene frequency (table 2) of Chinese population.The particularly important is, the 3bp disappearance that wherein is positioned at the exons 1 place is phase shift mutation, only detects in 2 routine patients with gastric cancer, does not detect in normal control, is the pathologic sudden change.
Table 2CDH1 detection in Gene Mutation and case-control analytical results
*New mutant
ξThe bilateral chi square test or the definite probability inspection of Fei Xieer of genotype distribution or gene frequency.
Embodiment 2: the CDH1 gene missense mutation that detects is to the analysis of protein function influence
Adopt SIFT to analyze (the Sorting Intolerant from Tolerant, http://sift.jcvi.org/) and PolyPhen (=Polymorphism Phenotyping, http://genetics.bwh.harvard.edu/pph/), analyze monamino acid that missense mutation causes and change influence the CDH1 protein function.The scoring threshold value that SIFT is given is 0.5, promptly thinks＜0.5 influence proteinic function probably, and 〉=0.5 then is tolerable.The PolyPhen instrument to judge the influence that suddenlys change for protein structure and function, when a minute difference surpasses 1.5, thinks to change the sudden change of function by the PSIC scoring difference of coded amino acid before and after the calculating sudden change.This research SIFT analyze to show the CDH1 that detects c.1888C＞G (L630V) sudden change score is 0.02, and PolyPhen divides difference to reach 1.748, so this sudden change may influence the CDH1 protein function.And V202I and T340A are optimum change (table 3).
Table 3SIFT and PolyPhen analyze the consequence of CDH1 gene missense mutation
Embodiment 3: the functional consequence analysis of the CDH1 gene promoter region mutation that detects
Adopt two luciferase reporter gene methods to detect the functional consequence of gene promoter region mutation.Make up various PGL3-CDH1 promoter region mutation type plasmids, transfection Hela cell according to the plain enzymic activity of the relative fluorescence of expression product, is assessed the promoter activity intensity of each mutated genes.
(1) make up various PGL3-CDH1 promoter region mutation type plasmids:
According to the sequence of CDH1 gene promoter area, design has the primer of double enzyme site.Pcr amplification comprises the zone from transcripting start point-345 to+271bp.Upstream primer is PF:5 '-ATGC
CTCGAGCCATCTCCAAAACGAACAAAC-3 ' (SEQ ID NO:39), ' ATGC ' for protection sequence, '
CTCGAG' be the recognition sequence of XhoI restriction endonuclease.Downstream primer is PR:5 '-ATGC
AAGCTTGAAGGGAAGCGGTGACGAC-3 ' (SEQ ID NO:40), ' ATGC ' for protection sequence, '
AAGCTT' be the recognition sequence of HindIII restriction endonuclease.Reaction system is genomic dna (100ng/ μ l) 2.0 μ l, dNTP Mixture (2.5mM) 3.0 μ l, the 10 * PCR buffer (Mg that has specific catastrophe point
2+Free) 2.0 μ l, MgCl
2(25mM) 2.0 μ l, upstream primer (10 μ M) 1.0 μ l, downstream primer (10 μ M) 1.0 μ l, ddH
2O 8.8 μ l, Taq archaeal dna polymerase (5Unit/ μ l) 0.2 μ l.Reaction conditions: 94 ℃ of pre-sex change 3min, 94 ℃ of sex change 30sec, 60 ℃ of renaturation 30sec, 72 ℃ are extended 1min, 40 circulations, 72 ℃ are extended 10min, 4 ℃ of preservations again.The PCR product is connected with pMD-18T (TaKaRa Dalian) carrier behind agarose gel electrophoresis rubber tapping purifying.Above-mentioned connection product is transformed into intestinal bacteria and clones.10 mono-clonals of picking carry out plasmid extraction and order-checking at random.The positive monoclonal plasmid that is defined as target plasmid through order-checking carries out the XhoI/HindIII double digestion, and pGL3-Basic (Promega Beijing) plasmid carries out double digestion simultaneously.Double digestion product electrophoresis, the rubber tapping purifying.Again target sequence is connected with the pGL3-Basic carrier, thereby makes up various PGL3-CDH1 promoter region mutation type plasmids.
(2) two luciferase reporter genes detect: the Hela cell is at 37 ℃ of DMEM substratum, 5%CO
2Following cultivation was gone down to posterity once in 2～3 days.At the cell of logarithmic phase growth,, be inoculated in 12 orifice plates with trysinization and counting.Inoculum density is 1 * 10
5The Hela cells/well.Add antibiotic-free and contain the DMEM culture medium culturing of 10%FBS to 70%-95% hole floorage.According to the liposome specification sheets with plasmid transfection to cell, continue to cultivate 24h.After getting 20 μ l cell pyrolysis liquids and 50 μ l LAR II mixing, last machine testing fluorescent value, the value that records is the value of Photinus pyralis LUC.Add 50 μ l Stop﹠amp again; Glo reagent mixes the back and surveys fluorescent value, and the value that records is the activity of ocean coelenteron luciferase.The activity of ocean coelenteron luciferase is represented transfection efficiency, so the value of the Photinus pyralis LUC in each hole equals the uciferase activity in each hole divided by the activity of ocean coelenteron luciferase.As standard value 1, the activity of wild-type and mutant promotor is compared with it respectively, can obtain the promoter activity intensity of each mutated genes with the uciferase activity of the unloaded plasmid of pGL3-Basic.
Through the detection of above-mentioned promoter region mutation, propose CDH1 promoter region-164del T ,-49G＞T sudden change increases reporter gene expression ,-160C＞A, c.48+6C＞T reduces reporter gene expression (Figure 11).
Embodiment 4: the exon that detects-intron intersection sudden change is to the impact analysis of RNA montage
Utilize the influence of ESEfinder (http://exon.cshl.edu/ESE/) and BDGP (http://www.fruitfly.org/seq_tools/splice-instrucs.html) prediction CDH1 gene intron, the missense mutation of exon juncture area to the RNA montage.ESEfinder seeks corresponding information, the potential ESE that exists in the forecasting sequence by analyzing the interaction of present known ESE and montage modulin SR protein from exon sequence.BDGP then stresses to assess the influence of sudden change to exon montage efficient.ESEfinder program (http://rulai.cshl.edu/tools/ESE) show CDH1 c.48+6T＞sudden change produces bigger influence to montage, lose as unborn SF2/ASF binding site on the sequence, increase the binding site of SRp55 newly, also reduced the binding ability of SC35 binding site in addition.And BDGP has shown that sudden change is slightly risen montage efficient.Similarly, CDH1 c.1320+7A＞G sudden change also produces considerable influence to montage, as increased the binding site of SRp55 newly, and increased the binding ability of SC35 binding site.And BDGP has shown that sudden change makes montage efficient slightly descend (table 4).Therefore this this two kinds of CDH1 sudden changes may be passed through to influence exon montage enhanser angle, thereby influence the normal montage of RNA.
Table 4CDH1 transgenation is for the analysis of RNA shear inference
Claims (5)
1. one kind
CDH1The detection in Gene Mutation test kit is characterized in that comprising following composition:
(1)
CDH1Gene PCR amplification and 19 pairs of sequencing primers; (2) pcr amplification reagent;
Said 19 pairs of primer sequences are as follows:
The 1st pair
F:?5'-cgaacccagtggaatcagaac-3’?R:?5'-gaagggaagcggtgacgac-3’
The 2nd pair
F:?5'-?acccggttccatctaccttt?-3’ R:?5'-?tgtgggagtgcaatttctcg?-3’
The 3rd pair
F:?5'-?ctttaatctgtccaatttccta?-3’ R:?5'-?ccactgtattcagcgtgac?-3’
The 4th pair
F:?5'-?gggactccacctacagaa?-3’ R:?5'-?cagggatgcttgctaaac?-3’
The 5th pair
F:?5'-?cgctgtctggctaggttg?-3’ R:?5'-?ctctgccaaatccctccc?-3’
The 6th pair
F:?5'-?ggaaaagacccagtgttg?-3’ R:?5'-?atcctgggtggatgttac?-3’
The 7th pair
F:5'-?ccttctcccatgttttcttcc?-3’ R:?5'-?gtattcagattctcattagtgg?-3’
The 8th pair
F:?5'-?cagcttgtctaaaccttcatc?-3’ R:?5'-?cctccacaccctctggatc?-3’
The 9th pair
F:?5'-?tcctggtcctgacttggt?-3’ R:?5'-?cctttctttggaaaccct?-3’
The 10th pair
F:?5'-?tagcagtcttggtactttg?-3’ R:?5'-?tgtgaggatgccagtttc?-3’
The 11st pair
F:5'-?tattaccaaaagcaacagt?-3’ R:?5'-?tcaggaggcacaaagatg?-3’
The 12nd pair
F:5'-tctcaccacctccacagc-3’ R:?5'-cacagataaagggaataaaaaa-3’
The 13rd pair
F:?5'-?ccagagcttgtccccgttc?-3’ R:?5'-?gtcgaggcagcaaaggctc?-3’
The 14th pair
F:?5'-?gggattcattactgttgcc?-3’ R:?5'-?attgggaggaaggtctgc?-3’
The 15th pair
F:?5'-?agcctcaggtcataaacat?-3’ R:?5'-?aagggacaaggaagcaag?-3’
The 16th pair
F:?5'-?ttcctcccctggtctcatc?-3’ R:?5'-?tcaaaggctgagtcacttgc?-3’
The 17th pair
F:?5'-?ttgctctgtctcccccacc?-3’ R:?5'-?tccgaataaagagatcaccac?-3’
The 18th pair
F:?5'-?agtgaaggcatcatccaa?-3’ R:?5'-?ctcctcctgagcttagag?-3’
The 19th pair
F:?5'-?tgcttttgtcccttcttc?-3’ R:?5'-?cgtgatttctgcatttcc?-3’。
2. test kit according to claim 1 is characterized in that, also comprises restriction enzyme
Nae I
3. test kit according to claim 2 is characterized in that, also has sepharose.
4. test kit according to claim 1 is characterized in that, also comprises LC Green Plus
+Fluorescence dye.
5. the said test kit of claim 1 is used for detecting
CDH1Gene mutation body.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010550700XA CN101974643B (en) | 2010-11-18 | 2010-11-18 | Epithelial type cadherin expressed gene CDH1 mutation detection kit and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010550700XA CN101974643B (en) | 2010-11-18 | 2010-11-18 | Epithelial type cadherin expressed gene CDH1 mutation detection kit and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101974643A true CN101974643A (en) | 2011-02-16 |
| CN101974643B CN101974643B (en) | 2012-04-04 |
Family
ID=43574550
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201010550700XA Expired - Fee Related CN101974643B (en) | 2010-11-18 | 2010-11-18 | Epithelial type cadherin expressed gene CDH1 mutation detection kit and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101974643B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102787173A (en) * | 2012-09-06 | 2012-11-21 | 南京大学 | Alternative splicing detection kit for epithelial cadherin expression gene CDH1 pre-mRNA and application of alternative splicing detection kit |
| CN102888406A (en) * | 2011-07-22 | 2013-01-23 | 武汉淘智生命科技有限公司 | Human idiopathic basal ganglia calcification disease-causing gene and coding protein thereof |
| CN106609307A (en) * | 2017-01-10 | 2017-05-03 | 北京艾克伦医疗科技有限公司 | Detection kit for diagnosis of patients with stomach cancer based on multiple genes |
| CN110541028A (en) * | 2019-08-27 | 2019-12-06 | 深圳市宝安区妇幼保健院 | Method for detecting FUS gene mutation and TARDBP gene mutation |
| CN111004849A (en) * | 2019-12-13 | 2020-04-14 | 北京艾迪康医学检验实验室有限公司 | Primer, method and kit for detecting multiple site mutations of CDH1 gene |
-
2010
- 2010-11-18 CN CN201010550700XA patent/CN101974643B/en not_active Expired - Fee Related
Non-Patent Citations (4)
| Title |
|---|
| 《Clinical Cancer Research》 20050801 Gianpaolo Suriano et al. Characterization of a Recurrent Germ Line Mutation of the E-Cadherin Gene: Implications for Genetic Testing and Clinical Management 5401-5409 1-5 第11卷, 第15期 2 * |
| 《Human Molecular Genetics》 19991231 Frances M. Richards et al. Germline E-cadherin gene (CDH1) mutations predispose to familial gastric cancer and colorectal cancer 607-610 1－5 第8卷, 第4期 2 * |
| 《中国消化杂志》 20040630 朱正纲等 胃癌/乳腺癌先证者家族性癌症综合征家系CDH1突变分析及病理学特征 344-348 1－5 第24卷, 第6期 2 * |
| 《癌变 畸变 突变》 20051105 张元颖等 E-Cadherin(CDH1)基因胚系突变与胃癌风险的研究 350-353 1－5 第17卷, 第6期 2 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102888406A (en) * | 2011-07-22 | 2013-01-23 | 武汉淘智生命科技有限公司 | Human idiopathic basal ganglia calcification disease-causing gene and coding protein thereof |
| CN102787173A (en) * | 2012-09-06 | 2012-11-21 | 南京大学 | Alternative splicing detection kit for epithelial cadherin expression gene CDH1 pre-mRNA and application of alternative splicing detection kit |
| CN102787173B (en) * | 2012-09-06 | 2014-06-18 | 南京大学 | Alternative splicing detection kit for epithelial cadherin expression gene CDH1 pre-mRNA and application of alternative splicing detection kit |
| CN106609307A (en) * | 2017-01-10 | 2017-05-03 | 北京艾克伦医疗科技有限公司 | Detection kit for diagnosis of patients with stomach cancer based on multiple genes |
| CN110541028A (en) * | 2019-08-27 | 2019-12-06 | 深圳市宝安区妇幼保健院 | Method for detecting FUS gene mutation and TARDBP gene mutation |
| CN111004849A (en) * | 2019-12-13 | 2020-04-14 | 北京艾迪康医学检验实验室有限公司 | Primer, method and kit for detecting multiple site mutations of CDH1 gene |
| CN111004849B (en) * | 2019-12-13 | 2022-08-26 | 南京艾迪康医学检验所有限公司 | Primer, method and kit for detecting multiple site mutations of CDH1 gene |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101974643B (en) | 2012-04-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101974643B (en) | Epithelial type cadherin expressed gene CDH1 mutation detection kit and application thereof | |
| CN104031978B (en) | A kind of test kit based on the transgenation of ARMS fluorescence quantitative PCR detection and method | |
| CN106755388B (en) | A kind of improved ARMS primer construction (Super-ARMS) and its application method | |
| CN102618624B (en) | Kit for screening deaf gene of Chinese populations, and use method thereof | |
| CN102242211A (en) | Method and kit for detecting mutant nucleic acid sequence | |
| CN105039514A (en) | Detection kit for human BRAF gene V600E mutation | |
| CN107034277A (en) | A kind of method for detecting low abundance gene mutation | |
| WO2020249111A1 (en) | Method and kit for detecting genome editing and application thereof | |
| CN107058538A (en) | The kit and application of a kind of Primer composition and its composition | |
| CN107488711A (en) | The method and its kit of the genotype detection of point mutation | |
| CN101974642A (en) | Mismatch repair gene MLH1 mutation detection kit and application thereof | |
| CN109234370B (en) | MUT gene mutation detection kit | |
| CN104694622A (en) | Probe for detecting K-ras gene mutation, primer, kit and method | |
| CN103421908B (en) | Kit used for screening CYP21A2 gene of Chinese population | |
| Sampaio et al. | Controlled reduction of genomic heterozygosity in an industrial yeast strain reveals wide cryptic phenotypic variation | |
| CN111304329A (en) | Kit for detecting mutation of BRAF gene V600E site | |
| US20130217020A1 (en) | Methods for improving sensitivity and specificity of screening assays of kras codons 12 and 13 mutations | |
| CN101918592B (en) | EXO1 promotor polymorphism associated with exceptional life expectancy in humans | |
| CN110373454A (en) | A kind of kit and method of joint-detection EGFR genetic mutation | |
| CN105506101A (en) | AS-PCR (allele-specific polymerase chain reaction) primer design method, gene polymorphism detection method and kit | |
| Ostrovsky et al. | Characterization of HPSE gene single nucleotide polymorphisms in Jewish populations of Israel | |
| Flanagan et al. | The human POLH gene is not mutated, and is expressed in a cohort of patients with basal or squamous cell carcinoma of the skin | |
| CN104928356A (en) | Rapid sensitive breast cancer susceptibility gene SNP detection method based on PCR primer 3' terminal nucleotide dideoxy modification | |
| CN110438224B (en) | Primer, kit and detection method for UGT1A1 gene polymorphism detection | |
| CN104450911B (en) | Kit for detecting related gene FMR1 of fragile X chromosome syndrome (FRAX) and application of kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120404 Termination date: 20131118 |