CN104404044A - Detection method and applications for single nucleotide polymorphisms in exon region of ANRIL (Antisense Noncoding RNA in the INK4 Locus) gene related to susceptibility of coronary artery disease - Google Patents
Detection method and applications for single nucleotide polymorphisms in exon region of ANRIL (Antisense Noncoding RNA in the INK4 Locus) gene related to susceptibility of coronary artery disease Download PDFInfo
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Abstract
The invention relates to the technical field of genes relating to a coronary artery disease in a biotechnology center and particularly relates to a detection method and applications for single nucleotide polymorphisms in an exon region of an ANRIL(Antisense Noncoding RNA in the INK4 Locus) gene related to the susceptibility of the coronary artery disease. The invention provides the gene ANRIL relating to the susceptibility of the coronary artery disease and polymorphisms (rs10965215 and rs10738605) thereof, a method for detecting the polymorphisms as well as the applications of the polymorphisms in the aspects of prevention, assistant diagnosis, treatment and the like for the coronary artery disease. A kit for genetic diagnosis of the coronary artery disease is constructed by using the gene related to the coronary artery disease and nucleotide sequences for the polymorphisms of the gene; the kit can be applied to assistant diagnosis of the coronary artery disease and the judgment on the susceptibility of the coronary artery disease, so that the prevention, early-stage diagnosis and treatment for the coronary artery disease are facilitated.
Description
Technical field
The present invention relates to biotechnology center flesh infarct genes involved technical field, be specifically related to relevant to myocardial infarction susceptible
aNRILthe detection method of gene extron subarea mononucleotide polymorphic site and application thereof.
Background technology
Myocardial infarction (coronary artery disease) is that current China human adult heart disease is in hospital and first dead reason, and its M & M is still in rising trend.Large-scale retrospective study estimates that the year two thousand thirty China's patients with myocardial infarction will increase to 2,300 ten thousand.Along with the quickening of China's aging population trend, change and the atherosclerosis Related Risk Factors of people life style continue to increase, the senile cardiovascular disorder such as myocardial infarction also in lasting ascendant trend, and will become one of important diseases threatening China's public health.Attested myocardial infarction Major Risk Factors comprises at present: hypertension, smoking, hyperlipemia, diabetes, Overweight and obesity, age and sex etc.Research in recent years finds, in some individualities, often show obvious family history, and prompting inherited genetic factors plays a significant role in the process of bringing out myocardial infarction.Single nucleotide polymorphism (single nucleotide polymorphism, SNP) is the individual important inherited genetic factors to disease inheritance susceptible of impact.
SNP refers to the DNA sequence polymorphism in genomic level caused by single nucleotide diversity.SNP is between ethnic group, one of the physical basis of heredity of interindividual variation.The theoretical basis of SNP research is allelic association, and it refers to that the significance of the marker allele incidence of disease trait changes, and represents the deviation of the allelotrope relevant to disease trait in random generation.When the frequency of a genetic marker is in patients significantly more than non-patient, namely show this mark and disease association.The effect that SNP plays in disease gene location mainly comprises: one is find the SNP that causes a disease in disease locating area, the appearance of this SNP may directly results in the change of gene on transcriptional level and translation skill, namely change the composition structure of gene expression amount or gene product protein, thus cause certain disease to occur or make the individual environment susceptible special to certain.Two be SNP as a genetic marker, with disease or phenotype close linkage.
Long-chain non-coding RNA (long non-coding RNA, lncRNA) is the functional RNA molecule of a class transcript length more than 200nt, and they generally do not participate in protein coding.LncRNA mainly realizes the regulation and control to genetic expression from many levels such as epigenetic, transcriptional control and post-transcriptional controls, participate in regulating the multiple life process such as cell proliferation, differentiation and apoptosis, and play vital effect in the generation evolution of cardiovascular disorder.In recent years, lncRNA also more and more receives people's concern as the effect of a kind of functional RNA molecule in cardiovascular disorder.ANRIL (antisense noncoding RNA in the INK4 locus, ANRIL) be the antisense non-coding RNA that INK4a-ARF-INK4b gene cluster transcribes out, the site that in chromosome 9p 21 region, coronary artery disease genetic predisposition is the strongest, and closely related with myocardial infarction.Nearest research shows, ANRIL is high expression level in coronary artery disease peripheral blood in patients PBMCs and atherosclerotic plaque.By organizing the analysis of equal samples to coronary artery disease peripheral blood in patients PBMCs and atherosclerotic plaque, find ANRIL and atherosclerosis closely related, the rising of its expression level is directly related with coincident with severity degree of condition, and the gene expression abundance of this prompting ANRIL is directly related with the formation of coronary atherosclerosis.Also studies have found that,
aNRILthe risk allelotrope of non-coding region SNPs as rs1333049, rs10757274, rs2383206, rs2383297 and rs10757278 of gene can significantly improve the gene expression abundance of ANRIL, and and p14
aRF, p15
iNK4band p16
iNK4agene expression abundance closely related, thus play a significant role in atherosclerotic forming process, and cause coronary artery disease further, point out these SNP can by affecting the expression of ANRIL, and then promote the formation and development of Coronary Atherosclerotic Plaque, and significantly increase the onset risk of myocardial infarction.
It is above-mentioned that these are right
aNRILits non-coding region SNPs is mainly concentrated on the association study of myocardial infarction susceptible.Relevant so far
aNRILthere is not been reported for the SNPs in gene extron subarea and the relation of myocardial infarction inheritance susceptible.The exon 1 SNPs of non-coding RNA often through the space structure directly affecting this non-coding RNA, and then affects its stability and expression, therefore in disease-susceptible humans, more has Research Significance.
Summary of the invention
An object of the present invention is for the deficiencies in the prior art, provides the ANRIL gene relevant to myocardial infarction susceptible.
Two of object of the present invention is for the deficiencies in the prior art, provides the detection method of the ANRIL gene extron subarea mononucleotide polymorphic site relevant to myocardial infarction susceptible.
Three of object of the present invention is for the deficiencies in the prior art, is provided for the primer of the mononucleotide polymorphic site detecting the ANRIL gene extron subarea relevant to myocardial infarction susceptible.
Four of object of the present invention is for the deficiencies in the prior art, is provided for the specific probe of the mononucleotide polymorphic site detecting the ANRIL gene extron subarea relevant to myocardial infarction susceptible.
Five of object of the present invention is for the deficiencies in the prior art, is provided for the test kit of the mononucleotide polymorphic site detecting the ANRIL gene extron subarea relevant to myocardial infarction susceptible.
Six of object of the present invention is for the deficiencies in the prior art, provides the mononucleotide polymorphic site in the ANRIL gene extron subarea relevant to myocardial infarction susceptible whether having to the auxiliary diagnosis of myocardial infarction with to individuality the application that myocardial infarction susceptibility judges.
One of to achieve these goals, the present invention adopts following technical scheme:
The ANRIL gene relevant to myocardial infarction susceptible is provided, described in
aNRILthe nucleotide sequence of gene has the sequence shown in SEQ ID NO:1 and SEQ ID NO:2;
Wherein, in the sequence shown in SEQ ID NO:1, the 269th is G;
In sequence shown in SEQ ID NO:2, the 300th is C.
Described
aNRILthe mononucleotide polymorphic site in gene extron subarea is: rs10965215, and its allelotrope point is to being the Nucleotide of the 269th in the sequence shown in G/A(and SEQ ID NO:1) and described
aNRILthe mononucleotide polymorphic site in gene extron subarea is: rs10738605, and its allelotrope point is to being the Nucleotide of the 300th in the sequence shown in C/G(and SEQ ID NO:2);
Described mononucleotide polymorphic site rs10965215 with rs10738605 is relevant to myocardial infarction susceptible degree.
To achieve these goals two, the present invention adopts following technical scheme:
There is provided the detection method of the ANRIL gene extron subarea mononucleotide polymorphic site relevant to myocardial infarction susceptible described above, it comprises the following steps:
Step one, determines
aNRILthe mononucleotide polymorphic site (rs10965215 and rs10738605) in gene extron subarea, the i.e. Nucleotide of the 300th in the Nucleotide of the 269th and the sequence shown in SEQ ID NO:2 in sequence shown in SEQ ID NO:1;
Step 2, adopt polymerase chain reaction-ligase chain reaction (LCR) (PCR-LDR) Protocols in Molecular Biology detect detect in sample
aNRILwhether gene extron subarea exists the type of single nucleotide polymorphism and mononucleotide polymorphic.
In technique scheme, in described step 2, described polymerase chain reaction system is: each described sample carries out two totals and amasss amplified reaction into 20ul system, amplify the sequence shown in SEQ ID NO:1 and SEQ ID NO:2 respectively, and comprise: prepared genomic dna diluent 1ul, 2mM dNTP 2ul, 3mM MgCl
20.6ul, the corresponding each 1.0ul of forward and reverse amplimer, 1 × Buffer 2ul, the Taq archaeal dna polymerase 0.2ul of 1 unit, ddH
2o 12.2ul;
The amplified reaction that described polymerase chain reaction program is: sequence shown in SEQ ID NO:1 was 95 DEG C of denaturations 2 minutes, 94 DEG C of sex change are after 30 seconds, carry out 40 50 DEG C annealing 1 point 30 seconds, 65 DEG C extend the amplification cycles of 30 seconds, finally carry out 65 DEG C of 10 minutes fill; The amplified reaction of sequence shown in SEQ ID NO:2 was 95 DEG C of denaturations 2 minutes, and 94 DEG C of sex change are after 30 seconds, carried out 40 53 DEG C annealing 1 point 30 seconds, and 72 DEG C extend the amplification cycles of 30 seconds, finally carry out 72 DEG C of 10 minutes fill.
In technique scheme, in described step 2, described ligase chain reaction (LCR) system comprises in total amount 10 μ L: PCR product 4ul, specific probe 1ul, 1 × Buffer 1ul, the ligase enzyme 0.05ul of 2 units, ddH
2o 4ul;
Described ligase chain reaction (LCR) program is: react 95 DEG C of denaturations after 2 minutes, carries out 40 94 DEG C of sex change 15s, the circulating reaction of 50 DEG C of reaction 25s.
To achieve these goals three, the present invention adopts following technical scheme:
Be provided for the primer of the mononucleotide polymorphic site detecting the ANRIL gene extron subarea relevant to myocardial infarction susceptible described above, described primer specifically comprises:
(1) amplimer of sequence shown in SEQ ID NO:1 (comprising rs10965215):
Forward primer: 5 '-GGATGTTTTGCAGGACTATT-3 ' (SEQ ID NO:3)
Reverse primer: 5 '-GGAATCATCACAGCATGGAC-3 ' (SEQ ID NO:4)
(2) amplimer of sequence shown in SEQ ID NO:2 (comprising rs10738605):
Forward primer: 5 '-TTTTCCAGTGGTGTTTCTAAATAA-3 ' (SEQ ID NO:5)
Reverse primer: 5 '-CCTCTGATGGTTTCTTTGGAGT-3 ' (SEQ ID NO:6).
To achieve these goals four, the present invention adopts following technical scheme:
Be provided for the specific probe of the mononucleotide polymorphic site detecting the ANRIL gene extron subarea relevant to myocardial infarction susceptible described above, comprise following two group-specific probes:
First group:
Detect the FAM probe of the polymorphic site (rs10965215) in sequence shown in SEQ ID NO:1:
5’-GTGGCAAATAGTCCTGCAAAACATCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3’ (SEQ ID NO: 7)
Detect the G probe of the polymorphic site (rs10965215) in sequence shown in SEQ ID NO:1:
5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCTCTCTTGGAATCCTTTGAAATGTC-3’ (SEQ ID NO: 8)
Detect the A probe of the polymorphic site (rs10965215) in sequence shown in SEQ ID NO:1:
5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCTCTCTTGGAATCCTTTGAAATGTT-3’ (SEQ ID NO: 9)；
Second group:
Detect the FAM probe of the polymorphic site (rs10738605) in sequence shown in SEQ ID NO:2:
5’-CACACCTAACAGTGATGCTTGAACCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3’ (SEQ ID NO: 10)
Detect the C probe of the polymorphic site (rs10738605) in sequence shown in SEQ ID NO:2:
5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTACTGACTCGGGAAAGGATTCCAG-3’ (SEQ ID NO: 11)
Detect the G probe of the polymorphic site (rs10738605) in sequence shown in SEQ ID NO:2:
5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTACTGACTCGGGAAAGGATTCCAC-3’ (SEQ ID NO: 12)。
To achieve these goals five, the present invention adopts following technical scheme:
Be provided for the test kit of the mononucleotide polymorphic site detecting the ANRIL gene extron subarea relevant to myocardial infarction susceptible, it is characterized in that: it comprises the primer of the mononucleotide polymorphic site for detecting the ANRIL gene extron subarea relevant to myocardial infarction susceptible described above, and the specific probe of the mononucleotide polymorphic site for detecting the ANRIL gene extron subarea relevant to myocardial infarction susceptible described above.
To achieve these goals six, the present invention adopts following technical scheme:
The mononucleotide polymorphic site in the ANRIL gene extron subarea relevant to myocardial infarction susceptible is provided whether to have to the auxiliary diagnosis of myocardial infarction with to individuality the application that myocardial infarction susceptibility judges, described in
aNRILthe mononucleotide polymorphic site in gene extron subarea is rs10965215 and rs10738605 described above.
Compared with prior art, beneficial effect is in the present invention:
(1) present invention is disclosed
aNRILthe mononucleotide polymorphic site (rs10965215 and rs10738605) in gene extron subarea and the dependency of myocardial infarction susceptible degree, and the purposes of this polymorphism in screening myocardial infarction Susceptible population.Wherein, the loci C of loci G and rs10738605 of rs10965215 all can increase the onset risk of individual myocardial infarction.
(2) utilize the nucleotide sequence of the ANRIL gene relevant to myocardial infarction susceptible provided by the present invention and polymorphic site thereof, build test kit myocardial infarction Susceptible population being carried out to genetics screening; This test kit can be applied to the auxiliary diagnosis to patients with myocardial infarction, whether has myocardial infarction susceptibility assess individuality.
(3) active drug that it is target that the present invention can screen with rs10965215 and rs10738605 polymorphic site, is about to
aNRILmononucleotide polymorphic site rs10965215 and rs10738605 in gene extron subarea screens as the potential target spot of medicine and designs medicine, finds out and has adjustment
aNRILthe bioactive molecule of genetic expression, promotes the discovery of new treatment myocardial infarction medicine, thus is conducive to prevention, the early diagnosis and therapy of myocardial infarction.
(4) method that the present invention sets up can be used for analyst's
aNRILthe G/A allelotype of exon 1 mononucleotide polymorphic site rs10965215 in gene order and the C/G allelotype of rs10738605, be applied to the auxiliary diagnosis of myocardial infarction and to individuality, whether there is myocardial infarction susceptibility and judge, thus being conducive to the prevention and therapy of myocardial infarction.
(5) primer provided by the invention and specific probe is utilized can to detect the polymorphic site of in the polymorphic site of in the sequence shown in SEQ ID NO:1 the 269th and the sequence shown in SEQ ID NO:2 the 300th special, efficiently.
(6) utilize sequence and the methods of genotyping of the ANRIL gene relevant to myocardial infarction susceptible provided by the invention and polymorphic site thereof, the detection kit of myocardial infarction being carried out to molecular genetics diagnosis can be built.
(7) due to the ANRIL gene relevant to myocardial infarction susceptible provided by the invention probably also take part in other with propagation, apoptosis etc. cellular activity to lack of proper care the pathologic process of being correlated with, comprise tumour etc., therefore, the present invention is to furtheing investigate from now on
aNRILthe relation of gene and other diseases provides experience and application foundation.
Accompanying drawing explanation
Fig. 1 is
aNRILthe LDR order-checking electrophorogram of the polymorphic site rs10965215 of gene.
Fig. 2 is
aNRILthe LDR order-checking electrophorogram of the polymorphic site rs10738605 of gene.
Embodiment
In order to make technical problem solved by the invention, technical scheme and beneficial effect clearly understand, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
embodiment 1.
The ANRIL gene relevant to myocardial infarction susceptible, the nucleotide sequence of this ANRIL gene has the sequence shown in SEQ ID NO:1 and SEQ ID NO:2;
Wherein, in the sequence shown in SEQ ID NO:1, the 269th is G;
In sequence shown in SEQ ID NO:2, the 300th is C.
Wherein, should
aNRILthe mononucleotide polymorphic site in gene extron subarea is: rs10965215, its allelotrope point is to being the Nucleotide of the 269th in the sequence shown in G/A(and SEQ ID NO:1) and the mononucleotide polymorphic site in this ANRIL gene extron subarea be: rs10738605, its allelotrope point is to being the Nucleotide of the 300th in the sequence shown in C/G(and SEQ ID NO:2);
This mononucleotide polymorphic site rs10965215 with rs10738605 is relevant to myocardial infarction susceptible degree.
embodiment 2.
The detection method of the ANRIL gene extron subarea mononucleotide polymorphic site relevant to myocardial infarction susceptible, it comprises the following steps:
Step one, determines the mononucleotide polymorphic site (rs10965215 and rs10738605) in ANRIL gene extron subarea, i.e. the Nucleotide of the 300th in the Nucleotide of the 269th and the sequence shown in SEQ ID NO:2 in sequence shown in SEQ ID NO:1;
Step 2, adopt polymerase chain reaction-ligase chain reaction (LCR) (PCR-LDR) Protocols in Molecular Biology detect detect in sample
aNRILwhether gene extron subarea exists the type of single nucleotide polymorphism and mononucleotide polymorphic.
Wherein, in step 2, polymerase chain reaction system is: each sample carries out two totals and amasss amplified reaction into 20ul system, amplify the sequence shown in SEQ ID NO:1 and SEQ ID NO:2 respectively, and comprise: prepared genomic dna diluent 1ul, 2mM dNTP 2ul, 3mM MgCl
20.6ul, the corresponding each 1.0ul of forward and reverse amplimer, 1 × Buffer 2ul, the Taq archaeal dna polymerase 0.2ul of 1 unit, ddH
2o 12.2ul;
The amplified reaction that polymerase chain reaction program is: sequence shown in SEQ ID NO:1 was 95 DEG C of denaturations 2 minutes, and 94 DEG C of sex change are after 30 seconds, carried out 40 50 DEG C annealing 1 point 30 seconds, and 65 DEG C extend the amplification cycles of 30 seconds, finally carry out 65 DEG C of 10 minutes fill; The amplified reaction of sequence shown in SEQ ID NO:2 was 95 DEG C of denaturations 2 minutes, and 94 DEG C of sex change are after 30 seconds, carried out 40 53 DEG C annealing 1 point 30 seconds, and 72 DEG C extend the amplification cycles of 30 seconds, finally carry out 72 DEG C of 10 minutes fill.
Wherein, in step 2, ligase chain reaction (LCR) system comprises in total amount 10 μ L: PCR product 4ul, specific probe 1ul, 1 × Buffer 1ul, the ligase enzyme 0.05ul of 2 units, ddH
2o 4ul;
Ligase chain reaction (LCR) program is: react 95 DEG C of denaturations after 2 minutes, carries out 40 94 DEG C of sex change 15s, the circulating reaction of 50 DEG C of reaction 25s.
embodiment 3.
For detecting the primer of the mononucleotide polymorphic site in the ANRIL gene extron subarea relevant to myocardial infarction susceptible, this primer specifically comprises:
(1) amplimer of sequence shown in SEQ ID NO:1 (comprising rs10965215):
Forward primer: 5 '-GGATGTTTTGCAGGACTATT-3 ' (SEQ ID NO:3)
Reverse primer: 5 '-GGAATCATCACAGCATGGAC-3 ' (SEQ ID NO:4)
(2) amplimer of sequence shown in SEQ ID NO:2 (comprising rs10738605):
Forward primer: 5 '-TTTTCCAGTGGTGTTTCTAAATAA-3 ' (SEQ ID NO:5)
Reverse primer: 5 '-CCTCTGATGGTTTCTTTGGAGT-3 ' (SEQ ID NO:6).
embodiment 4.
For detecting the specific probe of the mononucleotide polymorphic site in the ANRIL gene extron subarea relevant to myocardial infarction susceptible, comprise following two group-specific probes:
First group:
Detect the FAM probe of the polymorphic site (rs10965215) in sequence shown in SEQ ID NO:1:
5’-GTGGCAAATAGTCCTGCAAAACATCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3’ (SEQ ID NO: 7)
Detect the G probe of the polymorphic site (rs10965215) in sequence shown in SEQ ID NO:1:
5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCTCTCTTGGAATCCTTTGAAATGTC-3’ (SEQ ID NO: 8)
Detect the A probe of the polymorphic site (rs10965215) in sequence shown in SEQ ID NO:1:
5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCTCTCTTGGAATCCTTTGAAATGTT-3’ (SEQ ID NO: 9)；
Second group:
Detect the FAM probe of the polymorphic site (rs10738605) in sequence shown in SEQ ID NO:2:
5’-CACACCTAACAGTGATGCTTGAACCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3’ (SEQ ID NO: 10)
Detect the C probe of the polymorphic site (rs10738605) in sequence shown in SEQ ID NO:2:
5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTACTGACTCGGGAAAGGATTCCAG-3’ (SEQ ID NO: 11)
Detect the G probe of the polymorphic site (rs10738605) in sequence shown in SEQ ID NO:2:
5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTACTGACTCGGGAAAGGATTCCAC-3’ (SEQ ID NO: 12)。
embodiment 5.
For detecting the test kit of the mononucleotide polymorphic site in the ANRIL gene extron subarea relevant to myocardial infarction susceptible, it comprises the primer of the mononucleotide polymorphic site for detecting the ANRIL gene extron subarea relevant to myocardial infarction susceptible of embodiment 3, and the specific probe of the mononucleotide polymorphic site for detecting the ANRIL gene extron subarea relevant to myocardial infarction susceptible of embodiment 4.
embodiment 6.
Whether the mononucleotide polymorphic site in the ANRIL gene extron subarea relevant to myocardial infarction susceptible has to the auxiliary diagnosis of myocardial infarction with to individuality the application that myocardial infarction susceptibility judges, should
aNRILthe mononucleotide polymorphic site in gene extron subarea is rs10965215 and rs10738605 of embodiment 1.
experiment
One,
the collection of blood sample and the extraction of genomic dna
The 286 parts of myocardial infarction samples collected from First People's Hospital of Foshan City and Subsidiary Hospital of Guangdong Medical College, 62.03 ± 12.04 years old mean age (standard deviation), wherein 222 examples are the male sex.Myocardial infarction diagnosis standard is with reference to International Society of Cardiology and WHO clinical name " name of ischemic heart disease and Case definition ", all patients are all according to typical clinical manifestation, specific ECG change and myocardium enzyme measurement of concetration find, and all underwent coronary radiography confirms.Control group is through heart ECT(heart radioisotope scanning), coronary artery CTA(CT imaging in coronary artery) or coronarography etc. inspection get rid of the individuality of myocardial infarction, have 646 parts, 61.58 ± 12.28 years old mean age (standard deviation), wherein 378 examples are the male sex.All myocardial infarction patients and normal control individuals are South China's Chinese Han Population of consanguinity-less relation, and get rid of congenital heart disease, myocardosis, major Liver and kidney disease, its essential characteristic is in table 1.
Extracting test kit (TIANGEN) after the above-mentioned blood preparation extraction genomic dna collected by TIANamp poba gene group, is 20ng/ul by unified for sample DNA dilution, as DNA profiling.
The essential characteristic of table 1 control group and myocardial infarction group
Wherein, in table 1 a for correcting age, sex, smoking, drink, the variable such as hypertension, diabetes and hyperlipidemia.
two, the somatotype of mononucleotide polymorphic site
The present invention adopts the single nucleotide polymorphism technology (PCR-LDR) based on polymerase chain reaction, right
aNRILits allelotrope point of the polymorphic site rs10965215(existed in gene extron subarea is to being G/A) and its allelotrope point of rs10738605(to being C/G) in case group and control group, carried out gene type.
PCR reaction is carried out on MJ PTC-200 instrument.
PCR reaction system and program as follows:
Each sample carries out two totals and amasss amplified reaction into 20ul system, amplifies the sequence shown in SEQ ID NO:1 and SEQ ID NO:2 respectively.The genomic dna diluent 1ul of preparation is comprised, 2mM dNTP 2ul, 3mM MgCl in system
20.6ul, the corresponding each 1.0ul of forward and reverse amplimer, 1 × Buffer 2ul, the Taq archaeal dna polymerase 0.2ul of 1 unit, ddH
2o 12.2ul.The amplified reaction of sequence shown in SEQ ID NO:1 was 95 DEG C of denaturations 2 minutes, and 94 DEG C of sex change are after 30 seconds, carried out 40 50 DEG C annealing 1 point 30 seconds, and 65 DEG C extend the amplification cycles of 30 seconds, finally carry out 65 DEG C of 10 minutes fill.The amplified reaction of sequence shown in SEQ ID NO:2 was 95 DEG C of denaturations 2 minutes, and 94 DEG C of sex change are after 30 seconds, carried out 40 53 DEG C annealing 1 point 30 seconds, and 72 DEG C extend the amplification cycles of 30 seconds, finally carry out 72 DEG C of 10 minutes fill.
The amplimer of sequence (comprising rs10965215) shown in SEQ ID NO:1:
Forward primer: 5 '-GGATGTTTTGCAGGACTATT-3 ' (SEQ ID NO:3)
Reverse primer: 5 '-GGAATCATCACAGCATGGAC-3 ' (SEQ ID NO:4)
The amplimer of sequence (comprising rs10738605) shown in SEQ ID NO:2:
Forward primer: 5 '-TTTTCCAGTGGTGTTTCTAAATAA-3 ' (SEQ ID NO:5)
Reverse primer: 5 '-CCTCTGATGGTTTCTTTGGAGT-3 ' (SEQ ID NO:6)
Be including of 80bp by Ligase detection reaction (LDR) method to the length that obtains of increasing
aNRILshown in SEQ ID NO:1, sequence and length are including of 181bp
aNRILpolymorphic site in the DNA fragmentation of sequence shown in SEQ ID NO:2 carries out gene type.Reaction system is 10ul, comprises PCR product 4ul, specific probe 1ul, 1 × Buffer 1ul, the ligase enzyme 0.05ul of 2 units, ddH
2o 4ul.Reaction after 2 minutes, carries out 40 94 DEG C of sex change 15s 95 DEG C of denaturations, the circulating reaction of 50 DEG C of reaction 25s.Shown in SEQ ID NO:1, the LDR fragment length of sequence is respectively 166bp and 168bp.Shown in SEQ ID NO:2, the LDR fragment length of sequence is respectively 178bp and 180bp.Product is through the order-checking of ABI 377 electrophoresis, and application of results GeneMapper 4.0 software carries out data analysis.As shown in Figure 1, LDR order-checking electrophoresis sectional drawing display rs10965215 single nucleotide polymorphism type AG genotype is bimodal, AA and GG genotype is unimodal.As shown in Figure 2, LDR order-checking electrophoresis sectional drawing display rs10738605 single nucleotide polymorphism Type C G genotype is bimodal, CC and GG genotype is unimodal.
Specific probe is as follows:
Detect the FAM probe of the polymorphic site (rs10965215) in sequence shown in SEQ ID NO:1:
5’-GTGGCAAATAGTCCTGCAAAACATCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3’ (SEQ ID NO: 7)
Detect the G probe of the polymorphic site (rs10965215) in sequence shown in SEQ ID NO:1:
5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCTCTCTTGGAATCCTTTGAAATGTC-3’ (SEQ ID NO: 8)
Detect the A probe of the polymorphic site (rs10965215) in sequence shown in SEQ ID NO:1:
5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCTCTCTTGGAATCCTTTGAAATGTT-3’ (SEQ ID NO: 9)
Detect the FAM probe of the polymorphic site (rs10738605) in sequence shown in SEQ ID NO:2:
5’-CACACCTAACAGTGATGCTTGAACCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3’ (SEQ ID NO: 10)
Detect the C probe of the polymorphic site (rs10738605) in sequence shown in SEQ ID NO:2:
5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTACTGACTCGGGAAAGGATTCCAG-3’ (SEQ ID NO: 11)
Detect the G probe of the polymorphic site (rs10738605) in sequence shown in SEQ ID NO:2:
5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTACTGACTCGGGAAAGGATTCCAC-3’ (SEQ ID NO: 12)。
The present invention is by right
aNRILsomatotype is carried out in the SNPs site (rs10965215, rs76521274, rs76184305, rs10738605 and rs78766516) that the gene extron subarea minimum gene frequency of Chinese han population is greater than 10%, finds that the G allelotrope of rs10965215 SNP and the C allelotrope of rs10738605 SNP all can increase the onset risk of individual myocardial infarction.
three,
aNRILthe correlation analysis of gene extron subarea mononucleotide polymorphic site rs10965215 and rs10738605 and myocardial infarction
Statistical method: utilize the genotype of goodness-of-fit χ2-test,chi-square test polymorphic site to distribute and whether meet Hardy-Weinberg balance.Use SPSS specialty statistics software, select chi square test to carry out statistical analysis to the genotype distribution frequency of polymorphic site in patient's group and control group sample.Add up the dependency of each polymorphic site and myocardial infarction with unconditional logistic regression analysis, and correct by the myocardial infarction such as hypertension, smoking Hazard Factor, calculate odds ratio (OR) and 95% fiducial interval (CI) of relative risk.Statistical significant difference level set is
p<0.05.
Statistics: rs10965215 and rs10738605 polymorphic site genotype and the loci distribution frequency of normal healthy controls group sample and patients with myocardial infarction group are as shown in table 2:
Rs10965215 and the rs10738605 polymorphic site genotype of table 2 normal healthy controls group sample and patients with myocardial infarction group and loci distribution frequency
Wherein, in table 2 a for correcting age, sex, smoking, drink, the variable such as hypertension, diabetes and hyperlipidemia.
According to table 2 distribution frequency, rs10965215 and rs10738605 all meets Hardy-Weinberg balance in the genotype frequency distribution of control group sample.As seen from Table 2,
aNRILthe G loci of Polymorphism site rs10965215, the distribution frequency significance (P=0.020) in myocardial infarction patient colony is greater than its distribution frequency in control group colony; The C loci of rs10738605, the distribution frequency significance (P=0.019) in myocardial infarction patient colony is greater than its distribution frequency in control group colony.Analyze rs10965215 further by dominant models to find, relative AA genotype individuals, being distributed in control group and case group of AG/GG genotype individuals have more marked difference (
p=0.030), wherein the danger of AG/GG genotype individuals trouble myocardial infarction is 1.45 times of AA genotype individuals.Analyze rs10738605 further by dominant models to find, relative GG genotype individuals, being distributed in control group and case group of GC/CC genotype individuals have more marked difference (
p=0.008), wherein the danger of GC/CC genotype individuals trouble myocardial infarction is 1.58 times of CC genotype individuals.
four, detection kit
Preparation detects the test kit of myocardial infarction susceptibility, wherein containing following oligonucleotide primer pair, includes for specific amplification from the genome sample of detected object
aNRILthe genomic fragment of sequence.
The amplimer of sequence (comprising rs10965215) shown in SEQ ID NO:1:
Forward primer: 5 '-GGATGTTTTGCAGGACTATT-3 ' (SEQ ID NO:3)
Reverse primer: 5 '-GGAATCATCACAGCATGGAC-3 ' (SEQ ID NO:4)
The amplimer of sequence (comprising rs10738605) shown in SEQ ID NO:2:
Forward primer: 5 '-TTTTCCAGTGGTGTTTCTAAATAA-3 ' (SEQ ID NO:5)
Reverse primer: 5 '-CCTCTGATGGTTTCTTTGGAGT-3 ' (SEQ ID NO:6)
This test kit also comprises two group-specific probes, for detecting the allelotype of rs10965215 and rs10738605 polymorphic site in sample.
Detect the FAM probe of the polymorphic site (rs10965215) in sequence shown in SEQ ID NO:1:
5’-GTGGCAAATAGTCCTGCAAAACATCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3’ (SEQ ID NO: 7)
Detect the G probe of the polymorphic site (rs10965215) in sequence shown in SEQ ID NO:1:
5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCTCTCTTGGAATCCTTTGAAATGTC-3’ (SEQ ID NO: 8)
Detect the A probe of the polymorphic site (rs10965215) in sequence shown in SEQ ID NO:1:
5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCTCTCTTGGAATCCTTTGAAATGTT-3’ (SEQ ID NO: 9)
Detect the FAM probe of the polymorphic site (rs10738605) in sequence shown in SEQ ID NO:2:
5’-CACACCTAACAGTGATGCTTGAACCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3’ (SEQ ID NO: 10)
Detect the C probe of the polymorphic site (rs10738605) in sequence shown in SEQ ID NO:2:
5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTACTGACTCGGGAAAGGATTCCAG-3’ (SEQ ID NO: 11)
Detect the G probe of the polymorphic site (rs10738605) in sequence shown in SEQ ID NO:2:
5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTACTGACTCGGGAAAGGATTCCAC-3’ (SEQ ID NO: 12)。
Finally should be noted that; above embodiment is only in order to illustrate technical scheme of the present invention; but not limiting the scope of the invention; although done to explain to the present invention with reference to preferred embodiment; those of ordinary skill in the art is to be understood that; can modify to technical scheme of the present invention or equivalent replacement, and not depart from essence and the scope of technical solution of the present invention.
gene order table
<110> Guangdong Medical College
<120> is relevant to myocardial infarction susceptible
aNRILthe detection method of gene extron subarea mononucleotide polymorphic site and application thereof
<160> 2
<210> SEQ ID NO: 1
<211> 533
<212> DNA
<213>
ANRIL
<400> SEQ ID NO: 1
GGTGACAAAACCATTGGTAGACAGTGGTTGAGAAACAGAATAGTCTCAGGATATCACTCCGTAGATTTATTCATTAATTAAAAAGAGAAAATGTGCTTTGAGAGAGAGAAAGCTATTACCGTCTTTATCAAATAGGAGAGCCTGATCATGTGTGGTCTGAAGTTTATCTAATGGGATTCCTGATGGAATGTTTAGTCTGAATCTAATCACATAGAGACTTGTCTGACAAATCCAGATTTTTTGGATGTTTTGCAGGACTATTTGCCAC
GACATTTCAAAGGATTCCAAGAGAGAATATTGGTGTCCATGCTGTGATGATTCCTCAGCTCCTCTCATCTGATCTCCGTCCTGGCCCCCATGACTTTCTTTGTGGTAGTTAGGGTGTGGTATGTGCCACTGAGGCCCACACCTATTGCTGTAAGTGCTGTTTGGGAAACCATCATCTTTCAGGTCTGTGTGATAAAAGAAGAGCCTTGGGGAAATGTTCTCTTCCAAATTTAATCTTTACATTATTAGAAAATATTTTGATGACC
<210> SEQ ID NO: 2
<211> 534
<212> DNA
<213>
ANRIL
<400> SEQ ID NO: 2
TGACAATGAGAAATATTTAATGGCAAACTTAGTCTTCTAATTTGAAAATGGAAATCATAACAGTTCTTGCCTCTTAGGGATAGTGTGAGACAAGTGAAATAATCCATGTAAGAGGTATAGTACTATGCTTGCCATTCTTTAAGAGCTCAACAAATATTCACTTTTTACCTATTAGTATCAATCTTAATTCTAAAATTCTATTATTTAATATTTTCCAGTGGTGTTTCTAAATAATATCTAATGACTAGGCTAATACACTATGTGGTTCTTCTAGGGTTCAAGCATCACTGTTAGGTGTG
CTGGAATCCTTTCCCGAGTCAGTACTGCTTTCTAGAAGAAAACCGGGGAGATCTATTTGGAATGTATCTAACTCCAAAGAAACCATCAGAGGTAACAGGTAGGAGATATGAAACGACCTTTTAGATATGAACCCTAATTGAATAAAAGTTGCCAAACAACTGTTCCCAAACATCTAAAGAAGAGTTTTAGTCTAAGTGGAATGGCTGGAGAGTATGGGAAGAGTTCTTTCCTACT
Claims (9)
1. relevant to myocardial infarction susceptible
aNRILgene, is characterized in that: described in
aNRILthe nucleotide sequence of gene has the sequence shown in SEQ ID NO:1 and SEQ ID NO:2;
Wherein, in the sequence shown in SEQ ID NO:1, the 269th is G;
In sequence shown in SEQ ID NO:2, the 300th is C.
2. the ANRIL gene relevant to myocardial infarction susceptible according to claim 1, is characterized in that: described in
aNRILthe mononucleotide polymorphic site in gene extron subarea is: rs10965215, and its allelotrope point is to being the Nucleotide of the 269th in the sequence shown in G/A(and SEQ ID NO:1) and described
aNRILthe mononucleotide polymorphic site in gene extron subarea is: rs10738605, and its allelotrope point is to being the Nucleotide of the 300th in the sequence shown in C/G(and SEQ ID NO:2);
Described mononucleotide polymorphic site rs10965215 with rs10738605 is relevant to myocardial infarction susceptible degree.
3. the detection method of the ANRIL gene extron subarea mononucleotide polymorphic site relevant to myocardial infarction susceptible described in claim 1-2 any one, is characterized in that: it comprises the following steps:
Step one, determines
aNRILthe mononucleotide polymorphic site (rs10965215 and rs10738605) in gene extron subarea, the i.e. Nucleotide of the 300th in the Nucleotide of the 269th and the sequence shown in SEQ ID NO:2 in sequence shown in SEQ ID NO:1;
Step 2, adopt polymerase chain reaction-ligase chain reaction (LCR) (PCR-LDR) Protocols in Molecular Biology detect detect in sample
aNRILwhether gene extron subarea exists the type of single nucleotide polymorphism and mononucleotide polymorphic.
4. the detection method of the ANRIL gene extron subarea mononucleotide polymorphic site relevant to myocardial infarction susceptible according to claim 3, it is characterized in that: in described step 2, described polymerase chain reaction system is: each described sample carries out two totals and amasss amplified reaction into 20ul system, amplify the sequence shown in SEQ ID NO:1 and SEQ ID NO:2 respectively, and comprise: prepared genomic dna diluent 1ul, 2mM dNTP 2ul, 3mM MgCl
20.6ul, the corresponding each 1.0ul of forward and reverse amplimer, 1 × Buffer 2ul, the Taq archaeal dna polymerase 0.2ul of 1 unit, ddH
2o 12.2ul;
The amplified reaction that described polymerase chain reaction program is: sequence shown in SEQ ID NO:1 was 95 DEG C of denaturations 2 minutes, 94 DEG C of sex change are after 30 seconds, carry out 40 50 DEG C annealing 1 point 30 seconds, 65 DEG C extend the amplification cycles of 30 seconds, finally carry out 65 DEG C of 10 minutes fill; The amplified reaction of sequence shown in SEQ ID NO:2 was 95 DEG C of denaturations 2 minutes, and 94 DEG C of sex change are after 30 seconds, carried out 40 53 DEG C annealing 1 point 30 seconds, and 72 DEG C extend the amplification cycles of 30 seconds, finally carry out 72 DEG C of 10 minutes fill.
5. the detection method of the ANRIL gene extron subarea mononucleotide polymorphic site relevant to myocardial infarction susceptible according to claim 3, it is characterized in that: in described step 2, described ligase chain reaction (LCR) system comprises in total amount 10 μ L: PCR product 4ul, specific probe 1ul, 1 × Buffer 1ul, the ligase enzyme 0.05ul of 2 units, ddH
2o 4ul;
Described ligase chain reaction (LCR) program is: react 95 DEG C of denaturations after 2 minutes, carries out 40 94 DEG C of sex change 15s, the circulating reaction of 50 DEG C of reaction 25s.
6. require the primer of the mononucleotide polymorphic site in the ANRIL gene extron subarea relevant to myocardial infarction susceptible described in 1-2 any one for test right, it is characterized in that: described primer specifically comprises:
(1) amplimer of sequence shown in SEQ ID NO:1 (comprising rs10965215):
Forward primer: 5 '-GGATGTTTTGCAGGACTATT-3 ' (SEQ ID NO:3)
Reverse primer: 5 '-GGAATCATCACAGCATGGAC-3 ' (SEQ ID NO:4)
(2) amplimer of sequence shown in SEQ ID NO:2 (comprising rs10738605):
Forward primer: 5 '-TTTTCCAGTGGTGTTTCTAAATAA-3 ' (SEQ ID NO:5)
Reverse primer: 5 '-CCTCTGATGGTTTCTTTGGAGT-3 ' (SEQ ID NO:6).
7. require the specific probe of the mononucleotide polymorphic site in the ANRIL gene extron subarea relevant to myocardial infarction susceptible described in 1-2 any one for test right, it is characterized in that: comprise following two group-specific probes:
First group:
Detect the FAM probe of the polymorphic site (rs10965215) in sequence shown in SEQ ID NO:1:
5’-GTGGCAAATAGTCCTGCAAAACATCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3’ (SEQ ID NO: 7)
Detect the G probe of the polymorphic site (rs10965215) in sequence shown in SEQ ID NO:1:
5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCTCTCTTGGAATCCTTTGAAATGTC-3’ (SEQ ID NO: 8)
Detect the A probe of the polymorphic site (rs10965215) in sequence shown in SEQ ID NO:1:
5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCTCTCTTGGAATCCTTTGAAATGTT-3’ (SEQ ID NO: 9)；
Second group:
Detect the FAM probe of the polymorphic site (rs10738605) in sequence shown in SEQ ID NO:2:
5’-CACACCTAACAGTGATGCTTGAACCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3’ (SEQ ID NO: 10)
Detect the C probe of the polymorphic site (rs10738605) in sequence shown in SEQ ID NO:2:
5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTACTGACTCGGGAAAGGATTCCAG-3’ (SEQ ID NO: 11)
Detect the G probe of the polymorphic site (rs10738605) in sequence shown in SEQ ID NO:2:
5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTACTGACTCGGGAAAGGATTCCAC-3’ (SEQ ID NO: 12)。
8. for detecting the test kit of the mononucleotide polymorphic site in the ANRIL gene extron subarea relevant to myocardial infarction susceptible, it is characterized in that: it comprises the primer of the mononucleotide polymorphic site for detecting the ANRIL gene extron subarea relevant to myocardial infarction susceptible according to claim 6, and the specific probe of the mononucleotide polymorphic site for detecting the ANRIL gene extron subarea relevant to myocardial infarction susceptible according to claim 7.
9. whether the mononucleotide polymorphic site in the ANRIL gene extron subarea relevant to myocardial infarction susceptible has to the auxiliary diagnosis of myocardial infarction with to individuality the application that myocardial infarction susceptibility judges, it is characterized in that: described in
aNRILthe mononucleotide polymorphic site in gene extron subarea is rs10965215 and rs10738605 according to claim 2.
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| CN102747072A (en) * | 2012-06-07 | 2012-10-24 | 中国医学科学院阜外心血管病医院 | Coronary heart disease susceptibility substantially-associated single nucleotide polymorphism (SNP) sites of susceptible region chr5p15, and applications thereof |
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