CN1519374A - Polymorphism of 19th exon in TRPC7 gene - Google Patents

Abstract

A single nucleotide polymorphism (SNP) for the extron No.19 of TRPC7 gene and a method for analyzing the SNP and activity of said extron No.19 are disclosed. Said SNP is A->G polymorphism at the site No.232 in its sequence shown by SEQ ID No.1 and can cause the change Ile(I)->Val(V) at the site No.4 of coding protein, so changing protein activity.

Description

The ten No. nine exon polymorphism of TRPC7 gene

Technical field

The present invention relates to the single nucleotide polymorphism of the ten No. nine exon of TRPC7 gene.The invention still further relates to the method for the allelic mutation of analyzing the ten No. nine exon of TRPC7 gene.

Background technology

TRPC7 (transient receptor potential-related channels) is the calciphorin of high expression level in a kind of human brain.This albumen has 7 to wear the film functional zone.This proteic TRPC7 gene of encoding has 32 exons.The investigator thinks, this gene very likely is the candidate gene of some genetic diseasess such as hereditary hearing impairment, Knobloch syndromes or the like.(Genomics?1998?Nov?15；54(1)：124-31)

Before the application, also there is not the relevant report of the ten No. nine exon polymorphism of TRPC7 gene.

Summary of the invention

Purpose of the present invention just provides the polymorphism and the detection method thereof of the ten No. nine exon of TRPC7 gene.

In a first aspect of the present invention, the method for the single nucleotide polymorphism of the ten No. nine exon of a kind of people of detection TRPC7 gene is provided, it comprises step:

(a) determine the 232nd Nucleotide in sequence shown in the SEQ ID NO:1 of the ten No. nine exon of people TRPC7 gene;

(b) detect whether there is single nucleotide polymorphism in described position.

In a preference, described single nucleotide polymorphism is to have G at the 232nd.

In a second aspect of the present invention, a kind of isolating nucleic acid is provided, it has the sequence shown in the SEQ ID NO:1, and the 232nd is G.

In a third aspect of the present invention, a kind of allele specific nucleic acid primer is provided, its length is 15-50bp, and hybridizes specifically and amplify shown in the SEQ ID NO:1 that contains the ten No. nine exon of people TRPC7 gene the amplified production of the 232nd single nucleotide polymorphism in the sequence.

In a fourth aspect of the present invention, a kind of allele specific oligonucleotide probe is provided, its length is 15-50bp, and hybridizes specifically and detect shown in the SEQ ID NO:1 that contains the ten No. nine exon of people TRPC7 gene the 232nd single nucleotide polymorphism in the sequence.

A kind of test kit comprises:

(1) primer of the ten No. nine exon of specific amplification people TRPC7 gene is right,

(2) detect the ten No. nine exon of amplified production and normal TRPC7 gene and compare the reagent that whether exists variation required, the 232nd single nucleotide polymorphism in the sequence shown in the described SEQ ID NO:1.

Another kind of diagnostic kit comprises: the oligonucleotide probe that the primer that the present invention is above-mentioned and/or the present invention are above-mentioned.

Embodiment

The inventor is by extensive and deep research, had been found that in the coded protein sequence of the ten No. nine exon of TRPC7 gene the 4th Ile (I)-Val (V) polymorphic (be last 232 A-of mRNA〉G polymorphism), finished the present invention on this basis.

According to normal people TRPC7 gene sequencing result, found this SNP.Find that by analysis this SNP causes the 4th amino acids by Ile (I)-Val (V).Because this is the amino acid whose displacement of different properties, therefore, can cause the ten No. nine coded protein-active of exon of TRPC7 gene to change.

The cDNA sequence of the ten No. nine exon of TRPC7 gene is listed in SEQ ID NO:1, and wherein open reading frame is the 223-375 position, and its coded aminoacid sequence is listed in SEQ ID NO:2.The genome sequence of the ten No. nine exon of TRPC7 gene can obtain from GenBank.

Those skilled in the art will appreciate that a large amount of analytical technologies can be used for detecting whether the site exists single nucleotide polymorphism described in the ten No. nine exon of TRPC7 gene.These technology comprise (but being not limited to): dna sequencing, sequencing by hybridization; Enzymatic mispairing cutting, heteroduple analysis, dot blot, oligonucleotide arrays (DNA chip), Mini-sequencing, Taqman technology, molecular beacon etc., sex change high performance liquid chromatography (DHPLC).

On the other hand, detection method of the present invention is used to assess the individual susceptibility of suffering from the ten No. nine exon relative disease of TRPC7 gene.

Being used for specimen of the present invention and being not particularly limited, for detecting SNP, can be DNA or the mRNA that extracting goes out from samples such as blood, tissue.For detect the ten No. nine exon of TRPC7 gene active for, can anyly contain the sample of the ten No. nine exon of TRPC7 gene, as blood etc.

The primer and the probe that are used for the inventive method or detection kit design according to the cDNA sequence (sequence of SEQ ID NO:1) or the genome sequence of the ten No. nine exon of TRPC7 gene, and the synthetic technology of available routine is synthesized and got final product.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1

The extracting of gene and order-checking

Extract DNA with conventional phenol chloroform method from people's blood, concentration correction is used for conventional pcr amplification to 20ng/ul.Primer is:

Adopted primer is arranged: 5 '-acggttatgt ctgacccctc t-3 ' (SEQ ID NO:3)

Antisense primer: 5 '-ccagcctctc tcctgcttc-3 ' (SEQ ID NO:4)

The product of the 454bp that amplifies carries out dna sequencing behind the purifying.

Embodiment 2

The acquisition of SNP

The TRPC7 gene is directly checked order.Each sample sequence of measuring is compared, thereby draw the difference of sequence, obtain SNP.

Embodiment 3

Detection kit

Preparation one detects the detection kit of TRPC7 gene-correlation disease susceptibility, and wherein, it is right to contain the following primer that amplifies 232 SNP:

Adopted primer is arranged: 5 '-acggttatgt ctgacccctc t-3 ' (SEQ ID NO:3)

Antisense primer: 5 '-ccagcctctc tcctgcttc-3 ' (SEQ ID NO:4)

Amplified production and normal control are carried out stratographic analysis with sex change high performance liquid chromatograph (DHPLC), can detect 232 A-easily G type SNP.

All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

SEQCENCE?LISTING

＜110〉Research Center of Shanghai Human Genome

＜120〉the ten No. nine exon polymorphism of TRPC7 gene

<130>030115

<160>4

<170>PatentIn?version?3.1

<210>1

<211>454

<212>DNA

<213>Homo?sapiens

<220>

<221>CDS

<222>(223)..(375)

<223>

<400>1

acggttatgt?ctgacccctc?ttgctctttg?tggtgctagc?acaaagatgt?cggcagtcca 60

cgagggtgtg?ggggcagccc?caggactgct?cctgggagcc?tcctgcatgc?cgtgtctgtg 120

ctgtgagtgg?cagtgctgtc?tccaccgctg?ctgggcctgc?ctctgggccc?agtgagcatc 180

gggggccagg?agagtgtagc?ccacacaatc?tctgtcctgc?ag?atg?aag?gac?atc 234

Met?Lys?Asp?Ile

1

ttc?ttc?ttc?ctc?ttc?ctg?ctg?gct?gtg?tgg?gtg?gtg?tcc?ttc?ggg?gtg 282

Phe?Phe?Phe?Leu?Phe?Leu?Leu?Ala?Val?Trp?Val?Val?Ser?Phe?Gly?Val

5 10 15 20

gcc?aag?cag?gcc?atc?ctc?atc?cac?aac?gag?cgc?cgg?gtg?gac?tgg?ctg 330

Ala?Lys?Gln?Ala?Ile?Leu?Ile?His?Asn?Glu?Arg?Arg?Val?Asp?Trp?Leu

25 30 35

ttc?cga?ggg?gcc?gtc?tac?cac?tcc?tac?ctc?acc?atc?ttc?ggg?cag 375

Phe?Arg?Gly?Ala?Val?Tyr?His?Ser?Tyr?Leu?Thr?Ile?Phe?Gly?Gln

40 45 50

atcccgggct?acatcgacgg?taggagccgg?gcgccatggg?agctcgggtg?gtgctgccgg 435

gaagcaggag?agaggctgg 454

<210>2

<211>51

<212>PRT

<213>Homo?sapiens

<400>2

Met?Lys?Asp?Ile?Phe?Phe?Phe?Leu?Phe?Leu?Leu?Ala?Val?Trp?Val?Val

1 5 10 15

Ser?Phe?Gly?Val?Ala?Lys?Gln?Ala?Ile?Leu?Ile?His?Asn?Glu?Arg?Arg

20 25 30

Val?Asp?Trp?Leu?Phe?Arg?Gly?Ala?Val?Tyr?His?Ser?Tyr?Leu?Thr?Ile

35 40 45

Phe?Gly?Gln

50

<210>3

<211>21

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>3

acggttatgt?ctgacccctc?t 21

<210>4

<211>19

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>4

ccagcctctc?tcctgcttc 19

Claims (7)

1. method that detects the single nucleotide polymorphism of the ten No. nine exon of people TRPC7 gene is characterized in that it comprises step:

(a) determine the 232nd Nucleotide in sequence shown in the SEQ ID NO:1 of the ten No. nine exon of people TRPC7 gene;

(b) detect whether there is single nucleotide polymorphism in described position.

2. the method for claim 1 is characterized in that, described single nucleotide polymorphism is to have G at the 232nd.

3. an isolating nucleic acid is characterized in that, it has the sequence shown in the SEQ ID NO:1, and the 232nd is G.

4. allele specific nucleic acid primer, it is characterized in that, its length is 15-50bp, and hybridizes specifically and amplify shown in the SEQ ID NO:1 that contains the ten No. nine exon of people TRPC7 gene the amplified production of the 232nd single nucleotide polymorphism in the sequence.

5. an allele specific oligonucleotide probe is characterized in that, its length is 15-50bp, and hybridizes specifically and detect shown in the SEQ ID NO:1 that contains the ten No. nine exon of people TRPC7 gene the 232nd single nucleotide polymorphism in the sequence.

6. diagnostic kit is characterized in that it comprises:

(1) primer of the ten No. nine exon of specific amplification people TRPC7 gene is right,

(2) detect the ten No. nine exon of amplified production and normal TRPC7 gene and compare the reagent that whether exists variation required, the 232nd single nucleotide polymorphism in the sequence shown in the described SEQ ID NO:1.

7. a diagnostic kit is characterized in that it comprises: described primer of claim 4 and/or the described oligonucleotide probe of claim 5.