CN1519338A - Polymorphism of 39th exon in PCNT gene - Google Patents

Abstract

A single nucleotide polymorphism (SNP) for the extron No.39 of PCNT gene and a method for analyzing the SNP and activity of said extron No.39 are disclosed. Said SNP is the A->G polymorphism at the site No.441 in its sequence shown by SEQ ID No.1 and can cause the change Gln(Q)->Arg(R) at site No.104 of coding protein, so changing protein activity.

Description

The 30 No. nine exon polymorphism of PCNT gene

Technical field

The present invention relates to the single nucleotide polymorphism of the 30 No. nine exon of PCNT gene.The invention still further relates to the method for the allelic mutation of analyzing the 30 No. nine exon of PCNT gene.

Background technology

(pericentrin PCNT) is a kind of biomass cells centrosome associated protein of high conservative to middle peripheral proteins.The investigator thinks, it is closely related with the forming process of centrosome microtubule tissue in the eukaryotic cell.(Cell?1994Feb?25；76(4)：639-50)。

Before the application, also there is not the relevant report of the 30 No. nine exon polymorphism of PCNT gene.

Summary of the invention

Purpose of the present invention just provides the polymorphism and the detection method thereof of the 30 No. nine exon of PCNT gene.

In a first aspect of the present invention, the method for the single nucleotide polymorphism of the 30 No. nine exon of a kind of people of detection PCNT gene is provided, it comprises step:

(a) determine the 441st Nucleotide in sequence shown in the SEQ ID NO:1 of the 30 No. nine exon of people PCNT 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 441st.

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 441st 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 IDNO:1 that contains the 30 No. nine exon of people PCNT gene the amplified production of the 441st 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 IDNO:1 that contains the 30 No. nine exon of people PCNT gene the 441st single nucleotide polymorphism in the sequence.

A kind of test kit comprises:

(1) primer of the 30 No. nine exon of specific amplification people PCNT gene is right,

(2) detect the 30 No. nine exon of amplified production and normal PCNT gene and compare the reagent that whether exists variation required, the 441st 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, have been found that in the coded protein sequence of the 30 No. nine exon of PCNT gene the 104th Gln (Q)-Arg (R) polymorphic (be last 441 A-of mRNA〉G polymorphism), finished the present invention on this basis.

According to normal people PCNT gene sequencing result, found this SNP.Find that by analysis this SNP causes the 104th amino acids by Gln (Q)-Arg (R).Because this is the amino acid whose displacement of different properties, therefore, can cause the 30 No. nine coded protein-active of exon of PCNT gene to change.

The cDNA sequence of the 30 No. nine exon of PCNT gene is listed in SEQ ID NO:1, and wherein open reading frame is the 131-472 position, and its coded aminoacid sequence is listed in SEQ ID NO:2.The genome sequence of the 30 No. nine exon of PCNT 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 30 No. nine exon of PCNT 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 30 No. nine exon relative disease of PCNT 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 30 No. nine exon of PCNT gene active for, can anyly contain the sample of the 30 No. nine exon of PCNT 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 30 No. nine exon of PCNT 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 '-tggctaatag ggtgcatttc a-3 ' (SEQ ID NO:3)

Antisense primer: 5 '-caccacccgg gacttctc-3 ' (SEQ ID NO:4)

The product of the 472bp that amplifies carries out dna sequencing behind the purifying.

Embodiment 2

The acquisition of SNP

The PCNT 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 PCNT gene-correlation disease susceptibility, and wherein, it is right to contain the following primer that amplifies 441 SNP:

Adopted primer is arranged: 5 '-tggctaatag ggtgcatttc a-3 ' (SEQ ID NO:3)

Antisense primer: 5 '-caccacccgg gacttctc-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 441 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.

SEQUENCE?LISTING

＜110〉Research Center of Shanghai Human Genome

＜120〉the 30 No. nine exon polymorphism of PCNT gene

<130>030115

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tggctaatag?ggtgcatttc?aaaaggtaga?attgctgggc?taaagtatat?aaacaaatgt 60

ggacaggaaa?accatgtaga?cacttttcct?cttgattcag?tgtctcccat?cgtatgtgtt 120

tgctgtctag?gag?ctg?cgg?gcg?tct?ttg?gag?aca?cag?cgt?gct?cag?agc 169

Glu?Leu?Arg?Ala?Ser?Leu?Glu?Thr?Gln?Arg?Ala?Gln?Ser

1 5 10

agt?cga?ctc?tgc?gtg?gca?ctg?aaa?cac?gag?cag?acg?gcc?aag?gac?aac 217

Ser?Arg?Leu?Cys?Val?Ala?Leu?Lys?His?Glu?Gln?Thr?Ala?Lys?Asp?Asn

15 20 25

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Leu?Gln?Lys?Glu?Leu?Arg?Ile?Glu?His?Ser?Arg?Cys?Glu?Ala?Leu?Leu

30 35 40 45

gct?cag?gag?cgg?agc?cag?ctc?tct?gag?ctc?cag?aag?gac?ctt?gcg?gct 313

Ala?Gln?GLu?Arg?Ser?Gln?Leu?Ser?Glu?Leu?Gln?Lys?Asp?Leu?Ala?Ala

50 55 60

gag?aag?agc?cgc?acc?ctg?gag?ctg?tca?gag?gcc?ttg?cgg?cac?gag?cgg 361

Glu?Lys?Ser?Arg?Thr?Leu?Glu?Leu?Ser?Glu?Ala?Leu?Arg?His?Glu?Arg

65 70 75

ctc?ctg?acc?gag?cag?ctg?agc?cag?agg?aca?cag?gag?gct?tgc?gtg?cac 409

Leu?Leu?Thr?Glu?Gln?Leu?Ser?Gln?Arg?Thr?Gln?Glu?Ala?Cys?Val?His

80 85 90

cag?gac?aca?cag?gcc?cat?cac?gct?ctg?ctg?cag?aag?ctg?aag?gag?gag 457

Gln?Asp?Thr?Gln?Ala?His?His?Ala?Leu?Leu?Gln?Lys?Leu?Lys?Glu?Glu

95 100 105

aag?tcc?cgg?gtg?gtg 472

Lys?Ser?Arg?Val?Val

110

<210>2

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Glu?Leu?Arg?Ala?Ser?Leu?Glu?Thr?Gln?Arg?Ala?Gln?Ser?Ser?Arg?Leu

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Cys?Val?Ala?Leu?Lys?His?Glu?Gln?Thr?Ala?Lys?Asp?Asn?Leu?Gln?Lys

20 25 30

Glu?Leu?Arg?Ile?Glu?His?Ser?Arg?Cys?Glu?Ala?Leu?Leu?Ala?Gln?Glu

35 40 45

Arg?Ser?Gln?Leu?Ser?Glu?Leu?Gln?Lys?Asp?Leu?Ala?Ala?Glu?Lys?Ser

50 55 60

Arg?Thr?Leu?Glu?Leu?Ser?Glu?Ala?Leu?Arg?His?Glu?Arg?Leu?Leu?Thr

65 70 75 80

Glu?Gln?Leu?Ser?Gln?Arg?Thr?Gln?Glu?Ala?Cys?Val?His?Gln?Asp?Thr

85 90 95

Gln?Ala?His?His?Ala?Leu?Leu?Gln?Lys?Leu?Lys?Glu?Glu?Lys?Ser?Arg

100 105 110

Val?Val

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＜213〉artificial sequence

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＜223〉primer

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caccacccgg?gacttctc 18

Claims (7)

1. method that detects the single nucleotide polymorphism of the 30 No. nine exon of people PCNT gene is characterized in that it comprises step:

(a) determine the 441st Nucleotide in sequence shown in the SEQ ID NO:1 of the 30 No. nine exon of people PCNT 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 441st.

3. an isolating nucleic acid is characterized in that, it has the sequence shown in the SEQ ID NO:1, and the 441st 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 30 No. nine exon of people PCNT gene the amplified production of the 441st 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 30 No. nine exon of people PCNT gene the 441st single nucleotide polymorphism in the sequence.

6. diagnostic kit is characterized in that it comprises:

(1) primer of the 30 No. nine exon of specific amplification people PCNT gene is right,

(2) detect the 30 No. nine exon of amplified production and normal PCNT gene and compare the reagent that whether exists variation required, the 441st 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.