CN1519345A - Polynmorphism of third exon in B3GALT5 gene - Google Patents

Abstract

A single nucleotide polymorphism (SNP) for the extron No.3 of B3GALT5 gene and a method for analyzing the SNP and activity of said extron No.3 are disclosed. Said SNP is C->T polymorphism at the site No.13 in its sequence shown by SEQ ID No.1 and can cause the change Thr(T)->Met(M) at the site No.4 of coding protein, so changing protein activity.

Description

No. three exon polymorphism of B3GALT5 gene

Technical field

The present invention relates to the single nucleotide polymorphism of No. three exon of B3GALT5 gene.The invention still further relates to the method for the allelic mutation of analyzing No. three exon of B3GALT5 gene.

Background technology

SLe-a antigen is widely used as the tumor marker of cancer of the stomach, intestinal cancer and carcinoma of the pancreas.The first step of synthetic sLe-a just needs N-acetylglucosamine-β-1, and (N-acetylglucosamine-beta-1,3-galactosyltransferase is B3GALT) as catalyzer for the 3-galactosyltransferase.The B3GALT that contains 5 exons is called as B3GALT5, and the investigator thinks that it most possibly is exactly the synthetic antigenic enzyme of I type Louis in aforementioned cancer.(J.Biol.Chem.274：12499-12507，1999.)

Before the application, also there is not the relevant report of No. three exon polymorphism of B3GALT5 gene.

Summary of the invention

Purpose of the present invention just provides the polymorphism and the detection method thereof of No. three exon of B3GALT5 gene.

In a first aspect of the present invention, the method for the single nucleotide polymorphism of No. three exon of a kind of people of detection B3GALT5 gene is provided, it comprises step:

(a) determine the 13rd Nucleotide in sequence shown in the SEQ ID NO:1 of No. three exon of people B3GALT5 gene;

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

In a preference, described single nucleotide polymorphism is to have T at the 13rd.

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 13rd is T.

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 No. three exon of people B3GALT5 gene the amplified production of the 13rd 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 No. three exon of people B3GALT5 gene the 13rd single nucleotide polymorphism in the sequence.

A kind of test kit comprises:

(1) primer of No. three exon of specific amplification people B3GALT5 gene is right,

(2) detect No. three exon of amplified production and normal B3GALT5 gene and compare the reagent that whether exists variation required, the 13rd 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 B3GALT5 gene protein sequence that No. three exon is coded the 4th Thr (T)-Met (M) polymorphic (be last 13 C-of mRNA〉T polymorphism), finished the present invention on this basis.

According to normal people B3GALT5 gene sequencing result, found this SNP.Find that by analysis this SNP causes the 4th amino acids by Thr (T)-Met (M).Because this is the amino acid whose displacement of different properties, therefore, can cause the B3GALT5 gene protein-active that No. three exon is coded to change.

The cDNA sequence of No. three exon of B3GALT5 gene is listed in SEQ ID NO:1, and wherein open reading frame is the 3-386 position, and its coded aminoacid sequence is listed in SEQ ID NO:2.The genome sequence of No. three exon of B3GALT5 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 No. three exon of B3GALT5 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 No. three exon relative disease of B3GALT5 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 No. three exon of B3GALT5 gene active for, can anyly contain the sample of No. three exon of B3GALT5 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 No. three exon of B3GALT5 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 '-ggaaagagag gatggtgaag g-3 ' (SEQ ID NO:3)

Antisense primer: 5 '-tacctgtccc acggatattc a-3 ' (SEQ ID NO:4)

The product of the 388bp that amplifies carries out dna sequencing behind the purifying.

Embodiment 2

The acquisition of SNP

The B3GALT5 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 B3GALT5 gene-correlation disease susceptibility, and wherein, it is right to contain the following primer that amplifies 13 SNP:

Adopted primer is arranged: 5 '-ggaaagagag gatggtgaag g-3 ' (SEQ ID NO:3)

Antisense primer: 5 '-tacctgtccc acggatattc a-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 13 C-easily T 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〉No. three exon polymorphism of B3GALT5 gene

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1 5 10 15

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50 55 60

ttt?tgt?cct?cag?gcg?gcg?ttt?gtg?atg?aaa?aca?gac?tca?gac?atg?ttc 239

Phe?Cys?Pro?Gln?Ala?Ala?Phe?Val?Met?Lys?Thr?Asp?Ser?Asp?Met?Phe

65 70 75

atc?aat?gtt?gac?tat?ctg?act?gaa?ctg?ctt?ctg?aag?aaa?aac?aga?aca 287

Ile?Asn?Val?Asp?Tyr?Leu?Thr?Glu?Leu?Leu?Leu?Lys?Lys?Asn?Arg?Thr

80 85 90 95

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100 105 110

cag?cca?ttc?agc?aag?tgg?ttt?gtc?agt?aaa?tct?gaa?tat?ccg?tgg?gac 383

Gln?Pro?Phe?Ser?Lys?Trp?Phe?Val?Ser?Lys?Ser?Glu?Tyr?Pro?Trp?Asp

115 120 125

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Arg

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Gly?Thr?Thr?Ser?Ser?Ala?Ala?Glu?Thr?Lys?Glu?Val?Asp?Gln?Glu?Ser

20 25 30

Gln?Arg?His?Gly?Asp?Ile?Ile?Gln?Lys?Asp?Phe?Leu?Asp?Val?Tyr?Tyr

35 40 45

Asn?Leu?Thr?Leu?Lys?Thr?Met?Met?Gly?Ile?Glu?Trp?Val?His?Arg?Phe

50 55 60

Cys?Pro?Gln?Ala?Ala?Phe?Val?Met?Lys?Thr?Asp?Ser?Asp?Met?Phe?Ile

65 70 75 80

Asn?Val?Asp?Tyr?Leu?Thr?Glu?Leu?Leu?Leu?Lys?Lys?Asn?Arg?Thr?Thr

85 90 95

Arg?Phe?Phe?Thr?Gly?Phe?Leu?Lys?Leu?Asn?Glu?Phe?Pro?Ile?Arg?Gln

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Claims (7)

1. method that detects the single nucleotide polymorphism of No. three exon of people B3GALT5 gene is characterized in that it comprises step:

(a) determine the 13rd Nucleotide in sequence shown in the SEQ ID NO:1 of No. three exon of people B3GALT5 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 T at the 13rd.

3. an isolating nucleic acid is characterized in that, it has the sequence shown in the SEQ ID NO:1, and the 13rd is T.

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 No. three exon of people B3GALT5 gene the amplified production of the 13rd 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 No. three exon of people B3GALT5 gene the 13rd single nucleotide polymorphism in the sequence.

6. diagnostic kit is characterized in that it comprises:

(1) primer of No. three exon of specific amplification people B3GALT5 gene is right,

(2) detect No. three exon of amplified production and normal B3GALT5 gene and compare the reagent that whether exists variation required, the 13rd 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.