CN101153335A

CN101153335A - Primer group, probe or probe group, method and reagent box and micro-array for predicting alcohol degradation ability and hangover possibility

Info

Publication number: CN101153335A
Application number: CNA2007100960129A
Authority: CN
Inventors: 白象铉; 吴祉令; 金淑荣; 李贞男; 郑钟石
Original assignee: Samsung Electronics Co Ltd
Current assignee: Samsung Electronics Co Ltd
Priority date: 2006-09-27
Filing date: 2007-04-10
Publication date: 2008-04-02
Also published as: JP2008079604A; KR100818276B1

Abstract

The present invention provides a probe or probe group for expanding the oligonucleotide primer group of at least one target sequence selected from the acetaldehyde dehydrogenase 2(ALDH2)gene, cytochrome P4502E(CYP2E1)gene or alcohol dehydrogenase 2(ADH2)gene to hybridize with at least one target sequence selected form the ALDH2 gene, CYP2E1 gene or ADH2 gene, a tiny array fixed by the probe or the probe group, a method for forecasting the alcohol degradation capability and hangover by using the probe or the probe group, and a reagent kit containing the primer group and the probe or probe group for forecasting the alcohol degradation capability and hangover.

Description

Primer sets, probe or probe groups, method and test kit and the microarray of prediction alcohol degradation ability and hangover possibility

Technical background

The present invention relates to predict primer sets, probe or probe groups, method and the test kit of alcohol degradation ability and hangover possibility (hangover potential).More specifically, the present invention relates to the Oligonucleolide primers group that amplification is selected from least one target sequence of aldehyde dehydrogenase 2 (ALDH2), Cytochrome P450 2E1 (CYP2E1) and alcoholdehydrogenase 2 (ADH2), probe or probe groups with described at least one target sequence specific hybrid, fixed the microarray of described probe or probe groups, with the method for described probe or probe groups prediction alcohol degradation ability and hangover possibility with predict the test kit of alcohol degradation ability and hangover possibility with described primer sets and described probe or probe groups.

Association area is described

The main component of alcoholic beverage is an alcohol, and it is degraded to the known material acetaldehyde of being still drank after a night of causing by being present in alcoholdehydrogenase (ADH) in the health liver.Acetaldehyde is acetate and water (H by acetaldehyde dehydrogenase (ALDH) metabolism ₂O), discharge from health then.The Cytochrome P450 2E1 (CYP2E1) that is present in people's liver microsomes (microsome) is the enzyme of oxidation ethanol and acetaldehyde.Known CYP2E1 is taken in by long-term alcohol and induces, and therefore, thinks that it plays an important role in pure metabolism.

Single nucleotide polymorphism (SNP) is present in ADH2 gene, CYP2E1 gene and the ALDH2 gene.According to the common method that detects SNP, amplification SNP district and the district that is increased with Restriction Enzyme digestion.Yet this method has shortcoming, is that PCR and Restriction Enzyme react each all must carry out four times when measuring this four kinds of genotype of every increment, and all are analyzed to estimate and carry out.

Combination by CycleavePCR Core Kit and Cycleave people ALDH2 Typing Probe/PrimerSet (TaKaRa Code CY403) detects the SNP (487Glu (GAA) → Lys (AAA)) in the ALDH2 gene extron 12.Probe/primer sets is made up of primer sets and two probes that detect SNP, described primer sets be used to the increase zone of the pleomorphism site that comprises the ALDH2 gene, described probe promptly are used for the probe of the ROX mark that wild-type detects and are used for the probe of the FAM mark that mutant detects.

Except above-mentioned routine techniques, the primer sets that can predict alcohol degradation ability and hangover possibility was not disclosed.The probe that can predict alcohol degradation ability and hangover possibility was not disclosed yet.

Summary of the invention

The invention provides the primer sets that to predict alcohol degradation ability and hangover possibility.

The present invention also provides probe or the probe groups that can predict alcohol degradation ability and hangover possibility.

The present invention also provides the microarray of having fixed described probe or probe groups.

The present invention also provides the method with described primer sets prediction alcohol degradation ability and hangover possibility.

The present invention also provides the test kit that detects alcohol degradation ability and hangover possibility, and it comprises described primer sets and described probe or probe groups.

The accompanying drawing summary

Above-mentioned and other feature and advantage of the present invention are by to the detailed description of its exemplary embodiment with become more obvious with reference to the accompanying drawings, wherein:

Fig. 1 is the image that shows the results of hybridization of PCR product, described PCR product be to use the oligonucleotide probe that is fixed on according to an embodiment of the present invention on the chip and according to embodiments of the present invention primer sets obtain by PCR.

Summary of the invention

The invention provides the Oligonucleolide primers group that is selected from least one target sequence of aldehyde dehydrogenase 2 (ALDH2) gene, Cytochrome P450 2E1 (CYP2E1) gene and alcohol dehydrogenase 2 (ADH2) gene for amplification, described Oligonucleolide primers group comprises at least one the oligonucleotides group that is selected from lower group:

The oligonucleotides group, it comprises at least one oligonucleotides of selecting and at least one oligonucleotides of selecting from the group that another oligonucleotides forms from the group that oligonucleotides forms, oligonucleotides comprises the fragment such as at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:1 in the group that described oligonucleotides forms, and the oligonucleotides in the group that described another oligonucleotides forms comprises the fragment such as at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:2;

The oligonucleotides group, it comprises at least one oligonucleotides of selecting and at least one oligonucleotides of selecting from the group that another oligonucleotides forms from the group that oligonucleotides forms, oligonucleotides in the group that described oligonucleotides forms comprises the fragment such as at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:1, and the oligonucleotides in the group that described another oligonucleotides forms comprises the fragment such as at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:2;

The oligonucleotides group, it comprises at least one oligonucleotides of selecting and at least one oligonucleotides of selecting from the group that another oligonucleotides forms from the group that oligonucleotides forms, oligonucleotides in the group that described oligonucleotides forms comprises the fragment such as at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:3, and the oligonucleotides in the group that described another oligonucleotides forms comprises the fragment such as at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:4;

The oligonucleotides group, it comprises at least one oligonucleotides of selecting and at least one oligonucleotides of selecting from the group that another oligonucleotides forms from the group that oligonucleotides forms, oligonucleotides in the group that described oligonucleotides forms comprises the fragment such as at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:5, and the oligonucleotides in the group that described another oligonucleotides forms comprises the fragment such as at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:6;

The oligonucleotide group, it comprises at least one oligonucleotide selected and at least one oligonucleotide of selecting from the group that another oligonucleotide is formed from the group that oligonucleotide is formed, oligonucleotide in the group that described oligonucleotide is formed comprises the fragment as at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:7, and the oligonucleotide in the group that described another oligonucleotide is formed comprises the fragment as at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:8;

The oligonucleotide group, it comprises at least one oligonucleotide selected and at least one oligonucleotide of selecting from the group that another oligonucleotide is formed from the group that oligonucleotide is formed, oligonucleotide in the group that described oligonucleotide is formed comprises the fragment as at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:9, and the oligonucleotide in the group that described another oligonucleotide is formed comprises the fragment as at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:10;

The oligonucleotide group, it comprises at least one oligonucleotide selected and at least one oligonucleotide of selecting from the group that another oligonucleotide is formed from the group that oligonucleotide is formed, oligonucleotide in the group that described oligonucleotide is formed comprises the fragment as at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:11, and the oligonucleotide in the group that described another oligonucleotide is formed comprises the fragment as at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:12; With

The oligonucleotide group, it comprises at least one oligonucleotide selected and at least one oligonucleotide of selecting from the group that another oligonucleotide is formed from the group that oligonucleotide is formed, oligonucleotide in the group that described oligonucleotide is formed comprises the fragment as at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:13, and the oligonucleotide in the group that described another oligonucleotide is formed comprises the fragment as at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:14.

In the primer sets of the present invention, target sequence can be at least one target sequence that is selected from down group: the exon III district and the IX district of the exon XII district of ALDH2 gene, 5 '-control region of CYP2E1 gene and ADH2 gene.

Primer sets of the present invention can be to be used to increase the Oligonucleolide primers group in exon XII district of ALDH2 gene, and it comprises at least one the oligonucleotide group that is selected from down group:

The oligonucleotide group, it comprises at least one oligonucleotide selected and at least one oligonucleotide of selecting from the group that another oligonucleotide is formed from the group that oligonucleotide is formed, oligonucleotide in the group that described oligonucleotide is formed comprises the fragment as at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:1, and the oligonucleotide in the group that described another oligonucleotide is formed comprises the fragment as at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:2; With

The oligonucleotide group, it comprises at least one oligonucleotide selected and at least one oligonucleotide of selecting from the group that another oligonucleotide is formed from the group that oligonucleotide is formed, oligonucleotide in the group that described oligonucleotide is formed comprises the fragment as at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:3, and the oligonucleotide in the group that described another oligonucleotide is formed comprises the fragment as at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:4.

Of the present invention group can be to be used to increase the Oligonucleolide primers group of 5 '-control region of CYP2E1 gene, and it comprises at least one the oligonucleotide group that is selected from down group:

The oligonucleotide group, it comprises at least one oligonucleotide selected and at least one oligonucleotide of selecting from the group that another oligonucleotide is formed from the group that oligonucleotide is formed, oligonucleotide in the group that described oligonucleotide is formed comprises the fragment as at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:5, and the oligonucleotide in the group that described another oligonucleotide is formed comprises the fragment as at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:6; With

The oligonucleotide group, it comprises at least one oligonucleotide selected and at least one oligonucleotide of selecting from the group that another oligonucleotide is formed from the group that oligonucleotide is formed, oligonucleotide in the group that described oligonucleotide is formed comprises the fragment as at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:7, and the oligonucleotide in the group that described another oligonucleotide is formed comprises the fragment as at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:8.

Primer sets of the present invention can be the Oligonucleolide primers group that is used for the exon III district of amplifying ADH 2 genes, and it comprises at least one the oligonucleotide group that is selected from down group:

The oligonucleotide group, it comprises at least one oligonucleotide selected and at least one oligonucleotide of selecting from the group that another oligonucleotide is formed from the group that oligonucleotide is formed, oligonucleotide in the group that described oligonucleotide is formed comprises the fragment as at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:9, and the oligonucleotide in the group that described another oligonucleotide is formed comprises the fragment as at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:10; With

The oligonucleotide group, it comprises at least one oligonucleotide selected and at least one oligonucleotide of selecting from the group that another oligonucleotide is formed from the group that oligonucleotide is formed, oligonucleotide in the group that described oligonucleotide is formed comprises the fragment as at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:11, and the oligonucleotide in the group that described another oligonucleotide is formed comprises the fragment as at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:12.

Primer sets of the present invention can be the Oligonucleolide primers group that is used for the exon IX district of amplifying ADH 2 genes, it comprises the oligonucleotide group, described oligonucleotide group comprises at least one oligonucleotide selected and at least one oligonucleotide of selecting from the group that another oligonucleotide is formed from the group that oligonucleotide is formed, oligonucleotide in the group that described oligonucleotide is formed comprises the fragment as at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:13, and the oligonucleotide in the group that described another oligonucleotide is formed comprises the fragment as at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:14.

Primer sets of the present invention can be to be used to increase the Oligonucleolide primers group of at least one target sequence, described target sequence is selected from the exon XII district of ALDH2 gene, 5 '-control region of CYP2E1 gene and the exon III district and the IX district of ADH2 gene, described Oligonucleolide primers group comprises at least one the oligonucleotide group that is selected from down group oligonucleotide group: the oligonucleotide group, and it comprises the oligonucleotide and the oligonucleotide that has as the described nucleotide sequence of SEQ ID NO:2 that has as the described nucleotide sequence of SEQ ID NO:1; The oligonucleotide group, it comprises the oligonucleotide and the oligonucleotide that has as the described nucleotide sequence of SEQ ID NO:4 that has as the described nucleotide sequence of SEQ ID NO:3; The oligonucleotide group, it comprises the oligonucleotide and the oligonucleotide that has as the described nucleotide sequence of SEQID NO:6 that has as the described nucleotide sequence of SEQ ID NO:5; The oligonucleotide group, it comprises the oligonucleotide and the oligonucleotide that has as the described nucleotide sequence of SEQ ID NO:8 that has as the described nucleotide sequence of SEQ IDNO:7; The oligonucleotide group, it comprises the oligonucleotide and the oligonucleotide that has as the described nucleotide sequence of SEQ ID NO:10 that has as the described nucleotide sequence of SEQ ID NO:9; The oligonucleotide group, it comprises the oligonucleotide and the oligonucleotide that has as the described nucleotide sequence of SEQ ID NO:12 that has as the described nucleotide sequence of SEQ ID NO:11; With the oligonucleotide group, it comprises the oligonucleotide and the oligonucleotide that has as the described nucleotide sequence of SEQ ID NO:14 that has as the described nucleotide sequence of SEQ ID NO:13.

Primer sets of the present invention can be to be used to increase the exon XII district of ALDH2 gene, 5 '-control region and the exon III district of ADH2 gene and the Oligonucleolide primers group in IX district of CYP2E1 gene, this Oligonucleolide primers group comprises: the oligonucleotide group, and it comprises the oligonucleotide and the oligonucleotide that has as the described nucleotide sequence of SEQ ID NO:2 that has as the described nucleotide sequence of SEQ ID NO:1; The oligonucleotide group, it comprises the oligonucleotide and the oligonucleotide that has as the described nucleotide sequence of SEQ ID NO:4 that has as the described nucleotide sequence of SEQ ID NO:3; The oligonucleotide group, it comprises the oligonucleotide and the oligonucleotide that has as the described nucleotide sequence of SEQ IDNO:6 that has as the described nucleotide sequence of SEQ ID NO:5; The oligonucleotide group, it comprises the oligonucleotide and the oligonucleotide that has as the described nucleotide sequence of SEQ ID NO:8 that has as the described nucleotide sequence of SEQ ID NO:7; The oligonucleotide group, it comprises the oligonucleotide and the oligonucleotide that has as the described nucleotide sequence of SEQ ID NO:10 that has as the described nucleotide sequence of SEQ ID NO:9; The oligonucleotide group, it comprises the oligonucleotide and the oligonucleotide that has as the described nucleotide sequence of SEQ ID NO:12 that has as the described nucleotide sequence of SEQ ID NO:11; With the oligonucleotide group, it comprises the oligonucleotide and the oligonucleotide that has as the described nucleotide sequence of SEQ ID NO:14 that has as the described nucleotide sequence of SEQ ID NO:13.

For the gene of the metabolic key enzyme ADH2 of alcohol, some Aisa peoples (Oriental) have coding to have the atypia form (ADH2*2) of ADH2 gene of the enzyme of excellent activity, and this canonical form (ADH2*1) with the ADH2 gene is different.Can determine inherence (intrinsic) alcohol degradation ability in the individuality to the research of ADH2 gene expression characteristics, and according to the amount of drinking of genetype for predicting alcohol.

For in pure metabolism, help out and when ethanol exists inducible expression's enzyme CYP2E1, have polymorphism in 5 '-control region of CYP2E1 gene, and determine the expression level of CYP2E1 enzyme by the polymorphism of CYP2E1 gene.The canonical form of CYP2E1 gene (c1 gene) has shown low transcription speed.Yet the atypia form of CYP2E1 gene (c2 gene) is owing to high transcription speed has produced a lot of enzymes.CYP2E1 is about 10mM for the Km value of pure oxidation, and this is higher 10 times than ADH2.Therefore, known CYP2E1 only is responsible for 10% of the whole process of pure oxidation when hanging down determining alcohol, and is responsible for a big chunk of the whole process of pure oxidation when high determining alcohol.

Cause that for degraded the gene of the key enzyme ALDH2 of the material aldehyde of being still drank after a night, some Aisa peoples have the atypia form (ALDH2*2) of the ALDH2 gene of coding non-activity enzyme, this canonical form (ALDH2*1) with the ALDH2 gene is different.Can determine inherent alcohol degradation ability in the individuality to the research of ALDH2 gene expression characteristics, and according to the degree of genetype for predicting hangover possibility.

Term used herein " primer " refers to the single stranded oligonucleotide sequence with nucleic acid array complementation to be copied, and as the starting point of synthetic primer extension products.Determine that the length of primer and sequence are to be suitable for starting the synthetic of extension products.Preferably, primer is that about 5-50 Nucleotide is long.Can come suitably to determine the length and the sequence of primer according to the complicacy of target DNA or RNA and working conditions such as the temperature and the ionic strength of primer.

Among the present invention, based on the ADH2 and the CYP2E1 of known ability with degrading alcohol be responsible for the hereditary feature that degraded causes the key enzyme ALDH2 of the material aldehyde of being still drank after a night, design the exon XII district that to detect the ALDH2 gene, 5 '-control region and the exon III district of ADH2 gene and specificity (specific) primer sets in IX district of CYP2E1 gene, predicted alcohol degradation ability and hangover possibility.That is to say, two primer sets special have been designed to the exon XII district of ALDH2 gene, two primer sets special to 5 '-control region of CYP2E1 gene are to special two primer sets in the exon III district of ADH2 gene with to the special primer sets in exon IX district of ADH2 gene.

When carrying out PCR with primer sets of the present invention, target sequence district to be amplified is selected from the exon XII district of ALDH2 gene, 5 '-control region of CYP2E1 gene and the exon III district and the IX district of ADH2 gene.

Primer sets of the present invention comprises the sequence of prediction alcohol degradation ability and hangover possibility.The primer sets of exemplary embodiment sees the following form 1 according to the present invention.

Table 1: the primer sets of exemplary embodiment according to the present invention

The primer title	SEQ ID NO：	Explanation
The primer title	SEQ ID NO：	Explanation	ALDH2-fp1	1	The forward primer in the exon XII district of amplification ALDH2 gene
ALDH2-rp1	2	The reverse primer in the exon XII district of amplification ALDH2 gene	ALDH2-fp1	1
ALDH2-rp1	2		ALDH2-fp2	3	The forward primer in the exon XII district of amplification ALDH2 gene
ALDH2-rp2	4	The reverse primer in the exon XII district of amplification ALDH2 gene	ALDH2-fp2	3
ALDH2-rp2	4		CYP-fp1	5	The forward primer of 5 '-control region of amplification CYP2E1 gene
CYP-rp1	6	The reverse primer of 5 '-control region of amplification CYP2E1 gene	CYP-fp1	5
CYP-rp1	6		CYP-fp2	7	The forward primer of 5 '-control region of amplification CYP2E1 gene
CYP-rp2	8	The reverse primer of 5 '-control region of amplification CYP2E1 gene	CYP-fp2	7
CYP-rp2	8		ADH2-3-fp1	9	The forward primer in the exon III district of amplifying ADH 2 genes
ADH2-3-rp1	10	The reverse primer in the exon III district of amplifying ADH 2 genes	ADH2-3-fp1	9
ADH2-3-rp1	10		ADH2-3-fp2	11	The forward primer in the exon III district of amplifying ADH 2 genes
ADH2-3-rp2	12	The reverse primer in the exon III district of amplifying ADH 2 genes	ADH2-3-fp2	11
ADH2-3-rp2	12		ADH2-9-fp	13	The forward primer in the exon IX district of amplifying ADH 2 genes
ADH2-9-rp	14	The reverse primer in the exon IX district of amplifying ADH 2 genes	ADH2-9-fp	13

The primer sets of SEQ ID NO:1 and 2 primer sets and SEQ ID NO:3 and 4 be used to the to increase exon XII district of ALDH2 gene, the primer sets of SEQ ID NO:5 and 6 primer sets and SEQ ID NO:7 and 8 be used to increase 5 '-control region of CYP2E1 gene, the primer sets that the primer sets of SEQ ID NO:9 and 10 primer sets and SEQ ID NO:11 and 12 is used for the exon III district of amplifying ADH 2 genes and SEQ ID NO:13 and 14 is used for the exon IX district of amplifying ADH 2 genes.

In specification sheets, oligonucleotide as primer can comprise nucleotide analog, for example thiophosphoric acid (phosphorothioate), alkylthio phosphoric acid (alkylphosphorothioate) or peptide nucleic acid(PNA) or intercalating agent (intercalating agent).

The present invention also provide can with the oligonucleotide probe or the probe groups of at least one target sequence hybridization in the exon III district of 5 '-control region of the exon XII district that is selected from the ALDH2 gene, CYP2E1 gene and ADH2 gene and IX district, described oligonucleotide probe or probe groups are selected from down group:

Can with the oligonucleotide probe of the exon XII district of ALDH2 gene hybridization, it comprises at least one oligonucleotide that is selected from the group that oligonucleotide and complementary oligonucleotide thereof form, and the oligonucleotide in the group that described oligonucleotide and complementary oligonucleotide thereof are formed comprises as the fragment of at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:15 and the 11st Nucleotide of nucleotide sequence as described in comprising;

Can with the oligonucleotide probe of 5 '-control region of CYP2E1 gene hybridization, it comprises at least one oligonucleotide that is selected from the group that oligonucleotide and complementary oligonucleotide thereof form, and the oligonucleotide in the group that described oligonucleotide and complementary oligonucleotide thereof are formed comprises as the fragment of at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:17 and the 12nd Nucleotide of nucleotide sequence as described in comprising;

Can with the oligonucleotide probe of the exon III district of ADH2 gene hybridization, it comprises at least one oligonucleotide that is selected from the group that oligonucleotide and complementary oligonucleotide thereof form, and the oligonucleotide in the group that described oligonucleotide and complementary oligonucleotide thereof are formed comprises as the fragment of at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:19 and the 12nd Nucleotide of nucleotide sequence as described in comprising;

Can with the oligonucleotide probe of the exon IX district of ADH2 gene hybridization, it comprises at least one oligonucleotide that is selected from the group that oligonucleotide and complementary oligonucleotide thereof form, and the oligonucleotide in the group that described oligonucleotide and complementary oligonucleotide thereof are formed comprises as the fragment of at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:21 and the 12nd Nucleotide of nucleotide sequence as described in comprising; With

Can with the oligonucleotide probe of the exon IX district of ADH2 gene hybridization, it comprises at least one oligonucleotide that is selected from the group that oligonucleotide and complementary oligonucleotide thereof form, and the oligonucleotide in the group that described oligonucleotide and complementary oligonucleotide thereof are formed comprises as the fragment of at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:23 and the 13rd Nucleotide of nucleotide sequence as described in comprising.

Probe of the present invention or probe groups also can comprise at least one oligonucleotide that is selected from down group:

At least one oligonucleotide, it is selected from the group of being made up of oligonucleotide and complementary oligonucleotide thereof, and the oligonucleotide in the group that described oligonucleotide and complementary oligonucleotide thereof are formed comprises as the fragment of at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:16 and the 11st Nucleotide of nucleotide sequence as described in comprising;

At least one oligonucleotide, it is selected from the group of being made up of oligonucleotide and complementary oligonucleotide thereof, and the oligonucleotide in the group that described oligonucleotide and complementary oligonucleotide thereof are formed comprises as the fragment of at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:18 and the 12nd Nucleotide of nucleotide sequence as described in comprising;

At least one oligonucleotide, it is selected from the group of being made up of oligonucleotide and complementary oligonucleotide thereof, and the oligonucleotide in the group that described oligonucleotide and complementary oligonucleotide thereof are formed comprises as the fragment of at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:20 and the 12nd Nucleotide of nucleotide sequence as described in comprising;

At least one oligonucleotide, it is selected from the group of being made up of oligonucleotide and complementary oligonucleotide thereof, and the oligonucleotide in the group that described oligonucleotide and complementary oligonucleotide thereof are formed comprises as the fragment of at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:22 and the 12nd Nucleotide of nucleotide sequence as described in comprising; With

At least one oligonucleotide, it is selected from the group of being made up of oligonucleotide and complementary oligonucleotide thereof, and the oligonucleotide in the group that described oligonucleotide and complementary oligonucleotide thereof are formed comprises as the fragment of at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:24 and the 13rd Nucleotide of nucleotide sequence as described in comprising.

Probe of the present invention or probe groups can be can with the oligonucleotide probe or the probe groups of the exon XII district of ALDH2 gene hybridization, it comprises at least one oligonucleotide, described oligonucleotide is selected from the group of being made up of oligonucleotide and complementary oligonucleotide thereof, and the oligonucleotide in the group that described oligonucleotide and complementary oligonucleotide thereof are formed comprises as the fragment of at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:15 and the 11st Nucleotide of nucleotide sequence as described in comprising.

Probe of the present invention or probe groups can be can with the oligonucleotide probe or the probe groups of 5 '-control region of CYP2E1 gene hybridization, it comprises at least one oligonucleotide, described oligonucleotide is selected from the group of being made up of oligonucleotide and complementary oligonucleotide thereof, and the oligonucleotide in the group that described oligonucleotide and complementary oligonucleotide thereof are formed comprises as the fragment of at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:17 and the 12nd Nucleotide of nucleotide sequence as described in comprising.

Probe of the present invention or probe groups can be can with the oligonucleotide probe or the probe groups of the exon III district of ADH2 gene hybridization, it comprises at least one oligonucleotide, described oligonucleotide is selected from the group of being made up of oligonucleotide and complementary oligonucleotide thereof, and the oligonucleotide in the group that described oligonucleotide and complementary oligonucleotide thereof are formed comprises as the fragment of at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:19 and the 12nd Nucleotide of nucleotide sequence as described in comprising.

Probe of the present invention or probe groups can be can with the oligonucleotide probe or the probe groups of the exon IX district of ADH2 gene hybridization, it comprises at least one oligonucleotide, described oligonucleotide is selected from the group of being made up of oligonucleotide and complementary oligonucleotide thereof, and the oligonucleotide in the group that described oligonucleotide and complementary oligonucleotide thereof are formed comprises as the fragment of at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:21 and the 12nd Nucleotide of nucleotide sequence as described in comprising.

Probe of the present invention or probe groups can be can with the oligonucleotide probe or the probe groups of the exon IX district of ADH2 gene hybridization, it comprises at least one oligonucleotide, described oligonucleotide is selected from the group of being made up of oligonucleotide and complementary oligonucleotide thereof, and the oligonucleotide in the group that described oligonucleotide and complementary oligonucleotide thereof are formed comprises as the fragment of at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:23 and the 13rd Nucleotide of nucleotide sequence as described in comprising.

Probe of the present invention or probe groups can be can with the oligonucleotide probe or the probe groups of at least one target sequence hybridization, described target sequence is selected from down group: the exon III district and the IX district of the exon XII district of ALDH2 gene, 5 '-control region of CYP2E1 gene and ADH2 gene, and described oligonucleotide probe or probe groups comprise at least one oligonucleotide probe or the probe groups that is selected from down group:

Comprise and have as the oligonucleotide of the described nucleotide sequence of SEQ ID NO:15 and the oligonucleotide probe that can hybridize with the exon XII district of ALDH2 gene;

Comprise have as the oligonucleotide of the described nucleotide sequence of SEQ ID NO:17 and can with the oligonucleotide probe of 5 '-control region hybridization of CYP2E1 gene;

Comprise and have as the oligonucleotide of the described nucleotide sequence of SEQ ID NO:19 and the oligonucleotide probe that can hybridize with the exon III district of ADH2 gene;

Comprise and have as the oligonucleotide of the described nucleotide sequence of SEQ ID NO:21 and the oligonucleotide probe that can hybridize with the exon IX district of ADH2 gene; With

Comprise and have as the oligonucleotide of the described nucleotide sequence of SEQ ID NO:23 and the oligonucleotide probe that can hybridize with the exon IX district of ADH2 gene.

Probe of the present invention or probe groups can be can with 5 '-control region and the exon III district of ADH2 gene and the oligonucleotide probe or the probe groups of IX district hybridization of the exon XII district of ALDH2 gene, CYP2E1 gene, described oligonucleotide probe or probe groups comprise:

In probe of the present invention or probe groups, with in SEQ ID NO:15,17,19, the 21 and 23 relevant probes each specifically in conjunction with the exon XII district of ALDH2 gene, 5 '-control region and the exon III district of ADH2 gene and a district in the IX district of CYP2E1 gene, described district is present in and uses according to embodiments of the present invention primer sets by in the PCR product of pcr amplification.

In probe of the present invention or probe groups, be respectively mutant with SEQ ID NO:15,17,19,21 and 23 relevant probes with SEQ ID NO:16,18,20,22 and 24 relevant probes.Specifically, SEQ ID NO:15 and 16 the 11st Nucleotide are pleomorphism sites, and this pleomorphism site is G and be A in mutant (SEQ ID NO:16) in wild-type (SEQ ID NO:15).Therefore, when the exon XII district of ALDH2 gene is wild-type, this exon XII district's meeting with the relevant probe hybridization of SEQ ID NO:15, but the discord and the relevant probe hybridization of SEQ ID NO:16.If use primer sets according to embodiments of the present invention from the exon XII district amplification PCR products of individual ALDH2 gene with the relevant probe hybridization of SEQ ID NO:15, but discord and the relevant probe hybridization of SEQ ID NO:16, can determine that then this individuality has good acetaldehyde degradation capability, therefore less being still drank after a night.Based on this hybridization difference, can predict individual alcohol degradation ability and hangover possibility.

SEQ ID NO:17 and 18 the 12nd Nucleotide are pleomorphism sites, and this pleomorphism site is G and be C in mutant (SEQ ID NO:18) in wild-type (SEQ ID NO:17).Therefore, when 5 '-control region of CYP2E1 gene is wild-type, this 5 '-control region meeting with the relevant probe hybridization of SEQ ID NO:17, but the discord and the relevant probe hybridization of SEQ ID NO:18.If use primer sets according to embodiments of the present invention from 5 '-control region amplification PCR products of individual CYP2E1 gene with the relevant probe hybridization of SEQ ID NO:17, but discord and the relevant probe hybridization of SEQ ID NO:18 can determine that then this individuality has good alcohol degradation ability.Based on this hybridization difference, can predict individual alcohol degradation ability.

SEQ ID NO:19 and 20 the 12nd Nucleotide are pleomorphism sites, and this pleomorphism site is G and be A in mutant (SEQ ID NO:20) in wild-type (SEQ ID NO:19).Therefore, when the exon III district of ADH2 gene is wild-type, this exon III district's meeting with the relevant probe hybridization of SEQ ID NO:19, but the discord and the relevant probe hybridization of SEQ ID NO:20.If use primer sets according to embodiments of the present invention from the exon III district amplification PCR products of individual ADH2 gene with the relevant probe hybridization of SEQ ID NO:20, but discord and the relevant probe hybridization of SEQ ID NO:19, can determine that then this individuality has good alcohol degradation ability, because the mutant in the exon III district of ADH2 gene has better alcohol degradation ability than wild-type.Based on this hybridization difference, can predict individual alcohol degradation ability.

SEQ ID NO:21 and the 12nd Nucleotide of 22 and the 13rd Nucleotide of SEQ ID NO:23 and 24 are pleomorphism sites, and these pleomorphism sites are T and be G in mutant (SEQ ID NO:22 and 24) in wild-type (SEQ ID NO:21 and 23).Therefore, when the exon IX district of ADH2 gene is wild-type, this exon IX district's meeting with SEQ ID NO:21 or 23 relevant probe hybridizations, but the discord and SEQ ID NO:22 or 24 relevant probe hybridizations.If use primer sets according to embodiments of the present invention from the exon IX district amplification PCR products of individual ADH2 gene with SEQ ID NO:21 or 23 relevant probe hybridizations, but discord and SEQ ID NO:22 or 24 relevant probe hybridizations can determine that then this individuality has good alcohol degradation ability.Based on this hybridization difference, can predict individual alcohol degradation ability.

Individual alcohol degradation ability and the hangover possibility of the available prediction of probe groups according to embodiments of the present invention.The probe groups of exemplary embodiment of the present invention sees the following form 2.

Table 2: the probe groups of exemplary embodiment of the present invention

SEQ ID NO：	Probe	Wild-type/mutant	Target sequence
SEQ ID NO：	Probe	Wild-type/mutant	Target sequence	15	ALDH2-WP	Wild-type	The exon XII district of ALDH2 gene

16	ALDH2-MP	Mutant	The exon XII district of ALDH2 gene
16	ALDH2-MP	Mutant	The exon XII district of ALDH2 gene	17	CYP-WP	Wild-type	5 '-control region of CYP2E1 gene
18	CYP-MP	Mutant	5 '-control region of CYP2E1 gene	17	CYP-WP	Wild-type	5 '-control region of CYP2E1 gene
18	CYP-MP	Mutant	5 '-control region of CYP2E1 gene	19	ADH2-3-WP	Wild-type	The exon III district of ADH2 gene
20	ADH2-3-MP	Mutant	The exon III district of ADH2 gene	19	ADH2-3-WP	Wild-type	The exon III district of ADH2 gene
20	ADH2-3-MP	Mutant	The exon III district of ADH2 gene	21	ADH2-9-WP1	Wild-type	The exon IX district of ADH2 gene
22	ADH2-9-MP1	Mutant	The exon IX district of ADH2 gene	21	ADH2-9-WP1	Wild-type	The exon IX district of ADH2 gene
22	ADH2-9-MP1	Mutant	The exon IX district of ADH2 gene	23	ADH2-9-WP2	Wild-type	The exon IX district of ADH2 gene
24	ADH2-9-MP2	Mutant	The exon IX district of ADH2 gene	23	ADH2-9-WP2	Wild-type	The exon IX district of ADH2 gene

Term used herein " probe " refers to form with complementary strand target sequence hybridization the single-chain nucleic acid sequence of duplex molecule (heterozygote).

In the specification sheets, the oligonucleotide that is used as probe can comprise nucleotide analog, as thiophosphoric acid, alkylthio phosphoric acid or peptide nucleic acid(PNA), or intercalating agent.

The present invention also provides microarray, has wherein fixed the oligonucleotide probe or the probe groups of at least one embodiment of the present invention in substrate (substrate).

Term used herein " microarray " refers to be fixed on the high density arrays of suprabasil polynucleotide group.Here, every group of polynucleotide are the microarraies that are fixed in the prospective region of substrate.Microarray is well known in the art.At United States Patent (USP) 5,445, microarray is disclosed in 934 and 5,744,305, its open this paper that all incorporates into by reference.Used oligonucleotide probe or probe groups are as mentioned above in the microarray.

Term used herein " substrate " refers to and can keep hybridization characteristics and realize under the condition of low background hybridization and the substrate of oligonucleotide probe link coupled.Ground commonly used, described substrate can be droplet plate, film (for example nylon or soluble cotton), microballoon (pearl) or chip.Before nucleic acid probe being used for or being fixed in the substrate, can be with its modification so that probe stationary or raising hybridization efficiency.To the modification of nucleic acid probe can comprise homopolymeric tailing, with reactive functional groups such as aliphatic group, NH ₂Group, SH group or carboxyl coupling or with vitamin H, haptens or protein coupling.

The present invention also provides the method for prediction alcohol degradation ability and hangover possibility, and this method comprises:

Make at least one oligonucleotide probe or the probe groups of sample contact embodiment of the present invention, thereby make the target sequence and the probe sequence hybridization of sample; With

Hybridization degree between the target sequence of detection probes sequence and described sample.

Term used herein " hybridization " refers to that the combination of two complementary strands of nucleic acid is to form duplex molecule (heterozygote).

In the methods of the invention, hybridization can be carried out under high stringent hybridization condition.For example, high stringent hybridization condition can be to comprise equimolar Na ₂HPO ₄And NaH ₂PO ₄0.12M phosphate buffered saline buffer, 1mM EDTA and 0.02% sodium lauryl sulphate in, 65 ℃.

" severity " used herein is to be used for describing hybridization and the temperature of subsequent process and the term of solvent composition.Under high stringent hybridization condition, can form height homologous nucleic acid heterozygote.That is to say, can not form the insufficient heterozygote of complementary degree.Therefore, the severity of test conditions decision should be present between two nucleic acid chains the amount with the complementarity that forms heterozygote.Severity can be selected so that the stable difference maximization between the heterozygote of the heterozygote of probe-target and probe-non-target.

In the methods of the invention, sample can comprise use embodiment of the present invention primer sets as primer, and be derived from the PCR product that the DNA of blood, saliva etc. obtains by pcr amplification as template.DNA as template can be derived from blood, saliva etc., but the invention is not restricted to this.Also can use any sample that comprises gene according to the present invention.

Nucleic acid used in the inventive method can be selected from chromosomal DNA, cDNA and fragment thereof.

" PCR " used herein refers to the polymerase chain reaction, and be with polysaccharase from specificity in conjunction with the primer of target nucleic acid method to amplifying target nucleic acid.PCR is well known in the art and can uses commercially available test kit to carry out.The also available appropriate method known in the art of the amplification of target nucleic acid is carried out, described method for example ligase chain reaction (LCR), based on the amplification of nucleotide sequence, based on the amplification system of transcribing, strand displacement amplification, Q β replicative enzyme or other nucleic acid amplification method except that PCR.

In the inventive method, available detectable mark substance comes the labels targets sequence.For example, described mark substance can be fluorescent substance, phosphorescent material or radioactive substance, but the invention is not restricted to this.Preferably, described mark substance can be Cy-5 or Cy-3.5 ' when terminal primer with Cy-5 or Cy-3 mark came the amplified target sequence by PCR, available detectable mark substance came the labels targets sequence when using it.When carrying out PCR with radioactive substance, with radio isotope as ³²P or ³⁵S adds PCR solution, therefore with the labelled with radioisotope PCR product that mixes the PCR product.

In the methods of the invention, oligonucleotide probe or probe groups can be fixed in the substrate of microarray.Be fixed on suprabasil oligonucleotide probe of microarray or probe groups as mentioned above.

In the methods of the invention, the prediction to alcohol degradation ability and hangover possibility can make PCR product and the oligonucleotide probe or the probe groups hybridization of mark, and detect the signal of hybridizing the product generation and carry out by with detectable signal emitting material mark PCR product.Detectable signal can be optical signal or electrical signal, but the invention is not restricted to this.Optical active substance can be fluorescent substance or phosphorescent material.Fluorescent substance can be fluorescein, Cy-5 or Cy-3.The PCR product not mark or before or after hybridization with detectable signal emitting material mark.In the situation that the PCR product is not labeled, the hybridization between PCR product and oligonucleotide probe or the probe groups can be passed through electrical signal detection, but the invention is not restricted to this.

The present invention also provides the test kit of prediction alcohol degradation ability and hangover possibility, and it comprises the primer sets of embodiment of the present invention and the oligonucleotide probe or the probe groups of embodiment of the present invention.Test kit of the present invention can pass through the primer sets amplified target gene with embodiment of the present invention, and carries out the probe of amplified production and embodiment of the present invention or the hybridization between the probe groups, predicts individual alcohol degradation ability and hangover possibility.Test kit of the present invention can comprise the reagent that increases, and this reagent can comprise archaeal dna polymerase, dNTPs, damping fluid etc.Test kit of the present invention also can comprise the instruction manual that describes optimum reaction condition in detail.

Hereinafter, with reference to following embodiment the present invention is described more specifically.Following embodiment only for illustrative purposes rather than the intention limit the scope of the invention.

Embodiment

Embodiment 1: the primer of selecting to be used to predict alcohol degradation ability and hangover possibility

In embodiment 1, select to be used to predict the primer of alcohol degradation ability and hangover possibility.For this reason, obtain ALDH2 gene order, CYP2E1 gene order and ADH2 gene order from Genbank, and be used to predict the target sequence of alcohol degradation ability and hangover possibility with the DNASTAR program selection: the exon III district and the IX district of the exon XII district of ALDH2 gene, 5 '-control region of CYP2E1 gene and ADH2 gene.

The result, 7 groups of Oligonucleolide primers groups have been designed: have Oligonucleolide primers group as SEQ ID NO:1 and 2 described sequences, has Oligonucleolide primers group as SEQ ID NO:3 and 4 described sequences, has Oligonucleolide primers group as SEQ ID NO:5 and 6 described sequences, has Oligonucleolide primers group as SEQ ID NO:7 and 8 described sequences, has Oligonucleolide primers group as SEQ ID NO:9 and 10 described sequences, have as the Oligonucleolide primers group of SEQ ID NO:11 and 12 described sequences and have Oligonucleolide primers group as SEQ ID NO:13 and 14 described sequences.Each group in these Oligonucleolide primers groups can increase the exon XII district of ALDH2 gene, 5 '-control region and the exon III district of ADH2 gene and a district in the IX district of CYP2E1 gene.

Embodiment 2: the base that is used to predict alcohol degradation ability and hangover possibility with primer sets amplification of the present invention Cause

7 groups of primer sets with design among the embodiment 1 increase and are used to predict the gene of alcohol degradation ability and hangover possibility.

1) gDNA of extraction mouth cells

Collect saliva from each test subject, and extract gDNA from described saliva according to Qiagen DNA Mini Kit scheme.Then, with the described gDNA of Picogreen dsDNA reagent (molecular probe) quantitative analysis.For quantitative analysis, gDNA is carried out gel electrophoresis and palliating degradation degree and the nucleotide sequence of described gDNA are determined in order-checking (ABI3700).

2) target sequence amplification

With 1) in the gDNA of preparation increase by PCR in order to the primer of the embodiment 1 of Cy3 mark.Use 50 μ l reaction solns come the following PCR of carrying out, described reaction soln be by with 5 μ l 1) in the gDNA adding of the preparation mixture that comprises the Taq polysaccharase of forward and each 400nM of reverse primer, 200 μ M dNTP, 5 μ l10 * damping fluids and 1.25 units obtain: 95 ℃ of initial sex change in 120 seconds; 95 ℃ of sex change 20 seconds, 58 ℃ of annealing 20 seconds and 72 ℃ extend 20 seconds 35 take turns circulation; 72 ℃ of 180 seconds final extensions.Be used for chip then with PCR purification kit (Qiagen) purified pcr product.

Embodiment 3: detect the gene that uses primer sets amplification of the present invention on chip

In embodiment 3, allow PCR product that will obtain among the embodiment 2 and the probe hybridization that is fixed on the chip, measure the degree of probe-target hybridization and predict alcohol degradation ability and hangover possibility.

1) makes chip

For making chip, use SiO ₂Wafer (wafer) (thickness 1,000 ).Mixture with 20%3-aminopropyl ethoxy silane (Sigma) and 5%FC4430 applies described wafer, bakes 40 minutes in 120 ℃ in baking box, and cleans with tri-distilled water.

As the oligonucleotide probe solution that is ready to use in chip, by (80 μ M Bioneer) are dissolved in and prepare Nucleotide solution (ultimate density: 20 μ M) in 9mM PEG 10000 (Sigma) and 100% methane amide (Sigma) with the Nucleotide of amine-modification.Use microarray sample applicator (spotter) (Cartesian, pisys 5500) and SMP3 pin (pin) (Arrayit) described Nucleotide solution is imprinted on (print on) wafer, and be that insulation was fixed on Nucleotide on the wafer in 1 hour in the baking box of 70 ℃ of temperature, relative humidity (RH) 40% in condition setting.Thereby the described wafer of insulation is made chip in the 1-Methyl-2-Pyrrolidone solution of 50mM succinyl oxide then.

2) on chip, hybridize

With among the embodiment 2 amplification target DNA 94 ℃ of sex change 5 minutes, place immediately and obtain ssDNA on ice.Then, target DNA and 2 * hybridization buffer (12 * SSPET damping fluid) with 1: 1 mixed, and are applied to the gained mixture on the chip.Described chip is incubated 1 hour so that probe-target hybridization to take place at 48 ℃, clean stirring simultaneously in 5 minutes with lavation buffer solution I (3 * SSPET damping fluid), use lavation buffer solution II (1 * SSPET damping fluid) to clean then and stirred simultaneously in 5 minutes, dry 1 minute of 3000rpm.Then, obtain the intensity that image is also used Genepix 3.1 program measurement image with scanner (Axon).

3) test sample book classification

A group: 3 people, these people drink and flush (flushing) occurs behind one glass of soju and be still drank after a night

B group: 5 people, these people drink and change a day morning behind one bottle or the many bottles of soju and feel not bad

The C group: 3 people, these people flush occurs and are still drank after a night after drinking one bottle of soju

4) probe array on the chip

Shown in the probe array of point sample on chip sees the following form.

		Row
		Row								1	2	3	4	5	6	7	8
		OK	1	CYP-WP	CYP-WP	CYP-MP	CYP-MP	ALDH2- WP	ALDH2- WP	1	2	3	4	5	6	7	8	ALDH2- MP	ALDH2- MP
2	CYP-WP		1	CYP-WP	CYP-WP	CYP-MP	CYP-MP	ALDH2- WP	ALDH2- WP	CYP-WP	CYP-MP	CYP-MP	ALDH2- WP	ALDH2- WP	ALDH2- MP	ALDH2- MP		ALDH2- MP	ALDH2- MP
2	CYP-WP		3	ADH2-9 -WP2	ADH2-9 -WP2	ADH2-9 -MP2	ADH2-9 -MP2	ADH2-3 -WP	ADH2-3 -WP	CYP-WP	CYP-MP	CYP-MP	ALDH2- WP	ALDH2- WP	ALDH2- MP	ALDH2- MP	ADH2-3 -MP	ADH2-3 -MP
4	ADH2-9 -WP2		3	ADH2-9 -WP2	ADH2-9 -WP2	ADH2-9 -MP2	ADH2-9 -MP2	ADH2-3 -WP	ADH2-3 -WP	ADH2-9 -WP2	ADH2-9 -MP2	ADH2-9 -MP2	ADH2-3 -WP	ADH2-3 -WP	ADH2-3 -MP	ADH2-3 -MP	ADH2-3 -MP	ADH2-3 -MP

Fig. 1 shows probe that is fixed on the chip of the present invention and the figure that uses primer sets of the present invention by the PCR product results of hybridization of PCR acquisition.Among Fig. 1, last row is the result of A group, and middle row is the result of B group, and following row is the result of C group.The intensity of chip image shown in Figure 1 is summarised in the following table 3.

Table 3: the relative intensity of chip image

	S1	S2	S3	s4	S5	S6	S7	S8	S9	S10	S11
	S1	S2	S3	s4	S5	S6	S7	S8	S9	S10	S11	CYP	0.7	0.4	1.3	1.1	1.2	1.6	0.9	1.3	1.1	1.1	0.2
ALDH2	0.3	-1.2	0.4	1.8	2.0	2.1	1.8	2.2	0.7	0.5	0.4	CYP	0.7	0.4	1.3	1.1	1.2	1.6	0.9	1.3	1.1	1.1	0.2
ALDH2	0.3	-1.2	0.4	1.8	2.0	2.1	1.8	2.2	0.7	0.5	0.4	ADH2-3	0.6	-0.2	0.7	-0.2	-0.2	1.6	-0.9	-0.2	-0.1	-1.0	-0.3

In the last table 3,,, just mean that each gene is a wild type gene if wild-type surpasses 1 for the relative intensity (p) of mutant for CYP, ALDH2 and ADH2-3 gene.If p, means wild-type and mutant in 0 to 1 scope and exists with 1: 1 ratio.If it is mutated genes that p, means each gene less than 0.

For S2 individuality (person), ALDH2 is mutant (p＜0), and ADH2-3 is mutant (p＜0).From these results, can determine that the S2 individuality only drinks the smart beverage of a Glass Of Wine and will make drunk and occur immediately being still drank after a night.S4, S5, S7 and S8 individuality have shown the ALDH2 (p＞1.0) of wild-type and the ADH2-3 (p＜0) of mutant.From these results, can determine that S4, S5, S7 and S8 are individual even after having drunk, can not make drunk rapidly, and only can feel in the second day morning that any was still drank after a night more than one bottle soju.S9, S10 and S11 individuality have shown special-shaped (hetero-type) ALDH2 (0＜p＜1) and the ADH2-3 (p＜0) of mutant.This demonstration S9, S10 and S11 individuality can be felt to be still drank after a night after having drunk about one bottle of soju.

For individuality, we can say that they have the good capacity of drinking height alcoholic beverage (soju, whisky) with wild-type CYP (p＞0.5).

Therefore, available primer sets of the present invention and probe groups are predicted alcohol degradation ability and hangover possibility efficiently.

According to primer sets of the present invention and probe groups, can predict alcohol degradation ability and hangover possibility efficiently.

The method according to this invention can be predicted alcohol degradation ability and hangover possibility with high specificity.

Sequence table

＜110〉Samsung Electronics Co., Ltd (Samsung Electronics Co.Ltd.)

＜120〉primer sets, probe or probe groups, method and test kit and the microarray of prediction alcohol degradation ability and hangover possibility

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Claims

1. Oligonucleolide primers group, it is used at least one target sequence that amplification is selected from aldehyde dehydrogenase 2 (ALDH2) gene, Cytochrome P450 2E1 (CYP2E1) gene or alcoholdehydrogenase 2 (ADH2) gene, and described Oligonucleolide primers group comprises at least one the oligonucleotide group that is selected from down group:

The oligonucleotide group, it comprises at least one oligonucleotide selected and at least one oligonucleotide of selecting from the group that another oligonucleotide is formed from the group that oligonucleotide is formed, oligonucleotide in the group that described oligonucleotide is formed comprises the fragment as at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:1, and the oligonucleotide in the group that described another oligonucleotide is formed comprises the fragment as at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:2;

The oligonucleotide group, it comprises at least one oligonucleotide selected and at least one oligonucleotide of selecting from the group that another oligonucleotide is formed from the group that oligonucleotide is formed, oligonucleotide in the group that described oligonucleotide is formed comprises the fragment as at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:3, and the oligonucleotide in the group that described another oligonucleotide is formed comprises the fragment as at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:4;

The oligonucleotide group, it comprises at least one oligonucleotide selected and at least one oligonucleotide of selecting from the group that another oligonucleotide is formed from the group that oligonucleotide is formed, oligonucleotide in the group that described oligonucleotide is formed comprises the fragment as at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:5, and the oligonucleotide in the group that described another oligonucleotide is formed comprises the fragment as at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:6;

2. in the Oligonucleolide primers group of claim 1, wherein target sequence is the sequence that at least one is selected from down group: the exon III district and the IX district of the exon XII district of ALDH2 gene, 5 '-control region of CYP2E1 gene and ADH2 gene.

3. the Oligonucleolide primers group of claim 1, it is to be used to increase the Oligonucleolide primers group in exon XII district of ALDH2 gene, it comprises at least one the oligonucleotide group that is selected from down group:

4. the Oligonucleolide primers group of claim 1, it is to be used to increase the Oligonucleolide primers group of 5 '-control region of CYP2E1 gene, it comprises at least one the oligonucleotide group that is selected from down group:

5. the Oligonucleolide primers group of claim 1, it is the Oligonucleolide primers group that is used for the exon III district of amplifying ADH 2 genes, it comprises at least one the oligonucleotide group that is selected from down group:

6. the Oligonucleolide primers group of claim 1, it is the Oligonucleolide primers group that is used for the exon IX district of amplifying ADH 2 genes, it comprises the oligonucleotide group, described oligonucleotide group comprises at least one oligonucleotide selected and at least one oligonucleotide of selecting from the group that another oligonucleotide is formed from the group that oligonucleotide is formed, oligonucleotide in the group that described oligonucleotide is formed comprises the fragment as at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQID NO:13, and the oligonucleotide in the group that described another oligonucleotide is formed comprises the fragment as at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:14.

7. the Oligonucleolide primers group of claim 1, it is to be used to increase that at least one is selected from down the Oligonucleolide primers group of the target sequence of group: the exon III district and the IX district of the exon XII district of ALDH2 gene, 5 '-control region of CYP2E1 gene and ADH2 gene, described Oligonucleolide primers group comprises at least one oligonucleotide group of selecting from the group that following oligonucleotide group is formed: the oligonucleotide group, and it comprises the oligonucleotide and the oligonucleotide that has as the described nucleotide sequence of SEQ ID NO:2 that has as the described nucleotide sequence of SEQ ID NO:1; The oligonucleotide group, it comprises the oligonucleotide and the oligonucleotide that has as the described nucleotide sequence of SEQ ID NO:4 that has as the described nucleotide sequence of SEQ ID NO:3; The oligonucleotide group, it comprises the oligonucleotide and the oligonucleotide that has as the described nucleotide sequence of SEQ ID NO:6 that has as the described nucleotide sequence of SEQ ID NO:5; The oligonucleotide group, it comprises the oligonucleotide and the oligonucleotide that has as the described nucleotide sequence of SEQID NO:8 that has as the described nucleotide sequence of SEQ ID NO:7; The oligonucleotide group, it comprises the oligonucleotide and the oligonucleotide that has as the described nucleotide sequence of SEQ ID NO:10 that has as the described nucleotide sequence of SEQ IDNO:9; The oligonucleotide group, it comprises the oligonucleotide and the oligonucleotide that has as the described nucleotide sequence of SEQ ID NO:12 that has as the described nucleotide sequence of SEQ ID NO:11; With the oligonucleotide group, it comprises the oligonucleotide and the oligonucleotide that has as the described nucleotide sequence of SEQ ID NO:14 that has as the described nucleotide sequence of SEQ ID NO:13.

8. the Oligonucleolide primers group of claim 1, it is to be used to increase the exon XII district of ALDH2 gene, 5 '-control region and the exon III district of ADH2 gene and the Oligonucleolide primers group in IX district of CYP2E1 gene, this Oligonucleolide primers group comprises: the oligonucleotide group, and it comprises the oligonucleotide and the oligonucleotide that has as the described nucleotide sequence of SEQ ID NO:2 that has as the described nucleotide sequence of SEQID NO:1; The oligonucleotide group, it comprises the oligonucleotide and the oligonucleotide that has as the described nucleotide sequence of SEQ ID NO:4 that has as the described nucleotide sequence of SEQ ID NO:3; The oligonucleotide group, it comprises the oligonucleotide and the oligonucleotide that has as the described nucleotide sequence of SEQ ID NO:6 that has as the described nucleotide sequence of SEQ ID NO:5; The oligonucleotide group, it comprises the oligonucleotide and the oligonucleotide that has as the described nucleotide sequence of SEQ ID NO:8 that has as the described nucleotide sequence of SEQ ID NO:7; The oligonucleotide group, it comprises the oligonucleotide and the oligonucleotide that has as the described nucleotide sequence of SEQ ID NO:10 that has as the described nucleotide sequence of SEQ ID NO:9; The oligonucleotide group, it comprises the oligonucleotide and the oligonucleotide that has as the described nucleotide sequence of SEQ ID NO:12 that has as the described nucleotide sequence of SEQ ID NO:11; With the oligonucleotide group, it comprises the oligonucleotide and the oligonucleotide that has as the described nucleotide sequence of SEQ ID NO:14 that has as the described nucleotide sequence of SEQ ID NO:13.

9. oligonucleotide probe or probe groups, it can be selected from down the target sequence hybridization of group with at least one: the exon III district and the IX district of the exon XII district of ALDH2 gene, 5 '-control region of CYP2E1 gene and ADH2 gene, and described oligonucleotide probe or probe groups are selected from down group:

10. the oligonucleotide probe of claim 9 or probe groups, it also comprises at least one oligonucleotide that is selected from down group:

Be selected from least one oligonucleotide of the group that oligonucleotide and complementary oligonucleotide thereof form, the oligonucleotide in the group that described oligonucleotide and complementary oligonucleotide thereof are formed comprises as the fragment of at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:16 and the 11st Nucleotide of nucleotide sequence as described in comprising;

Be selected from least one oligonucleotide of the group that oligonucleotide and complementary oligonucleotide thereof form, the oligonucleotide in the group that described oligonucleotide and complementary oligonucleotide thereof are formed comprises as the fragment of at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:18 and the 12nd Nucleotide of nucleotide sequence as described in comprising;

Be selected from least one oligonucleotide of the group that oligonucleotide and complementary oligonucleotide thereof form, the oligonucleotide in the group that described oligonucleotide and complementary oligonucleotide thereof are formed comprises as the fragment of at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:20 and the 12nd Nucleotide of nucleotide sequence as described in comprising;

Be selected from least one oligonucleotide of the group that oligonucleotide and complementary oligonucleotide thereof form, the oligonucleotide in the group that described oligonucleotide and complementary oligonucleotide thereof are formed comprises as the fragment of at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:22 and the 12nd Nucleotide of nucleotide sequence as described in comprising; With

Be selected from least one oligonucleotide of the group that oligonucleotide and complementary oligonucleotide thereof form, the oligonucleotide in the group that described oligonucleotide and complementary oligonucleotide thereof are formed comprises as the fragment of at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:24 and the 13rd Nucleotide of nucleotide sequence as described in comprising.

11. the oligonucleotide probe of claim 9 or probe groups, its be can with the oligonucleotide probe or the probe groups of the exon XII district of ALDH2 gene hybridization, it comprises at least one oligonucleotide, this oligonucleotide is selected from the group that oligonucleotide and complementary oligonucleotide thereof are formed, and the oligonucleotide in the group that described oligonucleotide and complementary oligonucleotide thereof are formed comprises as the fragment of at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:15 and the 11st Nucleotide of nucleotide sequence as described in comprising.

12. the oligonucleotide probe of claim 9 or probe groups, its be can with the oligonucleotide probe or the probe groups of 5 '-control region of CYP2E1 gene hybridization, it comprises at least one oligonucleotide, this oligonucleotide is selected from the group that oligonucleotide and complementary oligonucleotide thereof are formed, and the oligonucleotide in the group that described oligonucleotide and complementary oligonucleotide thereof are formed comprises as the fragment of at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:17 and the 12nd Nucleotide of nucleotide sequence as described in comprising.

13. the oligonucleotide probe of claim 9 or probe groups, its be can with the oligonucleotide probe or the probe groups of the exon III district of ADH2 gene hybridization, it comprises at least one oligonucleotide, this oligonucleotide is selected from the group that oligonucleotide and complementary oligonucleotide thereof are formed, and the oligonucleotide in the group that described oligonucleotide and complementary oligonucleotide thereof are formed comprises as the fragment of at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:19 and the 12nd Nucleotide of nucleotide sequence as described in comprising.

14. the oligonucleotide probe of claim 9 or probe groups, its be can with the oligonucleotide probe or the probe groups of the exon IX district of ADH2 gene hybridization, it comprises at least one oligonucleotide, this oligonucleotide is selected from the group that oligonucleotide and complementary oligonucleotide thereof are formed, and the oligonucleotide in the group that described oligonucleotide and complementary oligonucleotide thereof are formed comprises as the fragment of at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:21 and the 12nd Nucleotide of nucleotide sequence as described in comprising.

15. the oligonucleotide probe of claim 9 or probe groups, its be can with the oligonucleotide probe or the probe groups of the exon IX district of ADH2 gene hybridization, it comprises at least one oligonucleotide, this oligonucleotide is selected from the group that oligonucleotide and complementary oligonucleotide thereof are formed, and the oligonucleotide in the group that described oligonucleotide and complementary oligonucleotide thereof are formed comprises as the fragment of at least 10 continuous nucleotides that exist in the described nucleotide sequence of SEQ ID NO:23 and the 13rd Nucleotide of nucleotide sequence as described in comprising.

16. the oligonucleotide probe of claim 9 or probe groups, its be can with the oligonucleotide probe or the probe groups of at least one target sequence hybridization, described target sequence is selected from down group: the exon III district and the IX district of the exon XII district of ALDH2 gene, 5 '-control region of CYP2E1 gene and ADH2 gene, and described oligonucleotide probe or probe groups comprise at least one oligonucleotide probe or the probe groups that is selected from down group:

Comprise and have as the oligonucleotide of the described nucleotide sequence of SEQ ID NO:19 and the oligonucleotide probe that can hybridize with the exon III district of ADH2 gene, its;

17. the oligonucleotide probe of claim 9 or probe groups, its be can with 5 '-control region and the exon III district of ADH2 gene and the oligonucleotide probe or the probe groups of IX district hybridization of the exon XII district of ALDH2 gene, CYP2E1 gene, described oligonucleotide probe or probe groups comprise:

18. microarray, the oligonucleotide probe or the probe groups of wherein in substrate, having fixed in the claim 9 to 17 each.

19. the method for prediction alcohol degradation ability and hangover possibility, this method comprises:

Make in the sample contact claim 9 to 17 each oligonucleotide probe or probe groups, thereby the target sequence of this sample and probe sequence are hybridized; With

Detect the hybridization degree of the target sequence of this probe sequence and described sample.

20. the method for claim 19, wherein said sample comprise primer sets that right to use profit requires in 1 to 8 each as primer, and the DNA that is derived from blood or saliva is as template, the PCR product that obtains by PCR.

21. the method for claim 19, wherein said target sequence comes mark with detectable mark substance.

22. the method for claim 21, wherein said mark substance are fluorescent substance, phosphorescent material or radioactive substance.

23. the method for claim 19, wherein said oligonucleotide probe or probe groups are fixed in the substrate of microarray.

24. the test kit of prediction alcohol degradation ability and hangover possibility, this test kit comprise in the claim 1 to 8 each primer sets and claim 9 to 17 in each oligonucleotide probe or probe groups.