CN102296121A - Kit for detecting phenylketonuria by using DNA methylation process - Google Patents
Kit for detecting phenylketonuria by using DNA methylation process Download PDFInfo
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- CN102296121A CN102296121A CN2011102663161A CN201110266316A CN102296121A CN 102296121 A CN102296121 A CN 102296121A CN 2011102663161 A CN2011102663161 A CN 2011102663161A CN 201110266316 A CN201110266316 A CN 201110266316A CN 102296121 A CN102296121 A CN 102296121A
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Abstract
The invention belongs to the technical field of diagnosis of disorder of amino acid metabolism and particularly relates to a kit for detecting phenylketonuria by using a DNA methylation process. When the kit disclosed by the invention is used, through the determination of the expression condition of a gene by detecting the methylation condition of a promoter region of an argininosuccinate synthetase (ASS) gene and the measurement of in vivo arginine content, the probability of having phenylketonuria can be predicted in advance, so that prevention measures can be taken in time to reduce pain in patients with phenylketonuria.
Description
Technical field
The invention belongs to disorder of amino acid metabolism medical diagnosis on disease technical field, be specifically related to a kind of test kit that utilizes the method detection pku of dna methylation.
Background technology
Pku (PKU) is a kind of autosomal recessive hereditary diseases, relates to being positioned at the Phenylalanine hydroxylase genetic flaw of No. 12 karyomit(e) on long-armed, causes Phenylalanine hydroxylase loss of activity in the liver, causes the phenylalanine metabolic disturbance.Pku is about 1/11800 at China's sickness rate.Pku belongs to single gene inheritance disease in heredity, be worldwide distribution, incidence in the crowd is with area, ethnic and different, as Japan 1: 70000, the U.S. 1: 12000～1: 18000, find that by amino acid analysis phenylketonuria patient's blood phenylalanine content exceeds 15 times of normal peoples.Amino acid is the masonry that constitutes the life mansion, and the variation that amino acid is formed in the various body fluid of human body can directly reflect the health level and the nutritional status of body, and relevant with multiple pathologic process.
Clinical amino acid analysis 25 routine PKU patients serums of the present inventor and 85 routine normal human serums, find that PKU patients serum arginine content is (0.014 ± 0.008), the normal human serum arginine content is both obvious differences of (0.129 ± 0.014) data presentation (p＜0.001), prompting PKU patient arginine dyssynthesis.Liver is the vitals of urea cycle, and urea cycle claims " ornithine cycle " again.Be a kind of detoxifcation mode of body to ammonia.It is that the more highly toxic ammonia that the metabolism of body internal protein produces is converted into the urea of low toxicity that its important biomolecule is learned meaning, thereby excretes.Liver is the major organs that mammal generates urea, because the effect of afgininosuccinate synthetase (ASS) makes arginine be hydrolyzed to ornithine and urea.And afgininosuccinate synthetase is one of key enzyme of urea cycle.Be methylation state by the promoter region CpG island of further discovering PKU patient's afgininosuccinate synthetase gene, this also possible explanation be the reason of arginine dyssynthesis.The CpG island is usually located at promoter region or first exon district of gene.In the healthy people's gene group, the CpG site in the CpG island normally is in non-methylation state, and the CpG site outside the CpG island is then normally methylated.This methylated form can be stable in fissional process reservation.The variation of table genetics research genetic expression, this variation can be by reduction division heredity, but do not include the change of correlation gene dna sequence dna.Epigenetic is a kind of gene function variation that does not have dna sequence dna to change and can be hereditary.Methylating is a kind of important form of table genetic modification, is the important tie between genotype and phenotype.Dna methylation is subjected to the regulation and control of dna methylation transferring enzyme, and in the process of cell proliferation and differentiation, and the methylation state of gene is entailed the offspring.The methylated difference of eukaryotic dna plays an important role in the regulate process of genetic expression in a variety of forms.Along with the continuous development and progress of dna methylation research method, the human secret of just accelerating to explore dna methylation.Dna methylation makes progress in the research of diseases such as heredity.Methylate and become the focus of biological study.
Summary of the invention
The object of the present invention is to provide a kind of test kit that utilizes the method detection pku of dna methylation.
The present invention also aims to provide a kind of mark that utilizes the method detection pku of dna methylation.
The present invention also aims to provide the method for utilizing dna methylation to detect the using method of the test kit of pku.
A kind of test kit that utilizes the method detection pku of dna methylation comprises dNTP, 10xPCR buffer, MgCl
2, ddH
2O, Taq archaeal dna polymerase, standard blood DNA, primer MSP-R, primer MSP-L, primer UMSP-R, primer UMSP-L, primer BSP-R1, primer BSP-L1, primer BSP-R2, primer BSP-L2; The nucleotide sequence of described primer MSP-R is shown in SEQ ID No.1 in the sequence table, the nucleotide sequence of primer MSP-L is shown in SEQ ID No.2 in the sequence table, the nucleotide sequence of primer UMSP-R is shown in SEQ ID No.3 in the sequence table, the nucleotide sequence of primer UMSP-L is shown in SEQ ID No.4 in the sequence table, the nucleotide sequence of primer BSP-R1 is shown in SEQ ID No.5 in the sequence table, the nucleotide sequence of primer BSP-L1 is shown in SEQ ID No.6 in the sequence table, the nucleotide sequence of primer BSP-L2 is shown in SEQ ID No.7 in the sequence table, and the nucleotide sequence of primer BSP-R2 is shown in SEQ ID No.8 in the sequence table; Above-mentioned primer is used to do methylation status of PTEN promoter, and during PCR, the pairing scheme of primer is:
Primer MSP-R, MSP-L pairing, primer UMSP-R, UMSP-L pairing, primer BSP-R1, BSP-L1 pairing, primer BSP-R2, BSP-L2 pairing, be used to detect ASS gene (GeneBank NG_011542, afgininosuccinate synthetase gene) promoter zone methylation situation.
Described standard blood DNA is to take from genomic dna and the processing of process bisulfite that no pku healthy human blood extracts.
A kind of mark that utilizes the method detection pku of dna methylation, described mark is ASS gene (GeneBank NG_011542, the afgininosuccinate synthetase gene) methylated promoter region, this zone comprise one or more methylated CpG island; The promoter region nucleotide sequence of described ASS (GeneBank NG_011542, afgininosuccinate synthetase gene) gene methylation is shown in SEQ ID No.9, SEQ ID No.10 in the sequence table and SEQ ID No.11.
Utilize the method for dna methylation to detect the using method of the test kit of pku, comprise the steps:
(1) from blood sample, extracts genomic dna;
(2) genomic dna is handled through bisulfite, made unmethylated cytosine(Cyt) all change uridylic into;
(3) utilize described test kit of claim 1 and primer pairing scheme, with ddH
2O, standard blood DNA, the blood sample genomic dna of handling through bisulfite are template, carry out the methylation status of PTEN promoter reaction;
(4) detect PCR result with 1.5% agarose gel electrophoresis, if with ddH
2O, standard blood DNA are that the PCR of template does not have amplified band, and be that the pcr amplification of template has amplified band with the blood sample genomic dna of handling through bisulfite, and amplified band is consistent with the stripe size of prediction, thinks that then the blood sample that is detected is from the phenylketonuria patient.
Beneficial effect of the present invention: adopt test kit of the present invention, judge the expression of gene situation by the situation that methylates that detects the ASS gene promoter area, the measurement of arginine content in the ligand, and then can predict the P of pku in advance, in time take preventive measures, alleviate phenylketonuria patient's misery.
Description of drawings
Fig. 1 is an arginic content in phenylketonuria patient and the normal human serum.
Fig. 2 is an Ass gene promoter area dna methylation pcr amplification (MSP);
Among the figure, 1 is ddH
2O contrast, 2-3 are that normal human serum DNA, 4-7 are phenylketonuria patient's serum DNA.
Fig. 3 is an Ass gene promoter area dna methylation pcr amplification (BSP).
Fig. 4 is that zone, Ass gene CpG island is at normal people and phenylketonuria patient's BSP sequencing result;
Among the figure, white point is the non-site that methylates of CpG, and stain is the CpG site that methylates, and the phenylketonuria patient methylates the site+45;-118.
Embodiment
The present invention will be further described below in conjunction with the drawings and specific embodiments.
The unaccounted experimental procedure of following examples is with reference to " molecular cloning experiment guide " third edition, or with reference to the specification sheets of corresponding reagent box.
The Taq archaeal dna polymerase that following examples are used is available from invitrogen company, and coli strain TOP10 is available from Beijing TIANGEN company, and DNA extraction kit is available from Qiagen company, and dna methylation kit is available from ZYMO RESEARCH company.
The 17 routine phenylketonuria patients that select Shanxi Province healthcare hospital for women ﹠ children to provide are experimental subjects.At 8 months～10 years old age, according to examination of newborn infant diseases method, baby due 72h gets heel portion blood, when finding blood phenylalanine concentration＞120 μ mol/L (2mg/dL), is diagnosed as hyperphenylalaninemia, does not all have complication and other underlying diseases.Get blood after head of a family's agreement by school's announcement.The physique amount is all normal.The full-automatic amino acidanalyser of 835 types (Hitachi, Ltd), amino acid standard substance (Sigma company).Get on an empty stomach venous blood 1mL of phenylketonuria patient, 3000r/min 15min gets 1: 1 mixing of sulfosalicylic acid of serum 0.5mL and 5%, shake well, and whizzer 10000r/min, 10min gets supernatant liquor, directly carries out analysis of amino acids.
Analyze 17 routine phenylketonuria patient's serum and 38 routine normal human serums, The data SPSS 10.0 softwares take statistics to learn and handle, and the t check is adopted in the Excel mapping, the results are shown in Figure 1, the arginine content in phenylketonuria patient's serum significantly is lower than the content of normal human serum.
Embodiment 2 design of primers
(1) according to ASS gene (GeneBank NG_011542, afgininosuccinate synthetase gene) sequence, screen the target sequence zone that methylates, its base sequence is shown in SEQ ID No.9, SEQ ID No.10 and SEQ ID No.11.
(2) according to methylate target sequence and the specific methylation site shown in SEQ ID No.9, SEQ ID No.10 and the SEQ ID No.11, use " MethPrimer " software, the specific primer sequence that designs the PCR that methylates is as follows:
MSP-R：5’-TAGGAGGTAAGTTTTTCGAAGGAC-3’(SEQ?ID?No.1)；
MSP-L：5’-GATTCCTAACCCTAACCCGC-3’(SEQ?ID?No.2)；
UMSP-R：5’-GTTAGGAGGTAAGTTTTTTGAAGGAT-3’(SEQ?ID?No.3)；
UMSP-L：5’-CAATTCCTAACCCTAACCCA-3’(SEQ?ID?No.4)；
BSP-R1：5’-TATTAGAGGTTATGGTTGGGGAG-3’(SEQ?ID?No.5)；
BSP-L1：5’-CCCAAATCTCCATATAAAAACTTCA-3’(SEQ?ID?No.6)；
BSP-R2：5’-GGTTTTGGGGGTTGTAGAAGGTT-3’(SEQ?ID?No.7)；
BSP-L2：5’-AAAACCCCTTCCCTCCTACCTC-3’(SEQ?ID?No.8)。
The extraction of DNA and the modification that methylates in embodiment 3 blood samples
Extract each 200 μ L of normal people and phenylketonuria patient's heel portion blood, carry out DNA extraction according to Qiagen kit test kit specification sheets, ultraviolet spectrophotometer is measured A260, calculates content.
The DNA that extracts is carried out the modification that methylates (bisulfite processing) of DNA according to dna methylation kit test kit specification sheets, make unmethylated cytosine(Cyt) all change uridylic into through the bisulfite processing, methylated cytosine(Cyt) remains unchanged, unmethylated cytosine(Cyt) all is converted into uridylic, and methylated cytosine(Cyt) is constant.
Embodiment 4 methylation status of PTEN promoter
The DNA and the ddH2O that extract and handle through bisulfite with no pku human blood are contrast template, and the DNA that phenylketonuria patient's blood extracts and handles through bisulfite is a template, adopt following proposal to do methylation status of PTEN promoter:
Primer MSP-R, MSP-L pairing, primer UMSP-R, UMSP-L pairing detects the situation that methylates of ASS gene promoter area, and reaction system is as follows:
The PCR reaction process is: 94 ℃, and 3min; 94 ℃ then, 30s; 55 ℃, 30s; 72 ℃, 60s reacts 32 circulations; 72 ℃ are extended 10min.
Amplified production is through 1.0% (massfraction) sepharose (0.5mg/L ethidium bromide) electrophoresis (as shown in Figure 2), primer MSP-R, MSP-L amplified production length are 116bp (SEQ ID No.9), and UMSP-R, UMSP-L amplified production length are 119bp (SEQ ID No.10).
Primer BSP-R1, BSP-L1 pairing, primer BSP-R2, BSP-L2 pairing carrying out nest-type PRC detects the situation that methylates of ASS gene promoter area, and reaction divides two-wheeled to carry out:
First round PCR, primer is BSP-R1, BSP-L1, reaction system is as follows:
The PCR reaction process is: 95 ℃, and 3min; 94 ℃ then, 30s; 62 ℃, 30s; 72 ℃, 60s reacts 32 circulations; 72 ℃ are extended 5min.
Amplified production is through 1.0% (massfraction) sepharose (0.5mg/L ethidium bromide) electrophoresis (as shown in Figure 3), and amplified production length is 950bp (SEQ ID No.11).
Second takes turns PCR, and primer is BSP-R2, BSP-L2, and template is 100 times of first round amplified production dilutions, and it is as follows to get 1 μ l reaction system:
The PCR reaction process is: 95 ℃, and 3min; 94 ℃ then, 30s; 62 ℃, 30s; 72 ℃, 60s reacts 32 circulations; 72 ℃ are extended 5min.
Amplified production is through 1.0% (massfraction) sepharose (0.5mg/L ethidium bromide) electrophoresis, and amplified production length is 721bp (SEQ ID No.12).
Embodiment 4 PCR product TA clone, bacterium transform and cloning and sequencing
Carry out according to Trans GenPpEASY2TVector test kit explanation that carrier connects and bacterium transforms, blue hickie screening positive clone and be template with hickie bacterium liquid utilizes universal primer to carry out pcr amplification, the segmental positive colony of screening insertion purpose.
PCR reaction system: 2.0 μ l, 10 * damping fluid, each 0.5 μ l of 0.5 μ l dNTP, upstream and downstream primer 10 μ mol/l, 0.25 μ l DNA Taq enzyme, 14.25 μ l ddH2O and 2 μ l templates.
PCR reaction conditions: 94 ℃ of pre-sex change 3min, 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 60s, 32 circulations; 72 ℃ are extended 10min.Amplified production is through 1.0% (massfraction) sepharose (0.5mg/L ethidium bromide) electrophoresis.Send the order-checking of TIANGEN biochemical technology (Beijing) company limited with positive colony bacterium liquid 100 μ l.
Sequencing result as shown in Figure 4, white point is the non-site that methylates of CpG, stain is the CpG site that methylates, the phenylketonuria patient methylates the site+45;-118.
Claims (4)
1. a test kit that utilizes the method detection pku of dna methylation comprises dNTP, 10xPCR buffer, MgCl
2, ddH
2O, Taq archaeal dna polymerase is characterized in that, also comprise standard blood DNA, primer MSP-R, primer MSP-L, primer UMSP-R, primer UMSP-L, primer BSP-R1, primer BSP-L1, primer BSP-R2, primer BSP-L2; The nucleotide sequence of described primer MSP-R is shown in SEQ ID No.1 in the sequence table, the nucleotide sequence of primer MSP-L is shown in SEQ ID No.2 in the sequence table, the nucleotide sequence of primer UMSP-R is shown in SEQ ID No.3 in the sequence table, the nucleotide sequence of primer UMSP-L is shown in SEQ ID No.4 in the sequence table, the nucleotide sequence of primer BSP-R1 is shown in SEQ ID No.5 in the sequence table, the nucleotide sequence of primer BSP-L1 is shown in SEQ ID No.6 in the sequence table, the nucleotide sequence of primer BSP-L2 is shown in SEQ ID No.7 in the sequence table, and the nucleotide sequence of primer BSP-R2 is shown in SEQ ID No.8 in the sequence table; Above-mentioned primer is used to do methylation status of PTEN promoter, and during PCR, the pairing scheme of primer is:
Primer MSP-R, MSP-L pairing, primer UMSP-R, UMSP-L pairing, primer BSP-R1, BSP-L1 pairing, primer BSP-R2, BSP-L2 pairing, be used to detect ASS gene (GeneBank NG_011542, afgininosuccinate synthetase gene) promoter zone methylation situation.
2. according to the described a kind of test kit that utilizes the method detection pku of dna methylation of claim 1, it is characterized in that described standard blood DNA is to take from genomic dna and the processing of process bisulfite that no pku healthy human blood extracts.
3. a method of utilizing dna methylation detects the mark of pku, it is characterized in that, described mark is the methylated promoter region of ASS gene (GeneBank NG_011542, afgininosuccinate synthetase gene), and this zone comprises one or more methylated CpG island; The promoter region nucleotide sequence of described ASS (GeneBank NG_011542, afgininosuccinate synthetase gene) gene methylation is shown in SEQ ID No.9, SEQ ID No.10 in the sequence table and SEQ ID No.11.
4. the described method of utilizing dna methylation of claim 1 detects the using method of the test kit of pku, it is characterized in that, comprises the steps:
(1) from blood sample, extracts genomic dna;
(2) genomic dna is handled through bisulfite, made unmethylated cytosine(Cyt) all change uridylic into;
(3) utilize described test kit of claim 1 and primer pairing scheme, with ddH
2O, standard blood DNA, the blood sample genomic dna of handling through bisulfite are template, carry out the methylation status of PTEN promoter reaction;
(4) detect PCR result with 1.5% agarose gel electrophoresis, if with ddH
2O, standard blood DNA are that the PCR of template does not have amplified band, and be that the pcr amplification of template has amplified band with the blood sample genomic dna of handling through bisulfite, and amplified band is consistent with the stripe size of prediction, thinks that then the blood sample that is detected is from the phenylketonuria patient.
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| CN2011102663161A CN102296121A (en) | 2011-09-09 | 2011-09-09 | Kit for detecting phenylketonuria by using DNA methylation process |
| CN201110449861.4A CN102424856B (en) | 2011-09-09 | 2011-12-29 | Kit for detecting phenylketonuria by use of DNA methylation method |
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| CN2011102663161A CN102296121A (en) | 2011-09-09 | 2011-09-09 | Kit for detecting phenylketonuria by using DNA methylation process |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102608329A (en) * | 2012-03-08 | 2012-07-25 | 郭健 | Maternal amniotic fluid phenylalanine detection method for phenylketonuria (PKU) antenatal diagnosis |
| CN110699449A (en) * | 2019-11-21 | 2020-01-17 | 深圳市龙华区人民医院 | Application of SYNPO gene methylation in asthenospermia diagnostic agent and kit |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6605432B1 (en) * | 1999-02-05 | 2003-08-12 | Curators Of The University Of Missouri | High-throughput methods for detecting DNA methylation |
| CN1187456C (en) * | 2001-11-27 | 2005-02-02 | 暨南大学 | CpG insular methylation test reagent kit and its application |
-
2011
- 2011-09-09 CN CN2011102663161A patent/CN102296121A/en not_active Withdrawn
- 2011-12-29 CN CN201110449861.4A patent/CN102424856B/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102608329A (en) * | 2012-03-08 | 2012-07-25 | 郭健 | Maternal amniotic fluid phenylalanine detection method for phenylketonuria (PKU) antenatal diagnosis |
| CN110699449A (en) * | 2019-11-21 | 2020-01-17 | 深圳市龙华区人民医院 | Application of SYNPO gene methylation in asthenospermia diagnostic agent and kit |
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| Publication number | Publication date |
|---|---|
| CN102424856B (en) | 2014-04-02 |
| CN102424856A (en) | 2012-04-25 |
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Open date: 20120201 |