CN110387409A - A kind of detection method of folic acid metabolism related gene polymorphism - Google Patents
A kind of detection method of folic acid metabolism related gene polymorphism Download PDFInfo
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Abstract
The present invention provides a kind of detection methods of folic acid metabolism related gene polymorphism, comprising: extracts the genomic DNA of subject；Configure single base extension system and the polymerase chain reaction PCR amplification system comprising genomic DNA；PCR amplification system is carried out amplification reaction, then carries out digestion process, then mixture slaking treated system and single base extension system, and carries out extension, finally carries out purification process；Product after detecting purification process using liquid chromatograph-mass spectrometer, determines the genotype of the specific SNP site of genomic DNA；Wherein, the elution mobile phase of liquid chromatograph-mass spectrometer is the distilled water containing 0.5%~5% (v/v) hexafluoroisopropanol and 0.03%~0.5% (v/v) triethylamine, and contain the methanol of 0.5%~5% (v/v) hexafluoroisopropanol and 0.03%~0.5% (v/v) triethylamine, type of elution is gradient elution, and the mass spectrograph in liquid chromatograph-mass spectrometer is TripleTOF series mass spectrograph.This programme being capable of sensitive, the easy SNP site progress Genotyping to target gene.
Description
Technical field
The present invention relates to technical field of biological, in particular to a kind of detection side of folic acid metabolism related gene polymorphism
Method.
Background technique
Folic acid be human body cell growth, division and reproductive process in required substance.Studies have shown that folic acid is in embryonic development
What early stage occurred apparently plays an important role in genetic programming twice.Have now found that the key enzyme of folic acid metabolism approach is deposited
In several genes polymorphic site, and these gene pleiomorphisms affect the activity of codase, and the folic acid in serum is caused to occur
Change.
Currently, generalling use polymerase chain when detecting the metabolic capability of patient's body folic acid by specific SNP site
Formula reaction PCR- restriction fragment length polymorphism analysis method is detected.But this method detection sensitivity is not high and operating procedure
It is cumbersome.
Summary of the invention
The embodiment of the invention provides a kind of detection methods of folic acid metabolism related gene polymorphism, can be sensitive, easy
Genotyping is carried out to the SNP site of target gene.
The embodiment of the invention provides a kind of detection methods of folic acid metabolism related gene polymorphism, comprising:
Extract the genomic DNA of subject；
Single base extension of the configuration comprising Single base extension primer sequence shown in SEQ ID NO.1~SEQ ID NO.3
Reaction system, and configure comprising specificity amplification primer sequence and the gene shown in SEQ ID NO.4~SEQ ID NO.9
The polymerase chain reaction PCR amplification system of group DNA；
Sequentially the PCR amplification system is carried out amplification reaction；
Digestion process sequentially is carried out to the system after amplified reaction；
Sequentially mixture slaking treated system and the single base extension system obtains mixed liquor, and to described
Mixed liquor carries out extension；
Purification process sequentially is carried out to the system after extension；
Product after detecting purification process using liquid chromatograph-mass spectrometer, determines the specific SNP of the genomic DNA
The genotype in site；
Wherein, the elution mobile phase of the liquid chromatograph-mass spectrometer is containing 0.5%~5% (v/v) hexafluoroisopropanol
With the distilled water of 0.03%~0.5% (v/v) triethylamine, and containing 0.5%~5% (v/v) hexafluoroisopropanol and 0.03%~
The methanol of 0.5% (v/v) triethylamine, type of elution is gradient elution, and the mass spectrograph in the liquid chromatograph-mass spectrometer is
TripleTOF series mass spectrograph.
Preferably,
In the liquid chromatograph-mass spectrometer,
The gradient elution of high performance liquid chromatograph includes: 0~0.5min, 2%B phase；0.5~2min, 2%~20%B phase；
2~6.5min, 20%~25%B phase；6.5~7min, 25%-90%B phase；7~8min, 90%B phase；8~8.1min, 90%
~2%B phase；8.1~10min, 2%B phase, wherein B phase be containing 0.5%~5% (v/v) hexafluoroisopropanol and 0.03%~
The methanol of 0.5% (v/v) triethylamine, A phase are containing 0.5%~5% (v/v) hexafluoroisopropanol and 0.03%~0.5% (v/v) three
The distilled water of ethamine.
Preferably,
The chromatographic condition of the high performance liquid chromatograph includes:
C18 reverse-phase chromatographic column, the column temperature of the C18 reverse-phase chromatographic column are as follows: 40~70 DEG C, sample volume are as follows: 10~50 μ l.
Preferably,
The mass spectrometric Mass Spectrometry Conditions of the TripleTOF series, comprising:
Using electric spray ion source ESI, polarity Negative, TOF MS scan pattern, spray voltage is -4000~-
5000V, temperature are 450~600 DEG C, and gas curtain gas is 10~30Psi, and atomization gas is 40~60Psi, and auxiliary gas is 40~60Psi,
Removing cluster voltage is -40~-80V, and entrance potential is -5~-25V, and vacuum degree is 2~6e-5Torr。
Preferably,
Single base extension primer sequence shown in SEQ ID NO.1~SEQ ID NO.3, comprising:
SEQ ID NO.1 show the Single base extension primer sequence in the site C677T of mthfr gene: 5 '-
AGAGGTGTCTGCGGGAG-3'；
SEQ ID NO.2 show the Single base extension primer sequence in the site A1298C of mthfr gene: 5 '-
AGGGAGCTGACCAGTGAAG-3'；
SEQ ID NO.3 show the Single base extension primer sequence in the site A66G of MTRR gene: 5 '-
CGCATCGCAGAAGAAAT-3’。
Preferably,
Specificity amplification primer sequence shown in SEQ ID NO.4~SEQID NO.9, comprising:
SEQ ID NO.4 show the forward primer sequence in the specificity amplification primer sequence in the site mthfr gene C677T
Column: 5 '-ACGTTGGATGGTCACCTGGATGGGAAAGATCC-3 '；
SEQ ID NO.5 show the reverse primer sequence in the specificity amplification primer sequence in the site mthfr gene C677T
Column: 5 '-ACGTTGGATGGTCTTCATCCCTCGCCTTGAAC-3 '；
SEQ ID NO.6 show the forward primer in the specificity amplification primer sequence in the site mthfr gene A1298C
Sequence: 5 '-ACGTTGGATGGAGACCTTCCTTGCAAATACATC-3 '；
SEQ ID NO.7 show the reverse primer sequence of the specificity amplification primer sequence in the site mthfr gene A1298C
It is classified as: 5 '-ACGTTGGATGGATCACTCACTTTGTGACCATTC-3 '；
SEQ ID NO.8 show the forward primer sequence of the specificity amplification primer sequence in the site MTRR Gene A 66G:
5'-ACGTTGGATGCGCTCTAACCTTATCGGATTCACTAAT-3'；
SEQ ID NO.9 show the reverse primer sequences of the specificity amplification primer sequence in the site MTRR Gene A 66G:
5’-ACGTTGGATGCAAGTGATGAGGAGGTTTCTGTTACTA-3’。
Preferably,
The mode of purification process is carried out to the system after extension, comprising: by resin, chromatography, ultrafiltration, molecular sieve
Any one or more mode remove the salt ion in the system after extension.
Preferably,
It is described that sequentially the PCR amplification system is carried out amplification reaction, comprising:
Using PCR instrument, 1~8min is preheated to the PCR amplification system at 92~98 DEG C；Sequentially become at 92~95 DEG C
0.17~2min of property；Sequentially anneal at 50~60 DEG C 0.17~3min；Sequentially extend 0.17~10min at 68~72 DEG C；
To be sequentially denaturalized at 92~95 DEG C 0.17~2min, extend 0.17 at 50~60 DEG C at 0.17~3min and 68~72 DEG C of annealing~
10min is recycled 25~45 times；Sequentially extend 0~15min eventually at 68~80 DEG C, the system after obtaining amplified reaction, and 2~
It is saved at 8 DEG C.
Preferably,
It is described that extension is carried out to the mixed liquor, comprising:
Using PCR instrument, 1~8min is preheated to the mixed liquor at 92~98 DEG C；It sequentially is denaturalized 5s at 92~98 DEG C,
Sequentially anneal at 50~60 DEG C 5~30s；Sequentially extend 5~30s at 70~81 DEG C；Sequentially will at 50~60 DEG C annealing 5~
Extend 5~30s at 30s and 70~80 DEG C to recycle 4~8 times；To be sequentially denaturalized at 92~98 DEG C 5s, annealing 5 at 50~60 DEG C~
Extend 5~30s at 30s and 70~80 DEG C to recycle 4~8 progress amplification cycles 25~40 times；Sequentially prolong eventually at 68~80 DEG C
0~10min is stretched, the system after obtaining extension, and saved at 2~8 DEG C.
Preferably,
It is described that digestion process sequentially is carried out to the system after amplified reaction, comprising:
The alkaline phosphatase of 0.5~2U and the corresponding alkaline phosphatase buffer of the alkaline phosphatase are added to amplification
In system after reaction；
Sequentially utilize PCR instrument, 10~60min of digestion process at 25~40 DEG C；
5~30min of heat denatured is sequentially carried out at 70~85 DEG C, the system after obtaining digestion process, and at 2~10 DEG C
Lower preservation.
Preferably,
The alkaline phosphatase, comprising: shrimp alkaline phosphotase, calf intestine alkaline phosphatase, escherichia coli alkaline phosphatase,
Any one in rat alkaline phosphatase and thermal sensitivity alkaline phosphatase.
Preferably,
The single base extension system, comprising:
5~7 parts of distilled water, 1~3 part of 10*PCR buffer, the dideoxyribonucleoside triphosphate mixture 1~3 of 20mmol
Part, 8~10 parts of the mixture and DNA of Single base extension primer sequence shown in SEQ ID NO.1~SEQ ID NO.3 are poly-
0.3~0.5 part of synthase, wherein each component is counted by volume, and the concentration of 0.3~0.5 part of the archaeal dna polymerase is 0.5~
The concentration of 2U/ μ l, every kind of Single base extension primer sequence in the mixture of 8~10 parts of Single base extension primer sequence is
1~20 μm of ol.
Preferably,
The PCR amplification system, comprising:
8~10 parts of distilled water, 4~6 parts of 10*PCR buffer, the MgCl of 25mmol23~5 parts, the deoxidation of 20mmol
1~2 part of ribonucleotide triphosphate mixture, specificity amplification primer sequence shown in SEQ ID NO.4~SEQ ID NO.9
9~11 parts of mixture, 1~2 part of archaeal dna polymerase and 19~21 parts of genomic DNA, wherein each component is counted by volume, and 9~11
Each specificity amplification primer sequence concentration in the mixture of the specificity amplification primer sequence of part is 0.1~2 μm of ol,
The concentration of 1~2 part of the archaeal dna polymerase is 0.5~2U/ μ l, the amount of 19~21 parts of the genomic DNA is 5~
500ng。
A kind of detection process of folic acid metabolism related gene polymorphism provided in an embodiment of the present invention is that can be based on subject
Genomic DNA, to the PCR amplification body comprising specificity amplification primer sequence shown in SEQ ID NO.4~SEQ ID NO.9
System carries out amplification reaction, and to amplify target gene segment, then digestion process is carried out, with the dezyribonucleoside in removal system
Triphosphoric acid dNTP, then by the system after digestion process and include Single base extension shown in SEQ ID NO.1~SEQ ID NO.3
The single base extension system of primer sequence is mixed, and carries out single base extension, is prolonged so that SNP site is special
The object that extends can carry out specific bond with 5 ' ends of the SNP site of genomic DNA, and be extended according to base pair complementarity principle
The base of target SNP genotype complementation, can be obtained extension products according to the genomic DNA template of subject, sequentially carry out again pure
Change processing recycles liquid chromatograph-mass spectrometer to be detected, due to different bases with the chaff interferent in removal system
Molecular weight is different, therefore can determine the genotype of analyzed SNP site according to the molecular weight of extension products after purification.By
Good in TripleTOF series mass spectrograph specificity, high sensitivity, high-throughput and easy to operate, the period is short, therefore can be realized
Sensitive, the easy SNP site to target gene carries out the purpose of Genotyping.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is the present invention
Some embodiments for those of ordinary skill in the art without creative efforts, can also basis
These attached drawings obtain other attached drawings.
Fig. 1 is a kind of process of the detection method for folic acid metabolism related gene polymorphism that one embodiment of the invention provides
Figure；
Fig. 2 is that the site C677T for the mthfr gene that one embodiment of the invention provides is the spectrogram of C677T-CC genotype；
Fig. 3 is that the site C677T for the mthfr gene that one embodiment of the invention provides is the spectrogram of C677T-CT genotype；
Fig. 4 is that the site C677T for the mthfr gene that one embodiment of the invention provides is the spectrogram of C677T-TT genotype；
Fig. 5 is that the site A1298C for the mthfr gene that one embodiment of the invention provides is the spectrum of A1298C-AA genotype
Figure；
Fig. 6 is that the site A1298C for the mthfr gene that one embodiment of the invention provides is the spectrum of A1298C-AC genotype
Figure；
Fig. 7 is that the site A1298C for the mthfr gene that one embodiment of the invention provides is the spectrum of A1298C-CC genotype
Figure；
Fig. 8 is that the site A66G for the MTRR gene that one embodiment of the invention provides is the spectrogram of A66G-AA genotype；
Fig. 9 is that the site A66G for the MTRR gene that one embodiment of the invention provides is the spectrogram of A66G-AG genotype；
Figure 10 is that the site A66G for the MTRR gene that one embodiment of the invention provides is the spectrogram of A66G-GG genotype.
Specific embodiment
In order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below in conjunction with the embodiment of the present invention
In attached drawing, technical scheme in the embodiment of the invention is clearly and completely described, it is clear that described embodiment is
A part of the embodiment of the present invention, instead of all the embodiments, based on the embodiments of the present invention, those of ordinary skill in the art
Every other embodiment obtained without making creative work, shall fall within the protection scope of the present invention.
As shown in Figure 1, the embodiment of the invention provides a kind of detection method of folic acid metabolism related gene polymorphism, packet
It includes:
Step 101: extracting the genomic DNA of subject；
Step 102: list of the configuration comprising Single base extension primer sequence shown in SEQ ID NO.1~SEQ ID NO.3
Base extension system, and configure comprising specificity amplification primer sequence shown in SEQ ID NO.4~SEQ ID NO.9 and
The polymerase chain reaction PCR amplification system of the genomic DNA；
Step 103: sequentially the PCR amplification system being carried out amplification reaction；
Step 104: digestion process sequentially being carried out to the system after amplified reaction；
Step 105: sequentially mixture slaking treated system and the single base extension system obtains mixed liquor,
And extension is carried out to the mixed liquor；
Step 106: purification process sequentially being carried out to the system after extension；
Step 107: the product after detecting purification process using liquid chromatograph-mass spectrometer determines the genomic DNA
The genotype of specific SNP site, wherein the elution mobile phase of the liquid chromatograph-mass spectrometer is containing 0.5%~5% (v/
V) distilled water of hexafluoroisopropanol and 0.03%~0.5% (v/v) triethylamine, and contain 0.5%~5% (v/v) hexafluoro isopropyl
The methanol of pure and mild 0.03%~0.5% (v/v) triethylamine, type of elution is gradient elution, the liquid chromatograph-mass spectrometer
In mass spectrograph be TripleTOF series mass spectrograph.
A kind of detection process of folic acid metabolism related gene polymorphism provided in an embodiment of the present invention is that can be based on subject
Genomic DNA, to the PCR amplification body comprising specificity amplification primer sequence shown in SEQ ID NO.4~SEQ ID NO.9
System carries out amplification reaction, and to amplify target gene segment, then digestion process is carried out, with the dezyribonucleoside in removal system
Triphosphoric acid dNTP, then by the system after digestion process and include Single base extension shown in SEQ ID NO.1~SEQ ID NO.3
The single base extension system of primer sequence is mixed, and carries out single base extension, is prolonged so that SNP site is special
The object that extends can carry out specific bond with 5 ' ends of the SNP site of genomic DNA, and be extended according to base pair complementarity principle
The base of target SNP genotype complementation, can be obtained extension products according to the genomic DNA template of subject, sequentially carry out again pure
Change processing recycles liquid chromatograph-mass spectrometer to be detected, due to different bases with the chaff interferent in removal system
Molecular weight is different, therefore can determine the genotype of analyzed SNP site according to the molecular weight of extension products after purification.By
Good in TripleTOF series mass spectrograph specificity, high sensitivity, high-throughput and easy to operate, the period is short, therefore can be realized
Sensitive, the easy SNP site to target gene carries out the purpose of Genotyping.
The chromatographic condition of the high performance liquid chromatograph includes:
C18 reverse-phase chromatographic column, the column temperature of the C18 reverse-phase chromatographic column are as follows: 40~70 DEG C, sample volume are as follows: 10~50 μ l.
Refer to 40 DEG C to 70 DEG C of any temperature for 40~70 DEG C, for example, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65
DEG C and 70 DEG C etc..In the range at any temperature, the result that detected is unanimous on the whole.
For sample volume, 10~50 μ l refer to any value within the scope of 10 μ l to 50 μ l, for example, 10 μ L, 15 μ L,
20 μ L, 25 μ L, 30 μ L, 35 μ L, 40 μ L, 45 μ L and 50 μ L etc..
Wherein, high for for liquid chromatograph-mass spectrometer used in detection folic acid metabolism related gene polymorphism
The gradient elution of effect liquid phase chromatogram instrument includes: 0~0.5min, 2%B phase；0.5~2min, 2%~20%B phase；2~6.5min,
20%~25%B phase；6.5~7min, 25%-90%B phase；7~8min, 90%B phase；8~8.1min, 90%~2%B phase；
8.1~10min, 2%B phase, wherein B phase is containing 0.5%~5% (v/v) hexafluoroisopropanol and 0.03%~0.5% (v/v) three
The methanol of ethamine, A phase are the distillation containing 0.5%~5% (v/v) hexafluoroisopropanol and 0.03%~0.5% (v/v) triethylamine
Water.
Wherein, refer to volume content 0.5%~5% containing 0.5%~5% (v/v) hexafluoroisopropanol.Containing 0.03%~
0.5% (v/v) triethylamine refers to volume content 0.03%~0.5%.
It is understood that the sum of A phase volume ratio and B phase volume ratio are 100% when elution.
The mass spectrometric Mass Spectrometry Conditions of the TripleTOF series, comprising:
Using electric spray ion source ESI, polarity Negative, TOF MS scan pattern, spray voltage is -4000~-
5000V, temperature are 450~600 DEG C, and gas curtain gas is 10~30Psi, and atomization gas is 40~60Psi, and auxiliary gas is 40~60Psi,
Removing cluster voltage is -40~-80V, and entrance potential is -5~-25V, and vacuum degree is 2~6e-5Torr。
For spray voltage, -4000~-5000V refers to any voltage that -4000V is arrived in -5000V range, than
Such as, -4000V, -4100V, -4200V, -4300V, -4400V, -4500V, -4600V, -4700V, -4800V, -4900V and -
5000V。
For temperature, 450~600 DEG C of any temperature referred within the scope of 450 DEG C to 600 DEG C, for example, 450 DEG C,
480 DEG C, 500 DEG C, 520 DEG C, 550 DEG C, 580 DEG C and 600 etc..
For gas curtain gas, 10~30Psi refers to any value in 10Psi to 30Psi range, for example, 10Psi,
15Psi, 20Psi, 25Psi and 30Psi.
For atomization gas, 40~60Psi refers to any temperature in 40Psi to 60Psi range, for example, 40Psi,
45Psi, 50Psi, 55Psi and 60Psi.
For auxiliary gas, 40~60Psi refers to any temperature in 40Psi to 60Psi range, for example, 40Psi,
45Psi, 50Psi, 55Psi and 60Psi.
For going for cluster voltage, -40~-80V refer to -40V to any voltage value in -80V range, for example, -
40V, -45V, -50V, -55V, -60V, -65V, -70V, -75V and -80 etc..
For entrance potential, -5~-25V refers to any voltage value that -5V is arrived in -20V range, for example, -5V, -
10V, -15V, -20V and -25V.
For vacuum degree, 2~6e-5Torr refers to 2e-5Torr to 6e-5Any value within the scope of Torr, for example,
2e-5Torr、3e-5Torr、4e-5Torr、5e-5Torr and 6e-5Torr。
It is understood that TripleTOF series mass spectrograph includes:4600 System、5600 System and same type mass spectrograph.
Specifically, after detecting using liquid chromatograph-mass spectrometer to product after purification, map can be obtained,
It is obtained after being deconvoluted using the Peakview software of Applied biosystems AB Sciex to spectrogram obtained
Analysis of spectra.
For the site C677T of mthfr gene in purified product, when molecular weight is only existed in analysis of spectra is 5576 ±
When the single base extension product of 3Da, the genotype that receptor gene organizes the site C677T of DNA is CC type；When in analysis of spectra
There are when the single base extension product that molecular weight is 5656 ± 3Da, the genotype that receptor gene organizes the site C677T of DNA is
TT type；It is tested when existing simultaneously the single base extension product that molecular weight is 5576 ± 3Da and 5656 ± 3Da in analysis of spectra
The genotype in the site C677T of person's genomic DNA is CT type；
For the site A1298C of mthfr gene in purified product, when molecular weight is only existed in analysis of spectra is 6195 ±
When the single base extension product of 3Da, the genotype that receptor gene organizes the site A1298C of DNA is AA type；When in analysis of spectra
When only existing the single base extension product that molecular weight is 6171 ± 3Da, receptor gene organizes the genotype in the site A1298C of DNA
For CC type；When existing simultaneously the single base extension product that molecular weight is 6195 ± 3Da and 6171 ± 3Da in analysis of spectra, by
The genotype in the site A1298C of examination person's genomic DNA is AC type；
It is 5481 ± 3Da when only existing molecular weight in analysis of spectra for the site A66G of MTRR gene in purified product
Single base extension product when, receptor gene organize DNA the site A66G genotype be AA type；When being only existed in analysis of spectra
When molecular weight is the single base extension product of 5497 ± 3Da, the genotype that receptor gene organizes the site A66G of DNA is GG type；
When existing simultaneously the single base extension product that molecular weight is 5481 ± 3Da and 5497 ± 3Da in analysis of spectra, receptor gene
The genotype in the site A66G of group DNA is AG type.
Fig. 2 shows the spectrograms that the site C677T of mthfr gene is C677T-CC genotype；
The site C677T that Fig. 3 shows mthfr gene is the spectrogram of C677T-CT genotype；
The site C677T that Fig. 4 shows mthfr gene is the spectrogram of C677T-TT genotype；
The site A1298C that Fig. 5 shows mthfr gene is the spectrogram of A1298C-AA genotype；
The site A1298C that Fig. 6 shows mthfr gene is the spectrogram of A1298C-AC genotype；
The site A1298C that Fig. 7 shows mthfr gene is the spectrogram of A1298C-CC genotype；
The site A66G that Fig. 8 shows MTRR gene is the spectrogram of A66G-AA genotype；
The site A66G that Fig. 9 shows MTRR gene is the spectrogram of A66G-AG genotype；
The site A66G that Figure 10 shows MTRR gene is the spectrogram of A66G-GG genotype.
Wherein, the abscissa of Fig. 2 to Figure 10 is the molecular weight after deconvoluting, and unit Da, ordinate is ion stream
Intensity.
Specifically, Single base extension primer sequence shown in SEQ ID NO.1~SEQ ID NO.3, comprising:
SEQ ID NO.1 show the Single base extension primer sequence in the site C677T of mthfr gene: 5 '-
AGAGGTGTCTGCGGGAG-3'；
SEQ ID NO.2 show the Single base extension primer sequence in the site A1298C of mthfr gene: 5 '-
AGGGAGCTGACCAGTGAAG-3'；
SEQ ID NO.3 show the Single base extension primer sequence in the site A66G of MTRR gene: 5 '-
CGCATCGCAGAAGAAAT-3’。
Specifically, specificity amplification primer sequence shown in SEQ ID NO.4~SEQ ID NO.9, comprising:
SEQ ID NO.4 show the forward primer sequence in the specificity amplification primer sequence in the site mthfr gene C677T
Column: 5 '-ACGTTGGATGGTCACCTGGATGGGAAAGATCC-3 '；
SEQ ID NO.5 show the reverse primer sequence in the specificity amplification primer sequence in the site mthfr gene C677T
Column: 5 '-ACGTTGGATGGTCTTCATCCCTCGCCTTGAAC-3 '；
SEQ ID NO.6 show the forward primer in the specificity amplification primer sequence in the site mthfr gene A1298C
Sequence: 5 '-ACGTTGGATGGAGACCTTCCTTGCAAATACATC-3 '；
SEQ ID NO.7 show the reverse primer sequence of the specificity amplification primer sequence in the site mthfr gene A1298C
It is classified as: 5 '-ACGTTGGATGGATCACTCACTTTGTGACCATTC-3 '；
SEQ ID NO.8 show the forward primer sequence of the specificity amplification primer sequence in the site MTRR Gene A 66G:
5'-ACGTTGGATGCGCTCTAACCTTATCGGATTCACTAAT-3'；
SEQ ID NO.9 show the reverse primer sequences of the specificity amplification primer sequence in the site MTRR Gene A 66G:
5’-ACGTTGGATGCAAGTGATGAGGAGGTTTCTGTTACTA-3’。
Wherein, the specific amplification in the site A66G in the site C677T, the site A1298C and MTRR gene of mthfr gene
Primer and Single base extension primer can design according to demand.According to the site C677T of mthfr gene, the site A1298C and
The site the A66G forward primer sequence and reverse primer sequences of MTRR gene synthesize corresponding specificity amplification primer, and specificity expands
The synthesis for increasing primer can be synthesized using solid-phase synthesis or commission Synbiotics AB and be detected forward and reverse primer；It is single
Base extension primer can entrust Synbiotics AB to synthesize and detect.
It should be noted that the site C677T of mthfr gene, the i.e. site rs1801133 of mthfr gene；MTHFR base
The site A1298C of cause, the i.e. site rs1801131 of mthfr gene；The site A66G of MTRR gene, i.e. MTRR gene
The site rs1801394.
In an embodiment of the present invention, the single base extension system, comprising:
5~7 parts of distilled water, 1~3 part of 10*PCR buffer, the dideoxyribonucleoside triphosphate mixture 1~3 of 20mmol
Part, 8~10 parts of the mixture and DNA of Single base extension primer sequence shown in SEQ ID NO.1~SEQ ID NO.3 are poly-
0.3~0.5 part of synthase, wherein each component is counted by volume, and the concentration of 0.3~0.5 part of the archaeal dna polymerase is 0.5~
The concentration of 2U/ μ l, every kind of Single base extension primer sequence in the mixture of 8~10 parts of Single base extension primer sequence is
1~20 μm of ol.
For the number of 10*PCR buffer, 1~3 part of any value referred within the scope of 1 part to 3 parts, for example, 1
Part, 1.5 parts, 2 parts, 2.5 parts and 3 parts.
For the number of dideoxyribonucleoside triphosphate mixture, 1~3 part of any number referred within the scope of 1 part to 3 parts
Value, for example, 1 part, 1.5 parts, 2 parts, 2.5 parts and 3 parts.
For the number of the mixture of Single base extension primer sequence, 8~10 parts refer within the scope of 8 parts to 10 parts
Any number, such as 8 parts, 8.5 parts, 9 parts, 9.5 and 10 parts.
For archaeal dna polymerase, 0.3~0.5 part of any value referred within the scope of 0.3 part to 0.5 part, for example, 0.3
Part, 0.4 part and 0.5 part.
For archaeal dna polymerase, 0.3~0.5 part of any value referred within the scope of 0.3 part to 0.5 part, for example, 0.3
Part, 0.4 part and 0.5 part.
Concentration for archaeal dna polymerase is for 0.5~2U/ μ l, and 0.5~2U/ μ l refers to 0.5U/ μ l to 2U/ μ l range
Interior any value, for example, 0.5U/ μ l, 1U/ μ l, 1.5U/ μ l and 2U/ μ l.
For the mixture of Single base extension primer sequence, 8~10 parts of arbitrary numbers referred within the scope of 8 parts to 10 parts
Value, such as 8 parts, 8.5 parts, 9 parts, 9.5 and 10 parts.
For the concentration of every kind of Single base extension primer sequence, 1~20 μm of ol refers within the scope of 1 μm of ol to 20 μm of ol
Any number, for example, 1 μm of ol, 5 μm of ol, 10 μm of ol, 15 μm of ol and 20 μm of ol.
In an embodiment of the present invention, the PCR amplification system, comprising:
8~10 parts of distilled water, 4~6 parts of 10*PCR buffer, 3~5 parts of the MgCl2 of 25mmol, the deoxidation of 20mmol
1~2 part of ribonucleotide triphosphate mixture, specificity amplification primer sequence shown in SEQ ID NO.4~SEQ ID NO.9
9~11 parts of mixture, 1~2 part of archaeal dna polymerase and 19~21 parts of genomic DNA, wherein each component is counted by volume, and 9~11
Each specificity amplification primer sequence concentration in the mixture of the specificity amplification primer sequence of part is 0.1~2 μm of ol,
The concentration of 1~2 part of the archaeal dna polymerase is 0.5~2U/ μ l, the amount of 19~21 parts of the genomic DNA is 5~
500ng。
For distillation, 8~10 parts of any numbers referred within the scope of 8 parts to 10 parts, such as 8 parts, 8.5 parts, 9 parts,
9.5 and 10 parts.
For 10*PCR buffer, 4~6 parts of any numbers referred within the scope of 4 parts to 6 parts, for example, 4 parts, 5 parts
And 6 parts.
For the number of the MgCl2 of 25mmol, 3~5 parts of any numbers referred within the scope of 3 parts to 5 parts, for example, 3
Part, 3.5 parts, 4 parts, 4.5 parts and 5 parts.
For the number of the deoxyribonucleoside triphosphate mixture of 20mmol, 1~2 part refers to 1 part to 2 parts range
Interior any number, for example, 1 part, 1.2 parts, 1.4 parts, 1.6 parts, 1.8 parts and 2 parts.
For the number of the mixture of specificity amplification primer sequence, 9~11 parts be can be within the scope of 9 parts to 11 parts
Any number, for example, 9 parts, 9.5 parts, 10 parts, 10.5 parts and 11 parts.
For the number of archaeal dna polymerase, 1~2 part of any number referred within the scope of 1 part to 2 parts, for example, 1 part,
1.2 parts, 1.4 parts, 1.6 parts, 1.8 parts and 2 parts.
For the number of genomic DNA, 19~21 parts of any numbers referred within the scope of 19 parts to 21 parts, for example,
19 parts, 19.5 parts, 20 parts, 20.5 parts and 21 parts.
For each specificity amplification primer sequence concentration, 0.1~2 μm of ol refers to 0.1 μm of ol to 2 μm of ol range
Interior any value, for example, 0.1 μm of ol, 0.5 μm of ol, 1 μm of ol, 1.5 μm of ol and 2 μm of ol.
For the concentration of archaeal dna polymerase, 0.5~2U/ μ l refers to any concentration within the scope of 0.5U/ μ l to 2U/ μ l,
For example, 0.5U/ μ l, 1U/ μ l, 1.5U/ μ l and 2U/ μ l etc..
For the amount of genomic DNA, 5~500ng refers to any value in 5ng to 500ng range, for example,
5ng, 50ng, 100ng, 150ng, 200ng, 250ng, 300ng, 350ng, 400ng, 450ng and 500ng.
It is in an embodiment of the present invention, described that sequentially the PCR amplification system is carried out amplification reaction, comprising:
Using PCR instrument, 1~8min is preheated to the PCR amplification system at 92~98 DEG C；Sequentially become at 92~95 DEG C
0.17~2min of property；Sequentially anneal at 50~60 DEG C 0.17~3min；Sequentially extend 0.17~10min at 68~72 DEG C；
To be sequentially denaturalized at 92~95 DEG C 0.17~2min, extend 0.17 at 50~60 DEG C at 0.17~3min and 68~72 DEG C of annealing~
10min is recycled 25~45 times；Sequentially extend 0~15min eventually at 68~80 DEG C, the system after obtaining amplified reaction, and 2~
It is saved at 8 DEG C.
Specifically, when carrying out pcr amplification reaction in order to prevent, part PCR amplification system is condensed, can be first to PCR
Amplification system carries out the pre-heat treatment, then carries out the reaction of degeneration (RD) of DNA profiling, sequentially carries out the special of DNA profiling and each site
Property amplimer annealing reaction, sequentially carry out the extension of the specificity amplification primer in each site, repeat circulation " become
Property-annealing-extension ", to amplify target gene fragment, then whole extension is carried out to system, to improve complete double-stranded DNA
Ratio.
It is in an embodiment of the present invention, described that digestion process sequentially is carried out to the system after amplified reaction, comprising:
The alkaline phosphatase of 0.5~2U and the corresponding alkaline phosphatase buffer of the alkaline phosphatase are added to amplification
In system after reaction；
Sequentially utilize PCR instrument, 10~60min of digestion process at 25~40 DEG C；
5~30min of heat denatured is sequentially carried out at 70~85 DEG C, the system after obtaining digestion process, and at 2~10 DEG C
Lower preservation.
Specifically, alkaline phosphatase and corresponding alkaline phosphatase buffer are added into the system after amplified reaction, into
Row digestion process and heat denatured can be led to by the concentration of pH value and salt ion in alkaline phosphatase buffer regulation system
Parlkaline phosphatase dephosphorylation, extra deoxyribonucleoside triphosphate dNTP in removal system.
Wherein, the alkaline phosphatase, comprising: shrimp alkaline phosphotase, calf intestine alkaline phosphatase, coli alkaline phosphorus
Any one in sour enzyme, rat alkaline phosphatase and thermal sensitivity alkaline phosphatase.
It is in an embodiment of the present invention, described that extension is carried out to the mixed liquor, comprising:
Using PCR instrument, 1~8min is preheated to the mixed liquor at 92~98 DEG C；It sequentially is denaturalized 5s at 92~98 DEG C,
Sequentially anneal at 50~60 DEG C 5~30s；Sequentially extend 5~30s at 70~81 DEG C；Sequentially will at 50~60 DEG C annealing 5~
Extend 5~30s at 30s and 70~80 DEG C to recycle 4~8 times；To be sequentially denaturalized at 92~98 DEG C 5s, annealing 5 at 50~60 DEG C~
Extend 5~30s at 30s and 70~80 DEG C to recycle 4~8 progress amplification cycles 25~40 times；Sequentially prolong eventually at 68~80 DEG C
0~10min is stretched, the system after obtaining extension, and saved at 2~8 DEG C.
Specifically, extension is being carried out comprising the system after single base extension system and digestion process in order to prevent
When, sectional interest condenses, and first can carry out the pre-heat treatment to single base extension system, then carry out " annealing-extension "
Reaction to extend the extension products of the Single base extension primer in target gene site, then carries out " annealing-extension " to system and follows
Ring reaction carries out whole extension, finally to increase the amount of the extension products of Single base extension primer.
In an embodiment of the present invention, the mode of purification process is carried out to the system after extension, comprising: pass through tree
Rouge, chromatography, ultrafiltration, any one or more mode in molecular sieve remove the salt ion in the system after extension, to go
Except the chaff interferent in system, the detection for influencing liquid chromatograph-mass spectrometer is avoided.
It should be noted that the mode of purification process can be only through any in resin, chromatography, ultrafiltration, molecular sieve
Mode, the salt ion being also possible in the form removal system that various ways combine, improve purification effect, for example, resin with
Chromatograph the mode that combines, the mode that resin is combined with ultrafiltration, the mode that chromatography is combined with molecular sieve, resin, ultrafiltration and
The mode etc. that molecular sieve combines.
It is described in detail below with detection method of several embodiments to folic acid metabolism related gene polymorphism.
Embodiment 1: the genomic DNA of subject is extracted
The genomic DNA 2ml of subject is extracted, the genomic DNA mode of extraction can be with buccal swab collection
Mouth desquamated cells, or the fresh peripheral blood/tissue samples collected use Tiangeng buccal swab genome DNA extracting reagent kit
(DP322) or blood/cell/tissue genome DNA extracting reagent kit (DP304) extracts, and using NP80-touch (Germany
IMPLEN the concentration and genomic DNA after purification of genomic DNA) are measured, wherein the OD260/ of the genomic DNA of subject
OD280 ratio is located in 1.6~2.2 range intervals, and concentration is located in 5~1000ng/ μ l range intervals, so as to what is extracted
The quality of genomic DNA meets test request.
Embodiment 2: configuration single base extension system
Configuration is mixed by the dideoxyribonucleoside triphosphate of 0.659 μ l of distilled water, 0.2 μ l of 10*PCR buffer, 20mmol
Mixture 0.9 the μ l and DNA of Single base extension primer sequence shown in 0.2 μ l of object, SEQ ID NO.1~SEQ ID NO.3
The single base extension system of 0.041 μ l of polymerase composition, wherein the concentration of archaeal dna polymerase is 1U/ μ l, Single base extension
The concentration of every kind of Single base extension primer sequence in the mixture of primer sequence is 10 μm of ol.
Wherein, SEQ ID NO.1 show the Single base extension primer sequence in the site C677T of mthfr gene, SEQ ID
NO.2 show the Single base extension primer sequence in the site A1298C of mthfr gene, and SEQ ID NO.3 show MTRR gene
The site A66G Single base extension primer sequence.
Embodiment 3: configuration PCR amplification system
Configuration by 0.9 μ l of distilled water, 0.5 μ l of 10*PCR buffer, 0.4 μ l of MgCl2 of 25mmol, 20mmol it is de-
Specificity amplification primer sequence shown in 0.1 μ l of oxygen ribonucleotide triphosphate mixture, SEQ ID NO.4~SEQ ID NO.9
1 μ l of mixture, 0.1 μ l of archaeal dna polymerase and 2 μ l of genomic DNA composition PCR amplification system, wherein specific amplification draws
Each specificity amplification primer sequence concentration in the mixture of object sequence is 2 μm of ol, and the concentration of archaeal dna polymerase is 1U/ μ
l。
Wherein, SEQ ID NO.4 show the forward direction in the specificity amplification primer sequence in the site mthfr gene C677T
Primer sequence, SEQ ID NO.5 show the reverse primer in the specificity amplification primer sequence in the site mthfr gene C677T
Sequence, SEQ ID NO.6 show the forward primer sequence in the specificity amplification primer sequence in the site mthfr gene A1298C
Column, SEQ ID NO.7 show the reverse primer sequences of the specificity amplification primer sequence in the site mthfr gene A1298C, SEQ
ID NO.8 show the forward primer sequence of the specificity amplification primer sequence in the site MTRR Gene A 66G, SEQ ID NO.9 institute
It is shown as the reverse primer sequences of the specificity amplification primer sequence in the site MTRR Gene A 66G.
Embodiment 4: PCR amplification system is carried out amplification reaction
Using PCR instrument, PCR amplification system is carried out amplification reaction according to the amplification program in following table 1.
Table 1
Embodiment 5: digestion process is carried out to the system after amplified reaction
The shrimp alkaline phosphotase of 1U and the corresponding alkaline phosphatase buffer of shrimp alkaline phosphotase are added to amplified reaction
It in system afterwards, and releases in PCR instrument, carries out 37 DEG C of digestion process 40min, then 75 DEG C of heat denatured 10min, last 4 DEG C
It saves.
Embodiment 6: single base extension is carried out
The single base extension system for pipetting 2 μ l embodiments 2 is added to mixture slaking treated in system, sufficiently mixed
It closes, obtains mixed liquor.Using PCR instrument, single base extension is carried out according to the response procedures in following table 2.
Table 2
Embodiment 7: purification process is carried out to the system after extension
The cations such as K+, Na+, Mg2+ in system after removing extension by resin desalination.
Embodiment 8: the product after detecting purification process using liquid chromatograph-mass spectrometer
High performance liquid chromatograph in liquid chromatograph-mass spectrometer elutes mobile phase are as follows:
Distilled water containing 0.5%~5% (v/v) hexafluoroisopropanol and 0.03%~0.5% (v/v) triethylamine, Yi Jihan
The methanol of 0.5%~5% (v/v) hexafluoroisopropanol and 0.03%~0.5% (v/v) triethylamine.
Gradient 0~0.5min, the 2%B phase of high performance liquid chromatograph；0.5~2min, 2%~20%B phase；2~
6.5min, 20%~25%B phase；6.5~7min, 25%-90%B phase；7~8min, 90%B phase；8~8.1min, 90%~
2%B phase；8.1~10min, 2%B phase, wherein B phase is containing 0.5%~5% (v/v) hexafluoroisopropanol and 0.03%~0.5%
(v/v) methanol of triethylamine, A phase are containing 0.5%~5% (v/v) hexafluoroisopropanol and 0.03%~0.5% (v/v) triethylamine
Distilled water.
C18 reverse-phase chromatographic column, column temperature are 55 DEG C, and sample volume is 20 μ l.
Mass spectrograph in liquid chromatograph-mass spectrometer is TripleTOF series mass spectrograph.
The mass spectrometric Mass Spectrometry Conditions of TripleTOF series, comprising:
Using electric spray ion source ESI, polarity Negative, TOF MS scan pattern, spray voltage is -4000~-
5000V, temperature are 450~600 DEG C, and gas curtain gas is 10~30Psi, and atomization gas is 40~60Psi, and auxiliary gas is 40~60Psi,
Removing cluster voltage is -40~-80V, and entrance potential is -5~-25V, and vacuum degree is 2~6e-5Torr。
The each embodiment of the present invention at least has the following beneficial effects:
1, in an embodiment of the present invention, can be based on the genomic DNA of subject, to including SEQ ID NO.4~SEQ
The PCR amplification system of specificity amplification primer sequence shown in ID NO.9 carries out amplification reaction, to amplify target gene piece
Section, then carries out digestion process, with the deoxyribonucleoside triphosphate dNTP in removal system, then by after digestion process system with
Single base extension system comprising Single base extension primer sequence shown in SEQ ID NO.1~SEQ ID NO.3 carries out
Mixing, and carries out single base extension, so that the special extension primer of SNP site can be with the SNP site of genomic DNA
5 ' ends carry out specific bond, and extend the base of target SNP genotype complementation according to base pair complementarity principle, according to tested
Extension products can be obtained in the genomic DNA template of person, sequentially carry out purification process again, with the chaff interferent in removal system, then benefit
It is detected with liquid chromatograph-mass spectrometer, since the molecular weight of different bases is different, is produced according to extension after purification
The molecular weight of object can determine the genotype of analyzed SNP site.Since TripleTOF series mass spectrograph specificity is good,
High sensitivity, high-throughput and easy to operate, the period is short, thus can be realized sensitive, the easy SNP site to target gene into
The purpose of row Genotyping.
It should be noted that, in this document, such as first and second etc relational terms are used merely to an entity
Or operation is distinguished with another entity or operation, is existed without necessarily requiring or implying between these entities or operation
Any actual relationship or order.Moreover, the terms "include", "comprise" or its any other variant be intended to it is non-
It is exclusive to include, so that the process, method, article or equipment for including a series of elements not only includes those elements,
It but also including other elements that are not explicitly listed, or further include solid by this process, method, article or equipment
Some elements.In the absence of more restrictions, the element limited by sentence " including a 〃 〃 ", it is not excluded that
There is also other identical factors in the process, method, article or apparatus that includes the element.
Finally, it should be noted that the foregoing is merely presently preferred embodiments of the present invention, it is merely to illustrate skill of the invention
Art scheme, is not intended to limit the scope of the present invention.Any modification for being made all within the spirits and principles of the present invention,
Equivalent replacement, improvement etc., are included within the scope of protection of the present invention.
SEQUENCE LISTING
<110>Beijing and conjunction medical diagnostic techniqu limited liability company
<120>a kind of detection method of folic acid metabolism related gene polymorphism
<160> 9
<170> PatentIn version 3.3
<210> 1
<211> 17
<212> DNA
<213>artificial sequence (Artificial sequence)
<220>
<223>primer
<400> 1
agaggtgtct gcgggag 17
<210> 2
<211> 19
<212> DNA
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agggagctga ccagtgaag 19
<210> 3
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<213>artificial sequence (Artificial sequence)
<220>
<223>primer
<400> 3
cgcatcgcag aagaaat 17
<210> 4
<211> 32
<212> DNA
<213>artificial sequence (Artificial sequence)
<220>
<223>primer
<400> 4
acgttggatg gtcacctgga tgggaaagat cc 32
<210> 5
<211> 32
<212> DNA
<213>artificial sequence (Artificial sequence)
<220>
<223>primer
<400> 5
acgttggatg gtcttcatcc ctcgccttga ac 32
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acgttggatg gagaccttcc ttgcaaatac atc 33
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<213>artificial sequence (Artificial sequence)
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acgttggatg gatcactcac tttgtgacca ttc 33
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<212> DNA
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acgttggatg cgctctaacc ttatcggatt cactaat 37
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<220>
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acgttggatg caagtgatga ggaggtttct gttacta 37
Claims (10)
1. a kind of detection method of folic acid metabolism related gene polymorphism characterized by comprising
Extract the genomic DNA of subject；
Single base extension of the configuration comprising Single base extension primer sequence shown in SEQ ID NO.1~SEQ ID NO.3
System, and configure comprising specificity amplification primer sequence and the genome shown in SEQ ID NO.4~SEQ ID NO.9
The polymerase chain reaction PCR amplification system of DNA；
Sequentially the PCR amplification system is carried out amplification reaction；
Digestion process sequentially is carried out to the system after amplified reaction；
Sequentially mixture slaking treated system and the single base extension system obtains mixed liquor, and to the mixing
Liquid carries out extension；
Purification process sequentially is carried out to the system after extension；
Product after detecting purification process using liquid chromatograph-mass spectrometer, determines the specific SNP site of the genomic DNA
Genotype；
Wherein, the elution mobile phase of the liquid chromatograph-mass spectrometer be containing 0.5%~5% (v/v) hexafluoroisopropanol and
The distilled water of 0.03%~0.5% (v/v) triethylamine, and containing 0.5%~5% (v/v) hexafluoroisopropanol and 0.03%~
The methanol of 0.5% (v/v) triethylamine, type of elution is gradient elution, and the mass spectrograph in the liquid chromatograph-mass spectrometer is
TripleTOF series mass spectrograph.
2. the detection method of folic acid metabolism related gene polymorphism according to claim 1, which is characterized in that
The chromatographic condition of the high performance liquid chromatograph includes:
C18 reverse-phase chromatographic column, the column temperature of the C18 reverse-phase chromatographic column are as follows: 40~70 DEG C, sample volume are as follows: 10~50 μ l.
3. the detection method of folic acid metabolism related gene polymorphism according to claim 1, which is characterized in that
The mass spectrometric Mass Spectrometry Conditions of the TripleTOF series, comprising:
Using electric spray ion source ESI, polarity Negative, TOF MS scan pattern, spray voltage is -4000~-
5000V, temperature are 450~600 DEG C, and gas curtain gas is 10~30Psi, and atomization gas is 40~60Psi, and auxiliary gas is 40~60Psi,
Removing cluster voltage is -40~-80V, and entrance potential is -5~-25V, and vacuum degree is 2~6e-5Torr。
4. the detection method of folic acid metabolism related gene polymorphism according to claim 1, which is characterized in that
Single base extension primer sequence shown in SEQ ID NO.1~SEQ ID NO.3, comprising:
SEQ ID NO.1 show the Single base extension primer sequence in the site C677T of mthfr gene: 5 '-
AGAGGTGTCTGCGGGAG-3'；
SEQ ID NO.2 show the Single base extension primer sequence in the site A1298C of mthfr gene: 5 '-
AGGGAGCTGACCAGTGAAG-3'；
SEQ ID NO.3 show the Single base extension primer sequence in the site A66G of MTRR gene: 5 '-
CGCATCGCAGAAGAAAT-3'；
And/or
Specificity amplification primer sequence shown in SEQ ID NO.4~SEQ ID NO.9, comprising:
SEQ ID NO.4 show the forward primer sequence in the specificity amplification primer sequence in the site mthfr gene C677T:
5'-ACGTTGGATGGTCACCTGGATGGGAAAGATCC-3'；
SEQ ID NO.5 show the reverse primer sequences in the specificity amplification primer sequence in the site mthfr gene C677T:
5'-ACGTTGGATGGTCTTCATCCCTCGCCTTGAAC-3'；
SEQ ID NO.6 show the forward primer sequence in the specificity amplification primer sequence in the site mthfr gene A1298C:
5'-ACGTTGGATGGAGACCTTCCTTGCAAATACATC-3'；
SEQ ID NO.7 show the reverse primer sequences of the specificity amplification primer sequence in the site mthfr gene A1298C are as follows:
5'-ACGTTGGATGGATCACTCACTTTGTGACCATTC-3'；
SEQ ID NO.8 show the forward primer sequence of the specificity amplification primer sequence in the site MTRR Gene A 66G: 5 '-A
CGTTGGATGCGCTCTAACCTTATCGGATTCACTAAT-3'；
SEQ ID NO.9 show the reverse primer sequences of the specificity amplification primer sequence in the site MTRR Gene A 66G: 5 '-A
CGTTGGATGCAAGTGATGAGGAGGTTTCTGTTACTA-3’。
5. the detection method of folic acid metabolism related gene polymorphism according to claim 1, which is characterized in that
The mode of purification process is carried out to the system after extension, comprising: pass through appointing in resin, chromatography, ultrafiltration, molecular sieve
One or more modes of anticipating remove the salt ion in the system after extension.
6. the detection method of folic acid metabolism related gene polymorphism according to claim 1, which is characterized in that
It is described that sequentially the PCR amplification system is carried out amplification reaction, comprising:
Using PCR instrument, 1~8min is preheated to the PCR amplification system at 92~98 DEG C；Sequentially it is denaturalized at 92~95 DEG C
0.17~2min；Sequentially anneal at 50~60 DEG C 0.17~3min；Sequentially extend 0.17~10min at 68~72 DEG C；It is suitable
It is secondary will be denaturalized at 92~95 DEG C 0.17~2min, extend 0.17 at 50~60 DEG C at 0.17~3min and 68~72 DEG C of annealing~
10min is recycled 25~45 times；Sequentially extend 0~15min eventually at 68~80 DEG C, the system after obtaining amplified reaction, and 2~
It is saved at 8 DEG C；
And/or
It is described that extension is carried out to the mixed liquor, comprising:
Using PCR instrument, 1~8min is preheated to the mixed liquor at 92~98 DEG C；5s is sequentially denaturalized at 92~98 DEG C, sequentially
Anneal 5~30s at 50~60 DEG C；Sequentially extend 5~30s at 70~81 DEG C；Sequentially by the 5~30s that anneals at 50~60 DEG C
Extend 5~30s at 70~80 DEG C to recycle 4~8 times；Sequentially 5s will be denaturalized at 92~98 DEG C, anneal at 50~60 DEG C 5~30s
Extend 5~30s at 70~80 DEG C to recycle 4~8 progress amplification cycles 25~40 times；Sequentially extend 0 eventually at 68~80 DEG C
~10min, the system after obtaining extension, and saved at 2~8 DEG C.
7. the detection method of folic acid metabolism related gene polymorphism according to claim 1, which is characterized in that
It is described that digestion process sequentially is carried out to the system after amplified reaction, comprising:
The alkaline phosphatase of 0.5~2U and the corresponding alkaline phosphatase buffer of the alkaline phosphatase are added to amplified reaction
In system afterwards；
Sequentially utilize PCR instrument, 10~60min of digestion process at 25~40 DEG C；
5~30min of heat denatured is sequentially carried out at 70~85 DEG C, the system after obtaining digestion process, and protected at 2~10 DEG C
It deposits.
8. the detection method of folic acid metabolism related gene polymorphism according to claim 7, which is characterized in that
The alkaline phosphatase, comprising: shrimp alkaline phosphotase, calf intestine alkaline phosphatase, escherichia coli alkaline phosphatase, rat
Any one in alkaline phosphatase and thermal sensitivity alkaline phosphatase.
9. according to claim 1 to the detection method of any folic acid metabolism related gene polymorphism in 8, feature exists
In,
The single base extension system, comprising:
5~7 parts of distilled water, 1~3 part of 10*PCR buffer, 1~3 part of the dideoxyribonucleoside triphosphate mixture of 20mmol,
8~10 parts of the mixture and archaeal dna polymerase of Single base extension primer sequence shown in SEQ ID NO.1~SEQ ID NO.3
0.3~0.5 part, wherein each component is counted by volume, and the concentration of 0.3~0.5 part of the archaeal dna polymerase is 0.5~2U/ μ l,
The concentration of every kind of Single base extension primer sequence in the mixture of 8~10 parts of Single base extension primer sequence is 1~20 μ
mol。
10. according to claim 1 to the detection method of any folic acid metabolism related gene polymorphism in 8, feature exists
In,
The PCR amplification system, comprising:
8~10 parts of distilled water, 4~6 parts of 10*PCR buffer, the MgCl of 25mmol23~5 parts, the deoxyribose core of 20mmol
1~2 part of guanosine triphosphate mixture, the mixture of specificity amplification primer sequence shown in SEQ ID NO.4~SEQ ID NO.9
9~11 parts, 1~2 part of archaeal dna polymerase and 19~21 parts of genomic DNA, wherein each component is counted by volume, 9~11 parts of spy
Each specificity amplification primer sequence concentration in the mixture of specific amplification primers sequence is 0.1~2 μm of ol, and 1~2 part
The concentration of the archaeal dna polymerase be 0.5~2U/ μ l, the amount of 19~21 parts of the genomic DNA is 5~500ng.
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| CN104946746A (en) * | 2015-05-28 | 2015-09-30 | 长沙湘资生物科技有限公司 | Folic acid heredity metabolism ability detection using mass spectrum |
| CN110157796A (en) * | 2019-05-30 | 2019-08-23 | 北京和合医学诊断技术股份有限公司 | A method of detection folic acid metabolism related gene polymorphism |
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| CN104946746A (en) * | 2015-05-28 | 2015-09-30 | 长沙湘资生物科技有限公司 | Folic acid heredity metabolism ability detection using mass spectrum |
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Application publication date: 20191029 |