CN102424849B - Human paraoxonase (PON) 2 gene 148 site G/A polymorphic detection kit - Google Patents
Human paraoxonase (PON) 2 gene 148 site G/A polymorphic detection kit Download PDFInfo
- Publication number
- CN102424849B CN102424849B CN 201110440646 CN201110440646A CN102424849B CN 102424849 B CN102424849 B CN 102424849B CN 201110440646 CN201110440646 CN 201110440646 CN 201110440646 A CN201110440646 A CN 201110440646A CN 102424849 B CN102424849 B CN 102424849B
- Authority
- CN
- China
- Prior art keywords
- sequence
- polymorphic
- enzyme
- site
- cut
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a human paraoxonase (PON) 2 gene 148 site G/A polymorphic detection kit. The detection kit comprises a forward primer and a reverse primer in a sequence near the amplified human PON2 gene polymorphic rs12026 site and a restriction enzyme, wherein the forward primer is shown in SEQ ID NO:12, the reverse primer is shown in SEQ ID NO:13, and the restriction enzyme is BsmAI. The invention has the advantages of simple operation process, low cost and wide operation range.
Description
The application is that application number is the dividing an application for the application for a patent for invention of " method of identifier PON2 gene pleiomorphism rs12026 " that " 200910227638.8 ", the applying date be on December 24th, 2009, denomination of invention.
One, technical field
The present invention relates to identify the method for single nucleotide polymorphism.More specifically, the present invention relates to the method for identifier PON2 gene pleiomorphism rs12026.
Two, background technology
SNP (single nucleotide polymorphism) refers to the mutant dna sequence that the change of single Nucleotide causes, comprises the forms such as displacement, insertion and disappearance of single base.SNP now has been widely used in the location of genetic linkage analysis, association analysis and the diseases predisposing gene of simple and complex disease, instructs the tumor susceptibility gene clone.Polymerase chain reaction-restriction fragment length polymorphic (PCR-RFLP) technology is the genotypic classical way of a kind of quick, easy, accurate, low-cost SNP of detection.This method principle is: restriction enzyme is that class identification DNA specific site is (common 4～6bp), and the enzyme that cuts at specific site.The specificity of restriction enzyme site means the same fragment sequence of the allelic complete digestion of specific DNA meeting generation.And the replacement of base or insertion, disappearance can produce or eliminate a specific restriction enzyme site, thereby change size and number that enzyme cutting back produces fragment.The difference of these endonuclease bamhi banding patterns is called restriction fragment length polymorphism.If SNP produces and has eliminated certain restriction endonuclease sites, then can enzyme be cut, electrophoresis is detected by the PCR product is carried out.This method major advantage is easy and simple to handle, and fast, endpoint is accurate.Main drawback is the selection of restriction enzyme site, and not all SNP site can use this method to differentiate, and the part restriction endonuclease is expensive.
(paraoxonase PON) is the aromatic alcohol of being combined with high-density lipoprotein (HDL) by the synthetic class of liver to paraoxonase.It just is found as far back as nineteen fifty-three, because can be named as paraoxonase by the neural malicious sterilant paraoxon of hydrolysis (paraoxon).Find that at present Mammals paraoxonase gene family has 3 member: PON1, PON2 and PON3, they have structural homology and similar enzymic activity.These three genes have the attach structure homology, and about 65% and 70% identity is arranged respectively on the level of amino acid levels and Nucleotide.
People PON2 gene is positioned at karyomit(e) No. 7, and the polymorphic C/G of the being nucleotide variation of this gene the 5th exon can cause the 148th amino acids variation (Gly/Ala), and it is polymorphic often to be designated as G148A, this polymorphic function that may influence PON2.PON2 gene and polymorphic sequence and relevant information can be consulted by NIH state-run medical library biology information technology center (http://www.ncbi.nlm.nih.gov), this polymorphic rs12026 that is numbered in snp database.Therefore, usually should be polymorphic in the crowd three kinds of PON2 gene genotype of existence: GG type (two allelotrope bases of human genome rs12026 polymorphic site are G), CG type (two allelotrope bases of human genome rs12026 polymorphic site are respectively C and G) and CC type (two allelotrope bases of human genome rs12026 polymorphic site are C).
The PCR-RFLP method is often adopted in the evaluation of people PON2 gene polymorphic rs12026 at present.Identifier PON2 gene polymorphic rs12026 uses restriction endonuclease Fnu4H I to identify often at present, expensive (can be with reference to the table 1 of back about the reference price of part restriction endonuclease) of this restriction endonuclease influenced the expense of experiment, is difficult to popularize in the laboratory and uses.And because the inherent defect of conventional P CR-RFLP method, those skilled in the art are difficult to select other restriction enzymes that people PON2 gene polymorphic rs12026 is identified usually.
Therefore, this area exists simple to operate, and cost is low, the demand of the method for the novel detection SNP that use range is wide.
Three, summary of the invention
The invention provides a kind of simple to operately, cost is low, the method for the identifier PON2 gene pleiomorphism rs12026 that use range is wide, and it may further comprise the steps:
A) provide human gene group DNA to be measured;
B) forward primer and the reverse primer of sequence near the use amplification people PON2 gene pleiomorphism rs12026 site are template with described human gene group DNA to be measured, carry out pcr amplification reaction, obtain amplified production;
C) use restriction enzyme that described amplified production is carried out enzyme and cut, obtain enzyme and cut product; And d) described enzyme is cut product and carry out electrophoresis, with identifier PON2 gene pleiomorphism rs12026, wherein, described reverse primer 3 ' end is adjacent with polymorphic site, change to bases G after the mispairing with the 4th base that base A matches in polymorphic site downstream by the T in the original series in the reverse primer, in order in amplified production, form the GAGAC structure with polymorphic G allelotrope, former sequence C AGAA or GAGAA change to CAGAC or GAGAC (wherein first base are that rs12026 is polymorphic after the mispairing, the 5th base is the base C that base mismatch G matches in the primer), wherein form restriction endonuclease BsmAI or the Alw26I identification that GAGAC sequence (with the GTCTC complementation) can be identified this sequence during for G when polymorphic allelotrope, and then, can judge polymorphic genotype according to fragment incision situation, more specifically, through sequential analysis and Literature Consult as can be known, the polymorphic method of this C/G of original detection can be used preceding four kinds of restriction endonucleases in the table 1, as by base mispairing PCR polymorphic site back the 4th bit base A being replaced with C, then this polymorphic can be by BsmAI restriction endonuclease and isoschizomers (isoschizomer thereof, derive from different plant species but can identify the restriction enzyme of same DNA sequence) identification, be that G allelotrope can and cut by BsmAI restriction endonuclease and isoschizomers identification thereof, and C allelotrope can not cut.
The reference of NEB company restriction endonuclease pricing information (www.neb-china.com obtains from http://) as restriction enzyme enzyme recognition site and price is provided in the following table 1.
Several restriction endonuclease recognition sequences of table 1 NEB company and price thereof
In one embodiment of the invention, forward primer constitutes by being selected from following nucleotide sequence: SEQ ID NO:1 (AGGTCCCGTAGTTATGTCTTGT), SEQ NO:7 (TGCCCAGAGGTCCCGTAG) and SEQ NO:12 (GTCCCGTAGTTATGTCTTGTAAA), in another embodiment of the invention, reverse primer constitutes by being selected from following nucleotide sequence: SEQ ID NO:2 (TCAGATGCAACAGAGAATT
GTCT), SEQ ID NO:8 (TTCAGATGCAACAGAGAATT
GTCT) and SEQ NO:13 (CAGATGCAACAGAGAATT
GTCT), the design of forward and reverse primer considers that mainly primer is to the influence of sensitivity, specific degree and the amplification efficiency of pcr amplification.Usually according to base complementrity pair principle design primer, it is closely complementary that the sequence of primer and template is wanted.Length is 15-30bp, the too short or long poor specificity that causes, and long its elongating temperature that also can cause is unfavorable for the PCR reaction greater than 74 ℃.The definite of reverse primer must must be contained the base mismatch G that matches mutually with the 4th the base A in polymorphic site downstream by its 3 ' end except mentioned above principle, in order in amplified production, form the GAGAC structure with polymorphic G allelotrope, be beneficial to BsmAI restriction endonuclease and isoschizomers thereof and carry out enzyme and cut evaluation.The length of amplified production mainly be by forward primer determine (because reverse primer is positioned near the polymorphic site downstream, its position is roughly definite), usually amplified production length is between the 100-200bp, so namely is beneficial to the amplification of PCR product, is beneficial to follow-up enzyme again and cuts evaluation.Otherwise, being unfavorable for amplification if product is too short, fragment length difference was less after long then enzyme was cut, and was unfavorable for differentiating.For example, in one embodiment of the invention, the nucleotide sequence that utilizes SEQ ID NO:1 can obtain following amplified production as the nucleotide sequence of forward primer and SEQ ID NO:2 as the PCR reaction of reverse primer:
aggtcccgta gttatgtctt gtaaattaac tctgttcttt caattcttta gatgacacag 60
tttatctctt tgttgtaaac cacccagaat tcaagaatac agtggaaatt tttaaatttg 120
aagaags
aga caattctctg ttgcatctga 150
(SEQ ID NO：4)
Underscore partly is respectively forward, the corresponding sequence of reverse primer in the amplification after product sequence, and the s of 127bp place represents the polymorphic SNP of being of C/G site rs12026.This amplified production length is 150bp, can occur 150bp, 121bp, three kinds of segments of 29bp (wherein 29bp can not see in electrophorogram) after the BsmAI enzyme is cut.Enzyme is cut the back genotype and judged: the GG type is 121bp, and the CG type has 121bp and two kinds of fragments of 150bp, and the CC type is 150bp.
For the evaluation that enzyme is cut product, use agarose gel electrophoresis and polyacrylamide gel electrophoresis usually, consider based on cost and convenience, use agarose gel electrophoresis more common.In the present invention, for the amplified production of above-mentioned 150bp, the condition of agarose gel electrophoresis is that enzyme is cut product in the 2.5%-4% sepharose under the 3-8V/cm condition, electrophoresis 40-80 minute, and the evaluation of under ultraviolet lamp, taking pictures.
In another preferred embodiment of the present invention, described restriction enzyme is selected from BsmAI, Alw26I and their isoschizomers, and more preferably described restriction enzyme is BsmAI, Alw26I.This mainly is based on taking all factors into consideration of cost and cutting efficiency.
In the present invention, the condition of pcr amplification reaction is not subjected to special restriction, as long as can obtain amplified production.Significant parameter-annealing temperature of PCR and time, depend on length, based composition and the concentration thereof of primer, also have the length of target sequence.If the PCR annealing temperature is crossed low then is prone to non-special product, the too high amplified production output that then influences finally need be determined with experiment.The PCR time of extending determines that according to product length 20s-60s gets final product usually.In order to improve pcr amplification efficient, and then further improve and identify efficient, in a preferred embodiment of the present invention, the pcr amplification condition that adopts is:
The pre-sex change of 95 degree is after 3 minutes; Carry out 30 following cyclic amplifications: 95 degree sex change 30 seconds, 56 degree annealing 30 seconds, 72 degree extended 30 seconds; 72 degree extend 5min after 30 loop ends;
For the selection of DNA to be measured, the not special restriction of the present invention.Both can be to obtain genomic dna from the body fluid of human body or tissue, can also be through the genome of degradation treatment in advance.For convenience of implementation, the genomic dna that preferably extracts from blood usually.Wherein said tissue comprises and containing in the human body all or the tissue of PON2 gene at least, and with whether to express this PON2 gene irrelevant.The method of extracting DNA from body fluid and/or tissue is well known to a person skilled in the art, and can be with reference to molecular biology manual commonly used, and for example " molecular cloning " the 2nd edition carries out.
In addition, also provide test kit for the identification of people PON2 gene pleiomorphism rs12026 in the present invention, it comprises amplification people's PON2 gene pleiomorphism rs12026 forward primer and reverse primer, reverse primer 3 ' is terminal adjacent with polymorphic site, change to bases G after the mispairing with the 4th base that base A matches in polymorphic site downstream by the T in the original series in the reverse primer, former sequence C AGAA or GAGAA change to CAGAC or GAGAC (wherein first base are that rs12026 is polymorphic after the mispairing, the 5th base is the base C that base mismatch G matches in the primer), wherein form restriction endonuclease BsmAI or the Alw26I identification that GAGAC sequence (with the GTCTC complementation) can be identified this sequence during for G when polymorphic allelotrope.As previously mentioned, this test kit is simple to operate, and cost is low, and use range is wide.
In another embodiment of the invention, also comprise restriction enzyme and their isoschizomerss such as BsmAI, Alw26I in the described test kit, like this can be conveniently to the detection of amplified production.Further, preferred described restriction enzyme is BsmAI, Alw26I.This mainly is based on the cost of restriction enzyme and taking all factors into consideration of cutting efficiency.
Certainly, it will be understood by those skilled in the art that in test kit to comprise other compositions, for example be used for the specification sheets of enforcement evaluation etc.
Four, description of drawings
Fig. 1 has described among the embodiment 1 the polymorphic PCR product of people PON2 gene (rs12026) and has cut the rear electrophoresis photo through the BsmAI enzyme.Wherein M is: dna molecular amount mark; The electrophoresis result of swimming lane 1:CC type sample; The electrophoresis result of swimming lane 2 and 3:CG type; The electrophoresis result of swimming lane 4:GG type sample.
Five, embodiment
Below by the specific embodiment of the present invention technical scheme of the present invention is described in detail.Wherein, need to prove that the present invention also is subject to embodiment described below and embodiment never in any form.
The nucleotide sequence that utilizes SEQ ID NO:1 below describes in detail concept of the present invention as the nucleotide sequence of forward primer and SEQ ID NO:2 as reverse primer as an example.
In one embodiment of the invention, the present invention detects the method for people PON2 gene polymorphic rs12026, and step is:
(1), to extract human gene group DNA to be measured be template, the human gene group DNA that described human gene group DNA's template obtains for the human body any part;
(2), the pcr amplification genomic dna, the templet gene group DNA that extracts is carried out pcr amplification, obtain to contain polymorphic near the PCR product of sequence:
(3), use restriction enzyme to carry out endonuclease reaction to pcr amplification product, get enzyme and cut product; And
(4), the product after enzyme is cut carries out agarose gel electrophoresis, identifier PON2 gene polymorphic rs12026.
Below each key step is described in detail
(1) design of primers is with synthetic:
Search PON2 gene order and SNP information in http://www.ncbi.nlm.nih.gov website, NIH state-run medical library biology information technology center, determine PON2 polymorphic site nucleotide variation data;
The portion gene sequence (601bp) that contains the polymorphic rs12026 of PON2
(SEQ ID NO：3)：
The s of 301bp place represents the polymorphic SNP of being of C/G site rs12026 in the gene order;
Introduce the primer of base mismatch according to above-mentioned sequences Design, determine position and the length of forward and reverse primer according to the sequence situation, specific as follows:
Forward primer 5 ' AGGTCCCGTAGTTATGTCTTGT 3 ' (SEQ ID NO:1),
Reverse primer 5 ' TCAGATGCAACAGAGAATT
GTCT 3 ' (SEQ ID NO:2);
Underscore G base is that base mismatch is used for creating restriction enzyme site in the downstream primer, sequence changes to CAGAC or GAGAC by CAGAA or GAGAA (wherein first base is that C/G is polymorphic) near polymorphic in the PCR product after the mispairing, and BsmAI identification GAGAC sequence (with the complementation of GTCTC sequence) is polymorphicly can cut during for G allelotrope, judges polymorphic genotype according to fragment incision situation;
According to forward primer sequence and reverse primer sequence synthesized primer thing, synthetic described primer can use method as known in the art, and for example solid-phase synthesis also can be entrusted bio-engineering corporation synthetic and detection forward and reverse primer.It is that 10 μ mol/L concentration are standby that synthetic forward, reverse primer are diluted;
(2) preparation pcr amplification product
Pcr amplification uses the reaction system of 15-100 μ l to be: 2 * PCR MIX consumption is the described human gene group DNA to be detected of 7.5-50 μ l, 50-2000ng and 10 μ mol/L forwards, each 0.1-10 μ l of reverse primer as half of pcr amplification reaction system volume, be supplemented to 15-100 μ l with the sterilization distilled water, mixing namely gets the pcr amplification reaction system;
Wherein, the mixed solution of described 2 * PCR MIX PCR reaction usefulness that is 2 times of concentration is made;
With the pcr amplification reaction system after 94-95 ℃ of pre-sex change 3-5 minute; Carry out 30-35 following circulation: 94-95 ℃ of sex change 20-60s, 50-65 ℃ of annealing 20-60s, 72 ℃ are extended 20-60s; 72 ℃ are extended 5-10min after 30-35 the loop ends, namely get pcr amplification product, the corresponding middle base 175bp-324bp place of portion gene sequence (SEQ ID NO:3) that contains the polymorphic rs12026 of PON2, its position;
Amplification back sequence (its position is corresponding to contain base 175-324 place in the portion gene sequence of the polymorphic rs12026 of PON2, common 150bp) is (SEQ ID NO:4):
aggtcccgta gttatgtctt gtaaattaac tctgttcttt caattcttta gatgacacag 60
tttatctctt tgttgtaaac cacccagaat tcaagaatac agtggaaatt tttaaatttg 120
aagaags
aga caattctctg ttgcatctga 150
Underscore partly is forward, the corresponding sequence of reverse primer in the 150bp sequence amplification after product sequence, and the s of 127bp place represents the polymorphic SNP of being of C/G site rs12026.
(3) endonuclease reaction
Pcr amplification product cut at 10-30 μ l enzyme carry out endonuclease reaction in the system, the described 10-30 μ l enzyme system of cutting is: restriction enzyme 1-10U and the 10 * enzyme cutting buffering liquid of the pcr amplification product of 2-15 μ l, identification GAGAC sequence (with the complementation of GTCTC sequence) account for 1/10 of cumulative volume, the sterilization distilled water is supplemented to 10-30 μ l, mixing, get enzyme and cut system, enzyme was cut system in 37 ℃ of water-bath 4-16 hours, namely get enzyme and cut product;
The restriction enzyme of described identification GAGAC sequence (with the complementation of GTCTC sequence) is BsmAI, Alw26I and isoschizomers thereof;
Can form after PCR product enzyme was cut when this polymorphic site contained G allelotrope and cut fragment 121bp (this fragment downstream sequence is cut because of enzyme and formed sticky end than 4 bases of upstream sequence increase) sequence (SEQ ID NO:5):
aggtcccgta gttatgtctt gtaaattaac tctgttcttt caattcttta gatgacacag 60
tttatctctt tgttgtaaac cacccagaat tcaagaatac agtggaaatt tttaaatttg 120
a 121
Enzyme is cut the back and is cut that underscore partly is the corresponding sequence of forward primer in the fragment 121bp sequence;
Can form after PCR product enzyme was cut when this polymorphic site contained G allelotrope and cut fragment 29bp (this fragment downstream sequence is cut because of enzyme and formed sticky end than 4 bases of upstream sequence minimizing) sequence (SEQ ID NO:6):
agaagsagac aattctctgt tgcatctga 29
The s of 6bp place represents the polymorphic SNP of being of C/G site rs12026;
(2) agarose gel electrophoresis, determine gene pleiomorphism:
Enzyme is cut product in 3% sepharose under the 3-8V/cm condition, electrophoresis 40-80 minute, the evaluation of under ultraviolet lamp, taking pictures, wherein, for different genotype, after the amplification of PON2 site the 150bp segment appears, through enzyme cut, 150bp appears in electrophoresis, three kinds of segments of 121bp, 29bp, genotype basis for estimation 150bp and 121bp fragment have or not judgement: the GG type is 121bp, and the CG type has 150bp and two kinds of fragments of 121bp, and the CC type is 150bp; Identify that through order-checking the result of sequencing result and use prior art detection gained is identical.
Be to implement some embodiment of the present invention below.
Embodiment 1
1 materials and methods
1.1 main agents and instrument
Reagent: 2 * PCR mix (MBI company), restriction enzyme BsmAI (NEB company), agarose (BBI company), primer is synthetic by Shanghai Sangon company.
Instrument: 9600 type PCR instrument (PE company), miniature electrophoresis chamber (Pharmacia Biotech, EPS1000), Gel Doc 2000 gel imaging instrument (Bio-RAD company).
The order-checking of PCR product is given birth to worker's biotechnology company limited by Shanghai and is finished.
1.2 sequence is searched and design of primers
Search PON2 gene order and SNP information in the NCBI website, determine PON2 polymorphic site nucleotide variation information in conjunction with relevant document, the design primer, specific as follows:
Forward primer 5 ' AGGTCCCGTAGTTATGTCTTGT 3 ' (SEQ ID NO:1),
Reverse primer 5 ' TCAGATGCAACAGAGAATT
GTCT 3 ' (SEQ ID NO:2);
1.3 extract DNA as DNA to be measured from whole blood sample
The EDTA anticoagulant tube is gathered human peripheral whole blood blood sample 300 μ l, and using the RelaxGene Blood DNA System of TIANGEN company poba gene group DNA extraction system (main component is cell pyrolysis liquid CL, Proteinase K, damping fluid FG, elution buffer TB) to extract Whole Blood Genomic DNA is human gene group DNA's template to be measured:
300 μ l peripheral bloods are transferred to centrifuge tube, add 750 μ l cell pyrolysis liquid CL, put upside down mixing 5 times;
Centrifugal 20 seconds of 10,000r/min abandons supernatant liquor;
Add the damping fluid mixed solution of 150 μ l in centrifuge tube, the damping fluid mixed solution is to be mixed and made at 100: 1 with volume ratio by damping fluid FG and Proteinase K, and vortex vortex mixer mixing to solution does not have agglomerate;
Then, at 65 ℃ of water-bath 10min, put upside down mixing therebetween 5 times;
The adding mass concentration is 99.9% Virahol 150 μ l, puts upside down abundant mixing to occurring thread or bunch shape genomic dna;
Centrifugal 3 minutes of 10,000r/min abandons supernatant liquor, and centrifuge tube is upside down on the clean thieving paper, guarantees that the DNA throw out is in pipe;
Adding mass concentration is 70% ethanol, 150 μ l, and vortex vibrated for 5 seconds, centrifugal 3 minutes of 10,000r/min, abandon supernatant liquor after, adding mass concentration again is 70% ethanol, 150 μ l, vortex vibrated for 5 seconds, centrifugal 3 minutes of 10,000r/min abandons supernatant liquor;
Centrifuge tube is upside down on the clean thieving paper stopped 5 minutes, guarantee that the DNA throw out is in pipe; Centrifuge tube is at room temperature left standstill, be dried to all evaporate clean (at least 5 minutes);
Add 200 μ l elution buffer TB again, vortex vibration 5 seconds, 65 ℃ were heated 10 minutes to 1 hour, during heating, flicked hydrotropy for several times, namely got Whole Blood Genomic DNA.
1.4PCR amplification and enzyme are cut evaluation
Human gene group DNA 100ng (preparation method is as mentioned above), each primer 0.2 μ mol/L, 1 * PCRmix, the sterilization distilled water is supplied 25 μ l reaction systems.The PCR reaction conditions is 94 ℃ of pre-sex change 5min at first, and 94 ℃ of sex change 30s get 56 ℃ of annealing 20s according to primer Tm value then, and 72 ℃ are extended 20s, and totally 30 circulations are extended 10min for back 72 ℃.Behind the pcr amplification, get PCR product 10 μ l, add 5U restriction endonuclease and 2 μ l, 10 * enzyme cutting buffering liquid and form 20 μ l reaction systems with the sterilization distilled water, after enzyme is cut 8h in 37 ℃ of water-baths, observe imaging in the gel imaging instrument behind the electrophoresis, the results are shown among Fig. 1.
2 results
2.1 enzyme is cut the result
Above-mentioned enzyme is cut product under 3% sepharose 8V/cm condition, electrophoresis 40 minutes, the evaluation of under ultraviolet lamp, taking pictures, wherein, the 150bp segment occurring after the amplification of PON2 site (is as the criterion with upstream sequence, as follows), cut rear electrophoresis through the BsmAI enzyme and 150bp, 121bp (downstream sequence is compared upstream sequence and produced four strand bases of sticky end increase because the MspI enzyme is cut), three kinds of segments (wherein can not see in the 29bp electrophorogram) of 29bp (downstream sequence is compared upstream sequence and reduced by four strand bases because the MspI enzyme is cut the generation sticky end) can occur.
As shown in Figure 1.Enzyme is cut the back genotype and judged: GG is fragment of 121bp, and CG has 121bp and two kinds of fragments of 150bp, and CC is fragment of 150bp.
2.2PON2 the gene polymorphic result identifies
Detected result finds that GG type, CG type and three kinds of genotype of CC type appear in PON2.In addition, to the evaluation of checking order of the polymorphic all types of PCR products of PON2, sequencing result and expected results are in full accord.
It is polymorphic that embodiment 2 peripheral blood whole blood samples are measured people PON2 gene rs12026:
Basic identical with the step of embodiment 1, the forward primer that the PCR reaction is adopted is SEQ ID NO:7, and reverse primer is SEQ ID NO:8, and annealing temperature is 57 ℃, and the restriction enzyme that adopts is Alw26I (MBI company);
Forward primer: 5 ' TGCCCAGAGGTCCCGTAG 3 ' (SEQ ID NO:7)
Reverse primer: 5 ' TTCAGATGCAACAGAGAATT
GTCT 3 ' (SEQ ID NO:8)
The result:
Amplification back sequence (its position is corresponding to contain base 168-325 place in the portion gene sequence of the polymorphic rs12026 of PON2, common 158bp), the amplified production sequence that obtains following (SEQ ID NO:9):
tgcccagagg tcccgtagtt atgtcttgta aattaactct gttctttcaa ttctttagat 60
gacacagttt atctctttgt tgtaaaccac ccagaattca agaatacagt ggaaattttt 120
aaatttgaag aags
agacaa ttctctgttg catctgaa 158
Underscore partly is forward, the corresponding sequence of reverse primer in the amplification after product sequence, and s represents the polymorphic SNP of being of C/G site rs12026.
Product sequence after enzyme is cut is as follows respectively:
Can form after PCR product enzyme was cut when this polymorphic site contained G allelotrope and cut fragment 128bp (this fragment downstream sequence is cut because of enzyme and formed sticky end than 4 bases of upstream sequence increase) sequence (SEQ ID NO:10):
tgcccagagg tcccgtagtt atgtcttgta aattaactct gttctttcaa ttctttagat 60
gacacagttt atctctttgt tgtaaaccac ccagaattca agaatacagt ggaaattttt 120
aaatttga 128
Can form after PCR product enzyme was cut when this polymorphic site contained G allelotrope and cut fragment 30bp (this fragment downstream sequence is cut because of enzyme and formed sticky end than 4 bases of upstream sequence minimizing) sequence (SEQ ID NO:11):
agaags
agac aattctctgt tgcatctgaa 30
Above-mentioned enzyme is cut product under 3% sepharose 6V/cm condition, electrophoresis 50 minutes, the evaluation of under ultraviolet lamp, taking pictures, wherein, the 158bp segment appears after the PON2 site amplification, through enzyme cut, 158bp appears in electrophoresis, three kinds of segments of 128bp, 30bp, genotype basis for estimation 158bp and 128bp fragment have or not judgement: the GG type is 128bp, the CG type has 158bp and two kinds of fragments of 128bp, and the CC type is 158bp.
It is polymorphic that embodiment 3 peripheral blood blood clot samples are measured people PON2 gene rs12026:
Basic identical with the step of embodiment 1, just adopt in the following method and extract DNA as DNA to be measured from peripheral blood blood clot sample.In addition, the forward primer that the PCR reaction is adopted is SEQ NO:12, and reverse primer is SEQ NO:13, and annealing temperature is 56 ℃.
Forward primer: 5 ' GTCCCGTAGTTATGTCTTGTAAA 3 ' (SEQ NO:12)
Reverse primer: 5 ' CAGATGCAACAGAGAATT
GTCT 3 ' (SEQ NO:13)
Extract DNA:
General blood collection tube is gathered human peripheral 400 μ l, and genomic dna is human gene group DNA's template to be measured in the use TIANGEN RelaxGene Blood DNA System of the company poba gene group DNA extraction system extraction peripheral blood blood clot;
Treat that serum is separated out the back separation of serum in the heparin tube, follow these steps to then carry out:
Sludged blood in the above-mentioned heparin tube is ground to the homogenate shape is placed in the centrifuge tube, add 750 μ l cell pyrolysis liquid CL, put upside down mixing 5 times;
Centrifugal 20 seconds of 10,000r/min abandons supernatant liquor;
Add 150 μ l damping fluid mixed solutions, the damping fluid mixed solution is to be mixed and made at 100: 1 with volume ratio by damping fluid FG and Proteinase K, and vortex vortex mixer mixing to solution does not have agglomerate;
Then, at 65 ℃ of water-bath 10min, put upside down mixing therebetween 6 times;
The adding mass concentration is 99.9% Virahol 150 μ l, puts upside down abundant mixing to occurring thread or bunch shape genomic dna;
Centrifugal 3 minutes of 10,000r/min abandons supernatant liquor, and centrifuge tube is upside down on the clean thieving paper, guarantees that the DNA throw out is in pipe;
Add mass concentration and be 70% ethanol 150 μ l, vortex vibrated for 5 seconds, centrifugal 3 minutes of 10,000r/min, abandon supernatant liquor after, add mass concentration again and be 70% ethanol 150 μ l, vortex vibrated for 5 seconds, centrifugal 3 minutes of 10,000r/min abandons supernatant liquor;
Centrifuge tube is upside down on the clean thieving paper stopped at least 5 minutes, guarantee that the DNA throw out is in pipe;
Centrifuge tube is at room temperature left standstill, be dried to all evaporate clean (at least 5 minutes);
Add 200 μ l elution buffer TB, vortex vibration 5 seconds, 65 ℃ were heated 10 minutes to 1 hour, during heating, flicked hydrotropy for several times, namely got genomic dna in the peripheral blood blood clot.
The result:
Amplification back sequence (its position is corresponding to contain base 177-323 place in the portion gene sequence of the polymorphic rs12026 of PON2, common 147bp), the amplified production sequence that obtains following (SEQ ID NO:14):
gtcccgtagt tatgtcttgt aaattaactc tgttctttca attctttaga tgacacagtt 60
tatctctttg ttgtaaacca cccagaattc aagaatacag tggaaatttt taaatttgaa 120
gaags
agaca attctctgtt gcatctg 147
Underscore partly is forward, the corresponding sequence of reverse primer in the amplification after product sequence, and s represents the polymorphic SNP of being of C/G site rs12026.
Product sequence after enzyme is cut is as follows respectively:
Can form after PCR product enzyme was cut when this polymorphic site contained G allelotrope and cut fragment 119bp (this fragment downstream sequence is cut because of enzyme and formed sticky end than 4 bases of upstream sequence increase) sequence (SEQ ID NO:15):
gtcccgtagt tatgtcttgt aaattaactc tgttctttca attctttaga tgacacagtt 60
tatctctttg ttgtaaacca cccagaattc aagaatacag tggaaatttt taaatttga 119
Can form after PCR product enzyme was cut when this polymorphic site contained G allelotrope and cut fragment 28bp (this fragment downstream sequence is cut because of enzyme and formed sticky end than 4 bases of upstream sequence minimizing) sequence (SEQ ID NO:16):
agaags
agac aattctctgt tgcatctg 28
Above-mentioned enzyme is cut product under 3% sepharose 5V/cm condition, electrophoresis 60 minutes, the evaluation of under ultraviolet lamp, taking pictures, wherein, the 147bp segment appears after the PON2 site amplification, through enzyme cut, 147bp appears in electrophoresis, three kinds of segments of 119bp, 28bp, genotype basis for estimation 147bp and 119bp fragment have or not judgement: the GG type is 119bp, the CG type has 147bp and two kinds of fragments of 119bp, and the CC type is 147bp.
It is polymorphic that embodiment 4 human lung tissue's samples are measured people PON2 gene rs12026
Basic identical with the step of embodiment 1, just adopt in the following method and extract DNA as DNA to be measured from lung tissue sample.
Use the excision lung tissue, it is human gene group DNA's template to be measured that phenol-chloroform method extracts the lung tissue genomic dna:
The lung tissue piece is thawed, and with physiological saline flush away blood stains, clip 0.1g, glass homogenizer grind first centrifuge tube of putting into 1.5ml after the broken lung tissue;
In first centrifuge tube, add 1ml DNA extraction damping fluid (10mmol/LtrisCl
2, 0.1mol/L EDTA, 20 μ g/ml Pancreatic RNases, 0.5% sodium lauryl sulphate) mixing adds 50 μ l mass concentrations and is 10% sodium lauryl sulphate again, and concentration is the Proteinase K 5.0 μ l of 20mg/ml, fully behind the mixing, in 56 ℃ of insulation 5h, every 2h shakes 1 time;
Be placed into room temperature, add saturated phenol 500 μ l, put upside down mixing, the centrifugal 10min of 10000r/min, water phase separated and organic phase are drawn second centrifuge tube that water that the upper strata contains nucleic acid places 1.5ml;
The isopyknic phenol of the water that contains nucleic acid in second centrifuge tube in adding and the pipe: chloroform: the mixed solution of primary isoamyl alcohol, wherein, phenol: chloroform: the mixed solution of primary isoamyl alcohol is by phenol: chloroform: primary isoamyl alcohol is mixed and made into volume ratio at 25: 24: 1, put upside down mixing, centrifugal 10 minutes of 10000r/min gets supernatant liquid and transfers in the 3rd centrifuge tube of 1.5ml;
In the 3rd centrifuge tube, add and the isopyknic chloroform of liquid in pipe: primary isoamyl alcohol is put upside down mixing with the mixed solution that volume ratio is mixed and made at 24: 1, and centrifugal 10 minutes of 10000r/min gets supernatant liquid in the 4th centrifuge tube of 1.5ml;
Add the dehydrated alcohol of-20 ℃ of precoolings of 2.5 times of volumes of clear liquid in the pipe in the 4th centrifuge tube, deposit D NA gets the DNA throw out;
With the DNA throw out, centrifugal 10 minutes of 12000r/min abandons ethanol;
The mass concentration that adds-20 ℃ again is 75% washing with alcohol, and centrifugal 5 minutes of 10000r/min removes ethanol, and is dry under 55 ℃, gets dry DNA throw out;
Add the dry DNA throw out of 100 μ l sterilization distilled water dissolving ,-20 ℃ of preservations namely get the lung tissue genomic dna, and are standby.
The result:
The gained enzyme is cut product under 3% sepharose 4V/cm condition, electrophoresis 70 minutes, the evaluation of under ultraviolet lamp, taking pictures, wherein, the 150bp segment appears after the PON2 site amplification, through enzyme cut, 150bp appears in electrophoresis, three kinds of segments of 121bp, 29bp, genotype basis for estimation 150bp and 121bp fragment have or not judgement: the GG type is 121bp, the CG type has 150bp and two kinds of fragments of 121bp, and the CC type is 150bp.
Identify that through order-checking the result of sequencing result and use prior art detection gained is identical.
From above-described embodiment as can be seen, the present invention is directed to and use PCR-RFLP to identify the shortcoming of the polymorphic rs12026 of PON2, namely use identification former polymorphic near the restriction endonuclease of sequence, factor such as the restriction endonuclease choice is little, and is expensive.The present invention overcomes this shortcoming by the mispairing primer that design comprises restriction enzyme site.This method can be thought the special applications of PCR-RFLP, its principle is the base alternative case design PCR primer according to the single base mutation site, wherein a primer is according to the contiguous sequences Design in mutational site, the artificial base mismatch of introducing, make a kind of mutant of primer 3 ' end and single base mutation form a restriction enzyme site behind pcr amplification, its PCR product can be analyzed with the method for similar PCR-RFLP.Because this method has been used primer 3 ' end mispairing technology, the PCR product carries out can carrying out genotypic evaluation work behind the restriction enzyme digestion and electrophoresis, therefore in practice, has very big handiness, and detection method is simple, cost is low, enzyme is cut the result and is easily differentiated, and is a kind of better method of carrying out genotype identification.The present invention redesigns this polymorphic sequence of new primer amplification by improving experimental technique, makes near sequence change this gene polymorphic in the amplified production, can use other restriction endonucleases and identify, thereby can select more cheap restriction endonuclease to finish detection.
Claims (1)
1. test kit for the identification of people PON2 gene pleiomorphism rs12026, it is characterized in that, the forward primer and reverse primer and the restriction enzyme that comprise near the sequence in amplification people PON2 gene pleiomorphism rs12026 site, described forward primer is shown in SEQ ID NO:12, described reverse primer is shown in SEQ ID NO:13, and described restriction enzyme is BsmAI.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110440646 CN102424849B (en) | 2009-12-24 | 2009-12-24 | Human paraoxonase (PON) 2 gene 148 site G/A polymorphic detection kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110440646 CN102424849B (en) | 2009-12-24 | 2009-12-24 | Human paraoxonase (PON) 2 gene 148 site G/A polymorphic detection kit |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009102276388A Division CN101812514B (en) | 2009-12-24 | 2009-12-24 | Method for identifying gene polymorphism rs12026 of human PON2 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102424849A CN102424849A (en) | 2012-04-25 |
| CN102424849B true CN102424849B (en) | 2013-09-04 |
Family
ID=45958877
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110440646 Expired - Fee Related CN102424849B (en) | 2009-12-24 | 2009-12-24 | Human paraoxonase (PON) 2 gene 148 site G/A polymorphic detection kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102424849B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117960248B (en) * | 2024-04-02 | 2024-06-21 | 江苏省中国科学院植物研究所 | Preparation method of bionic nano-enzyme MOF-808-Apt based on aptamer modification and method for detecting paraoxon by using bionic nano-enzyme MOF-808-Apt |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1766125A (en) * | 2005-10-18 | 2006-05-03 | 卫生部北京医院 | Method and detergent for predicting type 2 diabetes susceptibility using PON2 gene the nineth extron polymorphism |
| CN101294214A (en) * | 2007-04-27 | 2008-10-29 | 上海人类基因组研究中心 | coronary disease testing method and reagent kit |
-
2009
- 2009-12-24 CN CN 201110440646 patent/CN102424849B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1766125A (en) * | 2005-10-18 | 2006-05-03 | 卫生部北京医院 | Method and detergent for predicting type 2 diabetes susceptibility using PON2 gene the nineth extron polymorphism |
| CN101294214A (en) * | 2007-04-27 | 2008-10-29 | 上海人类基因组研究中心 | coronary disease testing method and reagent kit |
Non-Patent Citations (6)
| Title |
|---|
| PON2基因C311S、G148A多态性与脑卒中关系的研究;许宏伟 等;《中华医学遗传学杂志》;20070630;第24卷(第3期);摘要，第328页第一栏最后一段至第330页第一栏第一段 * |
| 双创造酶切位点聚合酶链反应_限制性片段长度多态性检测MGMT基因多态性的应用;王威 等;《卫生研究》;20080131;第37卷(第1期);第4页第二栏最后一段至第7页第一栏第3段 * |
| 应用创造酶切位点法检测单碱基突变;赵春江 等;《遗传》;20031231;第25卷(第3期);第328页材料和方法部分 * |
| 王威 等.双创造酶切位点聚合酶链反应_限制性片段长度多态性检测MGMT基因多态性的应用.《卫生研究》.2008,第37卷(第1期),第4-7页. |
| 许宏伟 等.PON2基因C311S、G148A多态性与脑卒中关系的研究.《中华医学遗传学杂志》.2007,第24卷(第3期),第328-330页. |
| 赵春江 等.应用创造酶切位点法检测单碱基突变.《遗传》.2003,第25卷(第3期),第327-329页. |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102424849A (en) | 2012-04-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103436610A (en) | PCR-RFLP rapid detection method for common sturgeons | |
| CN103436611A (en) | PCR-RFLP rapid detection method for common sturgeons | |
| EP3584324A1 (en) | Nucleic acid quantification products and processes | |
| CN101812513B (en) | Method for identifying gene polymorphism rs2274976 of human MTHFR | |
| CN106191253B (en) | Beijing duck based on GBS technology simplifies gene order surveying method | |
| CN102424849B (en) | Human paraoxonase (PON) 2 gene 148 site G/A polymorphic detection kit | |
| CN103436612A (en) | PCR-FRLP quick detecting method of common sturgeons | |
| CN106319071A (en) | Method for identifying human breast cancer LINC-ROR (Long Intergenic Non-Protein Coding RNA, Regulator Of Reprogramming) gene rs4801078 polymorphism by BsmAI | |
| KR102377677B1 (en) | InDel Markers for Discrimination of Cynanchum wilfordii and Cynanchum auriculatum and Method for Use thereof | |
| CN102433386B (en) | Human paraoxonase 2 (PON2) gene polymorphism (rs12026) detection kit | |
| CN102676655B (en) | Kit for identifying human methylene tetrahydrofolate reductase (MTHFR) gene polymorphism rs2274976 by using MspI | |
| CN101812514B (en) | Method for identifying gene polymorphism rs12026 of human PON2 | |
| CN102660638B (en) | Kit for identifying polymorphic rs2274976 of human MTHFR gene by BsrI | |
| CN102660639B (en) | Kit for identifying polymorphic rs2274976 of human MTHFR gene by BsrFI | |
| CN105695615A (en) | Method for identifying polymorphism of human breast cancer genes RAD51 rs7180135 by aid of BccI | |
| CN108823313A (en) | A kind of primer and kit detecting human breast carcinoma gene PCAT1 polymorphism rs4473999 | |
| CN102102129A (en) | Method for detecting single nucleotide polymorphism or small insertions and deletions by utilizing MutS proteins in genome range | |
| CN107365837A (en) | With BanII identification human gastric cancers ZNF33B 2:The method of 1 gene rs579501 polymorphisms | |
| CN107365838A (en) | With the method for BstUI surveyor's LINCRNA CCAT1 gene rs6470502 polymorphisms | |
| CN110438212B (en) | Enzyme digestion PCR kit and method for enrichment detection of specific gene fragment | |
| CN108085381B (en) | Application of gene mutation sites in preparation of reagent or kit for diagnosing chronic pancreatitis and evaluating prognosis | |
| CN105648068A (en) | Method for identifying human HOTAIR (HOX antisense intergenic RNA) gene polymorphism rs920778 by using MspI | |
| CN113604602A (en) | STR molecular marker of nematophagous fungi Arthrobotrya oligospora | |
| CN110452894A (en) | Target high temperature resistant II type restriction enzyme and its application in the site AGCT | |
| Wang et al. | Analysis of genotype polymorphism of tumor-related genes harbored in chromosome arm 1p and 8p in hepatocellular carcinoma patients by cSNP chip |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C53 | Correction of patent for invention or patent application | ||
| CB03 | Change of inventor or designer information |
Inventor after: Yang Yongli Inventor after: Yan Zhen Inventor after: Shi Nian Inventor after: Shi Xuezhong Inventor after: Lu Jie Inventor after: Ping Zhiguang Inventor after: Yao Wu Inventor after: Wang Wei Inventor after: Zhou Fang Inventor after: Li Chunyang Inventor before: Yang Yongli Inventor before: Yao Wu Inventor before: Wang Wei |
|
| COR | Change of bibliographic data |
Free format text: CORRECT: INVENTOR; FROM: YANG YONGLI YAO WU WANG WEI TO: YANG YONGLI SHI NIAN SHI XUEZHONG LU JIE PING ZHIGUANG YAO WU WANG WEI ZHOU FANG LI CHUNYANG YAN ZHEN |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130904 Termination date: 20181224 |