CN107058586A - A kind of GSTM1 genotyping detection methods based on quantitative PCR technique - Google Patents
A kind of GSTM1 genotyping detection methods based on quantitative PCR technique Download PDFInfo
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- CN107058586A CN107058586A CN201710426009.2A CN201710426009A CN107058586A CN 107058586 A CN107058586 A CN 107058586A CN 201710426009 A CN201710426009 A CN 201710426009A CN 107058586 A CN107058586 A CN 107058586A
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- gstm1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
Abstract
The invention discloses a kind of GSTM1 genotyping detection methods based on quantitative PCR technique, GSTM1 genetic fragments are expanded using the primer SEQ1 and SEQ2 of GSTM1 gene specifics, while the design GSTM1 gene specific TAQman probe SEQ3 Fam in the amplification region that defines of primer of GSTM1 gene specifics；ALbumin genetic fragments are expanded using ALbumin specific PCR primer SEQ4 and SEQ5, while the design ALbumin gene specific TAQman probe SEQ6 Vic in the amplification region that defines of primer of ALbumin gene specifics.Effectively PCR primer can be prevented to pollute, it is to avoid poisonous DNA coloring agents pollution, detection and interpretation of result process can be greatly simplified, detection flux is improved.
Description
Technical field
The present invention relates to technical field of gene detection, specifically a kind of GSTM1 partings detection side based on quantitative PCR technique
Method.
Background technology
GSTM1 is a kind of important metabolic enzyme, participates in internal life and the conversion of exogenous material and metabolism, assigns metabolin
New biological characteristics.There is polymorphism in mankind GSTM1, GSTM1 missings are a kind of common genotype.Different GSTM1 bases
Because the transformation function of the individually material of type has differences.GSTM1 Genotypings are used for the research of various biological character.Tradition
, it is use conventional gene specific PCR, agarose electrophoresis because GSTM1 deletion polymorphisms can be detected by regular-PCR more
Detection mode confirms GSTM1 genotype.Although easy, it is due to the necessary open pipe sample-adding of electrophoretic procedures and electrophoresis, easily causes
Laboratory pollution, it is difficult to ensure experimental reliability under high flux.Standard PCR electrophoretic analysis also is difficult to find heterozygote simultaneously
Genotype, accurate the determining of influence gene character analysis.
The content of the invention
It is an object of the invention to provide it is a kind of avoid the pollution of poisonous DNA coloring agents, improve detection flux based on quantitative
The GSTM1 genotyping detection methods of round pcr, to solve the problems mentioned in the above background technology.
To achieve the above object, the present invention provides following technical scheme：
A kind of GSTM1 genotyping detection methods based on quantitative PCR technique, are comprised the following steps that：
（1）Phase is used as using the normal homozygotes of people GSTM1, heterozygote and the deletion homozygote genomic DNA of two generation sequence verifications
Answer tester；
（2）GSTM1 genetic fragments are expanded using the primer SEQ1 and SEQ2 of GSTM1 gene specifics, while special in GSTM1 genes
Design GSTM1 gene specific TAQman probes SEQ3-Fam in the amplification region that the primer of the opposite sex is defined；If there is GSTM1 bases
Because then GSTM1 gene specifics TAQman probes SEQ3-Fam discharges FAM fluorescence signals and arrived by quantitative PCR instruments inspection；If not yet
There are GSTM1 fragments, then expanded without GSTM1, no FAM fluorescence signals release；
（3）ALbumin genetic fragments are expanded using ALbumin specific PCR primer SEQ4 and SEQ5, while in ALbumin
Design ALbumin gene specific TAQman probes SEQ6-Vic in the amplification region that the primer of gene specific is defined；If depositing
In qualified sample, then there are ALbumin amplifications, discharge Vic fluorescence signals, quantitative PCR instruments detect Vic fluorescence signals；If sample
This is unqualified, then sample is expanded without ALbumin, no Vic fluorescence signals release；
（4）Contrast amplification FAM/VIC signals confirm sample genotype to be detected；
The nucleotides sequence row number of the primer SEQ1 is as follows：5´-CCTGGCTGTCTAAACAGTCCT-3´；
The nucleotides sequence row number of the primer SEQ2 is as follows：5´-CACTCACTGTGCCTGCTCAT-3´；
The nucleotides sequence row number of the GSTM1 gene specifics TAQman probes SEQ3-Fam is as follows：
Fam-5´-TGCTGCCCATCCCTGCCCTCACAACCA-3´；
The nucleotides sequence row number of the primer SEQ4 is as follows：5´-ACTCCAAACCTGATGCTTCT-3´；
The nucleotides sequence row number of the primer SEQ5 is as follows：5´-CCTTAGGGCAGAATTATCCA-3´；
The nucleotides sequence row number of the ALbumin gene specifics TAQman probes SEQ6-Vic is as follows：
FAM-5´-CAGCCTGTTGCCCCTTTTAGAGTTCCTTTT-3´。
It is used as further scheme of the invention：The step（1）The middle normal homozygotes of people GSTM1, heterozygote and missing
The concentration of homozygote genomic DNA is 10ng/ μ l.
Compared with prior art, the beneficial effects of the invention are as follows：
The present invention realizes that stopped pipe one-time detection determines the mesh of GSTM1 gene copy numbers using the quantitative PCR based on TAQman probes
, solving conventional GSTM1 detections needs open pipe electrophoresis after PCR, and easily causes PCR primer in laboratory to pollute, it is difficult to high
The problem of flux stabilized obtains accurate genotypic results, while simplifying PCR detection method, prevents the behaviour of electrophoretic detection
Make the possibility that process is complicated, there is the environmental pollutions such as potential dna dyestuff danger and artifact's erroneous judgement；Can effectively it prevent
PCR primer pollutes, it is to avoid poisonous DNA coloring agents pollution, can greatly simplify detection and interpretation of result process, improve detection logical
Amount；In addition, the present invention also sets up a kind of quantitative mechanism, there is provided the technology path that identification finds heterozygote.
Brief description of the drawings
Fig. 1 is ALbumin gene magnifications and GSTM1 specific fragment amplification curve schematic diagrames in the embodiment of the present invention.
Embodiment
The technical scheme of this patent is described in more detail with reference to embodiment.
Referring to Fig. 1, a kind of GSTM1 genotyping detection methods based on quantitative PCR technique, are comprised the following steps that：
（1）The concentration of two generation sequence verifications is used for 10ng/ μ the l normal homozygotes of people GSTM1, heterozygote and deletion homozygote
Genomic DNA is used as corresponding tester；
（2）GSTM1 genetic fragments are expanded using the primer SEQ1 and SEQ2 of GSTM1 gene specifics, while special in GSTM1 genes
Design GSTM1 gene specific TAQman probes SEQ3-Fam in the amplification region that the primer of the opposite sex is defined；If there is GSTM1 bases
Because then GSTM1 gene specifics TAQman probes SEQ3-Fam discharges FAM fluorescence signals and arrived by quantitative PCR instruments inspection；If not yet
There are GSTM1 fragments, then expanded without GSTM1, no FAM fluorescence signals release；
（3）ALbumin genetic fragments are expanded using ALbumin specific PCR primer SEQ4 and SEQ5, while in ALbumin
Design ALbumin gene specific TAQman probes SEQ6-Vic in the amplification region that the primer of gene specific is defined；If depositing
In qualified sample, then there are ALbumin amplifications, discharge Vic fluorescence signals, quantitative PCR instruments detect Vic fluorescence signals；If sample
This is unqualified, then sample is expanded without ALbumin, no Vic fluorescence signals release；
（4）Contrast amplification FAM/VIC signals confirm sample genotype to be detected；
The nucleotides sequence row number of the primer SEQ1 is as follows：5´-CCTGGCTGTCTAAACAGTCCT-3´；
The nucleotides sequence row number of the primer SEQ2 is as follows：5´-CACTCACTGTGCCTGCTCAT-3´；
The nucleotides sequence row number of the GSTM1 gene specifics TAQman probes SEQ3-Fam is as follows：
Fam-5´-TGCTGCCCATCCCTGCCCTCACAACCA-3´；
The nucleotides sequence row number of the primer SEQ4 is as follows：5´-ACTCCAAACCTGATGCTTCT-3´；
The nucleotides sequence row number of the primer SEQ5 is as follows：5´-CCTTAGGGCAGAATTATCCA-3´；
The nucleotides sequence row number of the ALbumin gene specifics TAQman probes SEQ6-Vic is as follows：
FAM-5´-CAGCCTGTTGCCCCTTTTAGAGTTCCTTTT-3´。
The present invention judges in the presence of genetic fragment that educational circles unanimously thinks based on gene specific PCR combination TAQman probes can
The technical method leaned on.It is also the prompting sample quality and relative quantification that educational circles unanimously thinks using the amplification of ALbumin genetic contrasts
Reference frame.The technical solution adopted by the present invention uses the specific gene magnifications of GSTM1 and TAQman probes, while using
Control ALbumin genetic contrast amplifications can specify GSTM1 genotypic results.Based on TAQman probe quantitatives PCR method not
Need open pipe electrophoresis, effectively PCR primer can be prevented to pollute, it is to avoid the pollution of poisonous DNA coloring agents, can greatly simplify detection with
Interpretation of result process, improves detection flux.
Embodiment 1
（1）Test is prepared with normal human gene group DNA：Normal human mouth throat swab is taken, prepared by Chlex100 extractings, normal person
Genomic DNA concentration dilution is to 10ng/ μ l；
（2）PCR specific primers and probe design synthesis：Synthetic gene specific primer is designed according to people GSTM1 gene orders
SEQ1, SEQ2 and GSTM1 specificity T AQman Probe labellings Fam；It is special according to people ALbumin genes design reference gene
Property PCR primer SEQ4, SEQ5 and probe SEQ6 mark Vic；
（3）GSTM1 quantitative PCR detections：
1）Primed probe mixture is configured：
2）Reaction system：The μ l of primed probe mixture 1.5,2 times of TIANtoughGenotypingqPCRPreMix (Probe)
12.5 μ l, μ l, the ddH2O9.5 μ l of standard form 1.5.
3）Quantitative PCR apparatus：ABI7500
4）Reaction condition：95℃、2min；95 DEG C, 15s---60 DEG C, 32s, 42 circulations.
（4）Gene the result is read：
Referring to Fig. 1, Fig. 1 is people's GSTM1 normal detection results, ALbumin gene magnification curves, the amplification of GSTM1 specific fragments
Curve, abscissa is that ordinate is corresponding fluorescence signal with the period of 0-42 even number sign.
Vic, Fam signal is detected to be defined as expanding successfully；One of sample Fam/Vic and three check samples amplification Fam/
Vic ratios are 3-6, are defined as correspondence correspondence genotype；One of three samples or the multiple then the failure of an experiment of correspondence can not be corresponded to
The better embodiment to this patent is explained in detail above, but this patent is not limited to above-mentioned embodiment,
In the knowledge that one of ordinary skill in the art possesses, it can also be made on the premise of this patent objective is not departed from each
Plant change.
Claims (2)
1. a kind of GSTM1 genotyping detection methods based on quantitative PCR technique, it is characterised in that comprise the following steps that：
（1）Phase is used as using the normal homozygotes of people GSTM1, heterozygote and the deletion homozygote genomic DNA of two generation sequence verifications
Answer tester；
（2）GSTM1 genetic fragments are expanded using the primer SEQ1 and SEQ2 of GSTM1 gene specifics, while special in GSTM1 genes
Design GSTM1 gene specific TAQman probes SEQ3-Fam in the amplification region that the primer of the opposite sex is defined；If there is GSTM1 bases
Because then GSTM1 gene specifics TAQman probes SEQ3-Fam discharges FAM fluorescence signals and arrived by quantitative PCR instruments inspection；If not yet
There are GSTM1 fragments, then expanded without GSTM1, no FAM fluorescence signals release；
（3）ALbumin genetic fragments are expanded using ALbumin specific PCR primer SEQ4 and SEQ5, while in ALbumin
Design ALbumin gene specific TAQman probes SEQ6-Vic in the amplification region that the primer of gene specific is defined；If depositing
In qualified sample, then there are ALbumin amplifications, discharge Vic fluorescence signals, quantitative PCR instruments detect Vic fluorescence signals；If sample
This is unqualified, then sample is expanded without ALbumin, no Vic fluorescence signals release；
（4）Contrast amplification FAM/VIC signals confirm sample genotype to be detected；
The nucleotides sequence row number of the primer SEQ1 is as follows：5´-CCTGGCTGTCTAAACAGTCCT-3´；
The nucleotides sequence row number of the primer SEQ2 is as follows：5´-CACTCACTGTGCCTGCTCAT-3´；
The nucleotides sequence row number of the GSTM1 gene specifics TAQman probes SEQ3-Fam is as follows：
Fam-5´-TGCTGCCCATCCCTGCCCTCACAACCA-3´；
The nucleotides sequence row number of the primer SEQ4 is as follows：5´-ACTCCAAACCTGATGCTTCT-3´；
The nucleotides sequence row number of the primer SEQ5 is as follows：5´-CCTTAGGGCAGAATTATCCA-3´；
The nucleotides sequence row number of the ALbumin gene specifics TAQman probes SEQ6-Vic is as follows：
FAM-5´-CAGCCTGTTGCCCCTTTTAGAGTTCCTTTT-3´。
2. the GSTM1 genotyping detection methods according to claim 1 based on quantitative PCR technique, it is characterised in that the step
Suddenly（1）The concentration of the middle normal homozygotes of people GSTM1, heterozygote and deletion homozygote genomic DNA is 10ng/ μ l.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050255485A1 (en) * | 2004-05-14 | 2005-11-17 | Livak Kenneth J | Detection of gene duplications |
| CN101693920A (en) * | 2009-10-10 | 2010-04-14 | 上海中优医药高科技有限公司 | Glutathione-S-transferase genetype detection method |
| CN102586410A (en) * | 2011-01-18 | 2012-07-18 | 苏州科贝生物技术有限公司 | Reagent kit for quantitatively assessing long-term recurrence risks of breast cancer |
| CN103849685A (en) * | 2014-02-18 | 2014-06-11 | 南昌大学 | Simple method for simultaneously gene polymorphism of GSTM1, GSTT1 and GSTP1 by using multiple PCR (Polymerase Chain Reaction) |
-
2017
- 2017-06-08 CN CN201710426009.2A patent/CN107058586A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050255485A1 (en) * | 2004-05-14 | 2005-11-17 | Livak Kenneth J | Detection of gene duplications |
| CN101693920A (en) * | 2009-10-10 | 2010-04-14 | 上海中优医药高科技有限公司 | Glutathione-S-transferase genetype detection method |
| CN102586410A (en) * | 2011-01-18 | 2012-07-18 | 苏州科贝生物技术有限公司 | Reagent kit for quantitatively assessing long-term recurrence risks of breast cancer |
| CN103849685A (en) * | 2014-02-18 | 2014-06-11 | 南昌大学 | Simple method for simultaneously gene polymorphism of GSTM1, GSTT1 and GSTP1 by using multiple PCR (Polymerase Chain Reaction) |
Non-Patent Citations (4)
| Title |
|---|
| CHARLOTTE BRASCH-ANDERSEN等: "Possible Gene Dosage Effect of Glutathione-S-Transferases on Atopic Asthma: Using Real-Time PCR for Quantification of GSTM1 and GSTT1 Gene Copy Numbers", 《HUMAN MUTATION》 * |
| MARIA TIMOFEEVA等: "A multiplex real-time PCR method for detection of GSTM1 and GSTT1 copy numbers", 《CLINICAL BIOCHEMISTRY》 * |
| 周婧等: "实时荧光定量PCR技术在年龄相关性白内障患者GSTM1、GSTT1基因拷贝数变异检测中的应用", 《眼科新进展》 * |
| 杨庭松主编: "《胃癌治疗进展研究》", 31 May 2017, 同济大学出版社 * |
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Application publication date: 20170818 |