A kind of GSTM1 genotyping detection methods based on quantitative PCR techniqueTechnical field
The present invention relates to technical field of gene detection, specifically a kind of GSTM1 partings detection side based on quantitative PCR techniqueMethod.
Background technology
GSTM1 is a kind of important metabolic enzyme, participates in internal life and the conversion of exogenous material and metabolism, assigns metabolinNew biological characteristics.There is polymorphism in mankind GSTM1, GSTM1 missings are a kind of common genotype.Different GSTM1 basesBecause 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 moreDetection mode confirms GSTM1 genotype.Although easy, it is due to the necessary open pipe sample-adding of electrophoretic procedures and electrophoresis, easily causesLaboratory pollution, it is difficult to ensure experimental reliability under high flux.Standard PCR electrophoretic analysis also is difficult to find heterozygote simultaneouslyGenotype, 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 quantitativeThe 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 verificationsAnswer tester；
（2）GSTM1 genetic fragments are expanded using the primer SEQ1 and SEQ2 of GSTM1 gene specifics, while special in GSTM1 genesDesign GSTM1 gene specific TAQman probes SEQ3-Fam in the amplification region that the primer of the opposite sex is defined；If there is GSTM1 basesBecause then GSTM1 gene specifics TAQman probes SEQ3-Fam discharges FAM fluorescence signals and arrived by quantitative PCR instruments inspection；If not yetThere 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 ALbuminDesign ALbumin gene specific TAQman probes SEQ6-Vic in the amplification region that the primer of gene specific is defined；If depositingIn qualified sample, then there are ALbumin amplifications, discharge Vic fluorescence signals, quantitative PCR instruments detect Vic fluorescence signals；If sampleThis 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 missingThe 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 highThe problem of flux stabilized obtains accurate genotypic results, while simplifying PCR detection method, prevents the behaviour of electrophoretic detectionMake 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 preventPCR primer pollutes, it is to avoid poisonous DNA coloring agents pollution, can greatly simplify detection and interpretation of result process, improve detection logicalAmount；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 homozygoteGenomic 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 genesDesign GSTM1 gene specific TAQman probes SEQ3-Fam in the amplification region that the primer of the opposite sex is defined；If there is GSTM1 basesBecause then GSTM1 gene specifics TAQman probes SEQ3-Fam discharges FAM fluorescence signals and arrived by quantitative PCR instruments inspection；If not yetThere 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 ALbuminDesign ALbumin gene specific TAQman probes SEQ6-Vic in the amplification region that the primer of gene specific is defined；If depositingIn qualified sample, then there are ALbumin amplifications, discharge Vic fluorescence signals, quantitative PCR instruments detect Vic fluorescence signals；If sampleThis 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 canThe 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 contrastsReference frame.The technical solution adopted by the present invention uses the specific gene magnifications of GSTM1 and TAQman probes, while usingControl ALbumin genetic contrast amplifications can specify GSTM1 genotypic results.Based on TAQman probe quantitatives PCR method notNeed 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 withInterpretation 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 personGenomic 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 ordersSEQ1, SEQ2 and GSTM1 specificity T AQman Probe labellings Fam；It is special according to people ALbumin genes design reference geneProperty 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 fragmentsCurve, 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 eachPlant change.