CN113832219A - Nano-upgrade ultrahigh-flux SNP genotyping method based on PARMS technology - Google Patents
Nano-upgrade ultrahigh-flux SNP genotyping method based on PARMS technology Download PDFInfo
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- CN113832219A CN113832219A CN202010580782.6A CN202010580782A CN113832219A CN 113832219 A CN113832219 A CN 113832219A CN 202010580782 A CN202010580782 A CN 202010580782A CN 113832219 A CN113832219 A CN 113832219A
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Abstract
The invention relates to the technical field of molecular biology, and discloses a method for genotyping nano-grade ultrahigh-flux SNP (single nucleotide polymorphism) based on PARMS (parallel amplified polymorphic polymorphism) technology, which comprises the following steps: s1, preparing a PARMS reagent, diluting and mixing primers according to the instruction, and preparing a Primer mix reagent; s2, homogenizing the sample according to a certain concentration; s3, preparing 1 384-hole plate, marking as a Sample plate, adding 2 multiplied by PARMS master mix into each hole according to the MSND 96Samples 54Assays mode requirement, homogenizing the Sample, and then supplementing water to a certain amount; according to the invention, by using a PARMS technology, genotypes of 3 sites of 42 samples are detected on a wafer Smartchip nano-upgrade ultrahigh-flux QPCR platform (3 site genotypes of the 42 samples are known: 21 homozygous genotypes and 21 heterozygous genotypes are all available), and the detection result proves that the SNP typing effect of the method is good, the result interpretation is completely consistent with the known genotypes, and the detection success rate reaches 100%. And performing secondary repeatability test, wherein the consistency rate of the two parting results is 100 percent.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a nano-upgrade ultrahigh-flux SNP genotyping method based on a PARMS technology.
Background
SNP, which is called Single Nucleotide Polymorphisms throughout, refers to a DNA sequence polymorphism caused by a Single Nucleotide variation in a genome, i.e., a Single Nucleotide polymorphism. The SNP research has important biological significance and wide application, and can perform fine positioning of character genes, molecular assisted breeding, seed resource identification and the like in the agricultural field; in the medical field or clinical application, the molecular genetic mechanism research of diseases, the disease gene localization, the tumor and cancer screening, the drug sensitive or disease susceptible site screening and the like are mainly included.
The SNP genotyping technology is based on PCR amplification of a genome fragment containing SNP, and is mainly characterized by high accuracy, strong flexibility and large flux, and the methods mainly adopted in laboratories at present comprise a TaqMan probe method and a KASP technology. The TaqMan probe method is to respectively design PCR primers and a TaqMan probe capable of identifying single nucleotide difference sites aiming at different SNP sites on a genome, carry out real-time fluorescent PCR amplification and identify the SNP sites through fluorescent labeling of the probe. KASP is to respectively design specific PCR primers and a universal fluorescent probe capable of identifying single nucleotide difference sites aiming at different SNP sites on a genome by a touch-down PCR method, to perform real-time fluorescent PCR amplification by two PCR processes, and to identify the SNP sites by fluorescent labeling of the probe. Because the synthesis cost of the fluorescent probe is high, the TaqMan probe method needs to design a synthesis probe aiming at a single SNP site, the time cost and the reagent cost are high, and the KASP can be repeatedly used by synthesizing general probes in batches, so that the time and the cost are saved. The PARMS technical principle is similar to KASP, but the PARMS reagent has lower cost and better clustering effect of typing, is easy to automatically interpret by detection machine software, and reduces the misinterpretation of artificial interpretation. PARMS technology combined with Takara SmartChipTMThe Real-Time PCR System can improve the detection reaction flux to 5184, and each reaction only needs 100nl, thereby greatly saving reaction reagents and samples.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides a nano-upgrade ultrahigh-flux SNP genotyping method based on the PARMS technology, and solves the problems in the background technology.
(II) technical scheme
In order to achieve the purpose, the invention provides the following technical scheme: a nano-upgrade ultrahigh flux SNP genotyping method based on PARMS technology comprises the following steps:
s1, preparing a PARMS reagent, diluting and mixing primers according to the instruction, and preparing a Primer mix reagent;
s2, homogenizing the sample according to a certain concentration;
s3, preparing 1 384-hole plate, marking as a Sample plate, adding 2 multiplied by PARMS master mix into each hole according to the requirement of MSND 96Sample es 54assay mode, homogenizing the Sample, and then supplementing water to a certain amount;
s4, centrifuging the Sample plate, placing the Sample plate in a 384-hole plate placing area on the MSND, placing 1 hollow chip on a chip carrier, automatically distributing a Sample into a chip hole, sealing an intermediate film by the chip after the Sample is finished, centrifuging the chip under a proper temperature condition, and storing the chip under a certain temperature condition;
s5, preparing 1 384-well plate, labeled as Assay plate, and adding 2 XPAMS master mix, Primer mix, 2% BSA, 50mM MgCl into each well according to MSND 96Sample × 54Assay mode2Then supplementing water to a certain amount;
s6, centrifuging the Assay plate, placing the Assay plate in a 384-hole plate placing area on the MSND, taking the chip in the step 4, tearing off the intermediate film, placing the intermediate film on a chip carrier, automatically distributing reagents into the chip holes by the MSND, sealing a qPCR film on the finished chip, centrifuging under a proper temperature condition, and performing qPCR on-machine detection;
and S7, after qPCR is completed, the software can automatically analyze the SNP typing result.
Preferably, the sample concentration in the step S2 is 10 ng/. mu.l.
Preferably, the 2 × PARMS master mix volume in step S3 is 7.2 μ l, the homogenized sample volume is 2.88 μ l, and water is supplemented to 14.4 μ l.
Preferably, the step S5 is 2 × PARMS master mix, Primer mix, 2% BSA and 50mM MgCl2The amounts were 8.5. mu.l, 3.4. mu.l, 1.7. mu.l and 0.68. mu.l, respectively, and water was replenished to 17. mu.l.
Preferably, the centrifugation conditions of the Sample plate and the Assay plate in the step S4 and the step S6 are 2200rcf centrifugation for 2min, and the centrifugation conditions of the chip in the step S4 and the step S6 are 2200rcf centrifugation for 5min at 4 ℃.
Preferably, the chip in step S4 needs to be stored at 4 ℃.
Preferably, the qPCR program in step S6 is configured to:
the first step is as follows: at 57 ℃ for 1min (fluorescence collected);
the second step is that: 94 ℃ for 10 min;
the third step: 94 ℃ for 20 s;
the fourth step: at 65 deg.C for 1 min;
the fifth step: returning to the third step, 10 cycles, and cooling to 0.8 ℃ in each cycle;
and a sixth step: 94 ℃ for 20 s;
the seventh step: at 57 ℃ for 1min (fluorescence collected);
eighth step: returning to the fifth step, and performing 30 cycles;
the ninth step: at 57 ℃ 1mmin (fluorescence collected).
(III) advantageous effects
The invention provides a nano-upgrading ultrahigh-flux SNP genotyping method based on PARMS technology, which has the following beneficial effects:
according to the invention, by using a PARMS technology, genotypes of 3 sites of 42 samples are detected on a wafer Smartchip nano-upgrade ultrahigh-flux QPCR platform (3 site genotypes of the 42 samples are known: 21 homozygous genotypes and 21 heterozygous genotypes are all available), and the detection result proves that the SNP typing effect of the method is good, the result interpretation is completely consistent with the known genotypes, and the detection success rate reaches 100%. And carrying out secondary repeatability test, wherein the consistency rate of the two parting results is 100%; compared with the traditional real-time fluorescent quantitative PCR platform, the Wafergen SmartChip nanoupgrade ultra-high flux QPCR platform has three advantages. Firstly, in the same time, the maximum flux of the wafer SmartChip platform reaches 5184 reactions, and compared with 96 or 384 reactions of the traditional real-time fluorescence quantitative PCR platform, the flux is improved by 13.5-54 times. Secondly, compared with the conventional QPCR 20 microliter system, the wafer gene Smartchip platform 100 nanoliter system greatly saves the sample and reagent dosage, in other words, more SNP sites can be detected on the wafer gene Smartchip platform with the same sample dosage. Thirdly, the wafer SmartChip platform has high automation degree and less operation content of personnel, thereby not only greatly reducing the difference between boards and ensuring good uniformity, but also saving the labor cost and the time cost; compared with the traditional Taqman probe typing technology, the unique PARMS typing technology saves the reagent cost by 60 percent on the premise of ensuring the typing accuracy.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a technical scheme that: a nano-upgrade ultrahigh flux SNP genotyping method based on PARMS technology comprises the following steps:
s1, preparing a PARMS reagent, diluting and mixing primers according to the instruction, and preparing a Primer mix reagent;
s2, homogenizing the concentration of the sample according to 10 ng/mul;
s3, preparing 1 384-hole plate, marking as a Sample plate, adding 7.2 mu l of 2 x PARMS master mix into each hole according to the requirement of MSND 96Sample es 54assay mode, homogenizing the Sample by 2.88 mu l, and then supplementing water to 14.4 mu l;
s4, centrifuging a Sample plate 2200rcf for 2min, placing the Sample plate in a 384-hole plate placing area on MSND, placing 1 hollow chip on a chip carrier, automatically distributing a Sample into a chip hole, sealing an intermediate film on the chip after the completion, centrifuging the Sample plate for 5min at 2200rcf at 4 ℃, and storing the chip at 4 ℃;
s5, prepare 1 384 well plate, labeled as Assay plate, and add 2 XPAMS master mix 8.5 μ l, Pri mer mix 3.4 μ l, 2% BSA 1.7 μ l, 50mM MgCl per well as required by MSND 96Sample × 54Assay format20.68 μ l, then water was added to 17 μ l;
s6, centrifuging 2200rcf of the Assay plate for 2min, placing the Assay plate in a 384-hole plate placing area on the MSND, taking the chip in the step S4, tearing off the middle film and placing the middle film on a chip carrier, automatically distributing reagents into chip holes by the MSND, sealing a qPCR film on the chip after the completion, centrifuging 5min at 2200rcf at 4 ℃, detecting the qPCR on a computer, and setting a qPCR program as follows:
the first step is as follows: at 57 ℃ for 1min (fluorescence collected);
the second step is that: 94 ℃ for 10 min;
the third step: 94 ℃ for 20 s;
the fourth step: at 65 deg.C for 1 min;
the fifth step: returning to the third step, 10 cycles, and cooling to 0.8 ℃ in each cycle;
and a sixth step: 94 ℃ for 20 s;
the seventh step: at 57 ℃ for 1min (fluorescence collected);
eighth step: returning to the fifth step, and performing 30 cycles;
the ninth step: 1mmin (fluorescence collected) at 57 ℃;
and S7, after qPCR is completed, the software can automatically analyze the SNP typing result.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (7)
1. A nano-upgrade ultrahigh flux SNP genotyping method based on PARMS technology is characterized by comprising the following steps:
s1, preparing a PARMS reagent, diluting and mixing primers according to the instruction, and preparing a Primer mix reagent;
s2, homogenizing the sample according to a certain concentration;
s3, preparing 1 384-hole plate, marking As a Sample plate, adding 2 multiplied by PARMS master mix into each hole according to the MSND 96Samples 54As Samples mode requirement, homogenizing the Sample, and then supplementing water to a certain amount;
s4, centrifuging the Sample plate, placing the Sample plate in a 384-hole plate placing area on the MSND, placing 1 hollow chip on a chip carrier, automatically distributing a Sample into a chip hole, sealing an intermediate film by the chip after the Sample is finished, centrifuging the chip under a proper temperature condition, and storing the chip under a certain temperature condition;
s5, preparing 1 384-well plate, labeled as Assay plate, and adding 2 XPAMS master mix, Primer mix, 2% BSA, 50mM MgCl into each well according to the requirement of MSND 96Samples 54assays ays mode2Then supplementing water to a certain amount;
s6, centrifuging the Assay plate, placing the Assay plate in a 384-hole plate placing area on the MSND, taking the chip in the step 4, tearing off the intermediate film, placing the intermediate film on a chip carrier, automatically distributing reagents into the chip holes by the MSND, sealing a qPCR film on the finished chip, centrifuging under a proper temperature condition, and performing qPCR on-machine detection;
and S7, after qPCR is completed, the software can automatically analyze the SNP typing result.
2. The method of claim 1 for nano-liter scale ultra-high flux SNP genotyping based on PARMS technology, wherein: the sample concentration in the step S2 is 10 ng/. mu.l.
3. The method of claim 1 for nano-liter scale ultra-high flux SNP genotyping based on PARMS technology, wherein: the 2 XPAMS master mix volume in step S3 was 7.2. mu.l, the homogenized sample volume was 2.88. mu.l, and water was replenished to 14.4. mu.l.
4. The method of claim 1 for nano-liter scale ultra-high flux SNP genotyping based on PARMS technology, wherein: 2 XPAMS master mix, Primer mix, 2% BSA and 50mM MgCl in said step S52The amounts were 8.5. mu.l, 3.4. mu.l, 1.7. mu.l and 0.68. mu.l, respectively, and water was replenished to 17. mu.l.
5. The method of claim 1 for nano-liter scale ultra-high flux SNP genotyping based on PARMS technology, wherein: the centrifugation conditions of the Sample plate and the Assay plate in the step S4 and the step S6 are 2200rcf centrifugation for 2min, and the centrifugation conditions of the chip in the step S4 and the step S6 are 2200rcf centrifugation for 5min at 4 ℃.
6. The method of claim 1 for nano-liter scale ultra-high flux SNP genotyping based on PARMS technology, wherein: the chip in step S4 needs to be stored at 4 ℃.
7. The method of claim 1 for nano-liter scale ultra-high flux SNP genotyping based on PARMS technology, wherein: the qPCR program in step S6 is set to:
the first step is as follows: at 57 ℃ for 1min (fluorescence collected);
the second step is that: 94 ℃ for 10 min;
the third step: 94 ℃ for 20 s;
the fourth step: at 65 deg.C for 1 min;
the fifth step: returning to the third step, 10 cycles, and cooling to 0.8 ℃ in each cycle;
and a sixth step: 94 ℃ for 20 s;
the seventh step: at 57 ℃ for 1min (fluorescence collected);
eighth step: returning to the fifth step, and performing 30 cycles;
the ninth step: at 57 ℃ 1mmin (fluorescence collected).
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20140130885A1 (en) * | 2012-11-08 | 2014-05-15 | Wafergen, Inc. | Extraction of restrained liquid from wells |
| CN108179175A (en) * | 2018-01-23 | 2018-06-19 | 安徽微分基因科技有限公司 | A kind of KASP combinations buccal swab is without extraction in QPCR platforms into the method for pedestrian's SNP Genotypings |
| CN108384835A (en) * | 2018-01-23 | 2018-08-10 | 安徽微分基因科技有限公司 | A kind of efficient denier quantitative gene expression technology based on Wafergen Smartchip systems |
| CN108410958A (en) * | 2018-01-23 | 2018-08-17 | 安徽微分基因科技有限公司 | A kind of efficient denier genotyping technique based on Wafergen Smartchip systems |
| CN109825565A (en) * | 2019-01-22 | 2019-05-31 | 天津渤海水产研究所 | A kind of Cynoglossus semilaevis true and false milter discriminating method based on fluorescent molecule label system |
| CN110592682A (en) * | 2019-09-03 | 2019-12-20 | 张鸿 | High-throughput automatic method for constructing metagenome 16s rRNA gene library of microorganisms |
-
2020
- 2020-06-23 CN CN202010580782.6A patent/CN113832219A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20140130885A1 (en) * | 2012-11-08 | 2014-05-15 | Wafergen, Inc. | Extraction of restrained liquid from wells |
| CN108179175A (en) * | 2018-01-23 | 2018-06-19 | 安徽微分基因科技有限公司 | A kind of KASP combinations buccal swab is without extraction in QPCR platforms into the method for pedestrian's SNP Genotypings |
| CN108384835A (en) * | 2018-01-23 | 2018-08-10 | 安徽微分基因科技有限公司 | A kind of efficient denier quantitative gene expression technology based on Wafergen Smartchip systems |
| CN108410958A (en) * | 2018-01-23 | 2018-08-17 | 安徽微分基因科技有限公司 | A kind of efficient denier genotyping technique based on Wafergen Smartchip systems |
| CN109825565A (en) * | 2019-01-22 | 2019-05-31 | 天津渤海水产研究所 | A kind of Cynoglossus semilaevis true and false milter discriminating method based on fluorescent molecule label system |
| CN110592682A (en) * | 2019-09-03 | 2019-12-20 | 张鸿 | High-throughput automatic method for constructing metagenome 16s rRNA gene library of microorganisms |
Non-Patent Citations (2)
| Title |
|---|
| WANQIU CHEN ET AL.: "A Comprehensive Multi-Center Cross-platform Benchmarking Study of Single-cell RNA", BIORXIV, pages 109 - 110 * |
| 何燕等: "四川省稻田土壤的抗生素抗性基因多样性研究", 农业环境科学学报, vol. 39, no. 06, pages 1249 - 1258 * |
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