CN110819709A - Method for detecting CYP2C9 and VKORC1 gene polymorphism by fluorescent quantitative PCR (polymerase chain reaction) - Google Patents
Method for detecting CYP2C9 and VKORC1 gene polymorphism by fluorescent quantitative PCR (polymerase chain reaction) Download PDFInfo
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Abstract
The invention provides a primer probe combination for detecting CYP2C9 and VKORC1 gene polymorphism by fluorescent quantitative PCR, which comprises three groups of primers and a double probe shown in a sequence table; meanwhile, a method for detecting CYP2C9 and VKORC1 gene polymorphism by fluorescent quantitative PCR is provided. The invention adopts a real-time fluorescent quantitative PCR Taqman-MGB probe method to detect CYP2C9 and VKORC1 gene polymorphism, and adopts a wild type and mutant double-probe detection method, thereby not only improving specificity, but also reducing cost; the Taqman-MGB probe is superior to a common probe in the aspects of accuracy, repeatability, specificity, sensitivity and the like of an experimental result. Therefore, the CYP2C9 and VKORC1 gene polymorphism method established by the invention has the advantages of accurate result, high specificity and sensitivity, no toxicity, no pollution, low cost, rapidness, high efficiency and the like.
Description
Technical Field
The invention relates to the technical field of gene detection, in particular to a primer probe combination and a detection method for detecting CYP2C9 and VKORC1 gene polymorphism by fluorescent quantitative PCR (polymerase chain reaction).
Background
Warfarin is a vitamin K antagonist oral anticoagulant widely used clinically at present, and is used for anticoagulant treatment and prevention of venous thromboembolism, myocardial infarction, ischemic stroke, heart valve replacement operation, atrial fibrillation and the like. The warfarin preparation clinically used at present is a racemic mixture of S-warfarin and R-warfarin, wherein the anticoagulant activity of the S-warfarin is 2.7-3.8 times of that of the R-warfarin, and the S-warfarin is mainly metabolized by liver cytochrome P4502C 9(CYP2C9) in vivo. The coding region of CYP2C9 gene has Single Nucleotide Polymorphisms (SNPs), wherein the more important are CYP2C 9X 2 and CYP2C 9X 3, and the warfarin dosage required by individuals with the two mutations is obviously reduced, and the bleeding risk is increased. In addition, vitamin K epoxide cyclooxygenase complex 1(VKORC1) is the rate-limiting enzyme in the production of vitamin K-dependent coagulation factors in vivo, and warfarin competitively inhibits the action of this enzyme to achieve an anticoagulant effect. Therefore, in the clinical medication process, the patient should be tested for CYP2C9 and VKORC1 gene polymorphisms first, and warfarin individual medication differences of about 61% in age, sex and body weight are explained. At present, dosage formulas for regulating warfarin personalized medicine according to different genotypes and the combination of age and weight are relatively perfect.
At present, there are many methods for detecting polymorphisms of CYP2C9 and VKORC1 genes, such as direct sequencing, pyrosequencing, real-time fluorescence Quantitative PCR (QPCR), allele-specific Amplification (ARMS), and the like. Among them, the most commonly used sequencing method can directly detect the position and type of a mutation site, but the method has the disadvantages of complicated operation steps, long detection period, low sensitivity and easy pollution of an amplification product.
Disclosure of Invention
In order to solve the above defects, the technical purpose of the present invention is to provide a primer probe combination and a detection method for fluorescent quantitative PCR detection of CYP2C9 and VKORC1 gene polymorphisms, wherein the method employs a detection method of the primer and double probe combination, and has the advantages of accurate result, high specificity and sensitivity, no toxicity, no pollution, low cost, rapidness, high efficiency, etc.
In order to achieve the purpose, the invention is realized by the following technical scheme: a primer probe combination for detecting CYP2C9 and VKORC1 gene polymorphism by fluorescent quantitative PCR comprises three groups of primers and a double probe, wherein: nucleotide sequence of the first set of primer probes: the upstream primer is shown by SEQ ID NO.1, the downstream primer is shown by SEQ ID NO.2, the fluorescent probe 1 is shown by SEQ ID NO.3, and the fluorescent probe 2 is shown by SEQ ID NO. 4; nucleotide sequence of the second set of primer probes: the upstream primer is shown by SEQ ID NO.5, the downstream primer is shown by SEQ ID NO.6, the fluorescent probe 1 is shown by SEQ ID NO.7, and the fluorescent probe 2 is shown by SEQ ID NO. 8; nucleotide sequence of the third set of primer probes: the upstream primer is shown by SEQ ID NO.9, the downstream primer is shown by SEQ ID NO.10, the fluorescent probe 1 is shown by SEQ ID NO.11, and the fluorescent probe 2 is shown by SEQ ID NO. 12.
The primer probe combination of the invention is particularly aimed at CYP2C9 gene polymorphism: CYP2C9 x 2 site, CYP2C9 x 3 site, and VKORC1 gene polymorphism of CYP2C9 gene: the detection of VKORC1 gene-1639A & gtG site is specially designed, and specific gene segments of CYP2C9 gene and VKORC1 gene can be amplified with high efficiency, high specificity and accuracy. In the primer probe combination, the three groups of fluorescent probes are marked with fluorescent groups at the 5 'terminal region and are marked with quenching groups at the 3' terminal region, and the fluorescent groups of the fluorescent probe 1 and the fluorescent probe 2 in each group are different. Wherein the fluorescent group is selected from any one of FAM, TET, VIC, ROX, Texas Red-X, Cy3 and Cy 5; the quenching group is selected from TAMRA, BHQ, DABCYL, NFQ-MGB. According to a most preferred embodiment of the invention, the selection of the fluorescent probe is: the 5 ' end of the fluorescent probe 1 is marked with a fluorescent group of FAM, the 5 ' end of the fluorescent probe 2 is marked with a VIC fluorescent group, and the 3 ' ends of all the fluorescent probes are marked with quenching groups of NFQ-MGB.
The invention adopts a real-time fluorescent quantitative PCR Taqman-MGB probe method to detect CYP2C9 and VKORC1 gene polymorphism, and adopts a wild type and mutant double-probe detection method, thereby not only improving specificity, but also reducing cost; the Taqman-MGB probe is superior to a common probe in the aspects of accuracy, repeatability, specificity, sensitivity and the like of an experimental result. Therefore, the CYP2C9 and VKORC1 gene polymorphism method established by the invention has the advantages of accurate result, high specificity and sensitivity, no toxicity, no pollution, low cost, rapidness, high efficiency and the like.
Multiple tests prove that the primer probe combination provided by the invention has better specificity and amplification accuracy, can be applied to the preparation of a kit for detecting CYP2C9 and VKORC1 gene polymorphisms by fluorescent quantitative PCR, and can quickly and accurately obtain the results of CYP2C9 and VKORC1 gene polymorphisms after sample collection tests by preparing a finished kit.
The kit mainly comprises the primer probe combination provided by the invention, wherein the final concentration of the primer is preferably as follows: the final concentration of the upstream and downstream primers is 0.2-1.0 mu M; the final concentration of the fluorescent probes is 0.05-1.0. mu.M. Other PCR reagents may be selected according to conventional techniques, for example, a preferred embodiment further comprises DNA polymerase, buffer, uracil DNA glycosylase, dNTPs and ultrapure water.
When the finished product kit is manufactured, a reagent for extracting the DNA of a sample or a professional DNA extraction kit can be selectively configured according to actual needs; the DNA of the sample to be detected can be obtained more conveniently and quickly, and the convenience and the rapidity of the detection finished product kit are enhanced. The sample to be tested can be any sample of blood, cells, tissues or buccal swabs containing genomic DNA.
On the basis of the primer probe provided by the invention, the invention further provides a detection method for detecting CYP2C9 and VKORC1 gene polymorphism by fluorescent quantitative PCR, and the primer probe is adopted for carrying out fluorescent quantitative PCR detection.
According to a preferred embodiment, the detection method comprises the following specific steps:
(1) extracting genome DNA from a sample to be detected as an amplification template;
(2) preparing a fluorescent quantitative PCR reaction system containing the primer probe combination and the amplification template, wherein the final concentration of the upstream primer and the downstream primer in each reaction system is 0.2-1.0 mu M; the final concentration of the fluorescent probes is 0.05-1.0 mu M;
PCR amplification system 1(CYP2C9 x 2) comprising: 2 xTaqMan PCR reaction mixture (DNA polymerase, buffer solution, uracil DNA glycosylase, dNTPs (containing dUTP)), upstream and downstream primers (a first group of primer pairs) for amplifying CYP2C9 x 2 sites, wild type and mutant type specific fluorescent probes (a first group of fluorescent probe 1 and fluorescent probe 2), ultrapure water and a DNA template, wherein the volume of the reaction system is 10-50 μ l;
PCR amplification system 2(CYP2C9 x 3) comprising: 2 xTaqMan PCR reaction mixture (DNA polymerase, buffer solution, uracil DNA glycosylase, dNTPs (containing dUTP)), upstream and downstream primers (a second group of primer pairs) for amplifying CYP2C9 x 3 sites, wild type and mutant type specific fluorescent probes (a second group of fluorescent probe 1 and fluorescent probe 2), ultrapure water and a DNA template, wherein the volume of the reaction system is 10-50 μ l;
PCR amplification System 3(VKORC1-1639A > G) comprising: 2 xTaqMan PCR reaction mixture (DNA polymerase, buffer solution, uracil DNA glycosylase, dNTPs (containing dUTP)), upstream and downstream primers (a third group of primer pairs) for amplifying VKORC1-1639A & gtG sites, wild type and mutant type specific fluorescent probes (a third group of fluorescent probe 1 and fluorescent probe 2), ultrapure water and a DNA template, wherein the volume of the reaction system is 10-50 mu l;
(3) fluorescence PCR amplification reaction, conditions: pre-denaturation at 90-98 deg.C for 5-20 min; denaturation at 94-98 deg.C for 10-50 s; annealing at 55-69 deg.C for 60-90 s; extending for 15-300s at 68-72 ℃; 30-50 cycles;
(4) genotype interpretation:
the method comprises the following steps: after the amplification is finished, judging whether the experiment is successful or not according to the fact that the amplification curve is S-shaped or similar S-shaped and Ct is less than or equal to 30; then, the genotype is judged according to the following criteria: homozygous (mutant, wild): one curve Ct is less than or equal to 30 and is S-shaped or similar to S-shaped; the other curve Ct is more than 30, or no curve is generated; heterozygote type: the Ct of both curves is less than or equal to 30, and the curves are S-shaped or similar to S-shaped.
The second method comprises the following steps: after amplification was complete, genotypes were read automatically from the scatter plot, each point representing a sample and each data set representing one genotype (homozygous wild type, heterozygous mutant, homozygous mutant).
The invention is suitable for any common fluorescent quantitative PCR instrument on the market, and particularly comprises an ABI fluorescent quantitative PCR instrument (ABI7300, ABI7500, ABI ViiA7 and ABI Stepo plus (Stepo)); roche (Roche) fluorescent quantitative PCR instrument LightCycler 480; bio-rad (Berle) fluorescent quantitative PCR instruments CFX96TM and the like. Different fluorescent probes can be selected according to different PCR instruments for PCR amplification.
The detection method has the following advantages and effects:
(1) the two probes of the mutant type and the wild type are simultaneously added into a single tube, so that the operation is simple, the cost is reduced, the result analysis is clearer, the detection efficiency is improved, the detection of a large number of samples can be carried out, and the clinical operation is facilitated.
(2) The Taqman-MGB specific probe is used for detecting the gene polymorphism, so that the specificity is strong, and the result accuracy is high. The detection result is completely consistent with the sequencing result.
(3) The whole detection process is carried out in the same PCR tube, cover opening analysis is not needed, and aerosol pollution is not easily caused; electrophoresis analysis is not needed, and toxic and harmful reagents are not contacted.
(4) The entire detection process from sample reception, DNA extraction and PCR amplification can be completed within 4 hours. Can detect a large amount of samples simultaneously, improves the detection efficiency and saves the time cost.
The invention adopts a real-time fluorescent quantitative PCR Taqman-MGB probe method to detect CYP2C9 and VKORC1 gene polymorphism, and adopts a wild type and mutant double-probe detection method, thereby not only improving specificity, but also reducing cost. The MGB-Taqman probe is superior to a common probe in the aspects of accuracy, repeatability, specificity, sensitivity and the like of an experimental result. The detection method can quickly, accurately and efficiently detect the polymorphism of CYP2C 9X 2, CYP2C 9X 3 and VKORC1-1639A & gtG, so that the occurrence probability of false positive and false negative is minimized, and the detection method provides technical support for providing warfarin initial dosage according to the gene polymorphism results of CYP2C 9X 2, CYP2C 9X 3 and VKORC1-1639A & gtG.
Drawings
Fig. 1 shows three genotypes (TT homozygous mutant, CT heterozygous mutant, CC homozygous wild type) at CYP2C9 x 2 locus and blank.
Fig. 2 shows the distribution of the three genotypes at the CYP2C9 x 2 site.
Fig. 3 shows three genotypes (AC heterozygous mutant, AA homozygous wild type, CC homozygous mutant) and blank at CYP2C9 x 3 locus.
Fig. 4 shows the distribution of the three genotypes at the CYP2C9 x 3 site.
FIG. 5 shows three genotypes (AA homozygous mutant, GA heterozygous mutant, GG homozygous wild type) and blank for VKORC1-1639A > G locus.
FIG. 6 shows the distribution of three genotypes at VKORC1-1639A > G.
Detailed Description
To further illustrate the present invention, reference is made to the following examples. Specifically, the reagents used in the implementation of the invention are all commercial products, and the databases used in the implementation of the invention are all public online databases. The following examples are illustrative only and are not to be construed as limiting the invention.
Example 1
A preparation method of a DNA sample to be detected for detecting CYP2C9 and VKO C1 gene polymorphism comprises the following steps: the genomic DNA was extracted with a blood/cell/tissue genomic DNA extraction kit (DP304), and the concentration and purity of the DNA were determined using NP80-touch (IMPLEN, Germany), followed by preservation of the genomic DNA.
Example 2
1. Primer and probe design
According to the invention, specific primers and specific fluorescent probes are respectively designed according to base sequences of CYP2C9 x 2, CYP2C9 x 3 and VKORC1 gene loci, the specific primers are designed at two ends containing SNP loci through Primer Premier 5.0, and the most suitable primers are selected through experimental analysis; the specific probe is located in the region between a pair of primers, wherein the Tm should be between 65-70 ℃ and usually 5-10 ℃ higher than the primers. Wherein the 5 ' end of the probe for detecting the wild type is marked by a fluorescence reporter group (FAM), the 5 ' end of the probe for detecting the mutant type is marked by a fluorescence reporter group (VIC), the 3 ' end of the probe for detecting the mutant type is marked by a non-fluorescence quenching group (NFQ), and meanwhile, the probe is also linked with an MGB modifying group; in actual operation, the fluorescent reporter group can be replaced according to actual conditions, and only the inconsistency of the fluorescent reporter groups of the wild type probe and the mutant type probe is ensured.
The primer probe combinations of the present invention are shown in table 1:
TABLE 1 PCR primer Probe sequences
| Sequence numbering | Primer name | 5’-3’ |
| SEQ ID NO.1 | CYP2C9*2-F | TTTGGGATGGGGAAGAGGA |
| SEQ ID NO.2 | CYP2C9*2-R | TTTCTCAACTCCTCCACAAGGC |
| SEQ ID NO.3 | CYP2C9*2-P1 | FAM+ATTGAGGACCGTGTTCAAG+MGB |
| SEQ ID NO.4 | CYP2C9*2-P2 | VIC+CATTGAGGACTGTGTTCAAGA+MGB |
| SEQ ID NO.5 | CYP2C9*3-F | CTACACAGATGCTGTGGTGCAC |
| SEQ ID NO.6 | CYP2C9*3-R | TTCTGAArTTAATGTCACAGGTCACT |
| SEQ ID NO.7 | CYP2C9*3-P1 | FAM+CCAGAGATACATTGACCTTCT+MGB |
| SEQ ID NO.8 | CYP2C9*3-P2 | VIC+CCAGAGAIACCTTGACCTTCT+MGB |
| SEQ ID NO.9 | VKORC1-F | GAAGGGTAGGTGCAACAGTAAGG |
| SEQ ID NO.10 | VKORC1-R | CTTGTCTIAAACTCCTGACCTCAAG |
| SEQ ID NO.11 | VKORC1-P1 | FAM+CATTGGCCAGGTGC+MGB |
| SEQ ID NO.12 | VKORC1-P2 | VIC+CATTGGCCGGGTGC+MGB |
2. System preparation and PCR amplification
Amplifying CYP2C9 and VKORC1 gene target fragments through fluorescent quantitative PCR reaction, mixing primers (upstream and downstream primers) and probes (probe 1 and probe 2) according to a ratio of 1: 1 respectively, and amplifying the primers according to a reaction program shown in a table 2 and a reaction program shown in a table 3; blank control is set in the experimental process, and ultrapure water is used as a template.
TABLE 2 PCR amplification reaction System
| Reagent composition | Volume of |
| 2×TaqMan PCR Mix | 10μl |
| Probe Mix | 0.8μl |
| Primer Mix | 0.4μl |
| DNA | 1μl |
| Water (W) | 7.8μl |
TABLE 3 multiplex PCR reaction conditions
Remarking: is the location where the fluorescence signal was received.
In example 2, ABI7500 is selected for amplification, but the invention is not limited to ABI7500, and the invention is applicable to any fluorescent quantitative PCR instrument, and different fluorescent probes can be selected for detection according to different PCR instruments.
3. Result judgment
After the reaction is finished, the baseline and the threshold are automatically adjusted. After setting, the Ct value and curve condition of each sample were observed.
Ct is less than or equal to 30, and the curve is S-shaped or similar to S-shaped;
blank control Ct > 30, or no curve was generated.
4. Genotyping
Homozygous (mutant, wild): one curve Ct is less than or equal to 30 and is S-shaped or similar to S-shaped; the other curve Ct is > 30, or no curve is generated.
Heterozygote type: the Ct of both curves is less than or equal to 30, and the curves are S-shaped or similar to S-shaped.
The results are shown in fig. 1, and are three genotypes of CYP2C9 × 2 site: graphs of TT homozygous mutant, CT heterozygous mutant, CC homozygous wild type, and blank.
As shown in fig. 2, the distribution of the three genotypes at the CYP2C9 x 2 site.
As shown in fig. 3, three genotypes at CYP2C9 × 3 sites, respectively: plots of AC heterozygous mutant, AA homozygous wild type, CC homozygous mutant, and blank.
As shown in fig. 4, the distribution of the three genotypes at the CYP2C9 x 3 site.
As shown in FIG. 5, there are plots of the three genotypes (AA, AG, GG) and blank for VKORC1-1639A > G sites, respectively.
As shown in FIG. 6, the distribution of three genotypes at VKORC1-1639A > G sites.
Compared with the gold standard sequencing method, the detection result of the invention has the result coincidence rate of 100 percent, and the detection method has good specificity.
Example 3: configuration of the kit
Based on the above experimental results, this example provides a preferred kit for fluorescent quantitative PCR detection of polymorphisms of CYP2C9 and VKORC1 genes, which comprises the following reagents:
1. 2 × TaqMan PCR Mix: DNA polymerase, buffer, uracil DNA glycosylase, dNTPs (containing dUTP);
2. probe Mix: the final concentration of the three groups of fluorescent probes is 0.6 mu M;
3. primer Mix: three sets of primer pairs, F upstream primer (1.0. mu.M), R downstream primer (1.0. mu.M);
4. a sample DNA extraction reagent;
5. sample DNA collection kits (e.g., containing buccal swabs and swab kits);
6. ultrapure water.
The reagent is reasonably placed in the kit, the instructions (optionally reconfigured with a PCR test tube or a pore plate or a pipette) of the kit are put into the kit, the instructions comprise the step of collecting the DNA of the sample to be detected, the sample is put into a storage box after being collected, the step of extracting the DNA is carried out, and finally the PCR amplification process is carried out according to the detection method.
It should be noted that, in this document, 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. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other similar elements in a process, method, article, or apparatus that comprises the element.
The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements made to the technical solution of the present invention by those skilled in the art without departing from the spirit of the present invention shall fall within the protection scope defined by the claims of the present invention.
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Claims (10)
1. A primer probe combination for detecting CYP2C9 and VKORC1 gene polymorphism by fluorescent quantitative PCR (polymerase chain reaction), which is characterized by comprising three groups of primers and a double probe, wherein:
nucleotide sequence of the first set of primer probes: the upstream primer is shown by SEQ ID NO.1, the downstream primer is shown by SEQ ID NO.2, the fluorescent probe 1 is shown by SEQ ID NO.3, and the fluorescent probe 2 is shown by SEQ ID NO. 4; nucleotide sequence of the second set of primer probes: the upstream primer is shown by SEQ ID NO.5, the downstream primer is shown by SEQ ID NO.6, the fluorescent probe 1 is shown by SEQ ID NO.7, and the fluorescent probe 2 is shown by SEQ ID NO. 8; nucleotide sequence of the third set of primer probes: the upstream primer is shown by SEQ ID NO.9, the downstream primer is shown by SEQ ID NO.10, the fluorescent probe 1 is shown by SEQ ID NO.11, and the fluorescent probe 2 is shown by SEQ ID NO. 12.
2. The primer probe combination of claim 1, wherein the CYP2C9 gene polymorphism comprises CYP2C9 x 2 site, CYP2C9 x 3 site of CYP2C9 gene; the VKORC1 gene polymorphism comprises VKORC1 gene-1639A > G sites.
3. The primer-probe combination according to claim 1 or 2, wherein the fluorescent probes of the three groups are labeled with a fluorophore at a 5 '-terminal region and a quencher at a 3' -terminal region, and the fluorophores of the fluorescent probe 1 and the fluorescent probe 2 in each group are different from each other.
4. The primer probe combination of claim 3, wherein the fluorescent group is selected from FAM, TET, VIC, ROX, Texas Red-X, Cy3, Cy 5; the quenching group is selected from TAMRA, BHQ, DABCYL, NFQ-MGB.
5. The primer-probe combination of claim 3 or 4, wherein the fluorescent probes of the three groups have FAM fluorophore labeled at the 5 ' end of fluorescent probe 1, VIC fluorophore labeled at the 5 ' end of fluorescent probe 2, and NFQ-MGB quencher labeled at the 3 ' ends of all the fluorescent probes.
6. The primer probe combination of any one of claims 1-5 is applied to the preparation of a kit for detecting polymorphism of CYP2C9 and VKORC1 genes by fluorescent quantitative PCR.
7. A kit for fluorescent quantitative PCR detection of CYP2C9 and VKORC1 gene polymorphisms, which comprises the primer probe combination of any one of claims 1 to 5.
8. The kit of claim 7, further comprising a DNA polymerase, a buffer, a uracil DNA glycosylase, dNTPs comprising dUTP, and ultrapure water.
9. The kit according to claim 8, further comprising a test sample DNA extraction reagent or DNA extraction kit.
10. A detection method for detecting CYP2C9 and VKORC1 gene polymorphism by fluorescent quantitative PCR (polymerase chain reaction), which is characterized in that the primer-probe combination of any one of claims 1-5 is adopted to carry out the fluorescent quantitative PCR detection on a sample to be detected, and comprises the following steps:
(1) extracting genome DNA from a sample to be detected as an amplification template;
(2) preparing a fluorescent quantitative PCR reaction system containing the primer probe combination and the amplification template, wherein the final concentration of the upstream primer and the downstream primer in each reaction system is 0.2-1.0 mu M; the final concentration of the fluorescent probes is 0.05-1.0 mu M;
(3) fluorescence PCR amplification reaction, conditions: pre-denaturation at 90-98 deg.C for 5-20 min; denaturation at 94-98 deg.C for 10-50 s; annealing at 55-69 deg.C for 60-90 s; extending for 15-300s at 68-72 ℃; 30-50 cycles;
(4) genotype interpretation:
the method comprises the following steps: after the amplification is finished, judging whether the experiment is successful or not according to the fact that the amplification curve is S-shaped or similar S-shaped and Ct is less than or equal to 30; then, the genotype is judged according to the following criteria: homozygous (mutant, wild): one curve Ct is less than or equal to 30 and is S-shaped or similar to S-shaped; the other curve Ct is more than 30, or no curve is generated; heterozygote type: the Ct of both the two curves is less than or equal to 30, and the curves are S-shaped or similar to S-shaped;
the second method comprises the following steps: after amplification was complete, genotypes were read automatically from the scatter plot, each point representing a sample, and each data set representing one genotype: homozygous wild type, heterozygous mutant type, homozygous mutant type.
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