Taq Enzyme Mutants and Their Applications in SNP-Related Research

Abstract

The present invention belongs to the field of biochemistry, and specifically relates to a Taq enzyme mutant and its application in SNP-related research. The specific technical solution is as follows: a Taq enzyme mutant, which is mutated from wild-type C-Taq enzyme, and the amino acid sequence of wild-type C-Taq enzyme is shown in SEQ ID NO: 1. Based on the wild-type C-Taq enzyme, at least the following mutations are carried out: the threonine at position 506 of the wild-type C-Taq enzyme is mutated to lysine. The present invention analyzes the key active sites of wild-type C-Taq enzyme and performs site-directed mutagenesis, and uses multiple pairs of SNP genotyping primers for high and low GC target fragments to test the candidate mutant enzymes, and screens two enzymes with better discrimination ability for single-base mismatches. The single-base mismatch probe of the mutant enzyme provided by the present invention has a lower peak and higher genotyping accuracy. The mutant enzymes provided by the present invention can amplify well at high, medium and low GC sites, with wide universality; using these mutant enzymes can effectively save the cost of screening MGB probes.

Description

Technical Field

[0001] The present invention belongs to the field of biochemistry, and particularly relates to Taq enzyme mutants and their applications in SNP-related research. Background Art

[0002] Single Nucleotide Polymorphisms (SNPs) mainly refer to DNA sequence polymorphisms caused by variations of single nucleotides at the genomic level. Such variations are mainly caused by single-base transitions, transversions, insertions, deletions, etc. This is the most common type of human inheritable variation, accounting for more than 90% of all known polymorphisms. SNPs have the characteristics of high density, higher genetic stability, mainly existing in a dimorphic form in the population, and being easy to genotype, etc.; as the third-generation molecular markers, they are widely used in many fields such as molecular genetics, forensic physical evidence testing, disease diagnosis, and guiding personalized medication. Research has found that the polymorphisms of certain SNP loci are associated with the sensitivity of individuals to drugs, and SNP genotyping detection of the corresponding loci can guide patients to take personalized medications according to their different genotypes.

[0003] Currently, the commonly used methods for SNP detection include sequencing, PCR fluorescence probe method, ARMS-PCR, high-resolution melting curve, etc. The PCR fluorescence probe method is the most widely used due to its simple operation, rapidity, and easy result interpretation. The PCR fluorescence probe method requires designing two probes that are respectively complementary to the wild-type and mutant types at the SNP locus for genotyping detection. Due to their relatively long length, ordinary TaqMan probes are prone to single-base mismatches, so that the probes can also bind to the template, resulting in non-specific amplification results and affecting result interpretation. MGB modification can bind to the minor groove to increase the Tm value of the probe, thereby shortening the probe length and improving specificity, and is widely used in SNP genotyping. However, the cost of MGB probes is higher, and they also need to be frequently adjusted to screen for probes with low peak heights of mismatched probes and no impact on genotyping interpretation.

[0004] Therefore, there is an urgent need to develop a product with strong universality, large discrimination, and suitable for SNP genotyping to reduce the R & D cost. Summary of the Invention

[0005] The purpose of the present invention is to provide a universal SNP amplification enzyme and its application.

[0006] To achieve the above-mentioned invention object, the technical solution adopted by the present invention is as follows: a Taq enzyme mutant, which is mutated from wild-type C-Taq enzyme, and the amino acid sequence of wild-type C-Taq enzyme is shown as SEQ ID NO:1. At least the following mutations are carried out on the basis of wild-type C-Taq enzyme: the threonine at position 506 of the wild-type C-Taq enzyme is mutated into lysine.

[0007] Correspondingly, a Taq enzyme mutant, which is mutated from wild-type C-Taq enzyme, and the amino acid sequence of wild-type C-Taq enzyme is shown as SEQ ID NO:1. At least the following mutations are carried out on the basis of wild-type C-Taq enzyme: the threonine at position 506 of the wild-type C-Taq enzyme is mutated into lysine, and the glycine at position 510 of the wild-type C-Taq enzyme is mutated into arginine.

[0008] Correspondingly, the application of the Taq enzyme mutant in non-disease diagnosis and treatment in SNP research.

[0009] Preferably, the application in distinguishing single-base mismatches.

[0010] Preferably, the application in screening MGB probes.

[0011] The present invention has the following beneficial effects: by analyzing the key active sites of wild-type C-Taq enzyme and performing site-directed mutagenesis, and using multiple pairs of SNP genotyping primers and probes for high, low, and medium GC target fragments to test the candidate mutant enzymes, two enzymes with better ability to distinguish single-base mismatches are screened. The single-base mismatch probe of the mutant enzyme provided by the present invention has a lower peak and higher genotyping accuracy. The mutant enzymes provided by the present invention can amplify high, medium, and low GC sites well, with wide universality; using these mutant enzymes can effectively save the cost of screening MGB probes. Description of the Drawings

[0012] Figure 1 Schematic diagram of the qPCR detection results of amplifying the APOE-526 site using a universal SNP genotyping buffer for each Taq enzyme and mutant;

[0013] Figure 2 Schematic diagram of the result of C1-Taq enzyme amplifying the CYP2C19 wild-type template;

[0014] Figure 3 Schematic diagram of the result of C1-Taq enzyme amplifying the CYP2C19 mutant template;

[0015] Figure 4 Schematic diagram of the result of C1-Taq enzyme amplifying the AGTR1 wild-type template;

[0016] Figure 5 Schematic diagram of the result of amplifying the AGTR1 mutant template by C1-Taq enzyme;

[0017] Figure 6 Schematic diagram of the result of amplifying the UGT1A1 wild-type template by C1-Taq enzyme;

[0018] Figure 7 Schematic diagram of the result of amplifying the UGT1A1 mutant template by C1-Taq enzyme;

[0019] Figure 8 Schematic diagram of the result of amplifying the MTHFR wild-type template by C1-Taq enzyme;

[0020] Figure 9 Schematic diagram of the result of amplifying the MTHFR mutant template by C1-Taq enzyme;

[0021] Figure 10 Schematic diagram of the result of amplifying the CYP2D6 wild-type template by C1-Taq enzyme;

[0022] Figure 11 Schematic diagram of the result of amplifying the CYP2D6 mutant template by C1-Taq enzyme;

[0023] Figure 12 Schematic diagram of the result of amplifying the APOE wild-type template by C1-Taq enzyme;

[0024] Figure 13 Schematic diagram of the result of amplifying the APOE mutant template by C1-Taq enzyme. Detailed implementation manners

[0025] The present invention provides a Taq enzyme mutant: C-Taq enzyme mutant. The C-Taq enzyme mutant is mutated from the wild-type C-Taq enzyme, and the amino acid sequence of the wild-type C-Taq enzyme is shown in SEQ ID NO: 1. The mutation sites of the C-Taq enzyme mutant include: mutating the threonine (T) at position 506 of the wild-type C-Taq enzyme to lysine (K); preferably, the mutation sites further include: mutating the glycine (G) at position 510 of the wild-type C-Taq enzyme to arginine (R). The C-Taq enzyme mutant with both T506K and G510R mutations is named C1-Taq enzyme, and its amino acid sequence is shown in SEQ ID NO: 2. The C-Taq enzyme mutant with only T506K mutation is named C2-Taq enzyme, and its amino acid sequence is shown in SEQ ID NO: 3. At the same time, a control group is set: C3-Taq enzyme (only T506R mutation occurs, and its amino acid sequence is shown in SEQ ID NO: 4), C4-Taq enzyme (only G510K mutation occurs, and its amino acid sequence is shown in SEQ ID NO: 5), C5-Taq enzyme (only G510R mutation occurs, and its amino acid sequence is shown in SEQ ID NO: 6).

[0026] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. If not specifically specified, the technical means used in the embodiments are conventional means well-known to those skilled in the art. The data obtained are all the averages obtained after at least 3 repetitions, and all the repetitions obtained are valid data.

[0027] Example: Effect display of Taq enzyme mutant

[0028] 1. In this example, several SNP sites that are relatively common in the research and detection directions of SNPs are selected, and the GC content (the ratio of guanine and cytosine among the four DNA bases) is 30% - 75%. Primers and MGB probes are designed for these sites, as shown in Table 1 specifically, where P1 are all wild-type probes and P2 are mutant probes.

[0029] Table 1 Information comparison table of each SNP site

[0030]

[0031] Furthermore, the corresponding plasmid sequences are synthesized, as shown in Table 2 specifically.

[0032] Table 2 Corresponding plasmid sequences

[0033]

[0034] 2. Using the Taq enzyme mutant provided by the present invention and commercially available Taq enzymes, the APOE-526 locus was amplified using a universal SNP genotyping buffer respectively (when the common wild-type enzyme at the APOE-526 locus amplifies the mutant type, there will be strong non-specificity). The universal SNP genotyping buffer system is shown in Table 3, and the qPCR detection reaction system is shown in Table 4. The Taq enzyme in Table 4 refers to each Taq enzyme mutant or commercially available Taq enzyme. The commercially available Taq enzymes used in this example include: A-Taq enzyme, from Hieff® Taq DNA Polymerase DNA polymerase, product number: 10101; B-Taq enzyme, from Hieff® Taq DNA Polymerase DNA polymerase, product number: 13097. The qPCR detection reaction procedure is shown in Table 5.

[0035] Table 3 Universal buffer system

[0036]

[0037] Table 4 qPCR detection reaction system

[0038]

[0039] Table 5 qPCR detection reaction procedure

[0040]

[0041] The qPCR detection results are as Figure 1 shown. Figure 1 Among them, "APOE-526-P2" refers to the amplification curve of the APOE-526-P2 probe when each Taq enzyme amplifies the wild-type APOE template, and "APOE-526-P1" refers to the amplification curve of the APOE-526-P1 probe when each Taq enzyme amplifies the wild-type APOE template. It can be seen from Figure 1 the figure that: compared with the B-Taq enzyme, the Rn values amplified by each Taq enzyme mutant are close to it, but the non-specific peaks are lower; compared with the A-Taq enzyme, the non-specific peaks are basically the same, and the Rn value of the target peak is higher. It is proved that the Taq enzyme mutants provided by the present invention (especially C1-Taq and C2-Taq) can maintain good specificity while taking into account a relatively high fluorescence increase of the target peak.

[0042] 3. Select the C1-Taq enzyme with the best performance and further amplify the SNP site templates with different GC contents. The operation method, the general SNP genotyping buffer system, the qPCR detection reaction system and the reaction procedure are the same as those in step 2 of this example, except that APOE in the reaction system of step 2 is replaced with other SNP sites (the specific corresponding primer sequences are as shown in step 1), and the Taq enzyme is fixed as the C1-Taq enzyme. The amplification reaction results of each SNP site are shown in Table 6 corresponding to the attached drawings.

[0043] Table 6 Comparison table of amplification results of different SNP sites

[0044]

[0045] Figures 2 to 13 Among them, those ending with P1 refer to the amplification curves of the mutant probes of each SNP site when amplifying the templates of the corresponding SNP sites, and those ending with P2 refer to the amplification curves of the wild-type probes of each SNP site when amplifying the templates of the corresponding SNP sites. It can be seen that when the C1-Taq enzyme amplifies different SNP sites, the overall specificity is good, the fluorescence increase peak is also high, and there is no particularly low site, so it can be used as a general SNP amplification enzyme.

[0046] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations, variations, modifications, and substitutions made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A Taq enzyme mutant, characterized in that: The Taq enzyme mutant is mutated from wild-type C-Taq enzyme. The amino acid sequence of wild-type C-Taq enzyme is shown in SEQ ID NO:

1. The following mutations are made on the basis of wild-type C-Taq enzyme: the threonine at position 506 of the wild-type C-Taq enzyme is mutated to lysine.

2. A Taq enzyme mutant, characterized in that: The Taq enzyme mutant is mutated from wild-type C-Taq enzyme. The amino acid sequence of wild-type C-Taq enzyme is shown in SEQ ID NO:

1. The following mutations are made on the basis of wild-type C-Taq enzyme: the threonine at position 506 of the wild-type C-Taq enzyme is mutated to lysine, and the glycine at position 510 of the wild-type C-Taq enzyme is mutated to arginine.

3. Use of the Taq enzyme mutant according to claim 1 or 2 in non-disease diagnosis and treatment in SNP research, characterized in that: Application in differentiating single-base mismatches.

4. The application according to claim 3, characterized in that: Application in screening MGB probes.

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