CN112375808A - Blocker design and screening method of ARMS-TaqMan Blocker system - Google Patents

Blocker design and screening method of ARMS-TaqMan Blocker system Download PDF

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CN112375808A
CN112375808A CN202011295178.5A CN202011295178A CN112375808A CN 112375808 A CN112375808 A CN 112375808A CN 202011295178 A CN202011295178 A CN 202011295178A CN 112375808 A CN112375808 A CN 112375808A
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杨芳梅
徐红梅
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Herfei Ocgene Biotech Co ltd
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Abstract

The invention discloses a Blocker design and screening method of an ARMS-TaqMan Blocker system, wherein the design method comprises the steps of designing Blocker at a mutation site and covering the mutation site; blocker is completely matched with a wild template, the Tm value of the complete match is higher than that of ARMS primers matched with the wild template, and the allowable melting point difference is lower than 5 ℃; blocker is incompletely matched with the mutant template, and the Tm value of the incomplete match is consistent with or close to the Tm value of the ARMS primer and the mutant template; adding a plurality of irrelevant bases which are not matched with the template at the 3' end of the Blocker; the screening method comprises the steps of designing 3-10 Blockers according to the Blocker design principle, enabling Tm values matched with the mutant templates to present a gradually-increased gradient near the Tm value matched with the ARMS primer and the mutant template, verifying the influence of the gradient Blocker on the sensitivity and the specificity of a system through real-time PCR, and screening the Blocker which has no influence on the sensitivity and has the best specificity. The Blocker design and screening method provided by the invention reduces the cost and improves the specificity of the system.

Description

Blocker design and screening method of ARMS-TaqMan Blocker system
Technical Field
The invention relates to the technical field of tumor mutation detection, in particular to a Blocker design and screening method of an ARMS-TaqMan Blocker system.
Background
In recent years, the non-radioactive labeling of probes has been rapidly developed while receiving a great deal of attention, and has been widely used for nucleic acid sequencing, gene detection, disease diagnosis, and the like. The ARMS technique is a commonly used fluorescent probe method, and the basic principle is that if the 3' end base of a primer is not complementary to the template base, extension cannot be performed with a general thermostable DNA polymerase. Therefore, 3 primers are designed according to the known point mutation, and the 3' end base of the primers is respectively complementary with the mutant and normal template base, so that the template with a certain point mutation is distinguished from the normal template. Sometimes, in order to improve the specificity, a wild-type template amplification Blocker (packer) is added into the system to suppress the amplification of the wild-type template, although the method can suppress the wild type to a certain extent, due to the lack of accurate packer design software and effective screening rules, the suppression effect is not ideal; alternatively, mismatched bases can be introduced at the penultimate or third base at the 3' end of the ARMS primer, but introduction of mismatched bases generally sacrifices sensitivity. Therefore, the conventional ARMS-TaqMan Blocker system is difficult to simultaneously achieve sensitivity and specificity, and complex and tedious optimization work is often required.
The traditional competitive packer design principle is as follows: designing a Blocker at a mutation site, covering the mutation site, partially overlapping with an ARMS primer to play a role of position competition, and enabling the mutation site to be positioned at the middle position of the Blocker as much as possible so as to ensure that the melting point difference of the Blocker is larger (according to the principle that the melting point difference of the mutation site positioned in the middle is larger than that of the mutation site positioned at two sides); secondly, Blocker is completely matched with a wild template, the Tm value is about 5 ℃ higher than that of an ARMS primer matched with the wild template, so that the Blocker and the wild template are preferentially combined, and LNA, MGB and the like are added for modification sometimes in order to increase the melting point difference; thirdly, Blocker is not completely matched with the mutant template, and the Tm value is lower than that of the ARMS primer matched with the mutant template, so that the ARMS primer and the mutant template are preferentially combined; and fourthly, blocking modification, such as thio modification, amino modification, phosphorylation modification and the like, is required to be added at the 3' end of the Blocker to prevent extension.
The traditional Blocker design generally utilizes Tm value prediction software to predict Tm values, different prediction software predicts the Tm values greatly, and meanwhile, PCR components also have certain influence on the Tm values, so that the actual Tm values of the Blocker cannot be accurately evaluated, and the blocking effect of the Blocker is poor. Blocker designed according to a general design principle does not accord with an optimal condition, which is shown in that the Blocker is not completely matched with a mutant template, Tm values are all lower than those of ARMS primers matched with the mutant template, the Tm value predicted by software is only used for reference, and the actual Tm value is close to the prediction or greatly different, so that a plurality of Blockers are usually designed for screening, but the Blockers meeting the conditions of the third step are few, and in view of high modification cost, only 1-3 Blockers are generally designed for screening. If Tm value prediction is carried out through software, the low melting points of all the Blockers are probably lower than the Tm value of the matching of the ARMS primers and the mutant template, the probability of finding the optimal Blocker is extremely low, and good pressing effect can be achieved only when the low melting point of just one Blocker in the designed Blocker is actually consistent with or extremely close to the Tm value of the matching of the ARMS primers and the mutant template.
Disclosure of Invention
Aiming at the technical defects of the Blocker design of the existing ARMS-TaqMan Blocker system, the invention provides a Blocker design and screening method of the ARMS-TaqMan Blocker system.
The invention protects a Blocker design method of an ARMS-TaqMan Blocker system, wherein the Blocker is designed at a mutation site and covers the mutation site; blocker is completely matched with a wild template, the Tm value of the complete match is higher than that of ARMS primers matched with the wild template, and the allowable melting point difference is lower than 5 ℃; blocker is incompletely matched with the mutant template, and the Tm value of the incomplete match is consistent with or close to the Tm value of the ARMS primer and the mutant template; the 3' end of Blocker is added with a plurality of unrelated bases which do not match with the template.
According to the Blocker design principle, the mutation site is not strictly required to be positioned in the middle of the Blocker, the requirement of 5 ℃ of melting point difference is not required to be met, and the Blocker design principle is relatively easy to design as long as the melting point difference exists. Meanwhile, as the melting point difference of about 5 ℃ is not strictly required, LNA modification is not required, and LNA synthesis difficulty and extra cost are avoided.
The Blocker design principle of the invention ensures that the Tm value of the Blocker incompletely matched with the mutant template is consistent with or close to the Tm value of the ARMS primer matched with the mutant template, and because only under the Tm value, the Blocker in the system can not generate redundancy, the maximum pressing effect is achieved. This is quite different from the conventional competitive Blocker design principle.
According to the Blocker design principle, the 3 'end of the Blocker does not need to be added with special closed modification, but individual unrelated bases are added at the 3' end and are not matched with a template, so that extension cannot be carried out under the action of Taq DNA polymerase, special modification is not needed, and the cost is saved by dozens of times.
The invention also discloses a Blocker screening method of the ARMS-TaqMan Blocker system, which comprises the steps of designing 3-10 Blockers according to the Blocker design principle, wherein the low melting points of the Blockers are all near the Tm value matched with the ARMS primer mutation type template, the Blockers are higher and lower, different Blockers can have a base difference of 1 base, and the Tm values matched with the mutant type template are in a gradient which gradually increases near the Tm value matched with the ARMS primer mutation type template, so that the Tm value range is wide and dense, and the optimal Blocker cannot be missed; and then verifying the influence of the gradient Blocker on the sensitivity and the specificity of the system through real-time PCR, and screening the Blocker which has no influence on the sensitivity and has the best specificity.
The traditional Blocker design principle has high design cost, and the cost cannot be underestimated for a multi-site detection system. The Blocker design and screening principle of the invention has definite screening direction, can screen out the optimal Blocker at one time without repeated modification, is synthesized as common primers, does not need special modification and purification, and has low cost.
The invention also protects the application of the Blocker screening method of the ARMS-TaqMan Blocker system in tumor mutation detection.
Drawings
FIG. 1 is a comparison of Blocker gradient screening designed according to the design principles of the present invention and Blocker designed according to conventional design principles for site E545K;
FIG. 2 is a comparison of Blocker gradient screening designed according to the design principles of the present invention and Blocker designed according to conventional design principles for braf sites;
FIG. 3 is a comparison of Blocker gradient screening designed according to the design principles of the present invention and Blocker designed according to conventional design principles for the G719 site.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments. The embodiments of the present invention have been presented for purposes of illustration and description, and are not intended to be exhaustive or limited to the invention in the form disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art. The embodiment was chosen and described in order to best explain the principles of the invention and the practical application, and to enable others of ordinary skill in the art to understand the invention for various embodiments with various modifications as are suited to the particular use contemplated.
Description of the drawings: the primers, probes and Blocker used in the present invention were subjected to melting point prediction by the software TM Utility v1.3, and the sequence information of each primer, probe and Blocker is shown in Table 1, wherein F/R represents a primer, P represents a probe, B represents Blocker, FAM represents a fluorophore, BHQ1/MGB represents a quencher, PHO represents a phosphorylation modification, a box represents an LNA modification, a lowercase represents a base to be added manually, Tm1 represents a melting point when the primer/probe/Blocker is completely matched with a target sequence, and Tm2 represents a melting point when the primer/Blocker is not completely matched with the target sequence.
Figure BDA0002785055270000041
TABLE 1
Example 1 Blocker gradient screening at E545K site designed according to the design principles of the present invention and comparison with Blocker designed according to conventional design principles
First, primer, probe, Blocker design
The primers are designed according to the ARMS primer design principle, the length of the primers is 15-38nt, the GC content is 40-60%, the 3' end of the primers is free from hairpin structures, the matching of 4 bases is avoided between the primers, the base distribution is uniform, and the continuous GC or AT is avoided. The 3' end of the upstream ARMS primer is consistent with the mutation site, and the downstream primer is a universal primer and can amplify the wild template and the mutant template simultaneously. The sequence of the upstream primer E545K-F4 is shown as SEQ NO.01 in Table 1, and the sequence of the downstream primer E545K-R1 is shown as SEQ NO.02 in Table 1.
The probe is designed according to the design principle of a TaqMan probe, the length of the probe is 14-35nt, the GC content is 60-70%, the first base at the 5' end is prevented from being G, and the sequence of the probe E545K-P is shown as SEQ NO.03 in a table 1.
3 Blockers are designed according to the design principle of the invention, and are respectively E545K-B13, E545K-B18 and E545K-B19, and the sequences are respectively shown as SEQ NO.05-07 in Table 1.
1 Blocker E545K-B-1 is designed according to the conventional design principle, and the sequence is shown as SEQ NO.04 in Table 1.
Primer probes were biosynthesized in nature, and Blockers designed according to the present invention were purified by PAGE and solubilized to 100. mu.M with Tris-HCl (pH 8.0).
Second, PCR reaction system preparation
The PCR reaction solution was prepared according to the following Table 2, and deionized water was added to the reaction solution to a volume of 25. mu.l. Only adding pure water into the first group of reaction liquid, and not adding a Blocker; adding E545-B-1 into a second group of reaction liquid Blocker; adding E545-B13 into the third group of reaction solution Blocker; adding E545-B18 into the fourth group of reaction solution Blocker; a fifth set of reaction solutions, Blocker, was added with E545-B19.
PCR Components Final concentration
10 × Buffer (containing Mg)2+) 1×
dNTPs 0.16mM
E545K-F4 0.8μM
E545K-R1 0.8μM
E545K-P 0.4μM
Blocker 2μM
Taq enzyme 1U
TABLE 2
Third, sample preparation
E545K plasmid 10^3 copies/. mu.l and 10 ng/. mu.l wild type genome are mixed to obtain the standard substance with mutation rate of 50%, 50% of the standard substance is diluted with 10 ng/. mu.l wild type genome to obtain 10% of the standard substance, and 10 ng/. mu.l wild type genome is prepared for standby.
Fourthly, adding sample and operating the machine
The above five groups of reactions were added with 5. mu.l of 10% E545K plasmid standard and 10 ng/. mu.l of wild type genome, each group having 2 parallel channels and No Template Control (NTC) set. The computer program is as follows: 2min at 95 ℃ for 1 cycle; 5s at 95 ℃, 30s at 56 ℃ (no fluorescence collected), 15s at 72 ℃ and 10 cycles; 3s at 93 ℃, 30s at 56 ℃ (fluorescence collected), 30s at 60 ℃, and 35 cycles. The instrument used a SLAN96 fluorescent PCR instrument.
Fifth, analysis of experimental results
Referring to FIG. 1, the first set was without Blocker and the amplification curves are represented by black circles; the second group is added with E545-B-1, and an amplification curve is represented by a black positive triangle; the third group was added with E545-B13, and the amplification curves are represented by black squares; the fourth group is added with E545-B18, and the amplification curve is represented by black pentagram; E545-B19 was added to the fifth set, and the amplification curves were represented by black squares and NTC by white positive triangles.
As can be seen from FIG. 1, the fifth group plus E545-B19 (black rhombus) affects 10% of sensitivity, and the specificity of E545K-B18 (black pentagram) designed and screened according to the design principle of the invention is better than that of E545K-B-1 (black regular triangle) designed according to the traditional principle, and the CT value of 10 ng/. mu.l of wild-type sample is more posterior, which shows that the Blocker design and screening principle of the invention is better, so that the wild-type template can be compressed to a greater extent, and the specificity of the system is improved.
Example 2 Blocker gradient screening of braf sites designed according to the design principles of the present invention and comparison with Blocker designed according to conventional design principles
First, primer, probe, Blocker design
The primers are designed according to the ARMS primer design principle, the length of the primers is 15-38nt, the GC content is 40-60%, the 3' end of the primers is free from hairpin structures, the matching of 4 bases is avoided between the primers, the base distribution is uniform, and the continuous GC or AT is avoided. The 3' end of the upstream ARMS primer is consistent with the mutation site, and the downstream primer is a universal primer and can amplify the wild template and the mutant template simultaneously. The sequence of the upstream primer braf-F is shown as SEQ NO.08 in Table 1, and the sequence of the downstream primer braf-R is shown as SEQ NO.09 in Table 1.
The probe is designed according to the design principle of a TaqMan probe, the length of the probe is 14-35nt, the GC content is 60-70%, the first base at the 5' end is avoided to be G, and the sequence of the probe braf-P is shown as SEQ NO.10 in a table 1.
According to the design principle of the invention, 4 Blockers are designed, wherein the Blaf-B3, the Blaf-B4, the Blaf-B5 and the Blaf-B6 are respectively provided with sequences shown as SEQ NO.12-15 in Table 1.
1 Blocker, braf-B2 was designed according to the conventional design principle, and the sequence is shown in SEQ NO.11 of Table 1.
Primer probes were biosynthesized in nature, and Blockers designed according to the present invention were purified by PAGE and solubilized to 100. mu.M with Tris-HCl (pH 8.0).
Second, PCR reaction system preparation
PCR reaction solution preparation was performed according to the following Table 3, and deionized water was added to 25. mu.l. Only adding pure water into the first group of reaction liquid, and not adding a Blocker; adding braf-B2 into the second group of reaction solution Blocker, and adding braf-B3 into the third group of reaction solution Blocker; adding braf-B4 into the fourth group of reaction solution Blocker; adding braf-B5 into the fifth group of reaction solution Blocker; a sixth set of reaction solutions, Blocker plus braf-B6.
PCR Components Final concentration
10 x Buffer (containing Mg2+) 1×
dNTPs 0.16mM
braf-F 0.8μM
braf-R 0.8μM
braf-P 0.16μM
Blocker 0.8μM
Taq enzyme 1U
TABLE 3
Third, sample preparation
Mixing braf plasmid 10^3 copies/mu l and 10 ng/mu l wild type genome to obtain standard substance with mutation rate of 50%, diluting 50% of standard substance with 10 ng/mu l wild type genome to obtain 10% of standard substance, and preparing 20 ng/mu l wild type genome for later use.
Fourthly, adding sample and operating the machine
The above six groups of reactions were added with 5. mu.l of 10% standard and 20 ng/. mu.l of wild type genome, 2 parallel channels for each group and No Template Control (NTC) was set. The computer program is as follows: 2min at 95 ℃ for 1 cycle; 5s at 95 ℃, 30s at 56 ℃ (no fluorescence collected), 15s at 72 ℃ and 10 cycles; 3s at 93 ℃, 30s at 56 ℃ (fluorescence collected), 30s at 60 ℃, and 35 cycles. The instrument used a SLAN96 fluorescent PCR instrument.
Fifth, analysis of experimental results
Referring to FIG. 2, the first set was filled with pure water only and the amplification curves are indicated by black circles; a second set of braf-B2, the amplification curves are shown as black positive triangles; a third group, braf-B3, the amplification curves are shown as black squares; fourth group with braf-B4, the amplification curve is shown by black pentagram; a fifth panel was supplemented with braf-B5, and the amplification curves are shown as black inverted triangles; the sixth panel was supplemented with braf-B6, the amplification curves are shown as black squares and NTC as white triangles.
As can be seen from FIG. 2, braf-B4 (black pentagram) designed and screened according to the design principle of the invention has better specificity than braf-B2 (black regular triangle) designed according to the traditional design principle, and 20 ng/. mu.l of wild type sample has no CT value, which indicates that the Blocker design principle of the invention is better, and can press the wild type template to a greater extent, thereby improving the specificity of the system.
Example 3 Blocker gradient screening at position G719, designed according to the design principles of the present invention and comparison with Blocker designed according to conventional design principles
First, primer, probe, Blocker design
The primers are designed according to the ARMS primer design principle, the length of the primers is 15-38nt, the GC content is 40-60%, the 3' end of the primers is free from hairpin structures, the matching of 4 bases is avoided between the primers, the base distribution is uniform, and the continuous GC or AT is avoided. The 3' end of the upstream ARMS primer is consistent with the mutation site, and the downstream primer is a universal primer and can amplify the wild template and the mutant template simultaneously. The sequence of the upstream primer G719-F is shown as SEQ NO.16 in Table 1, and the sequence of the downstream primer G719-R is shown as SEQ NO.17 in Table 1.
The probe is designed according to the design principle of a TaqMan probe, the length of the probe is 14-35nt, the GC content is 60-70%, the first base at the 5' end is avoided to be G, and the sequence of the probe G719-P is shown as SEQ NO.18 in Table 1.
According to the design principle of the invention, 4 Blockers are designed, wherein the Blockers are respectively G719-B1, G719-B2, G719-B9 and G719-B10, and the sequences are respectively shown as SEQ NO.19-22 in Table 1.
1 Blocker, G719-B11 designed according to the conventional design principle, and the sequence is shown as SEQ NO.23 in Table 1.
Primer probes were biosynthesized in nature, and Blockers designed according to the present invention were purified by PAGE and solubilized to 100. mu.M with Tris-HCl (pH 8.0).
Second, PCR reaction system preparation
The PCR reaction solution was prepared according to the following Table 4, and deionized water was added thereto to make up 25. mu.l. Only adding pure water into the first group of reaction liquid, and not adding a Blocker; adding G719-B10 into a second group of reaction solution Blocker; G719-B9 is added into the third group of reaction solution Blocker; G719-B1 is added into the fourth group of reaction solution Blocker; G719-B2 is added into the fifth group of reaction solution Blocker; G719-B11 was added to the reaction solution of the sixth group Blocker.
PCR Components Final concentration
10 x Buffer (containing Mg2+) 1×
dNTPs 0.16mM
G719-F 0.8μM
G719-R 0.8μM
G719-P 0.08μM
G719-B 2μM
Taq enzyme 1U
TABLE 3
Third, sample preparation
Mixing the G719 plasmid 10^3 copies/mu l and 10 ng/mu l wild type genome to obtain a standard substance with a mutation rate of 50%, diluting 50% of the standard substance with 10 ng/mu l wild type genome to obtain 10% of the standard substance, and preparing 50 ng/mu l wild type genome for later use.
Fourthly, adding sample and operating the machine
The above six groups of reactions were added with 5. mu.l of 10% standard and 50 ng/. mu.l of wild type genome, 2 parallel channels for each group and No Template Control (NTC) was set. The computer program is as follows: 2min at 95 ℃ for 1 cycle; 5s at 95 ℃, 30s at 56 ℃ (no fluorescence collected), 15s at 72 ℃ and 10 cycles; 3s at 93 ℃, 30s at 56 ℃ (fluorescence collected), 30s at 60 ℃, and 35 cycles. The instrument used a SLAN96 fluorescent PCR instrument.
Fifth, analysis of experimental results
Referring to FIG. 2, the first set was only pure and the amplification curves are indicated by black circles; G719-B10 was added to the second set, and the amplification curves are shown as black positive triangles; the third group was added G719-B9 and the amplification curves are shown as black squares; G719-B1 was added to the fourth set, and the amplification curves were represented by black pentagons; G719-B2 was added to the fifth group, and the amplification curve was shown as a black inverted triangle; G719-B11 was added to the sixth set, and the amplification curve was represented by black squares and NTC by white triangles.
As can be seen from FIG. 3, G719-B1 (black pentagram) designed and screened according to the design principle of the present invention has better specificity than G719-B11 (black rhombus) designed according to the conventional design principle, and 50 ng/. mu.l of wild type sample has no CT value.
It is to be understood that the described embodiments are merely a few embodiments of the invention, and not all embodiments. All other embodiments, which can be derived by one of ordinary skill in the art and related arts based on the embodiments of the present invention without any creative effort, shall fall within the protection scope of the present invention.

Claims (4)

1. A Blocker design method of an ARMS-TaqMan Blocker system is characterized in that Blocker is designed at a mutation site and covered with the mutation site; blocker is completely matched with a wild template, the Tm value of the complete match is higher than that of ARMS primers matched with the wild template, and the allowable melting point difference is lower than 5 ℃; blocker is incompletely matched with the mutant template, and the Tm value of the incomplete match is consistent with or close to the Tm value of the ARMS primer and the mutant template; the 3' end of Blocker is added with a plurality of unrelated bases which do not match with the template.
2. A method for screening Blockers of an ARMS-TaqMan Blocker system is characterized in that 3-10 Blockers are designed according to the Blocker design principle of claim 1, Tm values of the Blockers matched with a mutant template show a gradually-increased gradient near the Tm value of an ARMS primer matched with the mutant template, and then the influence of the gradient Blockers on the sensitivity and the specificity of the system is verified through real-time PCR, so that the Blockers which have no influence on the sensitivity and have the best specificity are screened.
3. The Blocker screening method of claim 1, wherein different Blockers differ by 1 base.
4. Use of the Blocker screening method of ARMS-TaqMan Blocker system of claim 2 or 3 in tumor mutation detection.
CN202011295178.5A 2020-11-18 2020-11-18 Blocker design and screening method of ARMS-TaqMan Blocker system Pending CN112375808A (en)

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