阅读说明：本技术 一种ARMS-TaqMan Blocker体系的Blocker双链设计方法 (Method for designing Blocker double chain of ARMS-TaqMan Blocker system ) 是由 杨芳梅 徐红梅 于 2020-12-08 设计创作，主要内容包括：本发明公开了一种ARMS-TaqMan Blocker体系的Blocker双链设计方法,将Blocker设计在互补链上,形成非竞争性Blocker,与竞争性Blocker同时使用；非竞争性Blocker设计原则：将Blocker设计在突变位点处,覆盖突变位点；Blocker与野生型模板完全匹配；Blocker与突变型模板不完全匹配,不完全匹配的Tm值与退火温度一致或接近；Blocker的3’末端添加多个与模板不匹配的无关碱基。本发明创造性提出非竞争性Blocker设计原则,与竞争性Blocker同时使用,形成双链压制体系,可针对初期产生的少量非特异产物进行压制,在保证体系灵敏度不受影响的前提下,大大提高体系的特异性。(The invention discloses a method for designing a Blocker double chain of an ARMS-TaqMan Blocker system, which comprises the steps of designing Blocker on a complementary chain to form a non-competitive Blocker which is used together with a competitive Blocker; non-competitive Blocker design principle: designing Blocker at the position of a mutation site to cover the mutation site; blocker is completely matched with a wild template; blocker is incompletely matched with the mutant template, and the Tm value of the incomplete match is consistent with or close to the annealing temperature; the 3' end of Blocker is added with a plurality of unrelated bases which do not match with the template. The invention creatively provides a non-competitive packer design principle, and the non-competitive packer design principle is used together with a competitive packer to form a double-chain pressing system, so that a small amount of non-specific products generated in the initial stage can be pressed, and the specificity of the system is greatly improved on the premise of ensuring that the sensitivity of the system is not influenced.)

1. A method for designing a double-stranded Blocker of an ARMS-TaqMan Blocker system is characterized in that the Blocker is designed on a complementary strand to form a non-competitive Blocker which is used together with a competitive Blocker.

2. The method for designing a double-stranded Blocker of the ARMS-TaqMan Blocker system according to claim 1, wherein the non-competitive Blocker design principle is as follows: designing Blocker at the position of a mutation site to cover the mutation site; bloc ker is completely matched with a wild template; blocker is incompletely matched with the mutant template, and the Tm value of the incomplete match is consistent with or close to the annealing temperature; the 3' end of Blocker is added with a plurality of unrelated bases which do not match with the template.

3. The method for designing a double-stranded Blocker of the ARMS-TaqMan Blocker system according to claim 2, wherein the competitive Blocker design principle is as follows: designing Blocker at the position of a mutation site to cover 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.

4. Use of the double-stranded hold-down system of any one of claims 1-3 for the detection of a tumor mutation.

Technical Field

The invention relates to the technical field of tumor mutation detection, in particular to a Bloc ker double-chain design 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 Blocker design is designed at the position partially overlapped with the ARMS primer, plays a competitive role, but only can suppress the single strand of a non-specific template, if the specificity of the ARMS primer is not ideal, a small amount of non-specific products generated in the initial stage can become the template for subsequent amplification, and escape from the suppression effect of the Blocker, so that non-specific signals are increased.

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.

Disclosure of Invention

Aiming at the technical defects of the Blocker design of the existing ARMS-TaqMan Blocker system, the invention provides a non-competitive Blocker design which is used together with the competitive Blocker design to form the ARMS-TaqM an Blocker system pressed by double chains.

The invention provides a double-chain packer design method of an ARMS-TaqMan packer system, which is characterized in that a packer is designed on a complementary chain to form a non-competitive packer which is used together with a competitive packer.

Non-competitive Blocker design principle: designing Blocker at the position of a mutation site to cover the mutation site; blocker is completely matched with a wild template; blocker is incompletely matched with the mutant template, and the Tm value of the incomplete match is consistent with or close to the annealing temperature; the 3' end of Blocker is added with a plurality of unrelated bases which do not match with the template.

Competitive packer design principle: designing Blocker at the position of a mutation site to cover 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. The competitive packer design principle and the advantages thereof are described in detail in the invention patent application of 'a packer design and screening method of ARMS-TaqMan packer system' filed by the applicant at 11, 18, 2020, application number is 2020112951785, and the technical effect is only explained here, and the specific embodiment is not repeated.

The competitive packer design principle does not strictly require that the mutation site is located in the middle position of the packer, does not need to meet the requirement of 5 ℃ of melting point difference, only needs to have the melting point difference, and is relatively easier to design. 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 competitive Blocker design principle 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 the Blocker in the system can not generate redundancy only under the Tm value, thereby achieving the maximum suppression effect. This is quite different from the conventional competitive Blocker design principle.

According to the competitive Blocker design principle, the 3 'end of the Blocker does not need to be added with special blocking 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 TaqDNA polymerase, and special modification is not needed, so that the cost is saved by dozens of times.

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 double-chain pressing system in tumor mutation detection.

The invention creatively provides a non-competitive packer design principle, and the non-competitive packer design principle is used together with a competitive packer to form a double-chain pressing system, so that a small amount of non-specific products generated in the initial stage can be pressed, and the specificity of the system is greatly improved on the premise of ensuring that the sensitivity of the system is not influenced.

Drawings

FIG. 1 is a view of the double-strand suppression at E545K site according to the present invention;

FIG. 2 is a study of the double strand suppression of braf sites according to the invention;

FIG. 3 shows the double-strand suppression at the G719 site of the present invention.

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 TMUtilityv1.3, and the sequence information of each primer, probe and Blocker is shown in Table 1, wherein the lower case letters indicate bases to be added artificially, Tm1 indicates the melting point when the primer/probe/Blocker is completely matched with a target sequence, and Tm2 indicates the melting point when the primer/Blocker is not completely matched with a target sequence.

TABLE 1

Example 1E 545K site double-Strand suppression

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.

The competitive Blocker is E545K-B18, and the sequence is shown as SEQ NO.04 in the table 1; the noncompetitive packer is designed according to the design principle of the invention and has the sequence of E545K-B25, and the sequence is shown as SEQ NO.05 in the table 1.

The method comprises the steps that Blockers are obtained through screening, 3-10 Blockers are designed according to the Blocker design principle of the invention, the low melting points of the Blockers are all near the annealing temperature, are higher and lower than each other, and can have a difference of 1 base between different Blockers, so that the Tm values matched with the mutant templates of the Blockers present a gradient which is gradually increased near the annealing temperature, and the Tm values are wide and dense, and the best Blockers can not 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 Bloc ker which has no influence on the sensitivity and has the best specificity.

Second, PCR reaction system preparation

PCR reaction solution was prepared and deionized water was added to 25. mu.l. Only adding competitive Blocker E545-B18 into the first group of reaction solution, wherein the dosage reaches the maximum addition amount, and the specific components and the concentration refer to Table 2; competitive Block ERE545-B18 and non-competitive Block E545-B25 were added to the second set of reaction solution simultaneously, and the specific components and concentrations are shown in Table 3.

PCR Components	Dosage of
		10 × Buffer (containing Mg)2+)	1×
dNTPs	0.16mM
		E545K-F1	0.4μM
E545K-R1	0.4μM
		E545K-P	0.4μM
E545-B18	2μM
		Sodium chloride	32mM

TABLE 2

PCR Components	Dosage of
		10 × Buffer (containing Mg)2+)	1×
dNTPs	0.16mM
		E545K-F1	0.4μM
E545K-R1	0.4μM
		E545K-P	0.4μM
E545-B18	2μM
		E545-B25	0.32μM
Sodium chloride	32mM
		Taq enzyme	1U

TABLE 3

Third, sample preparation

E545K plasmid 10^3 copies/. mu.l and 10 ng/. mu.l wild type genome equal volume mixing, obtain mutation rate for 50% standard substance, will 50% standard substance with 10 ng/. mu.l wild type genome dilution, obtain 1% and 0.1% standard substance, prepare 10 ng/. mu.l wild type genome, spare.

Fourthly, adding sample and operating the machine

Both groups were supplemented with 5. mu.l of 1% and 0.1% standards and 10 ng/. mu.l of wild type genome, 2 parallel channels each 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. 1, a set of amplification curves of the first set to which only competitive Blocker E545-B18 was added is represented by black circles; the second group, one to which both competitive Blocker E545-B18 and non-competitive Blocker E545-B25 are added, is represented by black positive triangles; NTCs are represented by white positive triangles. As can be seen from FIG. 1, the specificity of the double stranded suppression system is better than that of the single stranded suppression, with CT values of 10 ng/. mu.l of wild type genome being more late.

Example 2 braf site double strand suppression study

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.06 in Table 1, and the sequence of the downstream primer braf-R is shown as SEQ N O.07 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.8 in a table 1.

The competitive Blocker is braf-B4, and the sequence is shown as SEQ NO.09 in the table 1; the noncompetitive Blocker is designed according to the design principle of the invention and is braf-B8, and the sequence is shown as SEQ NO.10 in Table 1; blocker was obtained by screening in the same manner as in example 1.

Second, PCR reaction system preparation

PCR reaction solution was prepared and deionized water was added to 25. mu.l. Only adding competitive Blockerbraf-B4 into the first group of reaction solution, wherein the dosage reaches the maximum addition amount, and the specific components and concentration refer to Table 3; competitive Blockerbraf-B4 and non-competitive Blockerbraf-B8 were added to the second group of reaction solution, and the specific components and concentrations were as shown in Table 4.

TABLE 3

PCR Components	Final concentration
		10 × Buffer (containing Mg)2+)	1×
dNTPs	0.16mM
		braf-F	0.8μM
braf-R	0.8μM
		braf-P	0.04μM
braf-B4	0.32μM
		braf-B8	0.6μM
Sodium chloride	20mM
		Taq enzyme	1U

TABLE 4

Third, sample preparation

Mixing braf plasmid 10^3 copies/mul with 10 ng/mul wild type genome to obtain standard substance with mutation rate of 50%, diluting 50% of standard substance with 10 ng/mul wild type genome to obtain 1% and 0.1% of standard substance, and preparing 100 ng/mul wild type genome for later use.

Fourthly, adding sample and operating the machine

Both groups were supplemented with 5. mu.l of 1% and 0.1% standards and 100 ng/. mu.l of wild type genome, 2 parallel channels each 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 competitive Blockberraf-B4, and the amplification curves are represented by black circles; competitive Blockberraf-B4 and non-competitive Blockberraf-B8 are added to the second group of reaction solution at the same time, and an amplification curve is represented by a black positive triangle; NTC is represented by white equilateral triangle. As can be seen from FIG. 2, the specificity of the double-stranded suppression system is better than that of the single-stranded suppression, the CT value of 100 ng/. mu.l wild-type genome is more posterior, and the non-specific fluorescence signal is lower.

Example 3 investigation of double Strand suppression at G719 site

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.11 in Table 1, and the sequence of the downstream primer G719-R is shown as SEQ NO.12 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.13 in Table 1.

The competitive Blocker is G719-B1, and the sequence is shown as SEQ NO.14 in the table 1; the non-competitive Blocker is designed according to the design principle of the invention and is G719-B6, and the sequence is shown as SEQ NO.15 in Table 1; blocker was obtained by screening in the same manner as in example 1.

Second, PCR reaction system preparation

PCR reaction solution was prepared and deionized water was added to 25. mu.l. Only competitive Blocker G719-B1 is added to the first group of reaction solution, the dosage reaches the maximum addition amount, and the specific components and concentrations refer to Table 5; competitive Blocker G719-B1 and non-competitive Blocker G719-B6 were added to the second group of reaction solutions at the same time, and the specific components and concentrations were as shown in Table 6.

PCR Components	Final concentration
		10 x Buffer (containing Mg2+)	1×
dNTPs	0.16mM
		G719-F	0.8μM
G719-R	0.8μM
		G719-P	0.04μM
G719-B1	0.2μM
		Sodium chloride	32mM

TABLE 5

TABLE 6

Third, sample preparation

Mixing the G719 plasmid 10^3 copies/ul and the wild type genome 10 ng/ul to obtain a standard substance with the mutation rate of 50%, diluting the standard substance with the wild type genome 10 ng/ul to obtain the standard substances of 1% and 0.1%, and preparing the wild type genome 10 ng/ul for later use.

Fourthly, adding sample and operating the machine

Both groups were added 5. mu.l of 1%, 0.1% standard and 10 ng/. mu.l wild type genome, 2 parallel channels each and No Template Control (NTC) was set. The computer program is as follows: 2min at 95 ℃ for one 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. 3, only competitive BlockG 719-B1 was added to the first set of reaction solutions, and the amplification curves are shown as black circles; competitive Blocker G719-B1 and non-competitive Blocker G719-B6 were added to the second reaction mixture, and the amplification curves are shown by black positive triangles; NTC is represented by white equilateral triangle. As can be seen from FIG. 3, the double-stranded suppression system 0.1% standard was not affected, specificity was better than single-stranded suppression, and there was no CT value at 10 ng/. mu.l wild-type genome.

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.