Primer modification design method
By designing and modifying the Tm values of upstream and downstream primers, the problem that primer design in the prior art is difficult to meet the Tm values requirements, which significantly improves the PCR amplification efficiency and ensures efficient binding of primers to the target sequence.
Patent Information
- Application Number
- CN202411666104.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-20
- Publication Date
- 2025-02-25
AI Technical Summary
Existing primer design software is difficult to design primers that meet the Tm value requirements, resulting in low PCR amplification efficiency and may have a negative impact on the PCR process.
By designing upstream and downstream primers for amplification and calculating their Tm values, LNA modification is performed for primers with lower Tm values, and their Tm values are adjusted to 55-65°C to improve the thermal stability of the binding of primers to the target sequence.
It significantly improves the efficiency of nucleic acid amplification, avoids the negative impact during PCR amplification, and ensures efficient binding of primers to the target sequence.
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Figure CN119506270A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of molecular biology, and in particular, relates to a design method for primer modification. Background Art
[0002] Primers and probes are one of the key factors that affect the success of nucleic acid amplification and the reliability of its results. However, in the primer design process, in order to meet the needs of high conservation or detection of specific targets, the primers designed by existing primer design software cannot meet the requirements of Tm value.
[0003] Locked Nucleic Acid (LNA) is a synthetic nucleic acid analog containing a bridged bicyclic sugar moiety. It has a similar phosphate backbone structure to DNA and RNA. It can form high-affinity hybrids with complementary LNA, sDNA, and RNA based on the principle of base complementary pairing. It can be used to modify primer probes and improve nucleic acid amplification efficiency. However, the results of using existing LNA to modify primers are uncontrollable and may even have a negative impact on the PCR amplification process. For example, it may affect the binding of enzymes, thereby affecting the amplification efficiency of PCR.
[0004] Therefore, there is a need in the art for a method for designing LNA modified primers, wherein the primers designed according to the method can maximize the amplification efficiency of the PCR reaction and will not produce various negative effects on the PCR amplification process. Summary of the invention
[0005] In view of this, in a first aspect, the present invention provides a method for designing primer modification, comprising the following steps:
[0006] S1, design upstream and downstream primers for amplification respectively;
[0007] S2, calculating the Tm values of the upstream and downstream primers designed in step S1, and comparing the Tm values of the upstream and downstream primers; and
[0008] S3, modifying the bases in the primers with lower Tm values compared in step S2 according to the following rules:
[0009] The base is cytosine DNA or guanine DNA, and the number of the bases is 3 to 5 starting from the second nucleotide at the 5' end of the primer.
[0010] Furthermore, the Tm value of the modified primer is 55-65°C, preferably 56-60°C.
[0011] Further, the modification is a modification of a locked nucleic acid or a derivative thereof. Preferably, the base is a cytosine DNA or a guanine DNA, and the number of the bases is 5 starting from the second nucleotide at the 5' end of the primer.
[0012] By using the method of the present invention, the Tm value of the primer is adjusted through LNA modification so that it can better bind to the target sequence, thereby improving the thermal stability of the binding between the primer and the target sequence and significantly improving the efficiency of nucleic acid amplification.
[0013] Furthermore, the number of nucleotides in the upstream and downstream primers is 15-30, preferably 18-22.
[0014] Furthermore, the Tm values of the modified upstream and downstream primers are 55-65°C, preferably 56-60°C.
[0015] In some specific embodiments, the primers can be designed by conventional primer design software, including but not limited to Primer 3, Beacon Design 7, Oligo 7, Primer 5, etc.
[0016] In a second aspect, the present invention provides an application of the design method described above in preparing a kit or device for detection.
[0017] In a third aspect, the present invention provides a detection composition, which includes upstream and downstream primers designed according to the above method.
[0018] Furthermore, the detection composition also includes a probe.
[0019] Furthermore, the probe may be modified with locked nucleic acid or its derivatives, or may not be modified with locked nucleic acid or its derivatives.
[0020] Furthermore, the detection composition also includes one or more of a nucleic acid extraction reagent and a nucleic acid amplification reagent.
[0021] The nucleic acid amplification reagent includes PCR Buffer, dNTPs, Mg 2+ , enzymes, etc.
[0022] In a fourth aspect, the present invention provides a design device for primer modification, comprising:
[0023] S1, primer design module, used to design upstream and downstream primers for amplification respectively;
[0024] S2, a primer comparison module, used to calculate the Tm values of the upstream and downstream primers designed by the S1 module, and compare the Tm values of the upstream and downstream primers; and
[0025] S3, a primer modification module, is used to modify the bases in the primers with lower Tm values compared in the module S2 according to the following rules:
[0026] The base is cytosine DNA or guanine DNA, and the number of the bases is 3 to 5 starting from the second nucleotide at the 5' end of the primer.
[0027] Furthermore, the Tm value of the modified primer is 55-65°C, preferably 56-60°C.
[0028] Further, the modification is a modification of a locked nucleic acid or a derivative thereof. Preferably, the base is a cytosine DNA or a guanine DNA, and the number of the bases is 5 starting from the second nucleotide at the 5' end of the primer.
[0029] In the S1 module, the number of nucleotides of the designed upstream and downstream primers is 15-30, preferably 18-22.
[0030] Furthermore, the Tm values of the designed upstream and downstream primers are 55-65°C, preferably 56-60°C.
[0031] The device also includes a nucleic acid extraction module, which is used to extract nucleic acid from the sample.
[0032] The device also includes a nucleic acid amplification module, which is used to amplify the nucleic acid of the sample by using the locked nucleic acid primers designed by the S3 module.
[0033] In a fifth aspect, the present invention provides a device, comprising:
[0034] at least one processor; and
[0035] a memory communicatively connected to at least one of the processors; wherein,
[0036] The memory stores instructions executable by the processor, and the instructions are used to be executed by the processor to implement any of the above-mentioned methods for designing primer modifications.
[0037] In some embodiments, the device further includes at least one input device and at least one output device; in the device, the processor, memory, input device, and output device are connected via a bus.
[0038] In a sixth aspect, a storage medium is provided, wherein the storage medium stores computer instructions, and the computer instructions are used to be executed by the computer to implement the primer modification design method described in any one of the above items.
[0039] In some embodiments, the storage medium is a computer-readable storage medium.
[0040] In a seventh aspect, the present invention provides a kit comprising:
[0041] Sample nucleic acid extraction reagents and amplification reagents; and
[0042] The apparatus, device or storage medium as described above. BRIEF DESCRIPTION OF THE DRAWINGS
[0043] Figure 1 The Ct values of the primers designed for the present invention and the comparative example primer set reaction system 1;
[0044] Figure 2 The Ct values of the primers designed for the present invention and the comparative example primer set reaction system 2;
[0045] Figure 3 The Ct values of the primers designed for the present invention and the comparative example primer set reaction system 3;
[0046] Figure 4 The Ct values of the primers designed for the present invention and the comparative example primer set reaction system 4;
[0047] Figure 5 Ct values of the primers designed for the present invention and the comparative example primer set reaction system 5. DETAILED DESCRIPTION
[0048] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are used to illustrate the present invention, rather than to limit the present invention.
[0049] Example 1: Locked nucleic acid primers designed by the design method used in the present invention to detect sample 1
[0050] Biotech H-TAQ enzyme (5U); Shengxiang Biotechnology PCR Buffer (S08); primers / probes synthesized in Bio-Tech and diluted to a working concentration of 40 pmol / μL by dissolving in TE; 1 mol / L Mg 2+ ; The target sequence plasmid was synthesized by Sangon Biotech (Shanghai) Co., Ltd.
[0051] The single 50 μL amplification system includes: 20.75 μL PCR buffer (S08), 2 μL 100 mmol / L dNTP (T), 0.6 μL 40 pmol / μL PCR upstream primer (SEQ ID NO.1-1), 0.6 μL 40 pmol / μL PCR downstream primer (SEQ1-3 to SEQ1-9), 0.1 μL 40 pmol / μL probe (SEQ ID NO.1-2), 1.6 μL H-TAQ enzyme, 0.4 μL 1 mol / L Mg 2+ , 3.95 μL purified water, 20 μL target sequence nucleic acid (SEQ1), the specific sequence is shown in Table 1.
[0052] PCR amplification program: 95°C for 8 min, 1 cycle; 95°C for 15 s, 57°C for 30 s, 45 cycles, reading the fluorescence signal once after each cycle; 25°C for 10 s, 1 cycle. The HBV target was set as the FAM channel, and the reaction system was set to 50 μL.
[0053] Table 1
[0054]
[0055]
[0056] The target sequence was SEQ1, and five locked nucleic acid modified reverse primers were used, with an annealing temperature of 57°C. The detection results are shown in Table 2 and Figure 1 As shown. The target sequence SEQ1, with the forward primer SEQ1-1, the TaqMan probe SEQ1-2, and the reverse primer example SEQ1-3 designed by the method of this patent, all have Ct values significantly lower than those of similar reverse primers (comparative examples SEQ1-4 to SEQ1-9). And the test results of comparative examples SEQ1-5 and SEQ1-9 show that the modification of LNA may have an adverse effect on the PCR reaction.
[0057] Table 2
[0058]
[0059] Example 2: Locked nucleic acid primers designed by the design method used in the present invention to detect sample 2
[0060] Feipeng TAQ enzyme (S03); Shengxiang Bio PCR Buffer (S15); Primers / probes synthesized at Bio-Tech (Shanghai) Co., Ltd. and diluted to a working concentration of 40 pmol / μL by TE; 1 mol / L Mg 2+ HBV target sequence plasmid was synthesized by Sangon Biotech (Shanghai) Co., Ltd.
[0061] The single 50μL amplification system includes: 22.55μL PCR buffer (S15), 1μL 100mmol / L dNTP (T), 0.5μL 40pmol / μL PCR upstream primer (SEQ2-3 to SEQ2-7), 0.5μL 40pmol / μL PCR downstream primer (SEQ2-1), 0.25μL 40pmol / μL probe (SEQ2-2), 2μL TAQ enzyme, 0.2μL 1mol / L Mg 2+ , 3 μL purified water, 20 μL target sequence nucleic acid (SEQ2), the specific sequence is shown in Table 3.
[0062] PCR amplification program: 95℃8min, 1 cycle; 95℃15s, 60℃30s, 45 cycles, reading the fluorescence signal once after each cycle; 25℃10s, 1 cycle. The HBV target was set as FAM channel, and the reaction system was set as 50μL.
[0063] Table 3
[0064]
[0065] The target sequence was SEQ2, and four locked nucleic acid-modified forward primers were used with an annealing temperature of 60°C.
[0066] like Figure 2 As shown in Table 4, the targeting sequence SEQ2, with the reverse primer SEQ2-1, the TaqMan probe SEQ2-2, and the forward primer example SEQ2-3 designed by the method of this patent, all have Ct values significantly lower than those of similar reverse primers (comparative examples SEQ2-4 to SEQ2-7).
[0067] Table 4
[0068]
[0069] Example 3: Locked nucleic acid primers designed by the design method used in the present invention to detect sample 3
[0070] Feipeng anstart taq enzyme (5U); Shengxiang Bio PCR Buffer (S15); primers / probes synthesized at Bio-Tech (Shanghai) Co., Ltd. and diluted to a working concentration of 40 pmol / μL by TE; 1 mol / L Mg 2+ ; The target sequence plasmid was synthesized by Sangon Biotech (Shanghai) Co., Ltd.
[0071] The single 50μL amplification system includes: 22.55μL PCR buffer (S15), 1μL 100mmol / L dNTP (T), 0.5μL 40pmol / μL PCR upstream primer (SEQ3-1), 0.5μL 40pmol / μL PCR downstream primer (SEQ3-3 to SEQ3-9), 0.25μL 40pmol / μL probe (SEQ3-2), 1.5μL 5Uanstart taq enzyme, 0.2μL 1mol / L Mg 2+ , 3 μL purified water, 20 μL target sequence nucleic acid (SEQ3), the specific sequence is shown in Table 5.
[0072] PCR amplification program: 95℃8min, 1 cycle; 95℃15s, 60℃30s, 45 cycles, reading the fluorescence signal once after each cycle; 25℃10s, 1 cycle. The HBV target was set as FAM channel, and the reaction system was set as 50μL.
[0073] Table 5
[0074]
[0075] The target sequence was SEQ3, and 5 locked nucleic acid-modified reverse primers were used with an annealing temperature of 60°C.
[0076] like Figure 3 As shown in Table 6, the Ct value of the targeted sequence SEQ3, with the forward primer SEQ3-1, the TaqMan probe SEQ3-2, and the reverse primer example SEQ3-3 designed by the method of this patent is significantly lower than that of the similar reverse primers (comparative examples SEQ3-4 to SEQ3-9).
[0077] Table 6
[0078]
[0079] Example 4: Locked nucleic acid primers designed by the design method used in the present invention to detect sample 4
[0080] Feipeng anstart taq enzyme (5U); Shengxiang Bio PCR Buffer (S15); primers / probes synthesized at Bio-Tech (Shanghai) Co., Ltd. and diluted to a working concentration of 40 pmol / μL by TE; 1 mol / L Mg 2+ ; The target sequence plasmid was synthesized by Sangon Biotech (Shanghai) Co., Ltd.
[0081] The single 50 μL amplification system includes: 17 μL PCR buffer (S15), 0.8 μL 100mmol / L dNTP (T), 0.4 μL 40pmol / μL PCR upstream primer (SEQ4-1), 0.4 μL 40pmol / μL PCR downstream primer (SEQ4-3 to 4-9), 0.125 μL 40pmol / μL probe (SEQ ID NO.4-2), 2 μL 5UTaq enzyme, 0.4 μL 1mol / L Mg 2+ , 8.875 μL purified water, 20 μL target sequence nucleic acid plasmid, the specific sequence is shown in Table 7.
[0082] PCR amplification program (general): 95°C for 8 min, 1 cycle; 95°C for 15 s, 58°C for 30 s, 45 cycles, reading the fluorescence signal once after each cycle; 25°C for 10 s, 1 cycle. The HBV target was set as the FAM channel, and the reaction system was set to 50 μL.
[0083] Table 7
[0084]
[0085] The target sequence was SEQ4, and 5 locked nucleic acid-modified reverse primers were used with an annealing temperature of 58°C.
[0086] like Figure 4 As shown in Table 8, the Ct value of the targeting sequence SEQ4, with the forward primer SEQ4-1, the TaqMan probe SEQ4-2, and the reverse primer example SEQ4-3 designed by the method of this patent is significantly lower than that of the similar reverse primers (comparative examples SEQ4-4 to SEQ4-9).
[0087] Table 8
[0088]
[0089]
[0090] Example 5: Locked nucleic acid primers designed by the design method used in the present invention to detect sample 5
[0091] Feipeng anstart taq enzyme (5U); Shengxiang Bio PCR Buffer (S15); primers / probes synthesized at Bio-Tech (Shanghai) Co., Ltd. and diluted to a working concentration of 40 pmol / μL by TE; 1 mol / L Mg 2+ ; The target sequence plasmid was synthesized by Sangon Biotech (Shanghai) Co., Ltd.
[0092] The single 50 μL amplification system includes: 17 μL PCR Buffer (S15), 0.8 μL 100 mmol / L dNTP (T), 0.4 μL 40 pmol / μL PCR downstream primer (SEQ5-1), 0.4 μL 40 pmol / μL PCR upstream primer (SEQ5-3 to 5-8), 0.125 μL 40 pmol / μL probe (SEQ ID NO.5-2), 2 μL 5UTaq enzyme, 0.4 μL 1 mol / L Mg 2+ , 8.875 μL purified water, 20 μL target sequence nucleic acid plasmid, the specific sequence is shown in Table 11.
[0093] PCR amplification program (general): 95°C for 8 min, 1 cycle; 95°C for 15 s, 57°C for 30 s, 45 cycles, reading the fluorescence signal once after each cycle; 25°C for 10 s, 1 cycle. The HBV target was set as the FAM channel, and the reaction system was set to 50 μL.
[0094] Table 9
[0095]
[0096] The target sequence was SEQ5, and three locked nucleic acid-modified forward primers were used with an annealing temperature of 57°C.
[0097] like Figure 5 As shown in Table 10, the Ct value of the targeting sequence SEQ5, with the reverse primer SEQ5-1, the TaqMan probe SEQ5-2, and the forward primer example SEQ5-3 designed by the method of this patent is significantly lower than that of the similar forward primer pair ratio (SEQ5-4 to SEQ5-8).
[0098] Table 10
[0099]
Claims
1. A method for designing primer modification, comprising the following steps: S1, design upstream and downstream primers for amplification respectively; S2, calculating the Tm values of the upstream and downstream primers designed in step S1, and comparing the Tm values of the upstream and downstream primers; and S3, modifying the bases in the primers with lower Tm values compared in step S2 according to the following rules: The base is cytosine DNA or guanine DNA, and the number of the bases is 3 to 5 starting from the second nucleotide at the 5' end of the primer.
2. The method according to claim 1, characterized in that In the step S3, the base is cytosine DNA or guanine DNA, and the number of the bases is 5 starting from the second nucleotide at the 5' end of the primer.
3. The method according to claim 1, characterized in that The Tm value of the modified primer is 55-65°C.
4. The method according to any one of claims 1 to 3, characterized in that The modification is modification of locked nucleic acid or its derivative.
5. Use of the method for designing primer modification according to any one of claims 1 to 4 in preparing a kit or device for detection.
6. A detection composition comprising upstream and downstream primers designed according to the primer modification design method according to any one of claims 1 to 4.
7. The detection composition according to claim 6, characterized in that The detection composition also includes a probe.
8. A design device for primer modification, comprising: S1, primer design module, used to design upstream and downstream primers for amplification respectively; S2, a primer comparison module, used to calculate the Tm values of the upstream and downstream primers designed by the S1 module, and compare the Tm values of the upstream and downstream primers; and S3, a primer modification module, is used to modify the bases in the primers with lower Tm values compared in the module S2 according to the following rules: The base is cytosine DNA or guanine DNA, and the number of the bases is 3 to 5 starting from the second nucleotide at the 5' end of the primer.
9. A device comprising: at least one processor; as well as a memory communicatively connected to at least one of the processors; wherein, The memory stores instructions executable by the processor, and the instructions are used to be executed by the processor to implement the method for designing a modified nucleic acid primer according to any one of claims 1 to 3. 10 . A storage medium storing computer instructions, wherein the computer instructions are used to be executed by the computer to implement the method for designing a modified nucleic acid primer according to claim 1 .