CN112980932B - Nucleotide for detecting gene mutation, composition and method thereof - Google Patents
Nucleotide for detecting gene mutation, composition and method thereof Download PDFInfo
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- 206010064571 Gene mutation Diseases 0.000 title claims abstract description 33
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- 150000001412 amines Chemical class 0.000 claims description 8
- 230000037430 deletion Effects 0.000 claims description 7
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
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Abstract
The invention discloses a nucleotide for detecting gene mutation, a composition and a method thereof, and relates to the technical field of mutation amplification, in particular to a nucleotide composition which comprises a first nucleic acid molecule and a second nucleic acid molecule, wherein the first nucleic acid molecule is designed to compete with a mutant AS primer in an ARMS primer for a sequence of a target sequence, the second nucleic acid molecule is complementary with the first nucleic acid molecule, and the nucleotide composition can reduce interference of wild DNA in a gene mutation detection process, reduce background signals in amplification and obviously improve detection specificity and sensitivity.
Description
Technical Field
The invention relates to the technical field of allele amplification, in particular to a nucleotide for detecting gene mutation, a composition and a method thereof.
Background
Common mutation sites of EGFR gene occur on exons 18, 19, 20 and 21, wherein the non-frameshift deletion mutation of exon 19 accounts for about 45%, the L858R point mutation of exon 21 accounts for 40-45%, and these two mutations are common mutations. In addition, EGFR gene has drug sensitive mutation and drug resistant mutation, and the drug sensitive mutation is sensitive to a certain target drug after mutation; the drug-resistant mutation is a mutation which is resistant to a certain targeting drug after mutation, such as T790M (2369C > T), and the mutation accounts for about 50% of the mutation frequency.
Approximately 50-60% of patients will have acquired drug resistance T790M after 1st line EGFR-TKI treatment, and the current three generation TKI Osemermertinib overcomes T790M, but is accompanied by more intricate C797S (2389T > A, 2390G > C).
The detection of EGFR genes T790M and C797S is clinically significant, and with the application of the three-generation TKI drugs and the further exploration of the clinical application of T790M and C797S mutant patients, EGFR targeting drugs can be listed as the accompanying diagnosis standard.
Currently, techniques for detecting targeted drug gene detection are mainly NGS and digital PCR techniques. The cost of the NGS detection technology is high, the period is long, the library can be built only after amplification and enrichment are carried out on the circulating tumor DNA sample, and the situation of base mismatch can be introduced in the amplification process; although the digital PCR has higher sensitivity and can absolutely quantify the sample without standard curve, the digital PCR instrument needs to use corresponding consumable materials and cannot be matched with the existing fluorescent quantitative PCR platform, so that the purchase cost of the instrument is increased and the sample detection cost is increased.
The detection technique of gene mutations can also employ ARMS technology (amplification refractory mutation system, mutation amplification system), also known as allele-specific amplification (allele specific amplification, ASA), which was first established by Newton et al for the detection of known mutations.
The basic principle of ARMS is that if the 3 'end base of the primer is not complementary with the template base, the 3' -5 'exonuclease correction activity is lacking by Taq enzyme, the 3' end mismatched base of the primer cannot be cut off, and the primer cannot extend normally due to the mismatch of the base, so that the primer cannot extend normally by using general heat-resistant DNA polymerase. Therefore, the PCR primer is designed according to the known point mutation, the 3' -end base of the PCR primer is complementary with the base of the mutated template, if the PCR can obtain the PCR fragment or the amplification curve with the corresponding length, the DNA sequence containing the mutation in the sample is indicated, if the PCR fragment or the amplification curve with the corresponding length cannot be obtained, the mutation sequence is not contained in the sample, and the template with the certain point mutation is distinguished from the normal template.
However, conventional ARMS techniques detect tumor DNA base mutations, which are largely dependent on the type of base mismatch, because there is a difference in the intensity of the different base mismatch types, i.e., a strong mismatch between purine and purine, a medium mismatch between pyrimidine and pyrimidine, a weak mismatch between pyrimidine and purine, or a weak mismatch between purine and purine. The mutation type of the base is related to the sensitivity and accuracy of ARMS technology detection, if the mismatch type of the base on the complementary strand of the mutated base and the wild type base is a strong mismatch, the detection sensitivity and accuracy are relatively high, and if the mismatch type of the base on the complementary strand of the mutated base and the wild type base is a weak mismatch, the detection sensitivity and specificity are low, which limits the application of ARMS technology in different mutations.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims at providing a nucleotide for detecting gene mutation, and a composition and a method thereof. The nucleotide composition can reduce the interference of wild DNA in the process of gene mutation detection, reduce the background signal in amplification and obviously improve the specificity and sensitivity of detection.
The invention is realized in the following way:
the present embodiments provide a nucleotide suitable for use with an ARMS primer and a first nucleic acid molecule to detect a genetic mutation; the first nucleic acid molecule is designed to compete with the mutant AS primer in the ARMS primer for the sequence of a target sequence taken from a region of the wild-type target gene sequence that covers the mutation site to be detected, the complementary region of the first nucleic acid molecule and the target sequence covers the mutation site to be detected, and the nucleotide comprises a second nucleic acid molecule; the full length or part thereof of the second nucleic acid molecule is complementary to the full length or part of the sequence of the first nucleic acid molecule, and the complementary region of the second nucleic acid molecule and the first nucleic acid molecule covers the mutation site to be detected.
The embodiment of the invention also provides application of the nucleotide in detection of gene mutation, wherein the nucleotide is matched with an ARMS primer and a first nucleic acid molecule.
Embodiments of the present invention provide a nucleotide composition comprising a nucleotide as described above and the first nucleic acid molecule.
The embodiment of the invention provides a reagent or a kit for detecting gene mutation, which comprises the nucleotide or the nucleotide composition.
The embodiment of the invention provides a method for detecting gene mutation, which is characterized in that the nucleotide composition is added into a reaction system for detecting target gene mutation.
The beneficial effects of the embodiment of the invention are as follows:
the invention provides a nucleotide composition comprising a first and a second nucleic acid molecule, the first nucleic acid molecule being designed to compete with a mutant AS primer in an ARMS primer for the sequence of a target sequence, the second nucleic acid molecule being complementary to the first nucleic acid molecule, the nucleotide composition being capable of reducing interference of wild type DNA during detection of a gene mutation, reducing background signal during amplification, and significantly improving specificity and sensitivity of detection.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the results of Ct value detection for test group 1 in test example 1;
FIG. 2 shows the results of Ct value detection for test group 2 in test example 1;
FIG. 3 shows the results of Ct value detection for test group 3 in test example 1;
FIG. 4 shows the results of Ct value detection for groups 1 to 4 in test example 2;
FIG. 5 shows the results of Ct value detection for groups 1 to 4 in test example 3;
FIG. 6 shows the results of CT value detection for groups 1 to 3 in test example 4.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The present embodiments provide a nucleotide suitable for use with an ARMS primer and a first nucleic acid molecule to detect a genetic mutation; the first nucleic acid molecule is designed to compete with the mutant AS primer in the ARMS primer for the sequence of a target sequence taken from a region of the wild-type target gene sequence that covers the mutation site to be detected, the complementary region of the first nucleic acid molecule and the target sequence covers the mutation site to be detected, and the nucleotide comprises a second nucleic acid molecule; the full length or part thereof of the second nucleic acid molecule is complementary to the full length or part of the sequence of the first nucleic acid molecule, and the complementary region of the second nucleic acid molecule and the first nucleic acid molecule covers the mutation site to be detected.
As used herein, "ARMS" is the amplification refractory mutation system, also known as a mutation-blocking amplification system.
Included in the ARMS primer is a mutant allele-specific primer (Mutant allele specific Primer, simply referred to AS mutant AS primer) that is complementary to the mutant base on the mutant template at the 3' end and is not complementary to the wild-type template, thereby distinguishing the wild-type template from the mutant template.
The above-mentioned "the sequence of the first nucleic acid molecule designed to compete with the mutant AS primer in the ARMS primer" means that: the mutant AS primer can be complementarily paired with the target sequence (containing the mutation to-be-detected site), and the first nucleic acid molecule can also be complementarily paired with the target sequence, wherein the first nucleic acid molecule and the target sequence are in competition, namely, the binding rate of the mutant AS primer and the target sequence can be reduced by adding the first nucleic acid molecule into a mixed system containing the specific primer and the target sequence.
In a mixed system of the first nucleic acid molecule and the second nucleic acid molecule, when the first nucleic acid molecule is contacted with the wild type target gene, the first nucleic acid molecule can be separated from the second nucleic acid molecule, and after separation, the first nucleic acid molecule and the wild type target gene are subjected to base complementary pairing so as to inhibit amplification of the wild type target gene by the primer; when the first nucleic acid molecule is contacted with the mutant target gene, the first nucleic acid molecule can be complementarily paired with the second nucleic acid molecule, so that the binding between the first nucleic acid molecule and the mutant target gene can be avoided or reduced, and the occurrence of false positive results can be avoided or reduced.
In alternative embodiments, the entire length of the first nucleic acid molecule is complementary to the target sequence.
In an alternative embodiment, the Tm value of the first nucleic acid molecule > the Tm value of the second nucleic acid molecule > the Tm value of the mutant AS primer.
Under the above-mentioned conditions for setting the Tm value, the tendency of the first nucleic acid molecule to bind to the wild-type target gene > the tendency of the first nucleic acid molecule to bind to the second nucleic acid molecule > the tendency of the first nucleic acid molecule to bind to the mutant target gene. I.e., when encountering a wild-type target gene, the first nucleic acid molecule preferentially performs base complementary pairing with the wild-type target gene; when a mutant target gene is encountered, the first nucleic acid molecule preferentially complementarily pairs with the second nucleic acid molecule, thereby avoiding or significantly reducing binding of the first nucleic acid molecule to the mutant target gene.
In an alternative embodiment, the Tm of the first nucleic acid molecule is 1℃to 15℃higher than the Tm of the second nucleic acid molecule. Specifically, the Tm value of the first nucleic acid molecule may be 1 ℃,2 ℃, 3 ℃, 4 ℃, 5 ℃, 6 ℃, 7 ℃, 8 ℃, 9 ℃, 10 ℃, 11 ℃, 12 ℃, 13 ℃, 14 ℃, or 15 ℃ higher than the Tm value of the second nucleic acid molecule.
In an alternative embodiment, the Tm of the first nucleic acid molecule is 1℃to 13℃higher than the Tm of the second nucleic acid molecule. Specifically, the Tm value of the first nucleic acid molecule may be 1 ℃,2 ℃, 3 ℃, 4 ℃, 5 ℃, 6 ℃, 7 ℃, 8 ℃, 9 ℃, 10 ℃, 11 ℃, 12 ℃, or 13 ℃ higher than the Tm value of the second nucleic acid molecule.
In alternative embodiments, the second nucleic acid molecule has a length that is less than the length of the first nucleic acid molecule.
In an alternative embodiment, the second nucleic acid molecule is 13 to 25nt.
In alternative embodiments, the second nucleic acid molecule is modified with an extension blocker;
in alternative embodiments, the extension blocker is modified at the 3' end of the second nucleic acid molecule;
in an alternative embodiment, the extension block comprises: amine (NH) 2 ) Biotin, ddNTPs, PEG and PO 4 One or more of the following.
In an alternative embodiment, the second nucleic acid molecule contains a modifying group that is at least one of a locked nucleic acid, a peptide nucleic acid, and a minor groove binder. The modification group can improve the Tm value of the nucleic acid molecule, enhance the binding stability of the nucleic acid molecule and the complementary sequence, and is beneficial to shortening the length of the nucleic acid molecule. In other embodiments, the second nucleic acid molecule may omit the modifying group.
In an alternative embodiment, the region of complementarity of the second nucleic acid molecule to the first nucleic acid molecule covers at least one of the 1 to 8 bases of the 5' end of the first nucleic acid molecule sequence.
The phrase "the complementary region covers at least one of 1 to 8 bases at the 5 '-end of the first nucleic acid molecule sequence" means that the complementary region of the second nucleic acid molecule and the first nucleic acid molecule is closer to the 5' -end of the first nucleic acid molecule sequence, and under this condition, the blocking effect of the second nucleic acid molecule on the first nucleic acid molecule is better, and the allele-specific amplification primer is prevented from being complementary to the wild-type sequence, thereby improving the specificity of the detection result.
In an alternative embodiment, the region of complementarity of the first nucleic acid molecule to the target sequence comprises a 5 'terminal complementary region, the 5' terminal complementary region covering at least one of the 1 to 8 bases of the 5 'end of the first nucleic acid molecule sequence, the mutation site to be detected being located in the 5' terminal complementary region.
The term "the 5' -end complementary region covers at least one of 1 to 8 bases at the 5' -end of the first nucleic acid molecule sequence" means that the 5' -end complementary region is closer to the 5' -end of the first nucleic acid molecule sequence, i.e., the position of the first nucleic acid molecule corresponding to the mutation site to be detected is closer to the 5' -end of the first nucleic acid molecule sequence.
In alternative embodiments, the genetic mutation comprises any of the following mutation patterns: substitution of one or several nucleotides, deletion of one or several nucleotides and insertion of one or several nucleotides.
In alternative embodiments, the gene of interest comprises an EGFR gene.
In an alternative embodiment, the mutation site to be detected is 2369c > t or 2390g > c.
In an alternative embodiment, when the mutation site to be detected is 2369C > T, the sequence of the second nucleic acid molecule is as shown in SEQ ID No.2, 11 or 12.
In an alternative embodiment, when the mutation site to be detected is 2390G > C, the sequence of the second nucleic acid molecule is shown as SEQ ID No. 4.
The embodiment of the invention also provides application of the nucleotide in detection of gene mutation, wherein the nucleotide is matched with an ARMS primer and a first nucleic acid molecule.
In this application, the nucleotide, first nucleic acid molecule and ARMS primer are as described in the previous embodiments and are not described in detail herein.
The embodiment of the present invention also provides a nucleotide composition, which includes the nucleotide and the first nucleic acid molecule described in the foregoing embodiments, and is not described herein.
In alternative embodiments, the first nucleic acid molecule and the first nucleic acid molecule are each present in single stranded form; alternatively, the first nucleic acid molecule is bound to the second nucleic acid molecule in a double stranded form.
In an alternative embodiment, the first nucleic acid molecule is modified with an extension blocker.
In an alternative embodiment, the extension blocker is modified at the 3' end of the first nucleic acid molecule.
In an alternative embodiment, the extension block comprises: amine (NH) 2 ) Biotin, ddNTPs, PEG and PO 4 One or more of the following.
In an alternative embodiment, the first nucleic acid molecule has a modification group attached thereto, the modification group being at least one of a locked nucleic acid, a peptide nucleic acid, and a minor groove binder.
In an alternative embodiment, the first nucleic acid molecule is 15 to 35nt.
In an alternative embodiment, when the mutation site to be detected is 2369C > T, the sequence of the first nucleic acid molecule is as shown in SEQ ID No.1, 13 or 14.
When the mutation site to be detected is 2390G > C, the sequence of the first nucleic acid molecule is shown as SEQ ID No. 3.
In an alternative embodiment, the nucleotide composition further comprises an ARMS primer for detecting the target gene.
In an alternative embodiment, the ARMS primer comprises: a first ARMS primer pair with sequences shown in SEQ ID No. 5-6 for detecting 2369C > T mutation; and/or a second ARMS primer pair with sequences shown in SEQ ID No. 7-8 for detecting 2390G > C mutation.
In an alternative embodiment, the nucleotide composition further comprises a probe.
In an alternative embodiment, the probe is a Taqman probe.
In an alternative embodiment, the probe comprises: a first probe for detecting 2369C > T mutation and having a sequence shown as SEQ ID No.9, and/or a second probe for detecting 2390G > C mutation and having a sequence shown as SEQ ID No. 10.
The embodiment of the invention also provides a reagent or a kit for detecting gene mutation, which comprises the nucleotide or the nucleotide composition according to the previous embodiment.
In alternative embodiments, the genetic mutation comprises any of the following mutation patterns: substitution of one or several nucleotides, deletion of one or several nucleotides and insertion of one or several nucleotides.
In addition, the embodiment of the invention also provides a method for detecting the mutation of the gene, which comprises the step of adding the nucleotide composition in the previous embodiment into a reaction system for detecting the mutation of the target gene.
In alternative embodiments, the genetic mutation comprises any of the following mutation patterns: substitution of one or several nucleotides, deletion of one or several nucleotides and insertion of one or several nucleotides.
In an alternative embodiment, the reaction system for detecting the mutation of the target gene is an AMRS-PCR reaction system.
In an alternative embodiment, when the first nucleic acid molecule in the nucleotide combination is present in single stranded form prior to performing the AMRS-PCR reaction, the method comprises annealing the first nucleic acid molecule to the second nucleic acid molecule and then adding to the reaction system.
In alternative embodiments, the concentration of the first nucleic acid molecule is less than the concentration of the second nucleic acid molecule.
In an alternative embodiment, the molar ratio of the first nucleic acid molecule to the second nucleic acid molecule is 1: (1.1-2.0); preferably 1:1.2.
in an alternative embodiment, the annealing is a gradient annealing, and the reaction conditions of the annealing are as follows: 94-96 ℃, 0.8-1.2 min, 84-86 ℃, 0.8-1.2 min, 74-76 ℃, 0.8-1.2 min, 64-66 ℃, 0.8-1.2 min, 54-56 ℃, 0.8-1.2 min, 44-46 ℃, 0.8-1.2 min, 34-36 ℃, 0.8-1.2 min, 24-26 ℃, 0.8-1.2 min, 14-16 ℃, 0.8-1.2 min, 3-5 ℃ and infinity.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1
This example provides a nucleotide composition for the T790M mutation (2369C > T) of EGFR gene comprising a first nucleic acid molecule, a second nucleic acid molecule, an ARMS primer and a probe, the sequence of the nucleotide composition being shown in Table 1.
TABLE 1 nucleic acid molecule sequences
Remarks:
1.V is a degenerate base, which may be substituted with G, A or C, in this embodiment A;
2. the Tm value of the first nucleic acid molecule is 83 ℃; the Tm value of the second nucleic acid molecule is 77.6 ℃; the Tm value of the upstream primer is 70 ℃; the Tm value of the downstream primer is 65 ℃;
3. the bolded and underlined bases in the first nucleic acid molecule and the second nucleic acid molecule correspond to mutation sites to be detected;
4. the mutant AS primer is an amplification primer specific for the mutant allele in the ARMS primer.
Example 2
This example provides a nucleotide composition for the EGFR gene C797S mutation (2390G > C) comprising a first nucleic acid molecule, a second nucleic acid molecule, an ARMS primer and a probe, the sequence of the nucleotide composition being shown in Table 2.
TABLE 2 nucleic acid molecule sequences
Remarks:
the mutant AS primer is a mutant allele-specific amplification primer in the ARMS primer.
Example 3
The nucleotide composition provided in this example includes the nucleotide compositions of example 1 and example 2.
Example 4
The present embodiment provides a method for detecting a gene mutation, comprising the steps of:
1. pretreatment:
pretreatment of T790M gene mutation
The nucleotide composition provided in example 1 was used to mix the first nucleic acid molecule and the second nucleic acid molecule at a molar ratio of 1:1.2, and the mixture was put into a conventional qualitative PCR instrument Biometra TRIO for gradient annealing heat treatment under the following reaction conditions:
95℃，1min→85℃，1min→75℃，1min→65℃，1min→55℃，1min→45℃，1min→35℃，1min→25℃，1min→15℃，1min→4℃，∞。
pretreatment of C797S Gene mutation
The nucleotide composition provided in example 2 was used to mix the first nucleic acid molecule and the second nucleic acid molecule at a molar ratio of 1:1.2, and the mixture was put into a conventional qualitative PCR instrument Biometra TRIO for gradient annealing heat treatment, as above.
AMRS-PCR reaction:
detection of T790M Gene mutation
And mixing the corresponding AMRS primer pair, the sample to be detected and the annealing products of the first nucleic acid molecule and the second nucleic acid molecule obtained in the pretreatment step to perform AMRS-PCR reaction, wherein the reaction system is shown in table 3, and the reaction conditions are shown in table 4.
Detection of C797S Gene mutation
The corresponding AMRS primer pair, the sample to be tested and the annealing products of the first nucleic acid molecule and the second nucleic acid molecule obtained in the pretreatment step are mixed for carrying out AMRS-PCR reaction, and the reaction system and the reaction conditions are the same as those described above (Table 3 and Table 4).
TABLE 3 reaction system
TABLE 4 reaction conditions
Test example 1
The effect of the Tm value of the second nucleic acid molecule in the nucleotide composition provided in example 1 on the detection of a gene mutation was verified.
Test procedure
Using the nucleotide composition provided in example 1, 3 sets of second nucleic acid molecules having different Tm values for the second nucleic acid molecules and 1 set of control sets without the second nucleic acid molecules were set.
The sequence of the second nucleic acid molecule of test set 1 is:
5’-GGCATGAGCTGCGTGATGA-3’-NH 2 -MGB(SEQ ID No.11)；
the sequence of the second nucleic acid molecule of test set 2 is:
5’-GCATGAGCTGCGTGATGA-3’-NH 2 -MGB(SEQ ID No.2)。
the sequence of the second nucleic acid molecule of test set 3 is:
5’-CATGAGCTGCGTGATGA-3’-NH 2 -MGB(SEQ ID No.12)。
the T790M mutant cell line and the normal wild type cell line are mixed according to different cell number ratios of 0.1% and 0% of the mutant cell line respectively, and DNA is extracted after mixing to be used as a template (a sample to be tested) containing 0.1% of the mutant ratio or no mutation.
Then, the gene mutation detection was performed on T790M by using the detection method provided in example 4, respectively, and then CT values and DeltaCT values of the DNA of the wild type cell line and the DNA of the 0.1% -T790M cell line were calculated.
Test results
TABLE 5 detection results
As shown in FIGS. 1 to 3, in combination with Table 5, the DNA templates of the same T790M mutation ratio (0.1%) were amplified, and the delta CT values of the amplification systems after adding the second nucleic acid molecules of different TM values were different.
Wherein, the test group 1 with the highest TM value has the least interference on the amplification of mutant DNA, but the inhibition effect on wild DNA is obviously reduced, and the delta CT value=9.1 has improved distinguishing effect compared with the control group;
test group 2 with medium TM had some interference with mutant templates, but had better inhibition effect on wild type, with maximum Δct compared to the other two second nucleic acid molecules, Δct=12.8, and best discrimination.
The test group 3 having the lowest TM value had some interference with the mutant template, but had the best inhibitory effect on the wild type, and the Δct value=10.2, and the discrimination effect was excellent.
Test example 2
The effect of the nucleotide composition provided in example 1 on the specificity of the T790M gene mutation detection was verified.
The T790M mutant cell line was used in combination with normal wild type cell lines at different cell number ratios of 0.1% and 0%, and DNA was extracted after mixing as a template (test sample) with or without mutation at a ratio of 0.1%.
The 4 test groups were set up as follows. The "first nucleic acid molecule" is abbreviated as "single-stranded Block", and a double-stranded form formed by binding the first nucleic acid molecule and the second nucleic acid molecule is abbreviated as "double-stranded Block", and the "no Block" is abbreviated as "no first nucleic acid and no second nucleic acid".
Group 1:0% t790m without Block;
group 2:0.1% t790m without Block;
group 3:0.1% T790M, single chain Block;
group 4:0.1% T790M, double strand block.
Then, the detection method provided in example 4 was used to detect the gene mutation of T790M, and then the DeltaCT values of 4 groups of samples to be tested were calculated.
Test results
TABLE 6 test results
Referring to FIG. 4, it can be seen from Table 6 that the specificity of adding double-stranded Block is better than that of single-stranded Blcok, and is significantly better than that of adding no Block, i.e., the addition of the first nucleic acid and/or the second nucleic acid can significantly improve the specificity of T790M gene mutation detection.
Test example 3
The effect of the nucleotide composition provided in example 2 on the specificity of detection of mutation in the C797S gene was verified.
The C797S mutant cell line was used in combination with a normal wild-type cell line at different cell number ratios of 0.1% and 0%, and DNA was extracted as a template (test sample) containing 0.1% mutation or no mutation after combination.
The 4 test groups were set up as follows. The first nucleic acid molecule is abbreviated as Block or single-stranded Block, and a double-stranded form formed by combining the first nucleic acid molecule and the second nucleic acid molecule is abbreviated as double-stranded Block, and the first nucleic acid and the second nucleic acid are not included, and are abbreviated as "no Block".
Group 1:0% c797s, no Block;
group 2:0.1% c797s, no Block;
group 3:0.1% c797s, double-stranded Block;
group 4:0.1% C797S, single chain block.
Then, the detection method provided in example 4 was used to detect the gene mutation of C797S, and then the DeltaCT values of 4 groups of samples to be tested were calculated, respectively.
Test results
TABLE 7 test results
Referring to FIG. 5, it can be seen from Table 7 that the specificity of adding double-stranded Block is better than that of single-stranded Blcok, and is significantly better than that of adding no Block, i.e., the specificity of gene mutation detection can be significantly improved by adding the first nucleic acid and/or the second nucleic acid.
Test example 4
Using the nucleotide composition provided in example 1, 3 sets of first nucleic acid molecules were set.
The sequence of the first nucleic acid molecule of test set 1 is:
5’-TCATCACGCAGCTCATGCC-3’-NH 2 -MGB (Tm value 83.0 ℃, first nucleic acid molecule in example 1);
the sequence of the first nucleic acid molecule of test set 2 is:
5’-CAGCTCATCACGCAGCTCAT-3’-NH 2 MGB (Tm 83.3 ℃ C., SEQ ID No. 13).
The sequence of the first nucleic acid molecule of test set 3 is:
5’-CCGTGCAGCTCATCACGC-3’-NH 2 MGB (Tm 82.9 ℃, SEQ ID No. 14).
The bolded and underlined bases in the sequence correspond to the mutation sites to be detected.
Then, the detection methods provided in example 4 were used to detect the wild type samples of T790M, and then CT values of 3 groups of wild type samples were calculated, respectively. The experimental results are shown in table 8 and fig. 6.
Table 8 test results
| Test group | Wild-type CT value |
| 1 | 32.5 |
| 2 | 30.3 |
| 3 | 26.8 |
As can be seen from Table 8 and FIG. 6, the closer the position of the first nucleic acid molecule corresponding to the mutation site to be detected is to the 5' end of the first nucleic acid molecule sequence, the better the blocking effect on the wild type; meanwhile, in the case of the second nucleic acid molecule being unchanged, the position of the region complementary to the first nucleic acid molecule relative to the first nucleic acid molecule changes, and therefore, the stronger the inhibition to the wild type, the greater the CT value when the region complementary to the second nucleic acid and the first nucleic acid molecule is near the 5' end of the first nucleic acid molecule.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
<110> Guangdong Fit biological Co., ltd
<120> a nucleotide for detecting gene mutation, composition and method thereof
<160> 14
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213> artificial sequence
<400> 1
tcatcacgca gctcatgcc 19
<210> 2
<211> 18
<212> DNA
<213> artificial sequence
<400> 2
gcatgagctg cgtgatga 18
<210> 3
<211> 23
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<213> artificial sequence
<400> 3
cggctgcctc ctggactatg tcc 23
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<211> 14
<212> DNA
<213> artificial sequence
<400> 4
ccaggaggca gccg 14
<210> 5
<211> 24
<212> DNA
<213> artificial sequence
<400> 5
cacctccacc gtgcavctca tctt 24
<210> 6
<211> 23
<212> DNA
<213> artificial sequence
<400> 6
ttgagcagct actgggagcc aat 23
<210> 7
<211> 21
<212> DNA
<213> artificial sequence
<400> 7
gcagctcatg cccttcggca c 21
<210> 8
<211> 21
<212> DNA
<213> artificial sequence
<400> 8
gcgatctgca cacaccagtt g 21
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<400> 9
agctcatgcc cttcggctgc c 21
<210> 10
<211> 28
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tggactatgt ccgggaacac aaagacaa 28
<210> 11
<211> 19
<212> DNA
<213> artificial sequence
<400> 11
ggcatgagct gcgtgatga 19
<210> 12
<211> 17
<212> DNA
<213> artificial sequence
<400> 12
catgagctgc gtgatga 17
<210> 13
<211> 20
<212> DNA
<213> artificial sequence
<400> 13
cagctcatca cgcagctcat 20
<210> 14
<211> 18
<212> DNA
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<400> 14
ccgtgcagct catcacgc 18
Claims (55)
1. A reagent or kit for detecting a mutation in a gene, comprising a nucleotide composition; the nucleotide composition comprises a second nucleic acid molecule and a first nucleic acid molecule; the nucleotide composition is suitable for use in combination with ARMS primers to detect gene mutations; the first nucleic acid molecule is designed to compete with a mutant AS primer in the ARMS primer for the sequence of a target sequence, the target sequence is taken from a region of a wild-type target gene sequence covering a mutation site to be detected, and a complementary region of the first nucleic acid molecule and the target sequence covers the mutation site to be detected;
the full length or part thereof of the second nucleic acid molecule is complementary to the full length or part of the sequence of the first nucleic acid molecule, and the complementary region of the second nucleic acid molecule and the first nucleic acid molecule covers the mutation site to be detected;
the full length of the first nucleic acid molecule is complementary to the target sequence; the complementary region of the first nucleic acid molecule and the target sequence comprises a 5 '-end complementary region, the 5' -end complementary region covers at least one of 1-8 bases of the 5 '-end of the first nucleic acid molecule sequence, and the mutation site to be detected is positioned in the 5' -end complementary region;
the Tm value of the first nucleic acid molecule > the Tm value of the second nucleic acid molecule > the Tm value of the mutant AS primer;
the nucleotide composition further comprises ARMS primers for detecting the target gene; the reaction system for detecting the mutation of the target gene is an AMRS-PCR reaction system.
2. The reagent or kit according to claim 1, wherein the Tm value of the first nucleic acid molecule is 1 to 15 ℃ higher than the Tm value of the second nucleic acid molecule.
3. The reagent or kit according to claim 2, wherein the Tm value of the first nucleic acid molecule is 1-13 ℃ higher than the Tm value of the second nucleic acid molecule.
4. The reagent or kit of claim 1, wherein the length of the second nucleic acid molecule is less than the length of the first nucleic acid molecule.
5. The reagent or kit according to claim 4, wherein the second nucleic acid molecule is 13 to 25nt.
6. The reagent or kit of claim 1, wherein the second nucleic acid molecule is modified with an extension blocker.
7. The reagent or kit according to claim 6, wherein the extension blocker is modified at the 3' end of the second nucleic acid molecule.
8. The reagent or kit according to claim 6, wherein the extension blocker is selected from the group consisting of: amines, biotin, ddNTPs, PEG and PO 4 One or more of the following.
9. The reagent or kit according to claim 1, wherein the second nucleic acid molecule further has a modification group attached thereto, the modification group being at least one of a locked nucleic acid, a peptide nucleic acid, and a minor groove binder.
10. The reagent or kit according to claim 1, wherein the complementary region of the second nucleic acid molecule and the first nucleic acid molecule covers at least one of 1 to 8 bases of the 5' end of the first nucleic acid molecule sequence.
11. The reagent or kit for detecting gene mutation according to any one of claims 1 to 10, wherein the gene mutation comprises any one of the following mutation modes: substitution of one or several nucleotides, deletion of one or several nucleotides and insertion of one or several nucleotides.
12. The reagent or kit according to claim 11, wherein the target gene comprises an EGFR gene.
13. The reagent or kit according to claim 12, wherein the mutation site to be detected is EGFR gene 2369C > T or EGFR gene 2390G > C.
14. The reagent or kit according to claim 13, wherein when the mutation site to be detected is EGFR gene 2369C > T, the sequence of the second nucleic acid molecule is shown in SEQ ID No.2, 11 or 12.
15. The reagent or kit according to claim 13, wherein when the mutation site to be detected is EGFR gene 2390G > C, the sequence of the second nucleic acid molecule is shown in SEQ ID No. 4.
16. The reagent or kit according to claim 1, wherein the first nucleic acid molecule and the first nucleic acid molecule are each present in single stranded form; alternatively, the first nucleic acid molecule is bound to the second nucleic acid molecule in a double stranded form.
17. The reagent or kit of claim 1, wherein the first nucleic acid molecule is modified with an extension blocker.
18. The reagent or kit of claim 17, wherein the extension blocker is modified at the 3' end of the first nucleic acid molecule.
19. The reagent or kit of claim 17, wherein the extension blocker is selected from the group consisting of: amines, biotin, ddNTPs, PEG and PO 4 One or more of the following.
20. The reagent or kit according to claim 1, wherein the first nucleic acid molecule has a modification group attached thereto, the modification group being at least one of a locked nucleic acid, a peptide nucleic acid, and a minor groove binder.
21. The reagent or kit according to claim 1, wherein the first nucleic acid molecule is 15 to 35nt.
22. The reagent or kit according to claim 1, wherein when the mutation site to be detected is EGFR gene 2369C > T, the sequence of the first nucleic acid molecule is shown in SEQ ID No.1, 13 or 14;
when the mutation site to be detected is EGFR gene 2390G > C, the sequence of the first nucleic acid molecule is shown as SEQ ID No. 3.
23. The reagent or kit according to claim 1, wherein the ARMS primer comprises: a first ARMS primer pair with sequences shown in SEQ ID No. 5-6 for detecting EGFR gene 2369C > T mutation; and/or a second ARMS primer pair with sequences shown in SEQ ID No. 7-8 for detecting EGFR gene 2390G > C mutation.
24. The reagent or kit of claim 1, wherein the nucleotide composition further comprises a probe.
25. The reagent or kit of claim 24, wherein the probe is a Taqman probe.
26. The reagent or kit according to claim 24, wherein the probe comprises: a first probe for detecting the EGFR gene 2369C > T mutation and having a sequence shown as SEQ ID No.9, and/or a second probe for detecting the EGFR gene 2390G > C mutation and having a sequence shown as SEQ ID No. 10.
27. The reagent or kit according to any one of claims 1 to 10, 12 to 26, wherein the genetic mutation comprises any one of the following mutation modes: substitution of one or several nucleotides, deletion of one or several nucleotides and insertion of one or several nucleotides.
28. The reagent or kit according to claim 1, wherein the method comprises annealing the first nucleic acid molecule to the second nucleic acid molecule before the AMRS-PCR reaction is performed when the first nucleic acid molecule in the nucleotide combination is present in single stranded form and then adding to the reaction system.
29. The reagent or kit of claim 28, wherein the concentration of the first nucleic acid molecule is less than the concentration of the second nucleic acid molecule.
30. The reagent or kit of claim 29, wherein the molar ratio of the first nucleic acid molecule to the second nucleic acid molecule is 1:1.1 to 2.0.
31. Use of a combination of a nucleotide composition and an ARMS primer for detecting a target gene in the preparation of a reagent or kit for detecting a gene mutation;
the nucleotide composition comprises a second nucleic acid molecule and a first nucleic acid molecule; the nucleotide composition is suitable for use in combination with ARMS primers to detect gene mutations; the first nucleic acid molecule is designed to compete with a mutant AS primer in the ARMS primer for the sequence of a target sequence, the target sequence is taken from a region of a wild-type target gene sequence covering a mutation site to be detected, and a complementary region of the first nucleic acid molecule and the target sequence covers the mutation site to be detected;
the full length or part thereof of the second nucleic acid molecule is complementary to the full length or part of the sequence of the first nucleic acid molecule, and the complementary region of the second nucleic acid molecule and the first nucleic acid molecule covers the mutation site to be detected;
the full length of the first nucleic acid molecule is complementary to the target sequence; the complementary region of the first nucleic acid molecule and the target sequence comprises a 5 '-end complementary region, the 5' -end complementary region covers at least one of 1-8 bases of the 5 '-end of the first nucleic acid molecule sequence, and the mutation site to be detected is positioned in the 5' -end complementary region;
the Tm value of the first nucleic acid molecule > the Tm value of the second nucleic acid molecule > the Tm value of the mutant AS primer;
the nucleotide composition further comprises ARMS primers for detecting the target gene; the reaction system for detecting the mutation of the target gene is an AMRS-PCR reaction system;
ARMS primers for detecting the target gene include: a first ARMS primer pair with sequences shown in SEQ ID No. 5-6 for detecting EGFR gene 2369C > T mutation; and/or a second ARMS primer pair with sequences shown in SEQ ID No. 7-8 for detecting EGFR gene 2390G > C mutation.
32. The use according to claim 31, wherein the Tm value of the first nucleic acid molecule is 1-15 ℃ higher than the Tm value of the second nucleic acid molecule.
33. The use according to claim 32, wherein the Tm value of the first nucleic acid molecule is 1-13 ℃ higher than the Tm value of the second nucleic acid molecule.
34. The use of claim 31, wherein the length of the second nucleic acid molecule is less than the length of the first nucleic acid molecule.
35. The use of claim 34, wherein the second nucleic acid molecule is 13 to 25nt.
36. The use of claim 31, wherein the second nucleic acid molecule is modified with an extension blocker.
37. The use of claim 36, wherein the extension blocker is modified at the 3' end of the second nucleic acid molecule.
38. The use of claim 36, wherein the extension blocker is selected from the group consisting of: amines, biotin, ddNTPs, PEG and PO 4 One or more of the following.
39. The use of claim 31, wherein the second nucleic acid molecule further has a modification group attached thereto, the modification group being at least one of a locked nucleic acid, a peptide nucleic acid, and a minor groove binder.
40. The use of claim 31, wherein the region complementary to the second nucleic acid molecule and the first nucleic acid molecule covers at least one of 1 to 8 bases of the 5' end of the first nucleic acid molecule sequence.
41. The use according to any one of claims 31 to 40, wherein the genetic mutation comprises any one of the following mutation patterns: substitution of one or several nucleotides, deletion of one or several nucleotides and insertion of one or several nucleotides.
42. The use of claim 41, wherein the gene of interest comprises an EGFR gene.
43. The use according to claim 42, wherein the mutation site to be detected is EGFR gene 2369C > T or EGFR gene 2390G > C.
44. The use according to claim 43, wherein when the mutation site to be detected is EGFR gene 2369C > T, the sequence of the second nucleic acid molecule is shown in SEQ ID No.2, 11 or 12.
45. The method of claim 43, wherein when the mutation site to be detected is EGFR gene 2390G > C, the sequence of the second nucleic acid molecule is shown in SEQ ID No. 4.
46. The use of claim 31, wherein the first nucleic acid molecule and the first nucleic acid molecule are each present in single stranded form; alternatively, the first nucleic acid molecule is bound to the second nucleic acid molecule in a double stranded form.
47. The use of claim 31, wherein the first nucleic acid molecule is modified with an extension blocker.
48. The use of claim 47, wherein the extension blocker is modified at the 3' end of the first nucleic acid molecule.
49. The use of claim 47, wherein the extension blocker is selected from the group consisting of: amines, biotin, ddNTPs, PEG and PO 4 One or more of the following.
50. The use of claim 31, wherein the first nucleic acid molecule has a modification group attached thereto, the modification group being at least one of a locked nucleic acid, a peptide nucleic acid, and a minor groove binder.
51. The use of claim 31, wherein the first nucleic acid molecule is 15 to 35nt.
52. The use according to claim 31, wherein when the mutation site to be detected is the EGFR gene 2369c > t, the sequence of the first nucleic acid molecule is as shown in SEQ ID No.1, 13 or 14;
when the mutation site to be detected is EGFR gene 2390G > C, the sequence of the first nucleic acid molecule is shown as SEQ ID No. 3.
53. The use of claim 31, wherein the nucleotide composition further comprises a probe.
54. The use of claim 53, wherein the probe is a Taqman probe.
55. The use of claim 53 wherein the probe comprises: a first probe for detecting the EGFR gene 2369C > T mutation and having a sequence shown as SEQ ID No.9, and/or a second probe for detecting the EGFR gene 2390G > C mutation and having a sequence shown as SEQ ID No. 10.
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| CN103255201A (en) * | 2012-02-16 | 2013-08-21 | 北京宏微特斯生物科技有限公司 | Method of detecting gene mutation based on Blocker primers and ARMS primers, and kit |
| CN104611427A (en) * | 2015-01-16 | 2015-05-13 | 江苏宏泰格尔生物医学工程有限公司 | AS-PCR (allele-specific polymerase chain reaction) primer design method, gene mutation detection method and kit |
| CN108048531A (en) * | 2017-11-16 | 2018-05-18 | 苏州吉玛基因股份有限公司 | A kind of super retardance fluorescence quantifying PCR method of highly sensitive detection rare mutation |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103255201A (en) * | 2012-02-16 | 2013-08-21 | 北京宏微特斯生物科技有限公司 | Method of detecting gene mutation based on Blocker primers and ARMS primers, and kit |
| CN104611427A (en) * | 2015-01-16 | 2015-05-13 | 江苏宏泰格尔生物医学工程有限公司 | AS-PCR (allele-specific polymerase chain reaction) primer design method, gene mutation detection method and kit |
| CN108048531A (en) * | 2017-11-16 | 2018-05-18 | 苏州吉玛基因股份有限公司 | A kind of super retardance fluorescence quantifying PCR method of highly sensitive detection rare mutation |
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