CN114525326A - Multi-SNP locus genotyping method based on nMALDI-TOF technology - Google Patents
Multi-SNP locus genotyping method based on nMALDI-TOF technology Download PDFInfo
- Publication number
- CN114525326A CN114525326A CN202111543725.1A CN202111543725A CN114525326A CN 114525326 A CN114525326 A CN 114525326A CN 202111543725 A CN202111543725 A CN 202111543725A CN 114525326 A CN114525326 A CN 114525326A
- Authority
- CN
- China
- Prior art keywords
- sample
- pcr
- reaction
- nmaldi
- probe
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000005516 engineering process Methods 0.000 title claims abstract description 21
- 238000003205 genotyping method Methods 0.000 title claims abstract description 21
- 239000000523 sample Substances 0.000 claims abstract description 82
- 238000000034 method Methods 0.000 claims abstract description 57
- 238000006243 chemical reaction Methods 0.000 claims abstract description 47
- 238000001514 detection method Methods 0.000 claims abstract description 31
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 22
- 238000002156 mixing Methods 0.000 claims abstract description 15
- 238000009396 hybridization Methods 0.000 claims abstract description 14
- 239000007795 chemical reaction product Substances 0.000 claims abstract description 13
- 239000011259 mixed solution Substances 0.000 claims abstract description 10
- 239000008213 purified water Substances 0.000 claims abstract description 9
- 108060002716 Exonuclease Proteins 0.000 claims abstract description 7
- 238000001976 enzyme digestion Methods 0.000 claims abstract description 7
- 102000013165 exonuclease Human genes 0.000 claims abstract description 7
- 239000000243 solution Substances 0.000 claims abstract description 6
- 238000012408 PCR amplification Methods 0.000 claims abstract description 5
- 239000012295 chemical reaction liquid Substances 0.000 claims abstract description 5
- 238000011033 desalting Methods 0.000 claims abstract description 5
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 claims abstract description 5
- 238000011068 loading method Methods 0.000 claims abstract description 4
- 230000002194 synthesizing effect Effects 0.000 claims abstract description 4
- 239000000203 mixture Substances 0.000 claims description 19
- 239000007853 buffer solution Substances 0.000 claims description 3
- 238000003752 polymerase chain reaction Methods 0.000 description 36
- 108020004414 DNA Proteins 0.000 description 18
- 230000000052 comparative effect Effects 0.000 description 15
- 150000002500 ions Chemical class 0.000 description 10
- 239000011347 resin Substances 0.000 description 8
- 229920005989 resin Polymers 0.000 description 8
- 108020004707 nucleic acids Proteins 0.000 description 6
- 102000039446 nucleic acids Human genes 0.000 description 6
- 150000007523 nucleic acids Chemical class 0.000 description 6
- 238000004949 mass spectrometry Methods 0.000 description 5
- 239000012528 membrane Substances 0.000 description 5
- 238000012986 modification Methods 0.000 description 5
- 230000004048 modification Effects 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 4
- 102200087133 rs2306283 Human genes 0.000 description 4
- 102200087157 rs4149056 Human genes 0.000 description 4
- 102200017284 rs7412 Human genes 0.000 description 4
- 108010000178 IGF-I-IGFBP-3 complex Proteins 0.000 description 3
- 239000003068 molecular probe Substances 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 102200017290 rs429358 Human genes 0.000 description 3
- 101150037123 APOE gene Proteins 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000005382 thermal cycling Methods 0.000 description 2
- 238000003260 vortexing Methods 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108091006731 SLCO1B1 Proteins 0.000 description 1
- 101150018278 SLCO1B1 gene Proteins 0.000 description 1
- 102100027233 Solute carrier organic anion transporter family member 1B1 Human genes 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000000752 ionisation method Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000007403 mPCR Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000005096 rolling process Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 238000001269 time-of-flight mass spectrometry Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
Abstract
The invention belongs to the technical field of SNP locus genotyping, and particularly relates to a multi-SNP locus genotyping method based on nMALDI-TOF technology. The method comprises the following steps: designing and synthesizing probes and PCR amplification primers aiming at a plurality of SNPs of the DNA of a sample to be detected; hybridizing the probe and the sample DNA; adding extension connection reaction liquid into the hybridization reaction product, uniformly mixing, and extending and connecting; adding mixed solution of exonuclease into the extension and connection reaction product, mixing uniformly, and performing enzyme digestion; adding the mixed solution of the primers S5& N7, purified water and PCR premixed solution into the enzyme digestion reaction product, uniformly mixing, and carrying out PCR reaction; and (3) desalting the PCR reaction product, and then loading the sample to MALDI-TOF-MS for detection to obtain a detection result. The invention can shorten the operation time by half on the basis of accurate result.
Description
Technical Field
The invention belongs to the technical field of SNP locus genotyping, and particularly relates to a multi-SNP locus genotyping method based on an nMALDI-TOF technology.
Background
The nucleic acid mass spectrometry detection technology is based on the MALDI-TOF principle, combines a primer extension analysis method and a base specific cleavage analysis method, performs special optimization aiming at the characteristics of double-stranded DNA, ensures that a sample does not generate or generates less fragment ions in the ionization process, can detect the molecular weight of nucleic acid in a biological sample, is an important means for researching Single Nucleotide Polymorphism (SNP) of a genome, and is widely applied to precise medical detection projects in recent years. The nucleic acid mass spectrometric detection is completed in four steps: firstly, extracting DNA of a sample and detecting the quality of the DNA; selecting gene locus, designing primer, synthesizing and inspecting quality; ③ 3 steps of reaction (PCR amplification, SAP reaction purification and single base extension reaction) of sample DNA; and fourthly, loading the reaction product to a special chip for detection by MALDI-TOF-MS to obtain a detection result. The method for detecting nucleic acid by using the flight time mass spectrometry has the advantages of stable experimental result, high resolution, high separation speed and less impurity interference compared with a sequencing method based on gel electrophoresis. The application of mass spectrometry in genotyping has a great deal of literature for deep analysis, and genotyping detection is widely applied to disease diagnosis and is a tool for genotyping analysis.
The conventional scheme for genotyping by applying the technology has the advantages that the operation time is over 7.3 hours when PCR amplification, SAP reaction purification and single base extension reaction are carried out, and the detection result cannot be obtained on the same day.
Disclosure of Invention
In order to solve the technical problems and shorten the operation time of genotyping, the invention provides a multi-SNP locus genotyping method based on nMALDI-TOF technology.
The invention adopts the following technical scheme:
a multi-SNP locus genotyping method based on nMALDI-TOF technology comprises the following steps:
s1, designing and synthesizing probes and PCR amplification primers aiming at a plurality of SNP sites of the DNA of a sample to be detected, wherein the probes are arch bridge probes and comprise probe binding regions at two ends and a probe arm in the middle, and the probe arm is a non-human sequence;
s2, hybridizing the probe and the sample DNA;
s3, adding extension connection reaction liquid into the hybridization reaction product of S2, uniformly mixing, and then extending and connecting;
s4, adding exonuclease VII into the reaction product of S3, uniformly mixing, and performing enzyme digestion;
s5, adding a mixed solution of primers S5 and N7, purified water and a PCR premixed solution into the reaction product of S4, uniformly mixing, and carrying out PCR reaction;
s6, desalting the reaction product of S5, loading the sample to a special chip, and detecting by MALDI-TOF-MS to obtain a detection result.
Further, the sequence of the probe arm is TAGATCGCTAAGGCGA.
Further, the sequences of the S5& N7 primers are as follows:
s5 primer: 5 '-TAGATCGC-3';
primer N7: 5 '-TAAGGCGA-3'.
Further, the hybridization reaction system of S2 is: x ul of sample DNA, 1ul of probe MIX, 1ul of hybridization buffer solution and purified water, wherein the amount of purified water is 10ul, and X represents the volume of 10-50 ng of DNA sample.
Further, the hybridization reaction procedure of S2 is: at 98 ℃ for 5 min; 60 ℃, 1.5 h; and keeping at 4 ℃.
Furthermore, the addition amount of the extension ligation reaction solution in S3 was 1ul, and the procedures of extension and ligation were as follows: 60 ℃ for 10 min; and keeping at 4 ℃.
Furthermore, the addition amount of the exonuclease mixture of S4 is 1ul, and the reaction procedure of the enzyme digestion is as follows: 30min at 37 ℃; at 95 ℃ for 10 min; and keeping at 4 ℃.
Furthermore, the amounts of the mixture of the primers S5& N7, the purified water and the PCR premix added to S5 were 25ul, 8.5ul and 3ul in this order, and the PCR reaction procedure was as follows: 30s at 98 ℃; 20 cycles of 98 ℃, 10s, 61 ℃, 30s, 72 ℃, 20 s; 72 ℃ for 5 min; and keeping at 4 ℃.
The invention has the following beneficial effects:
the invention uses molecular probe and rolling circle amplification technology to shorten the original experimental time by half, and can realize the detection in the same day; and the application of molecular probe technology can overcome the problem of non-specific combination of primers of multiplex PCR and realize detection of more gene loci.
Drawings
FIG. 1 is a schematic view of an arch bridge probe.
FIG. 2 shows the result of detection of rs2306283 site by the method (A) of comparative example 1 and the method (B) of example 1.
FIG. 3 shows the results of the detection of rs4149056 locus by the method (A) of comparative example 1 and the method (B) of example 1.
Fig. 4 shows the results of detection of the rs7412 locus by the method (a) of comparative example 1 and the method (B) of example 1.
FIG. 5 shows the results of detection of rs429358 site by the method (A) of comparative example 1 and the method (B) of example 1.
Detailed Description
The invention is described in detail below with reference to the figures and the specific embodiments, but the invention should not be construed as being limited thereto. The technical means used in the following examples are conventional means well known to those skilled in the art, and materials, reagents and the like used in the following examples can be commercially available unless otherwise specified.
The principle of genotyping using the nucleic acid time-of-flight mass spectrometry (nMALDI-TOF) technique:
the mass analyser of the mass spectrometer is an ion drift tube. Ions generated by the ion source are accelerated into the field-free drift tube and fly at a constant velocity towards the ion receiver. According to the principle that ions with different masses can be separated according to the m/z value, the larger the mass of the ions, the longer the time taken for the ions to reach the receiver, and the smaller the mass of the ions, the shorter the time taken for the ions to reach the receiver. The realization of the technology relies on the difference of quality between multiple PCR reactions, single base extension reactions and bases, and because the technical defects are influenced by multiple PCR, the method can not put sequences with high homology together for reaction, thereby limiting the number of detection sites of single-tube reaction. The optimized scheme is characterized in that the existing operation flow is abandoned, the molecular probe technology is adopted to hybridize with a sample to be detected, the probe style is modified, the traditional linear probe is modified into an arch bridge type probe, and all probe arms are connected by adopting non-human sequences. The probe pattern is shown in fig. 1.
Example 1: Multi-SNP locus genotyping method based on nMALDI-TOF technology
1. Hybridization reaction
1.1 taking out the ice box, the sample DNA to be detected, the probe MIX and the hybridization buffer solution, unfreezing at room temperature, uniformly mixing by vortex after unfreezing, instantly centrifuging, and placing on the ice box for later use. A corresponding number of PCR tubes were prepared and labeled.
1.2A hybridization reaction system (10ul) was prepared in 200ul PCR tubes according to the following table:
TABLE 1 hybridization reaction System
Note: when the sample is peripheral blood, "X" represents a volume of 10-50 ng DNA sample, and 50ng is recommended.
1.3 vortex or blow-beating and mixing evenly, performing instantaneous centrifugation, placing on a PCR instrument, and operating the following programs:
TABLE 2 hybridization reaction procedure
Note: the instrument run time was about 95 min.
2. Extension and connection
2.1 taking out the extension connection reaction liquid, unfreezing at room temperature, mixing uniformly by vortex after complete unfreezing, instantly centrifuging, and placing on an ice box for later use.
2.2 taking down the reaction tube from the PCR instrument, centrifuging, placing on an ice box, adding 1ul of extension connection reaction liquid, vortexing, centrifuging, placing on the PCR instrument, and running the following procedures:
TABLE 3 extension, ligation reaction procedure
Note: the instrument run time was about 10 min.
3. Enzyme digestion
3.1 taking out the mixed solution of the exonuclease VII, uniformly mixing the mixed solution by vortex, and instantly centrifuging the mixed solution and placing the mixed solution on an ice box for later use.
3.2 taking down the reaction tube from the PCR instrument, centrifuging, placing on an ice box, and adding 2ul of the mixed exonuclease (with the concentration of 1U/rxn) into the reaction tube.
3.3 vortex, centrifuge, place on PCR instrument, run the following program:
TABLE 4 digestion system
Note: the instrument run time was about 40 min.
4. PCR reaction
4.1 taking out the S5& N7 primer mixed solution (0.5 mu mol) and the PCR premixed solution, mixing the mixture evenly by vortex after complete thawing, centrifuging the mixture instantly, and placing the mixture on an ice box for later use.
4.2 remove the reaction tube from the PCR instrument, centrifuge, place on an ice box, and add the corresponding reagents according to the following table.
TABLE 5PCR reaction System addition Components and volumes
4.3 vortex mixing, instantaneous centrifugation, placing the reaction tube on a PCR instrument, and running the following procedures:
TABLE 6 PCR reaction procedure
Note: the instrument run time was about 40 min.
5. Sample desalting (the following procedure is for a 96-well plate, please adjust for the actual number of wells)
5.1 clean resin was spread flat on 96/15mg of sample plate and air dried for a minimum of 10 min.
5.2 Add 41ul of water to each well of the sample plate and centrifuge.
5.3 Add 15mg of clean resin: the sample is gently inverted in a volley manner, and the sample plate with the resin placed therein is inverted together with the sample plate (the two plates cannot be moved horizontally during the process), so that the resin falls into the hole.
5.4 seal the plate with membrane, put on the rotator and shake it up for 15 min.
5.5 plates were centrifuged for 5min at 3200g (4000 rmp for standard plate centrifuge).
And 5.6, placing the processed plate on a spotting instrument for spotting, and finishing the pretreatment.
Note: the optimized experimental scheme only needs to run for 185min, so that the running time of the instrument is greatly shortened, the on-day detection report of the sample on the same day can be realized, the scheme combines a probe technology, the multi-site one-tube reaction detection is realized, the influence of multiple PCR (polymerase chain reaction) is not required to be considered, and the site detection number can be greatly increased.
Comparative example 1: routine detection (nucleic acid mass spectrometry)
1. PCR reaction
1.1 prepare 1um (per primer) PCR primer mix containing forward and reverse primers for each SNP site in a multiplex reaction.
1.2 stock DNA samples were diluted to 10 ng/uL.
1.3 PCR mixtures were prepared according to the following table, to which DNA samples and controls were not added.
TABLE 7 PCR reaction System
Note: individual mechanical pipettors may need to reserve more excess volume; if less than 27, adjust the PCR enzyme to 0.5U/reaction, make up volume with HPLC grade water; the minimum/maximum amount of DNA template needs to be determined experimentally beforehand.
1.4 Add 2.2ul of PCR mix to each well, then add 3.3ul of DNA sample from the corresponding well to the reaction well. The plate was sealed with a PCR membrane and briefly centrifuged by vortexing (4000rpm5s)
1.5 Place 96-well plate on PCR instrument for thermal cycling:
TABLE 8 PCR reaction procedure
Note: the instrument run time was about 2.5 h.
2. SAP reaction
2.1 SAP blends were formulated according to Table 9.
TABLE 9 SAP blends
2.2 Add 2ul of SAP mix to each well (Total volume after mix addition: 7ul)
2.3 plates were sealed with membrane, vortexed and centrifuged (4000rpm5 s).
2.4 Place plate on PCR instrument for the following procedures:
TABLE 10 SAP reaction procedure
Note: the instrument run time was about 45 min.
3. Extension reaction
3.1 extension primer mix was prepared according to the "Linear adjustment method", and the volume of each primer was referred to the Excel table attached. 15ul of each extension primer (500uM) had been previously placed in wells A1 to C12 and was in dry powder form. Add 15ul PCR grade H2O into each well and mixed well. Different volumes of extension primers were then mixed into new tubes according to a linear Primer Regression table (linear Primer Regression spaadrame) and water was added to the target volume.
3.2, carrying out primary mass spectrometry on the mixed extension liquid, and adjusting the concentration of each extension according to the mass spectrogram.
3.3 iPLEX extension mix was prepared in a 1.5ml tube according to the following table.
TABLE 11 iPLEX extensional blends
3.4 Add 2ul of iPLEX extension mix per well and seal the plate with membrane, vortex and centrifuge (4000rpm5 s).
3.5 Place 96-well plate in PCR apparatus for thermal cycling as follows.
TABLE 12 elongation reaction procedure
Note: the instrument run time was about 3 h.
4. Sample desalting (the following procedure was set for one 96-well plate, please adjust for the number of actual wells.)
4.1 clean resin was spread flat on 96/15mg of sample plate and air dried for a minimum of 10 min.
4.2 Add 41ul of water to each well of the sample plate and centrifuge.
4.3 Add 15mg of clean resin: the sample is turned over gently in a volley manner, and the sample plate which are placed with the resin are turned over together (the two plates cannot move horizontally in the process), so that the resin falls into the hole.
4.4 seal the plate with the membrane and put on a rotator and shake it up and down for 15 min.
4.5 plates were centrifuged for 5min at 3200g (4000 rmp for standard plate centrifuge).
4.6 placing the processed plate on a spotting instrument for spotting, and finishing the preliminary treatment.
Note: the running time of the instrument in the conventional scheme needs 6.5h plus 1.5h of a manual operation part, the operation time for completing the experiment needs at least 8 h, and the time does not include the time of mass spectrometer detection, so that the requirement of the current report cannot be met.
Application examples
The SNP sites of SLCO1B1 and APOE genes were genotyped by the methods of example 1 and comparative example 1, respectively, with the SNP sites of SLCO1B1 gene including rs2306283 and rs4149056 and the SNP sites of APOE gene including rs7412 and rs 429358.
The probe sequences are shown in the following table, and the S5& N7 primer sequences are as follows:
s5 primer: TAGATCGC;
primer N7: TAAGGCGA.
Comparative example 1 relates to the presence of primers in a commercial test kit, and the kit has been certified for medical instruments (national instrument registration 20153400245) and is manufactured by friend Zhiyou medical science and technology.
TABLE 13 Probe sequences
Note: the probe arm sequence is underlined.
As a result:
1) FIG. 2A shows the result of detection of rs2306283 site by the method of comparative example 1 (fluorescence PCR method). Wherein the abscissa represents the CT value, the ordinate represents the fluorescence intensity, the FAM channel represents the wild type, the VIC channel represents the mutant type, and the ROX channel represents the quality control curve; fluorescence signals (rising curve) are detected in all three channels in the figure, which indicates that the site of the sample is heterozygous and the result is reliable.
Fig. 2B shows the result of detection of locus rs2306283 by the method of example 1. Wherein the abscissa represents the molecular mass size and the ordinate represents the signal intensity. Wherein, the position of the solid line I is the molecular mass position of the probe, the broken line II is the molecular mass position of the mutant type (G), the broken line III is the molecular mass position of the wild type (A), the position of the solid line I does not detect the probe signal, which indicates that the experimental probe is completely reacted, and both the broken line II and the broken line III detect the signal, which indicates that the site of the sample is heterozygous, which is consistent with the method of the comparative example 1.
2) FIG. 3A shows the result of detection of rs4149056 locus by the method of comparative example 1 (fluorescence PCR method). Wherein the abscissa represents the CT value and the ordinate represents the fluorescence intensity; in the figure, signals are detected by FAM channel and ROX channel, and no signal is detected by VIC channel, which indicates that the site of the sample is wild type, and the result is reliable.
Fig. 3B shows the result of the detection of the rs4149056 locus by the method of example 1. Wherein the abscissa represents the molecular mass size and the ordinate represents the signal intensity. Wherein, the dotted line I is the probe molecular mass position, the dotted line III is the wild type (T) molecular mass position, and the solid line II is the mutant type (C) molecular mass position; the position of the dotted line I in the figure does not detect a probe signal, which indicates that the experimental probe has completely reacted; the signal detected at the position of the dotted line III and the signal not detected at the position of the solid line II indicate that the sample is wild-type at this site. Consistent with the results of the comparative example 1 process.
3) FIG. 4A shows the result of detection of rs7412 locus by the method of comparative example 1 (fluorescence PCR method). Wherein the abscissa represents the CT value and the ordinate represents the fluorescence intensity; in the figure, the FAM channel and the ROX channel detect signals, and the VIC channel does not detect signals, which indicates that the site of the sample is wild type, and the result is reliable.
Fig. 4B shows the results of the detection of the rs7412 locus by the method of example 1. Wherein the abscissa represents the molecular mass size and the ordinate represents the signal intensity. Wherein, the position of the solid line I is the molecular mass position of the probe, the position of the dotted line III is the molecular mass position of the wild type (C), and the position of the dotted line II is the molecular mass position of the mutant type (T); the presence of a signal detected at position III of the dotted line and the absence of a signal detected at position II of the dotted line indicate that the site is wild type in the sample. Consistent with the results of the method of comparative example 1, the remaining positions of the dotted line are the molecular mass positions of other gene loci, regardless of the locus.
4) FIG. 5A shows the result of detection of rs429358 site by the method of comparative example 1 (fluorescence PCR method). Wherein the abscissa represents the CT value and the ordinate represents the fluorescence intensity; in the figure, signals are detected by FAM channel and ROX channel, and no signal is detected by VIC channel, which indicates that the site of the sample is wild type, and the result is reliable.
Fig. 5B shows the result of detecting rs429358 locus by the method of example 1. Wherein the abscissa represents the molecular mass size and the ordinate represents the signal intensity. Wherein, the dotted line I position is the probe molecular mass position, the dotted line III position is the wild type (T) molecular mass position, and the solid line II position is the mutant type (C) molecular mass position; in the figure, no probe signal is detected at the position of the dotted line I, which indicates that the experimental probe has completely reacted; the signal detected at the position of the dotted line III and the signal not detected at the position of the solid line II indicate that the sample is wild-type at this site. Consistent with the results of the comparative example 1 process.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Sequence listing
<110> Shenzhen second people's hospital (Shenzhen conversion medical research institute)
<120> multi-SNP locus genotyping method based on nMALDI-TOF technology
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 36
<212> DNA
<213> Artificial sequence
<400> 1
aaggtcgatg tagatcgcta aggcgacagt tacagg 36
<210> 2
<211> 36
<212> DNA
<213> Artificial sequence
<400> 2
tatgggagtc tagatcgcta aggcgaatac atgtgg 36
<210> 3
<211> 36
<212> DNA
<213> Artificial sequence
<400> 3
gctgcgtaag tagatcgcta aggcgatggt acactg 36
<210> 4
<211> 37
<212> DNA
<213> Artificial sequence
<400> 4
gagctgcagg tagatcgcta aggcgagtac tgcacca 37
Claims (8)
1. A multi-SNP locus genotyping method based on nMALDI-TOF technology is characterized by comprising the following steps:
s1, designing and synthesizing probes and PCR amplification primers aiming at a plurality of SNP sites of the DNA of a sample to be detected, wherein the probes are arch bridge probes and comprise probe binding regions at two ends and a probe arm in the middle, and the probe arm is a non-human sequence;
s2, hybridizing the probe and the sample DNA;
s3, adding extension connection reaction liquid into the hybridization reaction product of S2, uniformly mixing, and extending and connecting;
s4, adding exonuclease VII into the reaction product of S3, uniformly mixing, and performing enzyme digestion;
s5, adding a mixed solution of primers S5 and N7, purified water and a PCR premixed solution into the reaction product of S4, uniformly mixing, and carrying out PCR reaction;
s6, desalting the reaction product of S5, loading the sample to MALDI-TOF-MS for detection, and obtaining a detection result.
2. The method for genotyping multiple SNP sites based on nMALDI-TOF technology of claim 1, wherein the probe arm sequence is TAGATCGCTAAGGCGA.
3. The method for genotyping multiple SNP sites based on nMALDI-TOF technology of claim 2, wherein the primer sequences are as follows:
s5 primer: 5 '-TAGATCGC-3';
primer N7: 5 '-TAAGGCGA-3'.
4. The method for genotyping multiple SNP sites based on nMALDI-TOF technology of claim 3, wherein the hybridization reaction system of S2 is: x ul of sample DNA, 1ul of probe MIX, 1ul of hybridization buffer solution and purified water, wherein the amount of purified water is 10ul, and X represents the volume of 10-50 ng of DNA sample.
5. The method of claim 4, wherein the hybridization reaction procedure of S2 is as follows: at 98 ℃ for 5 min; 60 ℃, 1.5 h; and keeping at 4 ℃.
6. The method of claim 5, wherein the amount of the extension ligation reaction solution added in S3 is 1ul, and the procedures of extension and ligation are as follows: 10min at 60 ℃; and keeping at 4 ℃.
7. The nMALDI-TOF technology-based multi-SNP locus genotyping method according to claim 6, wherein the addition amount of the exonuclease VII of S4 is 1ul, and the reaction procedure of enzyme digestion is as follows: 30min at 37 ℃; at 95 ℃ for 10 min; and keeping at 4 ℃.
8. The method of claim 7, wherein the amounts of the mixed solution of the primers S5& N7, the purified water and the PCR premix added to S5 are 25ul, 8.5ul and 3ul in sequence, and the PCR reaction procedure is as follows: 30s at 98 ℃; 20 cycles of 98 ℃, 10s, 61 ℃, 30s, 72 ℃, 20 s; 5min at 72 ℃; and keeping at 4 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111543725.1A CN114525326A (en) | 2021-12-16 | 2021-12-16 | Multi-SNP locus genotyping method based on nMALDI-TOF technology |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111543725.1A CN114525326A (en) | 2021-12-16 | 2021-12-16 | Multi-SNP locus genotyping method based on nMALDI-TOF technology |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114525326A true CN114525326A (en) | 2022-05-24 |
Family
ID=81618419
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111543725.1A Pending CN114525326A (en) | 2021-12-16 | 2021-12-16 | Multi-SNP locus genotyping method based on nMALDI-TOF technology |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114525326A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101067156A (en) * | 2007-05-18 | 2007-11-07 | 中国人民解放军第三军医大学第一附属医院 | Multiple PCR method based on selective probe and application thereof |
| CN102719550A (en) * | 2012-07-10 | 2012-10-10 | 中国人民解放军第三军医大学第一附属医院 | Multi-RCA (rolling circle amplification) method based on split padlock probes |
| CN105274096A (en) * | 2015-09-07 | 2016-01-27 | 中国人民解放军第三军医大学第一附属医院 | Bridge type fluorescent probe with bridge type sequence zone doping into mismatched bases and application and method |
| CN109536605A (en) * | 2019-01-31 | 2019-03-29 | 深圳市第二人民医院 | The PCR primer combination and application of statins adverse reaction genotype polymorphism |
| CN112626184A (en) * | 2020-12-31 | 2021-04-09 | 深圳市第二人民医院(深圳市转化医学研究院) | Method for detecting methylation state of MGMT gene promoter |
-
2021
- 2021-12-16 CN CN202111543725.1A patent/CN114525326A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101067156A (en) * | 2007-05-18 | 2007-11-07 | 中国人民解放军第三军医大学第一附属医院 | Multiple PCR method based on selective probe and application thereof |
| CN102719550A (en) * | 2012-07-10 | 2012-10-10 | 中国人民解放军第三军医大学第一附属医院 | Multi-RCA (rolling circle amplification) method based on split padlock probes |
| CN105274096A (en) * | 2015-09-07 | 2016-01-27 | 中国人民解放军第三军医大学第一附属医院 | Bridge type fluorescent probe with bridge type sequence zone doping into mismatched bases and application and method |
| CN109536605A (en) * | 2019-01-31 | 2019-03-29 | 深圳市第二人民医院 | The PCR primer combination and application of statins adverse reaction genotype polymorphism |
| CN112626184A (en) * | 2020-12-31 | 2021-04-09 | 深圳市第二人民医院(深圳市转化医学研究院) | Method for detecting methylation state of MGMT gene promoter |
Non-Patent Citations (1)
| Title |
|---|
| 杨春晓: "SNP基因分型检测技术及应用进展", 中国药师, vol. 16, no. 6, pages 811 - 916 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN115678976B (en) | Kit for simultaneously detecting 26 deafness susceptibility gene mutation sites based on time-of-flight mass spectrum and application thereof | |
| CN107022641B (en) | Primer for detecting deafness gene and application thereof | |
| CN113025701B (en) | Early screening method and kit for non-alcoholic fatty liver disease susceptibility gene | |
| CN108841928B (en) | Human mitochondrial genome methylation detection kit and application thereof | |
| CN111334573A (en) | Gene detection kit for hypertension medication and use method | |
| CN111893216A (en) | Product for detecting DNA/RNA by nucleic acid mass spectrum and detection method | |
| CN111118145A (en) | Nucleic acid composition, kit and detection method for detecting cardiovascular disease medication related genes based on nucleic acid mass spectrometry technology | |
| CN110452958B (en) | Joint, primer and kit for methylation detection of micro-fragmented nucleic acid and application of joint and primer and kit | |
| EP3494236A1 (en) | Method for conducting early detection of colon cancer and/or of colon cancer precursor cells and for monitoring colon cancer recurrence | |
| CN114525326A (en) | Multi-SNP locus genotyping method based on nMALDI-TOF technology | |
| CN113025702B (en) | Early screening method and kit for ankylosing spondylitis susceptibility genes | |
| CN114836555A (en) | Bridged primer probe combination, kit and detection method for detecting helicobacter pylori point mutation by fluorescent quantitative PCR | |
| CN114540479A (en) | Composition, kit and detection method for detecting deafness-related gene SNP | |
| CN107312857A (en) | Neonate's achondroplasia and hypochondroplasis detection kit | |
| CN116515983B (en) | Typing kit, primer group and typing method for vitamin E metabolism related genes | |
| CN112592972A (en) | Early screening method and kit for diffuse toxic goiter susceptibility genes | |
| CN112029836A (en) | Nucleic acid composition and kit for detecting BRAF gene mutation and detection method of BRAF gene mutation | |
| CN112501283A (en) | Guiding method and kit for carbamazepine personalized medicine gene | |
| CN112831558B (en) | Early screening method and kit for Crohn disease susceptibility genes | |
| CN116179669B (en) | Typing kit, primer and typing method for vitamin B12 metabolism related genes | |
| CN114085895B (en) | Detection primer for rapidly detecting MSI and kit thereof | |
| CN117106865B (en) | High-sensitivity detection method based on double amplification and double LNA probe specific recognition DNA mutation | |
| CN116656804B (en) | Genotyping kit for hereditary hearing loss | |
| CN113106169B (en) | Primer system for detecting SARS-CoV-2 nucleic acid and its use | |
| KR102475292B1 (en) | Composition for Amplifying HLA genes and Uses thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |