CN117247998A - Multi-site gene mutation detection method based on ligase reaction - Google Patents
Multi-site gene mutation detection method based on ligase reaction Download PDFInfo
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Abstract
The invention discloses a multi-site gene mutation detection method based on ligase reaction, which comprises the steps of introducing three specific primers during amplification, performing multiplex PCR amplification, performing endonuclease double-enzyme digestion on a PCR amplification product to generate a plurality of sticky ends on double-stranded DNA, connecting the three genes into a DNA sequence smaller than 1000bp by using DNA ligase, and performing Sanger sequencing for multi-gene mutation site detection. Compared with the prior art, the invention introduces three specific primers during amplification, carries out double-enzyme digestion of endonuclease after multiplex PCR amplification, leads double-stranded DNA to generate a plurality of sticky ends, then uses DNA ligase to connect three genes into a DNA sequence smaller than 1000bp, can detect mutation sites of a plurality of genes at the same time, and realizes high-flux gene mutation detection. This greatly improves detection efficiency, saving time and resources.
Description
Technical Field
The invention belongs to the field of gene sequencing, and particularly relates to a multi-site gene mutation detection method based on ligase reaction.
Background
With the rapid development of molecular biology and genomics, the detection of genetic mutations has become an important tool for understanding genetic diseases, tumorigenesis mechanisms, and personalized medicine. Complex diseases such as hereditary diseases and cancers often involve mutations in a plurality of genes, which play an important role in the occurrence and progression of the disease.
Traditional methods of gene mutation detection, such as Sanger sequencing, are also known as chain termination or dideoxy methods. The principle of Sanger sequencing is to extend a single-stranded DNA template using a DNA polymerase and randomly add fluorescent-labeled dideoxynucleotides (ddNTPs) during extension, such that the extension reaction is terminated. DNA sequence information can be obtained by separating and detecting DNA fragments of different lengths. Usually for a single gene, and therefore are less efficient and take longer in the detection of multiple gene mutations. Sanger sequencing has the disadvantages of low efficiency, long time consumption, high cost, and is not suitable for large-scale DNA sequencing projects, and therefore can only be suitable for small-scale DNA sequencing projects, such as sequencing of individual genes, fragments or clones.
In addition, some gene mutations may exist simultaneously in different genes, and thus a high throughput method is required to detect mutations of a plurality of genes simultaneously.
The principle of the existing high-throughput sequencing technology is that DNA (or cDNA) is randomly fragmented and spliced to prepare a sequencing library, and corresponding signals are detected by carrying out extension reaction on tens of thousands of clones in the library, so that sequence information is finally obtained.
Although the existing high-throughput sequencing technology can quickly, accurately and efficiently acquire massive DNA sequence data, is suitable for applications such as whole genome sequencing, whole exon sequencing, target gene sequencing, transcriptome sequencing and methylation sequencing, and the like, the method still has the defects. If the detection threshold is high, specialized instruments and software are required, the cost is high, the data analysis is complex, and high-level bioinformatics knowledge and skills are required.
Accordingly, those skilled in the art have focused on developing a simple, convenient, quick and inexpensive method for detecting multiple mutation sites.
Disclosure of Invention
The invention aims to solve the defects in the prior art and provide a multi-site gene mutation detection method based on ligase reaction, which can overcome the problems and has simple operation flow and low cost.
In order to solve the technical problems, the invention adopts the technical method that: the invention discloses a multi-site gene mutation detection method based on ligase reaction, which comprises the steps of introducing three specific primers during amplification, performing multiplex PCR amplification, performing endonuclease double-enzyme digestion on a PCR amplification product to generate a plurality of sticky ends on double-stranded DNA, connecting the three genes into a DNA sequence smaller than 1000bp by using DNA ligase, and performing Sanger sequencing for multi-gene mutation site detection.
Further, the method comprises the following steps:
s1, extracting DNA from a tissue sample to be detected;
s2, carrying out multiplex PCR amplification on the DNA obtained in the step S1 by using three specific primers to obtain a PCR amplification product;
s3, purifying the PCR amplification product;
s4, carrying out enzyme digestion on the PCR amplified product after centering and purification by using endonuclease to obtain a DNA fragment containing a flat tail end;
s5, purifying the enzyme digestion product in the step S4;
s6, connecting the purified DNA fragments containing the flat tail ends with DNA fragments from different genomes by using DNA ligase to form heterozygous DNA molecules;
s7, purifying the heterozygous DNA molecules, and carrying out Sanger sequencing on the purified products.
Furthermore, the product length of the specific genes amplified by the three specific primers is not more than 330bp.
Further, the three specific primers are respectively Primer1, primer2 and Primer3; wherein, primer1 sequence is the forward sequence: 5'-TTTTGTCCTGTGCCGGTCTC-3'; reverse sequence: 5'-CCATTCAGCTCCAGTGCAT-3'.
Further, the Primer2 sequence, forward sequence: 5'-ACGTGTTTTGTCCTGTGCC-3'; reverse sequence: 5'-CCATTCAGCTCCAGTGCATTT-3'.
Further, the Primer3 sequence, forward sequence: 5'-GGGCAGGAATAAGGGCCAAC-3'; reverse sequence: 5'-GAGGAACCCAGACACGGGA-3'.
Further, the DNA ligase is a type of enzyme capable of catalyzing the formation of a phosphodiester bond between adjacent 5 'phosphate and 3' hydroxyl groups on two DNA strands, and can join blunt ends of double-stranded DNA and single-stranded nicks therein.
Further, the cleavage was performed using endonuclease, and the cleavage reaction system was configured to include 20. Mu.L RNase free dH 2 O contained 2.0. Mu.L of the DNA product, 5.0. Mu. L, ecoRI enzyme in 10 XEcoRI buffer, and 1.0. Mu.L.
Further, the enzyme digestion product is purified, which comprises the following steps:
SA1, placing the enzyme digestion reaction system into a PCR instrument after configuration, and incubating for 1h at 37 ℃; adding enzyme-free water to the PCR product to a total volume of 50. Mu.L, and purifying by using Agencourt AMPure XP magnetic beads;
SA2, mixing Agencourt AMPure XP magnetic beads by vortex oscillation, sucking volume of mu L to 90 mu L of PCR product, gently blowing 10 times by using a pipettor, fully mixing, and incubating at room temperature for 5min;
SA3, placing the uniformly mixed solution on a magnetic rack for 5min, and separating magnetic beads and liquid;
SA4 removing the liquid supernatant, washing with 200 μl of 80% ethanol for 2 times, each for 30s; discarding ethanol, and drying the magnetic beads;
SA4 adding 15 μl of enzyme-free water for dissolving, gently beating for 10 times with a pipette, mixing well, incubating at room temperature for 5min, placing on a magnetic rack for clarification, and sucking 15 μl to new PCR tube.
Further, the DNA ligase is used for connecting DNA fragments from different genomes, T4 DNA ligase of NEB is used, the purified enzyme digestion product is placed under the conditions of 10 Xligase Buffer and T4 DNA ligase for 16 ℃ overnight reaction, a multi-genome DNA fragment is generated, and then the connection product is purified.
The beneficial effects are that:
1. compared with the prior art, the invention introduces three specific primers during amplification, carries out double-enzyme digestion of endonuclease after multiplex PCR amplification, leads double-stranded DNA to generate a plurality of sticky ends, then uses DNA ligase to connect three genes into a DNA sequence smaller than 1000bp, can detect mutation sites of a plurality of genes simultaneously, and realizes high-flux gene mutation detection. This greatly improves detection efficiency, saving time and resources.
2. The ligase reaction can realize connection according to specific base sequence matching, thereby ensuring that a connection product is only generated when a target gene exists, ensuring the specificity and the accuracy of detection. Thus, it can be used to detect various types of genetic variations.
3. Compared with the existing gene mutation detection method, the connection product generated by the method can be analyzed by simple technologies such as electrophoresis, and the data processing and interpretation are relatively visual. In addition, the invention can be applied to a plurality of fields such as diagnosis of hereditary diseases, early screening and monitoring of cancers, individuation guidance of drug treatment, gene research and the like.
Drawings
FIG. 1 is a schematic flow chart of the present invention;
FIG. 2 is a schematic diagram showing the detection results of a multi-genome DNA mutation site according to an embodiment of the present invention.
Detailed Description
The invention is described in further detail below with reference to the accompanying drawings and detailed description.
Example 1
In the example, breast cancer tissue samples are selected, and the multi-gene mutation sites are detected.
1. Total DNA extraction
1) Before an experiment, heating the buffer solution B1 and B2 in the kit in a water bath at 37 ℃, dissolving the precipitate in the buffer solution, taking out the precipitate and storing the precipitate at room temperature;
2) Taking a small amount of tissue sample into a 1.5mL centrifuge tube, adding 100 mu L of buffer B1, and ensuring that the buffer can completely cover the sample;
3) Mortar (Tiangen catalog number: OSE-Y001) mashing the sample;
4) After the sample is smashed, 100 mu L of buffer B2 is added for shaking and mixing, and the mixture is centrifuged at 12 000rpm for 2min;
5) After centrifugation, 100 μl of supernatant was carefully aspirated into another clean 1.5mL centrifuge tube as a template for use;
2. multiplex PCR amplification
Preparing a multiple PCR reaction mixed solution on ice according to the following reaction components, fully and uniformly mixing, and then placing the mixed solution in a PCR instrument, wherein the reaction procedure is as follows: 98 ℃ for 2min;30 cycles (98 ℃,10s;58 ℃,15s;72 ℃,30 s); 72 ℃ for 5min;4 ℃, forever. Wherein the specific primer sequences used are described in the schemes.
3. PCR amplification product purification
1) To the PCR product was added enzyme-free water to a total volume of 50. Mu.L, followed by purification using Agencourt AMPure XP magnetic beads (Beckman Coulter, beverly, USA, #A 63880);
2) Vortex mix Agencourt AMPure XP beads and aspirate μl volume into 90 μl PCR product and gently blow 10 times with a pipette to mix thoroughly. Incubating for 5min at room temperature;
3) Placing the reaction tube on a magnetic rack for 5min, and separating magnetic beads and liquid;
4) The supernatant was removed and washed with 200. Mu.L of 80% ethanol 2 times for 30s each. The ethanol was discarded and the beads were dried.
5) Add 22. Mu.L of enzyme-free water to dissolve, gently blow 10 times with a pipette, mix well and incubate for 5min at room temperature.
6) The mixture was placed on a magnetic rack for clarification, and 20. Mu.L of the mixture was pipetted into a new PCR tube.
4. Endonuclease cleavage
The purified DNA product is digested to generate DNA fragments with flush ends, and the detailed flow is as follows:
4.1 configuration of cleavage reaction System
This was placed in a PCR apparatus and incubated at 37℃for 1h.
4.2 purification of the cleaved product
Step 1. To the PCR product, enzyme-free water was added to a total volume of 50. Mu.L, followed by purification using Agencourt AMPure XP magnetic beads (Beckman Coulter, beverly, USA, #A 63880);
step 2, vortex shaking and mixing Agencourt AMPure XP magnetic beads, sucking the volume of mu L to 90 mu L of PCR product, lightly blowing 10 times by using a pipettor, fully mixing, and incubating for 5min at room temperature;
step 3, placing the reaction tube on a magnetic rack for 5min, and separating magnetic beads and liquid;
step 4. Remove supernatant, wash with 200. Mu.L 80% ethanol 2 times, each for 30s. The ethanol was discarded and the beads were dried.
And 5, adding 15 mu L of enzyme-free water for dissolution, lightly blowing 10 times by using a pipette, fully mixing, and incubating for 5min at room temperature. The mixture was placed on a magnetic rack for clarification, and 15. Mu.L of the mixture was pipetted into a new PCR tube.
5. DNA ligase reaction
Using NEB T4 DNA ligase, the purified enzyme digestion product was placed in 10 Xligase Buffer and T4 DNA ligase conditions for 16℃overnight reaction to generate a multi-genome DNA fragment. The ligation product was then purified as follows:
step 1. To the PCR product, enzyme-free water was added to a total volume of 50. Mu.L, followed by purification using Agencourt AMPure XP magnetic beads (Beckman Coulter, beverly, USA, #A 63880);
step 2, vortex shaking and mixing Agencourt AMPure XP magnetic beads, sucking the volume of mu L to 90 mu L of PCR product, and lightly blowing 10 times by using a pipette for fully mixing. Incubating for 5min at room temperature;
step 3, placing the reaction tube on a magnetic rack for 5min, and separating magnetic beads and liquid;
step 4. Remove supernatant, wash with 200. Mu.L 80% ethanol 2 times, each for 30s. Discarding ethanol, and drying the magnetic beads; adding 22 mu L of enzyme-free water for dissolution, lightly blowing for 10 times by using a pipettor, fully mixing, and incubating for 5min at room temperature;
and 5, placing the mixture on a magnetic rack for clarification, and sucking 20 mu L of the mixture into a new PCR tube.
6. Sanger sequencing and analysis
Sanger sequencing was performed on the purified ligation product described above to analyze the multiple gene mutation sites. As a result, as shown in FIG. 2, the use of Sanger can detect mutation sites of a plurality of genes after ligation at the same time, thereby greatly improving the detection efficiency.
The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (10)
1. A method for detecting multi-site gene mutation based on ligase reaction is characterized in that: three specific primers are introduced during amplification, multiplex PCR amplification is carried out, then endonuclease double-enzyme cutting is carried out on PCR amplified products, double-stranded DNA is enabled to generate a plurality of sticky ends, then DNA ligase is used for connecting three genes into a DNA sequence smaller than 1000bp, and Sanger sequencing for detecting multi-gene mutation sites is carried out.
2. The method for detecting multiple site gene mutation based on ligase reaction according to claim 1, wherein: the method comprises the following steps:
s1, extracting DNA from a tissue sample to be detected;
s2, carrying out multiplex PCR amplification on the DNA obtained in the step S1 by using three specific primers to obtain a PCR amplification product;
s3, purifying the PCR amplification product;
s4, carrying out enzyme digestion on the PCR amplified product after centering and purification by using endonuclease to obtain a DNA fragment containing a flat tail end;
s5, purifying the enzyme digestion product in the step S4;
s6, connecting the purified DNA fragments containing the flat tail ends with DNA fragments from different genomes by using DNA ligase to form heterozygous DNA molecules;
s7, purifying the heterozygous DNA molecules, and carrying out Sanger sequencing on the purified products.
3. The method for detecting multiple site gene mutation based on ligase reaction according to claim 1, wherein: the length of the product of the specific genes amplified by the three specific primers is not more than 330bp.
4. The method for detecting multiple site gene mutation based on ligase reaction according to claim 1, wherein:
the three specific primers are respectively Primer1, primer2 and Primer3; wherein, primer1 sequence is the forward sequence: 5'-TTTTGTCCTGTGCCGGTCTC-3'; reverse sequence: 5'-CCATTCAGCTCCAGTGCAT-3'.
5. The method for detecting multiple site gene mutation based on ligase reaction according to claim 4, wherein: the Primer2 sequence, forward sequence: 5'-ACGTGTTTTGTCCTGTGCC-3'; reverse sequence: 5'-CCATTCAGCTCCAGTGCATTT-3'.
6. The method for detecting a multiple site gene mutation based on a ligase reaction according to claim 4 or 5, wherein: the Primer3 sequence, forward sequence: 5'-GGGCAGGAATAAGGGCCAAC-3'; reverse sequence: 5'-GAGGAACCCAGACACGGGA-3'.
7. The method for detecting multiple site gene mutation based on ligase reaction according to claim 1, wherein: the DNA ligase is a type of enzyme capable of catalyzing the formation of a phosphodiester bond between adjacent 5 '-phosphate and 3' -hydroxyl groups on two DNA double strands, and can be used for connecting the blunt ends of double-stranded DNA and single-stranded nicks therein.
8. The method for detecting multiple site gene mutation based on ligase reaction according to claim 1, wherein: the endonuclease was used for cleavage, and the cleavage reaction system was configured to include 20. Mu.L RNase free dH 2 O contained 2.0. Mu.L of the DNA product, 5.0. Mu. L, ecoRI enzyme in 10 XEcoRI buffer, and 1.0. Mu.L.
9. The method for detecting multiple site gene mutation based on ligase reaction according to claim 8, wherein: the enzyme digestion product is purified, and comprises the following steps:
SA1, placing the enzyme digestion reaction system into a PCR instrument after configuration, and incubating for 1h at 37 ℃; adding enzyme-free water to the PCR product to a total volume of 50. Mu.L, and purifying by using Agencourt AMPure XP magnetic beads;
SA2, mixing Agencourt AMPure XP magnetic beads by vortex oscillation, sucking volume of mu L to 90 mu L of PCR product, gently blowing 10 times by using a pipettor, fully mixing, and incubating at room temperature for 5min;
SA3, placing the uniformly mixed solution on a magnetic rack for 5min, and separating magnetic beads and liquid;
SA4 removing the liquid supernatant, washing with 200 μl of 80% ethanol for 2 times, each for 30s; discarding ethanol, and drying the magnetic beads;
SA4 adding 15 μl of enzyme-free water for dissolving, gently beating for 10 times with a pipette, mixing well, incubating at room temperature for 5min, placing on a magnetic rack for clarification, and sucking 15 μl to new PCR tube.
10. The method for detecting multiple site gene mutation based on ligase reaction according to claim 8, wherein: the DNA ligase is used for connecting DNA fragments from different genomes, T4 DNA ligase of NEB is used, the purified enzyme digestion product is placed in a 10X ligase Buffer and T4 DNA ligase condition for 16 ℃ overnight reaction, a multi-genome DNA fragment is generated, and then the connection product is purified.
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