CN114645075A - Detection method of true gene variation - Google Patents
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Abstract
The invention relates to the field of genetic disease gene detection, in particular to a true gene variation detection method, which comprises the following steps: designing a restriction enzyme probe which can only be combined with a pseudogene sequence of a target nucleic acid region, carrying out restriction enzyme digestion on the pseudogene of the target nucleic acid region by utilizing the restriction enzyme probe and a restriction endonuclease, and recovering a product after enzyme digestion; and (3) amplifying and recovering a product which is a true gene sequence, constructing a sequencing library, sequencing, and analyzing data to obtain a mutation site of the true gene. The detection method provided by the invention cuts the interference sequence under the action of the enzyme cutting probe and the endonuclease, reduces or completely eliminates the interference of pseudogenes, retains the required DNA sequence and improves the reliability of the detection result.
Description
Technical Field
The invention relates to the field of genetic disease gene detection, in particular to a true gene variation detection method.
Background
Since the human genome project was completed, scientists completed several sequencing works for the biological genome in succession, and the enormous data made scientists have comprehensive knowledge of the genome. Studies have shown that the human genome is approximately 30 billion base pairs, and less than 2% of the DNA sequences encode proteins, and over 98% of the remaining sequences are not involved in protein expression, of which pseudogenes are one.
Pseudogenes refer to sequences that are similar to functional genes, and current studies indicate that pseudogenes are produced mainly by two pathways: 1. when a cell replicates the whole genome before division, various mutations (insertion, confirmation, substitution or frame shift of bases) in the coding region or the regulatory region of a functional gene during DNA replication or chromosome exchange all result in the inability of the replicated gene to code, thereby losing normal function and becoming a pseudogene, which is called a repeat pseudogene; 2. transcription of DNA into mRNA followed by reverse transcription into cDNA and reintegration into the genome, in the process of which the insertion site is not suitable or the sequence is mutated and loses normal function, and this pseudogene is called a processing pseudogene or a backsite pseudogene.
Because the pseudogene and the true gene have high sequence similarity, the correct distinction between the pseudogene and the true gene becomes a key link for detecting true gene variation. Up to 8000 false genes have been found in the human genome, which can interfere with the detection of true genes in the corresponding true gene detection, thereby increasing the probability of false positives and false negatives.
At present, the existing means for detecting true genes mainly comprises long-fragment amplification, amplification of true genes through specific primers and further sequencing detection, but when the homology of true genes and false genes is high and fragments are long, the existing method cannot carry out whole-gene amplification and sequencing of true genes, the amplification stability is poor, and the occupation ratio of the false genes is high.
The existing second-generation sequencing method cannot discriminate true and false genes with high sequence homology due to short sequencing read length, and false gene sequence interference exists in data, so that mutation or false gene specific sites on the false genes can be mistaken for true gene mutation sites, or if the true gene mutation sites are consistent with the false gene specific sites, the sites can not be judged to be from the true genes or the false genes, and false negative and false positive detection can be caused.
Disclosure of Invention
In view of the above-mentioned disadvantages of the prior art, the present invention is directed to a method for detecting true genetic variation, which solves the problems of the prior art.
To achieve the above and other related objects, the present invention provides a method for detecting true genetic variation, comprising the steps of:
1) designing a restriction enzyme probe only capable of being combined with a pseudogene sequence of a target nucleic acid region, carrying out restriction enzyme digestion on the pseudogene of the target nucleic acid region by utilizing the restriction enzyme probe and a restriction endonuclease, and recovering a product after the restriction enzyme digestion;
2) amplifying the product recovered in the step 1) to obtain a true gene sequence, constructing a sequencing library, sequencing, and analyzing data to obtain a mutation site of the true gene.
As described above, the method for detecting true genetic variation according to the present invention has the following advantageous effects: the interference sequence is cut off under the action of the enzyme cutting probe and the endonuclease, the interference of the pseudogene is reduced or completely eliminated, the required DNA sequence is reserved, and the reliability of the detection result is improved.
Drawings
FIG. 1 shows a region within the exon No. 15 of the PKD1 gene.
FIG. 2 is a diagram showing the result of agarose gel electrophoresis of an amplification product of PKD1 gene.
FIG. 3 shows the electrophoretogram after enzyme digestion, which is DL50 Marker, experimental group, control group one, control group two and DL50 Marker from left to right.
FIG. 4 shows the sequencing results of the first generation of the control group.
FIG. 5 shows the results of the experimental group one generation sequencing.
FIG. 6-1 is a schematic diagram showing the design of a cleavage probe based on the sequence of a true gene, and FIG. 6-2 is a schematic diagram showing the design of a cleavage probe based on the sequence of a pseudogene.
Detailed Description
The invention provides a true gene variation detection method, which comprises the following steps:
1) designing a restriction enzyme probe only capable of being combined with a pseudogene sequence of a target nucleic acid region, carrying out restriction enzyme digestion on the pseudogene of the target nucleic acid region by utilizing the restriction enzyme probe and a restriction endonuclease, and recovering a product after the restriction enzyme digestion;
2) amplifying the product recovered in the step 1), wherein the obtained amplified product is the true gene, constructing a sequencing library and sequencing, and analyzing data to obtain the mutation site of the true gene.
The target nucleic acid region is a region where the differential sites of the true and false genes are located. In one embodiment, the nucleic acid region of interest is 200bp to 20kb in length. The target nucleic acid region is an amplification product obtained by PCR amplification.
In one embodiment, the region of interest cleaved in step 1) is an enriched region of interest, or pre-amplification product. The method for enriching a nucleic acid region of interest is selective PCR amplification of a nucleic acid region of interest from an initial pool of nucleic acids. PCR amplification refers to amplification by designing primers for specific sites.
In general, the specific site primer may be any primer that can specifically recognize the specific recognition region under the conditions for performing PCR (preferably, does not cause annealing or self-annealing between primers used in a single reaction vessel). The length of each primer is not particularly limited as long as it can specifically recognize the corresponding specific recognition region and hybridization between the primers does not occur.
Preferably, the method for designing the specific site primer is as follows: in the interval of 100 bp-10 kb of the upstream and downstream of the differential locus of the true and false genes, 15 bp-30 bp are respectively selected as the upstream and downstream primer sequences on the specific sequence of the true gene, the GC content of the primer sequences is 40% -70%, and the 3' end has no continuous G or C.
In one embodiment, the amplified product is electrophoresed, purified, and then digested in step 1).
The enzyme cutting probe has one or more of the following characteristics:
A. phosphorylating the 5' end or both ends of the enzyme digestion probe;
B. the length of the enzyme cutting probe is 10nt to 30 nt;
C. the first basic group at the 5' end of the enzyme digestion probe is T;
D. t at the 5' end of the enzyme digestion probe is the No. 1 position, and the true and false gene difference sites correspond to the No. 9-12 positions of the probe; preferably 10 to 11.
The length of the probe is selected from one or more of the following ranges: 10 to 13nt, 13 to 16nt, 16 to 20nt, 20 to 25nt, 25 to 30 nt. Preferably, it is selected from 13nt to 20 nt. More preferably, it is 15 to 17 nt.
In one embodiment, the T of the first base at the 5' end of the cleavage probe may be additionally added. That is, after selecting 15 bp-30 bp on the specificity sequence of the pseudogene as the probe sequence, if the first base is not T, T can be added at the position of the first base at the 5' end. The difference between the sequence of the enzyme-digested probe added with the T and the sequence digested by the T is less than 3 nt; preferably, the sequence difference is 0 to 1 nt. Except for the difference sequence, the enzyme cutting probe is reversely complementary with the sequence which is cut by the enzyme.
The enzyme cutting probe is designed according to the difference of the DNA sequence of the true and false gene of the target nucleic acid region and can only be combined with the false gene sequence of the target nucleic acid region.
In one embodiment, the method for designing the digestion probe is as shown in FIG. 6, 13 bp-20 bp of the pseudogene sequence is selected at the difference of the true and the pseudogene DNA sequences, and the selected sequence can be directly used as the probe sequence if the first base is T; if the first base is not T, then adding T at the position of the first base at the 5' end can be used as a probe sequence. Of course, it is also possible to select 15 bp-30 bp on the true gene sequence first, then replace the different bases of the true gene and the false gene in the selected sequence with the bases of the false gene, if the first base of the selected sequence is T, then it can be directly used as the probe sequence; if the first base is not T, then T is added at the position of the first base at the 5' end.
In one embodiment, the number of pairs of cleavage probes is one or more. Such as two, three, four or more pairs.
In one embodiment, the cleavage system used in step 1) comprises: restriction endonuclease, enzyme cutting probe, pre-amplification product of target nucleic acid region, buffer solution and aqueous medium. The pre-amplification product is obtained by PCR amplification of a target nucleic acid region.
In one embodiment, the concentration of the amplification product in the enzymatic digestion system is 5 ng/. mu.l to 7 ng/. mu.l based on the total volume of the enzymatic digestion system. Preferably, the concentration of the amplification product is 5.5-6.5 ng/. mu.l.
In one embodiment, the final concentration of the restriction endonuclease in the enzyme digestion system is 0.5-0.7 μ M based on the total volume of the enzyme digestion system. Preferably, the final concentration of the restriction endonuclease is 0.55-0.65. mu.M.
In one embodiment, the total final concentration of the sense strand and the antisense strand of the enzyme cutting probe in the enzyme cutting system is 5-7 μ M based on the total volume of the enzyme cutting system. Preferably, the total final concentration of the sense strand and the antisense strand is 5.5 to 6.5. mu.M.
In one embodiment, the ratio of the final concentration of restriction endonuclease to the final concentration of cleavage probe in the cleavage system is 1: 5-20; preferably, the ratio is 1: 8-15.
The buffer solution provides an optimal reaction system for in vitro reaction for enzyme digestion reaction, and may contain appropriate pH, appropriate ion concentration and the like. The buffer is generally available from commercial products.
The aqueous medium may generally serve as a dilution solvent. In one embodiment, the aqueous medium is selected from water.
In a preferred embodiment, the total volume of the digestion system is 50. mu.l, including a final concentration of 0.63. mu.M restriction endonuclease, 5. mu.L of 10 Xbuffer, a final concentration of 6. mu.M digestion probe, 300ng amplification product, and the remainder of the volume is made up with water. The media system can be scaled up or down on the basis of the above.
In one embodiment, the enzyme is cut at 85-99 deg.C for 15-30 min, and the temperature is decreased by 0.1 deg.C/s to 10 deg.C. In a preferred embodiment, the digestion conditions are 95 ℃ for 25min, 0.1 ℃ per second down to 10 ℃.
The restriction endonuclease is an enzyme that recognizes and attaches a specific deoxyribonucleotide sequence and cleaves the phosphodiester bond between two deoxyribonucleotides at a specific site in each strand.
In one embodiment, the restriction endonuclease is PfAgo. Pfago is an artificial restriction enzyme constructed from Argonaute protein (Pfago) extracted from Pyrococcus furiosus.
In one embodiment, the methods of sequencing library preparation and sequencing may employ methods commonly used in the art. The sequencing library is selected from a primary sequencing library or a secondary sequencing library. Accordingly, the sequencing is selected from one-generation sequencing or two-generation sequencing. In one embodiment, Nextseq 500 of the illumina platform is used for next generation sequencing.
Those skilled in the art will appreciate that the data analysis can be implemented using existing software functionality. Such as the existing Software haput 2, Tell-Seq Data Analysis Software, R Software.
The target nucleic acid region of the present application is derived from various biological samples isolated or obtained from a subject. Such as one or more of amniotic fluid, blood or blood products, umbilical cord blood, villi, cerebrospinal fluid, spinal fluid.
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
Example 1
This example selects a region in the exon 15 of the PKD1 gene where the pseudogene PKD1P2 and PKD1 are at different sites. As shown in FIG. 1, the first row sequence is PKD1 sequence, the second row sequence is PKD1P2 sequence, and the shaded sites are pseudogene specific sites.
Reagent: 2 Taq Mix (Vazyme, P212-01), Pfago enzyme, agarose gel DNA recovery kit (TIANGEN, DP219-02), DNA Clean Beads (Vazyme, N411-03)
Primer list:
| name (R) | Sequence of | Serial number |
| ago-7-F | 5'-GACTGCACCATGGACTTCGT-3' | SEQ ID NO.1 |
| ago-7-R | 5'-TCGGAGGTCTCCCAGCTCAGCCCCT-3' | SEQ ID NO.2 |
| Ago-7p | 5'-T(P)GAGGGTGACACTTGT(P)-3' | SEQ ID NO.3 |
| Ago-8p | 5'-T(P)CAACACAAGTGTCAC(P)-3' | SEQ ID NO.4 |
An amplification system:
| name of reagent | Volume (μ l) | Final concentration |
| 2*Taq Mix | 25 | 1* |
| ago-7F/R(10μM) | 1.5 | 300nM |
| Genomic DNA | 1 | 100ng |
| Water (W) | 22.5 | - |
| total | 50 | - |
Amplification conditions are as follows:
after the amplification, the amplification product was subjected to agarose gel electrophoresis at a gel concentration of 1% to detect whether a desired band was generated, as shown in FIG. 2. The obtained target band was purified by 2 × DNA Clean Beads, and then subjected to subsequent digestion.
Enzyme digestion system:
| experimental group | Volume/. mu.L | Final concentration |
| 10*buffer | 5 | - |
| PfAgo(4mg/mL) | 0.7 | 0.63μM |
| Ago-7P/8P(100μM) | 3 | 6μM |
| PKD1-ago-7 product (160bp) | 10.71 | 300ng |
| Water (W) | 30.59 | - |
| total | 50 | - |
| Control group one | Volume/. mu.L | Final concentration |
| 10*buffer | 5 | - |
| PfAgo(4mg/mL) | 0 | 0 |
| Ago-7P/8P(100μM) | 3 | 6μM |
| PKD1-ago-7 product (160bp) | 10.71 | 300ng |
| Water (W) | 31.29 | - |
| total | 50 | - |
| Control group two | Volume/. mu.L | Final concentration |
| 10*buffer | 5 | - |
| PfAgo(4mg/mL) | 0.7 | 0.63μM |
| Ago-7P/8P(100μM) | 0 | 0 |
| PKD1-ago-7 product (160bp) | 10.71 | 300ng |
| Water (W) | 33.59 | - |
| total | 50 | - |
Enzyme cutting conditions are as follows:
after the enzyme digestion is finished, performing electrophoresis detection to obtain an electrophoresis pattern, which is shown in FIG. 3.
The digested product was purified by 2-point DNA Clean Beads, and amplified with ago-7F/R primer
And (3) an amplification system:
| name of reagent | Volume (ul) | Final concentration |
| 2*Taq Mix | 25 | 1* |
| ago-7F/R(10μM) | 1.5 | 300nM |
| Enzyme digestion purified product | 5 | - |
| Water (W) | 18.5 | - |
| total | 50 | - |
Amplification conditions:
the obtained amplification product was directly subjected to the first-generation sequencing detection, and the obtained sequencing results are shown in fig. 4 and 5. The first-generation sequencing result shows that the pseudogene ratio of the control group is about 50%, and the experimental group has almost no pseudogene.
The obtained amplification product was directly subjected to second-generation sequencing detection, the sequencing depth was 10000 ×, and the obtained sequencing results are shown in the following table. The second-generation sequencing result shows that the pseudogene proportion of the control group is 38.4 percent, and the pseudogene proportion of the experimental group is 1.6 percent.
And (4) conclusion: according to the first-generation sequencing map and the second-generation sequencing statistical result, the enzyme digestion system can be used for excising the pseudogene, and the technology can be better applied to true and pseudogene detection.
The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the invention set forth herein, as well as variations of the methods of the invention, will be apparent to persons skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.
Sequence listing
<110> Shanghai Wehn biomedical science and technology, Inc
<120> a method for detecting true gene variation
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
gactgcacca tggacttcgt 20
<210> 2
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tcggaggtct cccagctcag cccct 25
<210> 3
<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
tgagggtgac acttgt 16
<210> 4
<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
tcaacacaag tgtcac 16
Claims (10)
1. A method for detecting true genetic variation, comprising the steps of:
1) designing a restriction enzyme probe only capable of being combined with a pseudogene sequence of a target nucleic acid region, carrying out restriction enzyme digestion on the pseudogene of the target nucleic acid region by utilizing the restriction enzyme probe and a restriction endonuclease, and recovering a product after the restriction enzyme digestion;
2) amplifying the product recovered in the step 1), wherein the obtained amplified product is the true gene, constructing a sequencing library and sequencing, and analyzing data to obtain the mutation site of the true gene.
2. The method of claim 1, wherein the digestion probe is designed based on the difference in DNA sequence between the true and false genes in the target nucleic acid region.
3. The detection method according to claim 1, wherein the enzymatic cleavage probe has one or more of the following characteristics:
A. phosphorylating the 5' end or both ends of the enzyme digestion probe;
B. the length of the restriction enzyme probe is 10nt to 30 nt;
C. the first basic group at the 5' end of the enzyme digestion probe is T;
D. t at the 5' end of the enzyme digestion probe is the No. 1 position, and the true and false gene difference sites correspond to the No. 9-12 positions of the probe; preferably, the number 10-11 position.
4. The detection method according to claim 1, wherein the length of the enzyme cutting probe is 13nt to 20 nt; preferably, the amount is 15 to 17 nt.
5. The detection method according to claim 1, wherein the difference between the sequence of the enzyme-cleaved probe and the sequence cleaved by the enzyme is less than 3 nt; preferably, the sequence difference is 0-1 nt; except for the difference sequence, the enzyme cutting probe is reversely complementary with the sequence which is cut by the enzyme.
6. The detection method according to claim 1, wherein the enzyme digestion system used in the step 1) comprises: restriction endonuclease, an enzyme cutting probe, a pre-amplification product of a target nucleic acid region and a buffer solution.
7. The detection method according to claim 6, further comprising one or more of the following features:
1) taking the total volume of the enzyme digestion system as a reference, wherein the concentration of a pre-amplification product in the enzyme digestion system is 5 ng/mu l-7 ng/mu l; preferably, the concentration of the amplification product is 5.5-6.5 ng/mu l;
2) based on the total volume of the enzyme digestion system, the final concentration of the restriction endonuclease in the enzyme digestion system is 0.5-0.7 mu M; preferably, the final concentration of the restriction endonuclease is 0.55-0.65. mu.M.
8. The detection method according to claim 6, wherein the total final concentration of the sense strand and the antisense strand of the enzyme cutting probe in the enzyme cutting system is 5-7 μ M on the basis of the total volume of the enzyme cutting system; preferably, the total final concentration of the sense strand and the antisense strand is 5.5 to 6.5. mu.M.
9. The detection method according to claim 6, wherein a ratio of a final concentration of the restriction endonuclease to a final concentration of the cleavage probe in the cleavage system is 1: 5-20; preferably, the ratio is 1: 8-15.
10. The method of detecting according to claim 1, wherein the restriction endonuclease is Pfago.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20190284547A1 (en) * | 2018-03-16 | 2019-09-19 | Purdue Research Foundation | Ngago-based gene-editing method and the uses thereof |
| CN109880891A (en) * | 2019-04-22 | 2019-06-14 | 上海交通大学 | The detection technique system and application of low abundance DNA mutation are enriched with based on nuclease coupling PCR principle |
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