CN111662968A - Detection method of PAM-sequence-free DNA based on CRISPR and application thereof - Google Patents
Detection method of PAM-sequence-free DNA based on CRISPR and application thereof Download PDFInfo
- Publication number
- CN111662968A CN111662968A CN202010583843.4A CN202010583843A CN111662968A CN 111662968 A CN111662968 A CN 111662968A CN 202010583843 A CN202010583843 A CN 202010583843A CN 111662968 A CN111662968 A CN 111662968A
- Authority
- CN
- China
- Prior art keywords
- crispr
- dna
- detection
- detection system
- cas
- 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
- 108020004414 DNA Proteins 0.000 title claims abstract description 79
- 238000001514 detection method Methods 0.000 title claims abstract description 64
- 108091033409 CRISPR Proteins 0.000 title abstract description 12
- 238000010354 CRISPR gene editing Methods 0.000 title abstract description 12
- 230000003321 amplification Effects 0.000 claims abstract description 27
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 27
- 238000007846 asymmetric PCR Methods 0.000 claims abstract description 20
- 102100035102 E3 ubiquitin-protein ligase MYCBP2 Human genes 0.000 claims abstract 2
- 239000000523 sample Substances 0.000 claims description 27
- 108700004991 Cas12a Proteins 0.000 claims description 17
- 108090000623 proteins and genes Proteins 0.000 claims description 15
- 102000004169 proteins and genes Human genes 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 9
- 238000010791 quenching Methods 0.000 claims description 6
- 230000000171 quenching effect Effects 0.000 claims description 6
- 102000008142 Cytochrome P-450 CYP1A1 Human genes 0.000 claims description 5
- 108010074918 Cytochrome P-450 CYP1A1 Proteins 0.000 claims description 5
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 5
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 5
- 238000000137 annealing Methods 0.000 claims description 3
- 238000004925 denaturation Methods 0.000 claims description 3
- 230000036425 denaturation Effects 0.000 claims description 3
- 101000860092 Francisella tularensis subsp. novicida (strain U112) CRISPR-associated endonuclease Cas12a Proteins 0.000 claims 2
- 102000053602 DNA Human genes 0.000 abstract description 32
- 108020004682 Single-Stranded DNA Proteins 0.000 abstract description 18
- 230000000694 effects Effects 0.000 abstract description 16
- 238000009825 accumulation Methods 0.000 abstract description 2
- 238000003776 cleavage reaction Methods 0.000 description 9
- 230000007017 scission Effects 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 4
- 102000039446 nucleic acids Human genes 0.000 description 4
- 108020004707 nucleic acids Proteins 0.000 description 4
- 150000007523 nucleic acids Chemical class 0.000 description 4
- 241001302239 Mycobacterium tuberculosis complex Species 0.000 description 3
- 101150055214 cyp1a1 gene Proteins 0.000 description 3
- 238000001917 fluorescence detection Methods 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 230000007018 DNA scission Effects 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 108020005004 Guide RNA Proteins 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 102000018120 Recombinases Human genes 0.000 description 2
- 108010091086 Recombinases Proteins 0.000 description 2
- 108091081021 Sense strand Proteins 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000000692 anti-sense effect Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000012154 double-distilled water Substances 0.000 description 2
- 238000001502 gel electrophoresis Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 1
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 206010036790 Productive cough Diseases 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 1
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 1
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 1
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 1
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000010457 gene scissor Methods 0.000 description 1
- 238000010362 genome editing Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 238000011901 isothermal amplification Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000012123 point-of-care testing Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011897 real-time detection Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 210000003802 sputum Anatomy 0.000 description 1
- 208000024794 sputum Diseases 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000008685 targeting Effects 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
- C12Q1/686—Polymerase chain reaction [PCR]
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a detection method and application of PAM-sequence-free DNA based on CRISPR, wherein the detection system comprises an asymmetric PCR amplification system and a CRISPR-Cas detection system; the asymmetric PCR amplification system comprises a forward and a reverse specific primer pair. According to the invention, the accumulation of single-stranded DNA in a PCR system is controlled by adjusting the proportion of the forward/reverse primers, the cis-form cutting activity of the CRISPR-Cas system is activated by using the generated reverse single-stranded DNA, the detection of the target double-stranded DNA is carried out, the restriction effect of a PAM sequence on the traditional CRISPR-Cas detection system is eliminated, and the application range of the CRISPR-Cas detection system is expanded.
Description
Technical Field
The invention belongs to the technical field of analytical chemistry, belongs to the technical field of nucleic acid detection, and relates to a detection method of DNA without a PAM sequence based on CRISPR and application thereof.
Background
Jennifer Doudna discovered in 2018, as shown in FIG. 1, when Cas12a protein, gRNA and target double-stranded DNA (target dsDNA) form a ternary complex structure, Cas12a protein not only cleaves target dsDNA in trans, but also cleaves nearby short-chain probe DNA (Probe ssDNA) in cis without difference, so that the short-chain probe DNA modified with a fluorophore and a quencher at both ends is designed, when the short-chain probe DNA is cleaved, fluorescence is generated, and the fluorescence intensity is proportional to the concentration of the target DNA within a certain time, and the target DNA can be subjected to rapid real-time fluorescence detection by using the characteristics of the CRISPR system (Chen J S, Ma E, Harrington L B, CRISPR-Cas12a target binding and fluorescent single-stranded activity [ J201360, Science, 8, 6387) 439). The cutting characteristics of the CRISPR-Cas12a system on target single-stranded DNA and non-target single-stranded DNA were studied systematically by Zhao national screen team of Chinese academy of sciences in 2018, 3/12.T.once again, the unique advantages of the CRISPR-Cas12a system in the field of nucleic acid detection (Li S Y, Cheng Q X, Liu J K, et al. CRISPR-Cas12a has both cis-andtran-cleavage activity on single-stranded DNA [ J ]. Cell research,2018,28(4): 491-. Then, the Zhang FengO group realizes the rapid detection of trace target DNA on paper-based materials by using a CRISPR-Cas12a system based on recombinase polymerase isothermal amplification technology (RPA), and the sensitivity can reach the order of aM/L (Gootenberg J S, Abudayyeh O, Kellner M J, et al. multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6[ J ] Science,2018,360 (6387: 439 and 444.). Chen believes that CRISPR-Cas12a can be a powerful tool for DNA detection from various sources, and the detect (DNA Endonuclease Targeted CRISPR transreporter) technique is potentially applicable to any POCT detection based on DNA analysis, including cancer and infectious diseases.
CN 110541022A discloses a Mycobacterium tuberculosis complex detection kit based on a CRISPR-Cas12a system, which improves the detection sensitivity by adopting a recombinase polymerase amplification technology, activates the bypass cleavage activity of Cas12a after the CRISPR-Cas12a specifically targets a Mycobacterium tuberculosis complex target sequence, can sensitively and specifically detect the Mycobacterium tuberculosis complex from sputum, and has the advantages of no invasion, frequent multiple detection, high detection speed and the like.
CRISPR targeting specificity is determined by two parts, one part is base pairing between RNA chimera (crRNA) and target DNA, and the other part is interaction between Cas protein and a short DNA sequence (usually at the 3' end of target DNA), which is called Protospacer Adjacent Motif (PAM), therefore, the CRISPR-Cas12a system strongly depends on the PAM sequence in the target DNA, i.e. when the target DNA does not contain PAM sequence (TTTN), Cas12a protein in the CRISPR-Cas12a system cannot bind to target DNA through gRNA, cis-cleavage activity cannot be activated, and the detection system will fail, which greatly limits the applicability of the system.
Therefore, in order to expand the applicable range of the CRISPR-Cas12a system without being limited by the PAM sequence, the traditional CRISPR-Cas12a system needs to be improved.
Disclosure of Invention
Aiming at the defects and practical requirements of the prior art, the invention provides a method for detecting DNA without a PAM sequence based on CRISPR and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a CRISPR-based PAM-sequence-free DNA detection system, which comprises an asymmetric PCR amplification system and a CRISPR-Cas detection system;
the asymmetric PCR amplification system comprises a forward and a reverse specific primer pair.
According to the invention, by utilizing the principle that the CRISPR-Cas12a/b system is not limited by a PAM sequence in the detection of single-stranded DNA, excessive reverse primers of the target DNA are added in the PCR process to carry out asymmetric PCR, a large amount of reverse target single-stranded DNA matched with crRNA is generated, and the homeotropic cleavage activity of the CRISPR-Cas12a/b system can be activated after the reverse target single-stranded DNA is combined with the crRNA, so that the detection of the target double-stranded DNA is realized.
Preferably, the molar amount of the forward primer in the specific primer pair is less than that of the reverse primer, preferably the molar ratio of the forward primer to the reverse primer is 1 (2-50), and may be, for example, 1:2, 1:3, 1:4, 1:5, 1:10, 1:20, 1:30, 1:40 or 1:50, preferably 1 (2-10).
In the invention, the molar ratio of the forward primer to the reverse primer is set to be 1 (2-50), so that not only is amplification of target double-stranded DNA completed, but also reverse target single-stranded DNA matched with crRNA can be generated, the homeotropic cleavage activity of a CRISPR-Cas12a/Cas12b system is activated, and the detection of the target double-stranded DNA is realized; if the molar ratio of the forward primer to the reverse primer is less than 1 (2-50), sufficient reverse target single-stranded DNA cannot be generated, and if the molar ratio of the forward primer to the reverse primer is more than 1 (2-50), amplification of the target double-stranded DNA cannot be completed.
Preferably, the asymmetric PCR amplification system further comprises a DNA polymerase, which may be, for example, LA Taq DNA polymerase (TAKaRa) or Pyrobest DNA polymerase (TAKaRa), dNTPs comprising dATP, dTTP, dCTP and dGTP, and a template.
Preferably, the CRISPR-Cas detection system comprises a Cas protein and a crRNA.
Preferably, the Cas protein comprises Cas12a, preferably any one or a combination of at least two of the equivalents AsCas12a, LbCas12a or FnCas12 a.
According to the present invention, the third gene scissors CRISPR-Cas12B developed by the recently Zhang Bow team has similar bypass cleavage activity to Cas12a protein (Strecker J, Jones S R, Koopal B, et al. engineering of CRISPR-Cas12B for human Genome editing [ J ]. Nature Communications,2019,10(1):212-212.), and the Liwei team has invented CDelection technology based on it for DNA detection and single base analysis (Teng F, Guo L, Cui T, et al. CDelection: CRISPR-Cas12B-based DNA detection with-interaction sensitivity and single-base specificity [ J ]. Genome Biology,2019,20 (1)). Based on the above references, Cas12b has the same biological activity as Cas12a, and can also achieve the effect of DNA cleavage, and the invention adopts Cas protein comprising Cas12b, preferably any one or a combination of at least two of the equivalent sources of aa Cas12b, BhCas12b or AkCas12 b. However, Cas12b is characterized by a smaller size and lower off-target rate than Cas12a, but since it is derived from thermotolerant acidophilic bacteria, the optimal active temperature is as high as 48 ℃, so it is clear that low temperature Cas12a has certain advantages in the field of rapid detection.
In the invention, the single-stranded DNA cleavage activity of the Cas12a/b protein is utilized, which is beneficial to realizing DNA detection independent of PAM sequence.
Preferably, the CRISPR-Cas detection system further comprises a probe.
Preferably, the 5 'end of the probe is modified with a fluorescent group, and the 3' end of the probe is modified with a quenching group.
In the invention, the fluorescence probe is cut by using the bypass cutting activity of the Cas12a/b protein, so that the real-time fluorescence detection of the target DNA is realized.
In a second aspect, the present invention provides a CRISPR-based method for detecting PAM-free sequence DNA, comprising the steps of:
(1) carrying out asymmetric PCR by adopting a specific primer pair of the target DNA;
(2) and detecting the amplification product by using a CRISPR-Cas detection system.
Preferably, the molar amount of the forward primer in the specific primer pair is less than that of the reverse primer, and preferably, the molar ratio of the forward primer to the reverse primer is 1 (2-50).
Preferably, the conditions of the asymmetric PCR are that denaturation is carried out for 30-40 s at the temperature of 93-95 ℃, annealing is carried out for 10-30 s at the temperature of 50-60 ℃, extension is carried out for 20-30 s at the temperature of 70-72 ℃, and circulation is carried out for 30-40 times.
Preferably, the CRISPR-Cas detection system contains a probe.
Preferably, the 5 'end of the probe is modified with a fluorescent group, and the 3' end of the probe is modified with a quenching group.
In a third aspect, the invention provides a CRISPR-based CYP1A1 detection kit, which comprises a forward primer shown as SEQ ID NO. 1 and a reverse primer shown as SEQ ID NO. 2;
SEQ ID NO:1:5’-AAGAGAAAGACCTCCCAGCGGGCAA-3’;
SEQ ID NO:2:5’-CACCCCTGATGGTGCTATCGACAAG-3’。
in the invention, the specific primer pair of CYP1A1 is designed according to the sense strand and the antisense strand of CYP1A1, wherein the sense strand of CYP1A1 is shown as SEQ ID NO. 5, and the antisense strand is shown as SEQ ID NO. 6;
SEQ ID NO:5:
5’-AAGAGAAAGACCTCCCAGCGGGCAATGGTCTCACCGATACACTTCCGCTTGCCCATGCCAAAGATAATCACCTTCTCACTTAACACCTTGTCGATAGCACCATCAGGGGTG-3’;
SEQ ID NO:6:
5’-CACCCCTGATGGTGCTATCGACAAGGTGTTAAGTGAGAAGGTGATTATCTTTGGCATGGGCAAGCGGAAGTGTATCGGTGAGACCATTGCCCGCTGGGAGGTCTTTCTCTT-3’。
preferably, the molar ratio of the forward primer to the reverse primer is 1 (2-50).
Preferably, the kit further comprises a DNA polymerase and dNTPs.
Preferably, the kit further comprises a Cas protein and a crRNA.
Preferably, the Cas protein comprises Cas12a, preferably any one or a combination of at least two of the equivalents AsCas12a, LbCas12a or FnCas12 a.
Preferably, the Cas protein comprises Cas12b, preferably any one or a combination of at least two of AaCas12b, BhCas12b or AkCas12b equivalent homologues.
Preferably, the crRNA is shown as SEQ ID NO. 3;
SEQ ID NO:3:
5’-UAAUUUCUACUCUUGUAGAUCAAUGGUCUCACCGAUACAC-3’。
preferably, the kit further comprises a probe.
Preferably, the 5 'end of the probe is modified with a fluorescent group, and the 3' end of the probe is modified with a quenching group.
Preferably, the probe is shown as SEQ ID NO. 4;
SEQ ID NO:4:5’-FAM-TTATT-BHQ1-3’。
in a fourth aspect, the present invention provides a use of the detection system of the first aspect for detecting DNA without PAM sequence.
Compared with the prior art, the invention has the following beneficial effects:
(1) according to the invention, by using the principle that the detection of the CRISPR-Cas12a/b system on the single-stranded DNA is not limited by a PAM sequence, an excessive reverse primer of the target DNA is added in the PCR process to carry out asymmetric PCR, so that a large amount of reverse target single-stranded DNA matched with the crRNA is generated, and the homeotropic cleavage activity of the CRISPR-Cas12a system or the CRISPR-Cas12b system can be activated after the reverse target single-stranded DNA is combined with the crRNA, so that the detection of the target double-stranded DNA is realized;
(2) according to the invention, the molar ratio of the forward primer to the reverse primer is set to 1 (2-50), so that not only is amplification of a target double-stranded DNA completed, but also a large amount of reverse target single-stranded DNA matched with crRNA can be generated, the homeotropic cleavage activity of a CRISPR-Cas12a system or a CRISPR-Cas12b system is activated, the detection of the target double-stranded DNA is realized, and the application range of the CRISPR-Cas12 detection system is expanded.
Drawings
FIG. 1 is a schematic diagram of a CRISPR-Cas12a detection system;
FIG. 2 is a gel electrophoresis of the asymmetric PCR amplification product, wherein the first lane shows DNA molecular weight (25-500 bp), the second lane shows the amplification product with excessive forward primer, and the third lane shows the amplification product with excessive reverse primer;
FIG. 3 is a real-time fluorescence curve of the amplification product.
Detailed Description
To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.
The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.
Example 1 asymmetric PCR amplification of CYP1A1 Gene
In the embodiment, CYP1A1 gene is taken as an example, an excessive reverse primer is adopted to carry out asymmetric PCR, the system is shown in Table 1, and a control experiment with excessive forward primers is set, wherein the forward primer is shown as SEQ ID NO. 1, the reverse primer is shown as SEQ ID NO. 2, and the template is shown as SEQ ID NO. 5-6;
TABLE 1 asymmetric PCR detection System
|
① Forward primer excess (2:1) | ② reverse primer excess (1:2) |
Pyrobest(5U/μL) | 1.2μL | 1.2μL |
10×Buffer | 10μL | 10μL |
ddH2O | 78μL | 78μL |
dNTP(2.5mM) | 9.6μL | 9.6μL |
Forward primer (50. mu.M) | 2.4μL | 1.2μL |
Reverse primer (50. mu.M) | 1.2μL | 2.4μL |
Stencil (1 ng/. mu.L) | 1.2μL | 1.2μL |
Nucleic acid amplification is carried out by adopting a Veriti PCR amplification instrument of Thermo Fisher Science company under the conditions of denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s and extension at 72 ℃ for 20s, and the cycle is carried out for 40 times; and carrying out agarose gel electrophoresis detection on the amplification product.
FIG. 2 shows a gel electrophoresis image of the amplification product, wherein the first lane is the DNA molecular weight (25-500 bp from bottom to top), the second lane is the amplification product with excess forward primer (forward primer: reverse primer: 2:1), and the third lane is the amplification product with excess reverse primer (forward primer: reverse primer: 1: 2).
Example 2
In this example, asymmetric PCR was carried out using an excess amount of reverse primer based on CYP1A1 gene in example 1, and the primer sequence, template sequence, amplification conditions, etc. used are the same as those in example 1, as shown in Table 2;
TABLE 2 asymmetric PCR detection System
Example 3 CRISPR-Cas12a detection of amplified products
The real-time fluorescence detection of the amplification products of the embodiments 1 and 2 is performed based on a CRISPR-Cas12a detection system, wherein the system is shown in Table 3, crRNA is shown in SEQ ID NO. 3, and a probe is shown in SEQ ID NO. 4;
TABLE 3 CRISPR-Cas12a detection System
Components | Dosage of |
Cas12a protein (1.0. mu.M) | 1.25μL |
crRNA(5.0μM) | 2.5μL |
Probe needle | 2.5μL |
10×Buffer | 5μL |
ddH2O | 15μL |
①, ②, ③, ④ or ⑤ | 25μL |
Real-time fluorescent quantitative detection is carried out by using a Spark enzyme-linked immunosorbent assay (ELIASA) of ENCO company.
As shown in fig. 3, which is a real-time fluorescence curve of an amplification product, it can be seen that only when the reverse primer is excessive and the PCR amplification product contains a large amount of reverse target single-stranded DNA, the CRISPR-Cas12a detection system is activated, and the Cas12a protein cleaves the fluorescent probe, thereby realizing real-time detection of the target dsDNA; with the increase of the proportion of the reverse primer to the forward primer, the signal appears more quickly, and the obvious difference appears only in 10-30 min.
In conclusion, the invention controls the accumulation of single-stranded DNA in a PCR system by regulating the proportion of forward/reverse primers, utilizes the generated reverse single-stranded DNA to activate the cis-cleavage activity of the CRISPR-Cas12a system, detects the target double-stranded DNA, eliminates the restriction of a PAM sequence on the traditional CRISPR-Cas12a detection system, and enlarges the application range of the CRISPR-Cas12a detection system.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
SEQUENCE LISTING
<110> southern university of science and technology
<120> detection method of PAM-sequence-free DNA based on CRISPR and application thereof
<130>20200615
<160>6
<170>PatentIn version 3.3
<210>1
<211>25
<212>DNA
<213> Artificial sequence
<400>1
aagagaaaga cctcccagcg ggcaa 25
<210>2
<211>25
<212>DNA
<213> Artificial sequence
<400>2
cacccctgat ggtgctatcg acaag 25
<210>3
<211>40
<212>DNA
<213> Artificial sequence
<400>3
uaauuucuac ucuuguagau caauggucuc accgauacac 40
<210>4
<211>5
<212>DNA
<213> Artificial sequence
<400>4
<210>5
<211>111
<212>DNA
<213> Artificial sequence
<400>5
aagagaaaga cctcccagcg ggcaatggtc tcaccgatac acttccgctt gcccatgcca 60
aagataatca ccttctcact taacaccttg tcgatagcac catcaggggt g 111
<210>6
<211>111
<212>DNA
<213> Artificial sequence
<400>6
cacccctgat ggtgctatcg acaaggtgtt aagtgagaag gtgattatct ttggcatggg 60
caagcggaag tgtatcggtg agaccattgc ccgctgggag gtctttctct t 111
Claims (10)
1. A PAM-sequence-free DNA detection system based on CRISPR is characterized by comprising an asymmetric PCR amplification system and a CRISPR-Cas detection system;
the asymmetric PCR amplification system comprises a forward and a reverse specific primer pair;
the molar amount of the forward primer in the specific primer pair is less than that of the reverse primer.
2. The detection system according to claim 1, wherein the asymmetric PCR amplification system further comprises DNA polymerase, dNTPs, and a template.
3. The detection system according to claim 1 or 2, wherein the CRISPR-Cas detection system comprises a Cas protein and a crRNA;
preferably, the Cas protein comprises Cas12a and/or Cas12 b;
preferably, the Cas protein comprises any one of AsCas12a, LbCas12a, FnCas12a, AaCas12b, BhCas12b or AkCas12b, or a combination of at least two thereof;
preferably, the CRISPR-Cas detection system further comprises a probe DNA;
preferably, the 5 'end of the probe DNA is modified with a fluorescent group, and the 3' end of the probe DNA is modified with a quenching group.
4. A method for detecting PAM-free sequence DNA based on CRISPR (clustered regularly interspaced short palindromic repeats), which is characterized by comprising the following steps:
(1) carrying out asymmetric PCR by adopting a specific primer pair of the target DNA;
(2) and detecting the amplification product by using a CRISPR-Cas detection system.
5. The detection method according to claim 4, wherein the specific primer pair has a smaller molar amount of forward primer than reverse primer;
preferably, the conditions of the asymmetric PCR are that denaturation is carried out for 30-40 s at the temperature of 93-95 ℃, annealing is carried out for 10-30 s at the temperature of 50-60 ℃, extension is carried out for 20-30 s at the temperature of 70-72 ℃, and circulation is carried out for 30-40 times.
6. The detection method according to claim 4 or 5, wherein the CRISPR-Cas detection system contains probe DNA;
preferably, the 5 'end of the probe DNA is modified with a fluorescent group, and the 3' end of the probe DNA is modified with a quenching group.
7. A CYP1A1 detection kit based on CRISPR is characterized by comprising a forward primer shown as SEQ ID NO. 1 and a reverse primer shown as SEQ ID NO. 2;
preferably, the molar ratio of the forward primer to the reverse primer is 1 (2-50);
preferably, the kit further comprises a DNA polymerase and dNTPs.
8. The kit of claim 7, further comprising a Cas protein and a crRNA;
preferably, the Cas protein comprises Cas12a and/or Cas12 b;
preferably, the Cas protein comprises any one of AsCas12a, LbCas12a, FnCas12a, AaCas12b, BhCas12b or AkCas12b, or a combination of at least two thereof;
preferably, the crRNA is shown as SEQ ID NO 3.
9. The kit of claim 7 or 8, wherein the kit further comprises a probe;
preferably, the 5 'end of the probe is modified with a fluorescent group, and the 3' end of the probe is modified with a quenching group;
preferably, the probe is shown as SEQ ID NO. 4.
10. Use of a detection system according to any one of claims 1 to 3 for the detection of DNA free of PAM sequences.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010583843.4A CN111662968A (en) | 2020-06-23 | 2020-06-23 | Detection method of PAM-sequence-free DNA based on CRISPR and application thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010583843.4A CN111662968A (en) | 2020-06-23 | 2020-06-23 | Detection method of PAM-sequence-free DNA based on CRISPR and application thereof |
Publications (1)
Publication Number | Publication Date |
---|---|
CN111662968A true CN111662968A (en) | 2020-09-15 |
Family
ID=72389571
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202010583843.4A Pending CN111662968A (en) | 2020-06-23 | 2020-06-23 | Detection method of PAM-sequence-free DNA based on CRISPR and application thereof |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN111662968A (en) |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112795673A (en) * | 2021-02-09 | 2021-05-14 | 上海市质量监督检验技术研究院 | CRISPR (clustered regularly interspaced short palindromic repeats) detection method for Cronobacter in food and kit thereof |
CN112980933A (en) * | 2020-12-30 | 2021-06-18 | 南方科技大学 | SNP (Single nucleotide polymorphism) typing detection method based on CRISPR-Cas (clustered regularly interspaced short palindromic repeats) system and application thereof |
CN113174433A (en) * | 2021-04-22 | 2021-07-27 | 中南大学 | Cas protein-based detection method |
CN113201586A (en) * | 2021-04-22 | 2021-08-03 | 中南大学 | Cas protein-based detection method |
CN113234856A (en) * | 2021-04-27 | 2021-08-10 | 华南理工大学 | DENV one-step nucleic acid detection method based on CRISPR/Cas12a and constant-temperature amplification |
CN113388691A (en) * | 2021-07-23 | 2021-09-14 | 中国疾病预防控制中心传染病预防控制所 | Nucleic acid detection method based on PCR amplification and CRISPR-Cas12a and application |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101140232A (en) * | 2006-09-08 | 2008-03-12 | 上海主健生物工程有限公司 | Reagent kit for detecting pulmonary cancer susceptibility with CYP1A1 gene and ERCC2 gene |
CN107109495A (en) * | 2014-11-11 | 2017-08-29 | 伊鲁米那股份有限公司 | Use the polynucleotide amplification of CRISPR cas systems |
CN107488710A (en) * | 2017-07-14 | 2017-12-19 | 上海吐露港生物科技有限公司 | A kind of purposes of Cas albumen and the detection method and kit of target nucleic acids molecule |
CN107488711A (en) * | 2017-07-27 | 2017-12-19 | 北京宏微特斯生物科技有限公司 | The method and its kit of the genotype detection of point mutation |
CN108034728A (en) * | 2018-02-07 | 2018-05-15 | 北京诺赛基因组研究中心有限公司 | SNP marker for detecting lung cancer susceptibility combines, primer combines and kit |
WO2020110127A1 (en) * | 2018-11-29 | 2020-06-04 | Yeda Research And Development Co. Ltd. | Methods of activating dysfunctional immune cells and treatment of cancer |
-
2020
- 2020-06-23 CN CN202010583843.4A patent/CN111662968A/en active Pending
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101140232A (en) * | 2006-09-08 | 2008-03-12 | 上海主健生物工程有限公司 | Reagent kit for detecting pulmonary cancer susceptibility with CYP1A1 gene and ERCC2 gene |
CN107109495A (en) * | 2014-11-11 | 2017-08-29 | 伊鲁米那股份有限公司 | Use the polynucleotide amplification of CRISPR cas systems |
CN107488710A (en) * | 2017-07-14 | 2017-12-19 | 上海吐露港生物科技有限公司 | A kind of purposes of Cas albumen and the detection method and kit of target nucleic acids molecule |
CN107488711A (en) * | 2017-07-27 | 2017-12-19 | 北京宏微特斯生物科技有限公司 | The method and its kit of the genotype detection of point mutation |
CN108034728A (en) * | 2018-02-07 | 2018-05-15 | 北京诺赛基因组研究中心有限公司 | SNP marker for detecting lung cancer susceptibility combines, primer combines and kit |
WO2020110127A1 (en) * | 2018-11-29 | 2020-06-04 | Yeda Research And Development Co. Ltd. | Methods of activating dysfunctional immune cells and treatment of cancer |
Non-Patent Citations (2)
Title |
---|
LINXIAN等: "HOLMESv2: A CRISPR-Cas12b-Assisted Platform for Nucleic Acid Detection and DNA Methylation Quantitation.", 《ACS SYNTH BIOL.》 * |
刘博等: "应用于染色体步移的PCR扩增技术的研究进展", 《遗传》 * |
Cited By (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112980933A (en) * | 2020-12-30 | 2021-06-18 | 南方科技大学 | SNP (Single nucleotide polymorphism) typing detection method based on CRISPR-Cas (clustered regularly interspaced short palindromic repeats) system and application thereof |
CN112980933B (en) * | 2020-12-30 | 2023-08-29 | 南方科技大学 | SNP (Single nucleotide polymorphism) typing detection method based on CRISPR-Cas (CRISPR-Cas) system and application of SNP typing detection method |
CN112795673A (en) * | 2021-02-09 | 2021-05-14 | 上海市质量监督检验技术研究院 | CRISPR (clustered regularly interspaced short palindromic repeats) detection method for Cronobacter in food and kit thereof |
CN112795673B (en) * | 2021-02-09 | 2022-03-01 | 上海市质量监督检验技术研究院 | CRISPR (clustered regularly interspaced short palindromic repeats) detection method for Cronobacter in food and kit thereof |
CN113174433A (en) * | 2021-04-22 | 2021-07-27 | 中南大学 | Cas protein-based detection method |
CN113201586A (en) * | 2021-04-22 | 2021-08-03 | 中南大学 | Cas protein-based detection method |
CN113201586B (en) * | 2021-04-22 | 2024-03-26 | 苏州淦江生物技术有限公司 | Cas protein-based detection method |
CN113174433B (en) * | 2021-04-22 | 2024-03-26 | 苏州淦江生物技术有限公司 | Cas protein-based detection method |
CN113234856A (en) * | 2021-04-27 | 2021-08-10 | 华南理工大学 | DENV one-step nucleic acid detection method based on CRISPR/Cas12a and constant-temperature amplification |
CN113234856B (en) * | 2021-04-27 | 2024-02-20 | 华南理工大学 | DENV one-step method nucleic acid detection method based on CRISPR/Cas12a and isothermal amplification |
CN113388691A (en) * | 2021-07-23 | 2021-09-14 | 中国疾病预防控制中心传染病预防控制所 | Nucleic acid detection method based on PCR amplification and CRISPR-Cas12a and application |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN111662968A (en) | Detection method of PAM-sequence-free DNA based on CRISPR and application thereof | |
CN111094588B (en) | Application of Cas protein, detection method of target nucleic acid molecule and kit | |
Bergkessel et al. | Colony PCR | |
US20210269866A1 (en) | Crispr effector system based amplification methods, systems, and diagnostics | |
JP6335918B2 (en) | Target enrichment without restriction enzymes | |
CN107034277B (en) | Method for detecting low-abundance gene mutation | |
Jia et al. | New applications of CRISPR/Cas9 system on mutant DNA detection | |
US11584955B2 (en) | Application of Cas protein, method for detecting target nucleic acid molecule and kit | |
WO2019178346A1 (en) | Enrichment of nucleic acids | |
US20210207203A1 (en) | Crispr double nickase based amplification compositions, systems, and methods | |
CN112795625A (en) | Method for detecting multiple nucleic acids based on CRISPR technology | |
EP4028515A1 (en) | Novel class 2 type ii and type v crispr-cas rna-guided endonucleases | |
CN111511925A (en) | Method for amplifying target nucleic acid and composition for expanding target nucleic acid | |
CN115335536A (en) | Compositions and methods for point-of-care nucleic acid detection | |
CN110923314B (en) | Primer for detecting SNP locus rs9263726, crRNA sequence and application thereof | |
Ballantyne et al. | Molecular crowding increases the amplification success of multiple displacement amplification and short tandem repeat genotyping | |
WO2013101290A1 (en) | Methods of using telomeres as markers for aging | |
WO2023203206A1 (en) | Multiplexable crispr-cas9-based virus detection method | |
CN102985560A (en) | A method of regulating oligonucleotide functionality | |
Maaroufi et al. | Development of a multiple internal control for clinical diagnostic real-time amplification assays | |
CN114250276B (en) | MicroRNA detection system and method based on exponential amplification reaction and Argonaute nuclease | |
Pan et al. | A novel whole genome amplification method using type IIS restriction enzymes to create overhangs with random sequences | |
WO2016159183A1 (en) | METHOD AND KIT FOR DETECTING BACTERIA OF GENUS Nitrobacter | |
CN105603055B (en) | Multiplex polymerase chain reaction method and application thereof | |
CN110423796A (en) | A method of improving nucleic acid in vitro amplified reaction efficiency |
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 |