CN110195102A - A kind of beta Thalassemia methods of genotyping - Google Patents
A kind of beta Thalassemia methods of genotyping Download PDFInfo
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Abstract
The invention discloses a kind of beta Thalassemia methods of genotyping.And the beta Thalassemia gene parting detecting reagent based on CRISPR/Cas12a is constructed based on this method.The technology is high to the specific good, high sensitivity of beta Thalassemia Genotyping detection, precision, additionally it is possible to realize multidigit point while detect, have great importance to the detection and screening of beta Thalassemia.Program testing cost is cheap, easy to operate simultaneously, has broad application prospects.
Description
Technical field
The invention belongs to Molecular Detection diagnostic techniques fields.More particularly, to a kind of beta Thalassemia Genotyping side
Method.
Background technique
Beta Thalassemia (Hemophilia) is since the missing or defect of beta globin (HBB) gene make beta globin chain
Synthesis be suppressed caused by hemolytic anemia, mostly caused by beta-globin point mutation.β chain person cannot be synthesized completely
Claim β 0, can partially synthetic β chain person (for normal 5%~30%) claim β+.Beta Thalassemia is the most common heredity in the world
One of, there are about more than 100,000,000 people to carry beta Thalassemia gene in the whole world according to statistics.
The gene diagnosis method of beta Thalassemia mainly has: Sanger PCR sequencing PCR, restriction fragment length polymorphism
(RFLP), reverse dot blot hybridization (RDB), ApoE gene (ARMS-PCR).The operation of Sanger sequencing approach is cumbersome,
Its application in clinical diagnosis is significantly limited, the detection flux of sequencing is also limited, and needs to testing result artificial
Analysis, analysis take time and effort.RFLP is to carry out endonuclease reaction by identification specific sequence site, and method simplicity is low in cost,
But its limitation is can only to detect to the limited mutation that can produce restriction enzyme site, due to incomplete or partially digested reaction
Also result in false positive or false negative result.Reverse dot blot hybridization technology the result is that with the naked eye interpretation, fault rate is high, often results in
A sample is repeatedly detected.ARMS-PCR need to design corresponding primer for each mutation, and every a pair of primer amplification condition all needs excellent
Change, when need to detect various mutations simultaneously if it is cumbersome, and will appear false positive or false negative result.
Market and social detection demand based on the poor dcc gene in ground, it is necessary to which poor dcc gene detection side a kind ofly is provided
Method.Applicants have found that not there is abrupt climatic change side beta globin genes (HBB) based on CRISPR/Cas12a system also at present
The relevant report of method.It finds and designs as suitable targets and prepare the gRNA of accurate, selectively targeted target gene and be
The key technology of CRISPR/Cas9 gene knockout, the gRNA of suitable targets and efficient, selectively targeted target HBB gene
It is the key that detection beta Thalassemia to be realized based on CRISPR/Cas12a, and further such that be based on CRISPR/Cas12a system
The editor of the HBB gene of system, modification, detection are possibly realized.
Summary of the invention
The technical problem to be solved by the present invention is to overcome the defect of existing beta Thalassemia Genotyping detection technique and not
Foot finds suitable targets and designs, prepares accurate, efficient, selectively targeted beta Thalassemia related mutation gene
GRNA, this is the key that CRISPR/Cas12a identification target gene；And it is constructed based on this kind of based on CRISPR/Cas12a system
The beta Thalassemia related mutation gene tester and detection kit of system.
The object of the present invention is to provide a kind of based on CRISPR/Cas12a detection beta Thalassemia gene mutation site
GRNA combination.
The beta Thalassemia Genotyping detection based on CRISPR/Cas12a that it is a further object of the present invention to provide a kind of
Method.
Yet another object of the invention is a kind of beta Thalassemia Genotyping detection examination based on CRISPR/Cas12a
Agent box.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
The present invention provides a kind of beta Thalassemia methods of genotyping, and this method is carried out based on CRISPR/Cas12a technology
The Genotyping of beta Thalassemia detects, and specifically carries out CRISPR detection using Cas12a albumen and gRNA；Detect target position
Point nucleotide sequence such as SEQ ID NO.1-3 any one or appoint it is several shown in.
The design principle of the gRNA are as follows: when choosing gRNA targeting sequence, targeting sequence 5 ' end should have 5 '-TTTN-
3 ' sequences, and target and do not form stable secondary structure between sequence itself, targeting sequence and remaining sequence.
Scheme may be selected as preferred, the sequence of the gRNA such as SEQ ID NO.4-8 is any shown.GRNA combination
It also should be within protection scope of the present invention.
Furthermore it is preferred that detection architecture includes: 2 μ l RPA products, LbCas12a, the 22.5nM gRNA of 45nM purifying,
The reporter dna chain of 100nM capable of emitting fluorescence when LbCas12a is cut, i.e., non-specific single stranded DNA fluorescence probe (DNAseAlert
QC System, Thermo Scientific), 0.5 μ l RNase inhibitor (Promega) and nuclease detect buffer
(20mM Tris,60mM NaCl,10mM MgCl 2,pH7.3)。
Preferably, program is detected are as follows: react 1.5 hours at 37 DEG C, fluorescence Dynamic testing 5 minutes primary.
The present invention also provides a kind of beta Thalassemia gene parting detecting reagent based on CRISPR/Cas12a simultaneously,
Including Cas12a albumen and above-mentioned gRNA.
In addition, above-mentioned Cas12a albumen is with endonuclease activity and with the Cas12a egg of attached cleavage activity
It is white.Such as LbCas12a, SsCas12a, ScCas12a, FnCas12a, AsCas12a etc..
The sequence of the ScCas12a is as shown in SEQ ID NO.9, the sequence of the SsCas12a such as SEQ ID NO.10
It is shown, sequence reference Addgene pMAL-his-LbCpf1-EC (Plasmid#79008) of the LbCas12a,
Sequence of the sequence of FnCas12a referring to Addgene 6-His-MBP-TEV-FnCpf1 (Plasmid#90094), AsCas12a
Referring to Addgene AsCpf1-2NLS (Plasmid#102565).
Detection while in order to realize to multiple beta Thalassemia gene mutation sites, at the beginning of being easy to implement quick multidigit point
The application scenarios of sieve, we have developed the Multiple detection systems for being directed to above-mentioned beta Thalassemia gene mutation target, to selection
A variety of beta Thalassemia gene mutation sequences are analyzed, and corresponding gRNA sequence design analysis, according to the phase of these sequences
Like property, G/C content, base homogeneity, whether there is or not formation second level hairpin structure, same reactions the parameters such as no cross reaction, to reaction
System and gRNA combination optimize.The solution of the present invention can be realized to 3 target site realities shown in SEQ ID NO.1-3
Existing multidigit point detects simultaneously, has great importance for the detection screening of beta Thalassemia.
The invention has the following advantages:
The present invention provides a kind of for targeting the gRNA of beta Thalassemia related mutation gene RNA, the present invention also provides
A kind of beta Thalassemia related mutation gene tester based on CRISPR/Cas12a system, detection kit.This hair
Bright detection method combines gRNA targets identification beta Thalassemia related mutation gene transcript RNA (target RNA sequence)
Advantage and when CRISPR/Cas12a compound detects target RNA sequence, compound can cut with detection label
The characteristics of reporter rna, release detectable signal, CRISPR/Cas12a system is applied in beta Thalassemia related mutation gene
In detection, high sensitivity, accuracy is high, specificity is good, can in 25-37 DEG C of realization at room temperature highly sensitive, high-precision molecule
Detection, detection limit value can reach A Moer grades (10-18Mole/L), realize target Single Molecule Detection.It is also able to achieve multidigit point simultaneously
It detects, clinical detection excellent effect, has great importance for the detection screening of beta Thalassemia simultaneously.
Moreover, program testing cost is cheap, supports without large scale equipment, is easy to operate, quick, realizing detection by bed,
It is a kind of detection method and detection kit with huge commercial application value convenient for extensive use and Rapid Popularization.
Detailed description of the invention
Fig. 1 is the different gRNA detection effects in embodiment 1 to beta-thalassemia mutation site.
Fig. 2 is gRNAs more to beta-thalassemia mutation site while detection effect in embodiment 2.
Specific embodiment
The present invention is further illustrated below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention
It limits in any form.It will be appreciated by persons skilled in the art that can be replaced using the alternative of this field routine
The conventional clone of Cas12a gene in the embodiment of the present invention, the building of recombinant expression carrier, the expression of Cas12a albumen and pure
Change, the amplification of target nucleotide/target gene segment and etc. one of or it is a variety of, to obtain similar or equivalent effect.
Unless stated otherwise, the present invention uses reagent, method and apparatus for the art conventional reagent, method and are set
It is standby.Unless stated otherwise, following embodiment agents useful for same and material are commercially available.
Embodiment 1 is detected based on the beta Thalassemia Genotyping of CRISPR/Cas12a system
1, CRISPR/Cas12a gene cloning and protein expression
Using the Cas12a protein gene for being originated from Lachnospiraceae bacterium, codon optimization makes base
Because being more suitable for expressing in mammalian cells.Cas12a protein gene cloning after optimization enters with 6-His histidine tag
PET28a plasmid, facilitates protein purification to express.The conversion of Cas12a Protein reconstitution expression vector, expression bacterium use BL21star
(DE3)。
After the conversion of Cas12a Protein reconstitution expression vector, it is pure to carry out protein expression, SDS-PAGE detection and gel column
Change, the Cas12a albumen after purification of acquisition puts -80 DEG C of preservations.
Specific protein expression condition are as follows: 0.5mMIPTG is added when cultivating bacterium solution OD600=0.6 and cultivates 4 hours.It collects
Thallus carries out protein purification.
Purification condition are as follows: thallus is resuspended in lysate (50mM Tris, pH8.0,300mM NaCl, 5% glycerol, 20mM
Imidazoles), it carries out ultrasonication (70% amplitude, 2s On/4s Off, 3 minutes, Sonics 750w Ultrasound Instrument), in centrifuge separation
Clear liquid is eluted with ni-sepharose purification with the lysate of the imidazoles containing 250mM, elution fraction is concentrated, with Superdex 200, Tricorn
10/300 gel chromatographic columns are purified.SDS-PAGE detection and gel column purification, the Cas12a albumen after purification of acquisition,
Put -80 DEG C of preservations.
2, target sequence expands
Target nucleotide can pass through PCR amplification, recombinase polymeric enzymatic amplification (RPA), NASBA isothermal duplication or ring mediation etc.
Temperature amplification (LAMP), strand displacement amplification (SDA), helicase dependent amplification (HDA) and nickase amplified reaction (NEAR) mode
Expand target DNA.
Recombinase polymeric enzymatic amplification RPA (Recombinase Polymerase Amplification):
RPA primer is designed using NCBI Primer blast, amplified fragments size is 80-120nt, the denaturation temperature of primer
Degree can be 54-67 DEG C, Opt=60, and length 30-35nt, Opt=32, G/C content is 40-60% in primer, according to designing sequence
Column synthetic DNA primer.
It refers to respectivelyBasic andIt is anti-that BasicRT (TwistDx) kit carries out RPA
It answers, unlike, before sample nucleic acid addition, the MgAc of 280mM, i.e. magnesium acetate is first added.It is reacted 30 minutes at 37 DEG C.
Glue separation and purifying (using MinElute gel extraction kit (Qiagen) kit), after purification
DsDNA be incubated overnight (use T7RNA polymerase (Thermo) kit) with 37 DEG C of T7 polymerase, then use
RNeasy mini kit (Qiagen) kits RNA, to obtain target nucleus RNA.
3, gRNA is prepared
GRNA primer sequence design principle: when choosing targeting sequence, targeting sequence 5 ' end should have 5 '-TTTN-3 ' sequence.
And stable secondary structure is not formed between targeting sequence itself, targeting sequence and remaining sequence, http can be passed through: //
Www.rgenome.net/cas-designer/ online software Computer Aided Design.
T7 primer sequence: TGTAATACGACTCACTATAGGG
GRNA primer construction:
5 '-targeting sequence-" ATCTACACTTAGTAGAAATTA "-CCCTATAGTGAGTCGTATTACA-3 '
Wherein " ATCTACACTTAGTAGAAATTA " sequence can be replaced " ATCTACAACAGTAGAAATTA " or
" ATCTACAACAGTAGAAATTA " or " ATCTACAACAGTAGAAATTA " or " GCATGAGAACCATGCATTTC " or
" ACCTAATTACTAGGTAATTT " or " ATCTACAAAAGTAGAAATCC " or " ATCTACAATAGTAGAAATTA " or
" ATCTACAAAGTAGAAATTAT " or " ATCTACAAACAGTAGAAATT ".
Referring to T7RNApolymerase kit (Thermo) kit specification, by the DNA fragmentation with T7 promoter, T7
Primer, the mixing of T7 polymerase, 37 DEG C of overnight incubations；RNeasy mini kit (Qiagen) is used again, obtains the gRNAs of purifying.
T7 primer sequence: TGTAATACGACTCACTATAGGG
T7gRNA primer sequence:
" targeting sequence " -5 '-ATCTACACTTAGTAGAAATTACCCTATAGTGAGTCGTATTACA-3 '
By a large amount of exploratory developments, one group of detection target gene and corresponding target based on CRISPR/Cas12a have been obtained
To gRNA sequence (SEQ ID NO.4-8, as shown in table 1), additionally it is possible to realize multidigit point while detect.
The target gene referred in 1 this patent of table and corresponding gRNA target sequence
4, detection of nucleic acids
Detection architecture includes: 2 μ l RPA products, the LbCas12a of 45nM purifying, 22.5nM gRNA, and 100nM exists
The reporter dna chain of LbCas12a capable of emitting fluorescence when cutting, i.e., non-specific single stranded DNA fluorescence probe (DNAseAlert QC
System, Thermo Scientific), 0.5 μ l RNase inhibitor (Promega) and nuclease detect buffer (20mM
Tris,60mM NaCl,10mM MgCl 2,pH 7.3)。
Reaction system is placed in fluorescence analyser (BioTek), reacts 1.5 hours under 37 DEG C (unless otherwise indicated), fluorescence
Dynamic testing 5 minutes primary.
Analysis reaction fluorescence data: the fluorescence data in order to calculate removal background facilitates the comparison between different condition, sample
The initial fluorescence of product is removed.Background fluorescence (no target nucleotide or without gRNA under conditions of) can be removed from sample, to obtain
Obtain the data of background correction fluorescence.
Testing result is as shown in Figure 1, the results showed that: the Cas12a albumen and designed gRNA can recognize cutting target
Site simultaneously generates fluorescence signal and possesses good specificity.
Embodiment 2 is detected simultaneously based on the beta Thalassemia polygenic mutation site of CRISPR/Cas12a system
Detection while in order to realize to multiple beta Thalassemia gene mutation sites, at the beginning of being easy to implement quick multidigit point
The application scenarios of sieve, we have developed the Multiple detection systems for being directed to above-mentioned beta Thalassemia gene mutation target, to selection
A variety of beta Thalassemia gene mutation sequences are analyzed, and corresponding gRNA sequence design analysis, according to the phase of these sequences
Like property, G/C content, base homogeneity, whether there is or not formation second level hairpin structure, same reactions the parameters such as no cross reaction, to reaction
System and gRNA combination optimize.GRNA in embodiment 1 can be realized multidigit point for corresponding target gene
It detects simultaneously.The present embodiment presents a kind of experiment of gRNA assembled scheme of beta Thalassemia polygenic mutation site primer
And result.
1, template prepares template selection preparation to be detected according to step 2 in embodiment 1, prepares SEQ ID NO.1, SEQ ID
NO.2, SEQ ID NO.3 are as template.
2, gRNA preparation and abrupt climatic change
After preparing gRNA according to the method for step 3 in embodiment 1,3 kinds of gRNA of corresponding templates is taken to mix by equal proportion
(SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.8) then prepares CRISPR/ according to the method for step 4 in embodiment 1
Cas12a detection architecture detects multiple gRNA method detection effect.Template in detection reaction is respectively SEQ ID NO.1, SEQ
ID NO.2, SEQ ID NO.3.Sample in experiment without target nucleic acid is negative control, and positive criteria product SEQ ID NO.1 is independent
Adding corresponding gRNA (SEQ ID NO.5) is positive control, and other conditions are constant.The system of configuration is placed on fluorescence analyser
(BioTek), it is reacted 1.5 hours under 37 DEG C (unless otherwise indicated), fluorescence Dynamic testing 5 minutes primary.
3, testing result is as shown in Figure 2.The experimental results showed that 3 heavy gRNA reaction systems are added in specific target gene
Afterwards, there is fluorescence generation, result is the positive.The beta Thalassemia based on CRISPR/Cas12a system for illustrating that experiment is established is more
Gene mutation site detects simultaneously good detection effect and specificity.
It will be appreciated by persons skilled in the art that can be implemented using the alternative replacement present invention of this field routine
The conventional clone of Cas12a gene, the building of recombinant expression carrier, the expression of Cas12a albumen and purifying, target nucleus glycosides in example
The amplification of acid/target gene segment and etc. one of or it is a variety of, to obtain similar or equivalent effect.
In addition above sequence shown in SEQ ID NO.9 and SEQ ID NO.10 is as follows:
The sequence of SEQ ID NO.9:(ScCas12a)
ATGCAGACCCTGTTTGAGAACTTCACAAATCAGTACCCAGTGTCCAAGACCCTGCGCTTTGAGCTGATC
CCCCAGGGCAAGACAAAGGACTTCATCGAGCAGAAGGGCCTGCTGAAGAAGGATGAGGACCGGGCCGAGAAGTATAA
GAAGGTGAAGAACATCATCGATGAGTACCACAAGGACTTCATCGAGAAGTCTCTGAATGGCCTGAAGCTGGACGGCC
TGGAGGAATACAAGACCCTGTATCTGAAGCAGGAGAAGGACGATAAGGATAAGAAGGCCTTTGACAAGGAGAAGGAG
AACCTGCGCAAGCAGATCGCCAATGCCTTCCGGAACAATGAGAAGTTTAAGACACTGTTCGCCAAGGAGCTGATCAA
GAACGATCTGATGTCTTTCGCCTGCGAGGAGGACAAGAAGAATGTGAAGGAGTTTGAGGCCTTCACCACATACTTCA
CCGGCTTCCACCAGAACCGCGCCAATATGTACGTGGCCGATGAGAAGAGAACAGCCATCGCCAGCAGGCTGATCCAC
GAGAACCTGCCAAAGTTTATCGACAATATCAAGATCTTCGAGAAGATGAAGAAGGAGGCCCCCGAGCTGCTGTCTCC
TTTCAACCAGACCCTGAAGGATATGAAGGACGTGATCAAGGGCACCACACTGGAGGAGATCTTTAGCCTGGATTATT
TCAACAAGACCCTGACACAGAGCGGCATCGACATCTACAATTCCGTGATCGGCGGCAGAACCCCTGAGGAGGGCAAG
ACAAAGATCAAGGGCCTGAACGAGTACATCAATACCGACTTCAACCAGAAGCAGACAGACAAGAAGAAGCGGCAGCC
AAAGTTCAAGCAGCTGTATAAGCAGATCCTGAGCGATAGGCAGAGCCTGTCCTTTATCGCCGAGGCCTTCAAGAACG
ACACCGAGATCCTGGAGGCCATCGAGAAGTTTTACGTGAATGAGCTGCTGCACTTCAGCAATGAGGGCAAGTCCACA
AACGTGCTGGACGCCATCAAGAATGCCGTGTCTAACCTGGAGAGCTTTAACCTGACCAAGATCTATTTCCGCTCCGG
CACCTCTCTGACAGACGTGAGCCGGAAGGTGTTTGGCGAGTGGAGCATCATCAATAGAGCCCTGGACAACTACTATG
CCACCACATATCCAATCAAGCCCAGAGAGAAGTCTGAGAAGTACGAGGAGAGGAAGGAGAAGTGGCTGAAGCAGGAC
TTCAACGTGAGCCTGATCCAGACCGCCATCGATGAGTACGACAACGAGACAGTGAAGGGCAAGAACAGCGGCAAAGT
GATCGTCGATTATTTTGCCAAGTTCTGCGACGATAAGGAGACAGACCTGATCCAGAAGGTGAACGAGGGCTACATCG
CCGTGAAGGATCTGCTGAATACACCCTGTCCTGAGAACGAGAAGCTGGGCAGCAATAAGGACCAGGTGAAGCAGATC
AAGGCCTTTATGGATTCTATCATGGACATCATGCACTTCGTGCGCCCCCTGAGCCTGAAGGATACCGACAAGGAGAA
GGATGAGACATTCTACTCCCTGTTCACACCTCTGTACGACCACCTGACCCAGACAATCGCCCTGTATAACAAGGTGC
GGAACTATCTGACCCAGAAGCCTTACAGCACAGAGAAGATCAAGCTGAACTTCGAGAACAGCACCCTGCTGGGCGGC
TGGGATCTGAATAAGGAGACAGACAACACAGCCATCATCCTGAGGAAGGAAAACCTGTACTATCTGGGCATCATGGA
CAAGAGGCACAATCGCATCTTTCGGAACGTGCCCAAGGCCGATAAGAAGGACTCTTGCTACGAGAAGATGGTGTATA
AGCTGCTGCCTGGCGCCAACAAGATGCTGCCAAAGGTGTTCTTTTCTCAGAGCAGAATCCAGGAGTTTACCCCTTCC
GCCAAGCTGCTGGAGAACTACGAAAATGAGACACACAAGAAGGGCGATAATTTCAACCTGAATCACTGTCACCAGCT
GATCGATTTCTTTAAGGACTCTATCAACAAGCACGAGGATTGGAAGAATTTCGACTTTAGGTTCAGCGCCACCTCCA
CCTACGCCGACCTGAGCGGCTTTTACCACGAGGTGGAGCACCAGGGCTACAAGATCTCTTTTCAGAGCATCGCCGAT
TCCTTCATCGACGATCTGGTGAACGAGGGCAAGCTGTACCTGTTCCAGATCTATAATAAGGACTTTTCCCCATTCTC
TAAGGGCAAGCCCAACCTGCACACCCTGTACTGGAAGATGCTGTTTGATGAGAACAATCTGAAGGACGTGGTGTATA
AGCTGAATGGCGAGGCCGAGGTGTTCTACCGCAAGAAGAGCATTGCCGAGAAGAACACCACAATCCACAAGGCCAAT
GAGTCCATCATCAACAAGAATCCTGATAACCCAAAGGCCACCAGCACCTTCAACTATGATATCGTGAAGGACAAGAG
ATACACCATCGACAAGTTTCAGTTCCACATCCCAATCACAATGAACTTTAAGGCCGAGGGCATCTTCAACATGAATC
AGAGGGTGAATCAGTTCCTGAAGGCCAATCCCGATATCAACATCATCGGCATCGACAGAGGCGAGAGGCACCTGCTG
TACTATGCCCTGATCAACCAGAAGGGCAAGATCCTGAAGCAGGATACCCTGAATGTGATCGCCAACGAGAAGCAGAA
GGTGGACTACCACAATCTGCTGGATAAGAAGGAGGGCGACCGCGCAACCGCAAGGCAGGAGTGGGGCGTGATCGAGA
CAATCAAGGAGCTGAAGGAGGGCTATCTGTCCCAGGTCATCCACAAGCTGACCGATCTGATGATCGAGAACAATGCC
ATCATCGTGATGGAGGACCTGAACTTTGGCTTCAAGCGGGGCAGACAGAAGGTGGAGAAGCAGGTGTATCAGAAGTT
TGAGAAGATGCTGATCGATAAGCTGAATTACCTGGTGGACAAGAATAAGAAGGCAAACGAGCTGGGAGGCCTGCTGA
ACGCATTCCAGCTGGCCAATAAGTTTGAGTCCTTCCAGAAGATGGGCAAGCAGAACGGCTTTATCTTCTACGTGCCC
GCCTGGAATACCTCTAAGACAGATCCTGCCACCGGCTTTATCGACTTCCTGAAGCCCCGCTATGAGAACCTGAATCA
GGCCAAGGATTTCTTTGAGAAGTTTGACTCTATCCGGCTGAACAGCAAGGCCGATTACTTTGAGTTCGCCTTTGACT
TCAAGAATTTCACCGAGAAGGCCGATGGCGGCAGAACCAAGTGGACAGTGTGCACCACAAACGAGGACAGATATGCC
TGGAATAGGGCCCTGAACAATAACAGGGGCAGCCAGGAGAAGTACGACATCACAGCCGAGCTGAAGTCCCTGTTCGA
TGGCAAGGTGGACTATAAGTCTGGCAAGGATCTGAAGCAGCAGATCGCCAGCCAGGAGTCCGCCGACTTCTTTAAGG
CCCTGATGAAGAACCTGTCCATCACCCTGTCTCTGAGACACAATAACGGCGAGAAGGGCGATAATGAGCAGGACTAC
ATCCTGTCCCCTGTGGCCGATTCTAAGGGCCGCTTCTTTGACTCCCGGAAGGCCGACGATGACATGCCAAAGAATGC
CGACGCCAACGGCGCCTATCACATCGCCCTGAAGGGCCTGTGGTGTCTGGAGCAGATCAGCAAGACCGATGACCTGA
AGAAGGTGAAGCTGGCCATCTCCAACAAGGAGTGGCTGGAGTTCGTGCAGACACTGAAGGGCAAAAGGCCGGCGGCC
ACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGGGATCCTACCCATACGATGTTCCAGATTACGCTTATCCCTACGA
CGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCC
The sequence of SEQ ID NO.10:(SsCas12a)
ATGCAGACCCTGTTTGAGAACTTCACAAATCAGTACCCAGTGTCCAAGACCCTGCGCTTTGAGCTGATC
CCCCAGGGCAAGACAAAGGACTTCATCGAGCAGAAGGGCCTGCTGAAGAAGGATGAGGACCGGGCCGAGAAGTATAA
GAAGGTGAAGAACATCATCGATGAGTACCACAAGGACTTCATCGAGAAGTCTCTGAATGGCCTGAAGCTGGACGGCC
TGGAGAAGTACAAGACCCTGTATCTGAAGCAGGAGAAGGACGATAAGGATAAGAAGGCCTTTGACAAGGAGAAGGAG
AACCTGCGCAAGCAGATCGCCAATGCCTTCCGGAACAATGAGAAGTTTAAGACACTGTTCGCCAAGGAGCTGATCAA
GAACGATCTGATGTCTTTCGCCTGCGAGGAGGACAAGAAGAATGTGAAGGAGTTTGAGGCCTTCACCACATACTTCA
CCGGCTTCCACCAGAACCGCGCCAATATGTACGTGGCCGATGAGAAGAGAACAGCCATCGCCAGCAGGCTGATCCAC
GAGAACCTGCCAAAGTTTATCGACAATATCAAGATCTTCGAGAAGATGAAGAAGGAGGCCCCCGAGCTGCTGTCTCC
TTTCAACCAGACCCTGAAGGATATGAAGGACGTGATCAAGGGCACCACACTGGAGGAGATCTTTAGCCTGGATTATT
TCAACAAGACCCTGACACAGAGCGGCATCGACATCTACAATTCCGTGATCGGCGGCAGAACCCCTGAGGAGGGCAAG
ACAAAGATCAAGGGCCTGAACGAGTACATCAATACCGACTTCAACCAGAAGCAGACAGACAAGAAGAAGCGGCAGCC
AAAGTTCAAGCAGCTGTATAAGCAGATCCTGAGCGATAGGCAGAGCCTGTCCTTTATCGCCGAGGCCTTCAAGAACG
ACACCGAGATCCTGGAGGCCATCGAGAAGTTTTACGTGAATGAGCTGCTGCACTTCAGCAATGAGGGCAAGTCCACA
AACGTGCTGGACGCCATCAAGAATGCCGTGTCTAACCTGGAGAGCTTTAACCTGACCAAGATGTATTTCCGCTCCGG
CGCCTCTCTGACAGACGTGAGCCGGAAGGTGTTTGGCGAGTGGAGCATCATCAATAGAGCCCTGGACAACTACTATG
CCACCACATATCCAATCAAGCCCAGAGAGAAGTCTGAGAAGTACGAGGAGAGGAAGGAGAAGTGGCTGAAGCAGGAC
TTCAACGTGAGCCTGATCCAGACCGCCATCGATGAGTACGACAACGAGACAGTGAAGGGCAAGAACAGCGGCAAAGT
GATCGCCGATTATTTTGCCAAGTTCTGCGACGATAAGGAGACAGACCTGATCCAGAAGGTGAACGAGGGCTACATCG
CCGTGAAGGATCTGCTGAATACACCCTGTCCTGAGAACGAGAAGCTGGGCAGCAATAAGGACCAGGTGAAGCAGATC
AAGGCCTTTATGGATTCTATCATGGACATCATGCACTTCGTGCGCCCCCTGAGCCTGAAGGATACCGACAAGGAGAA
GGATGAGACATTCTACTCCCTGTTCACACCTCTGTACGACCACCTGACCCAGACAATCGCCCTGTATAACAAGGTGC
GGAACTATCTGACCCAGAAGCCTTACAGCACAGAGAAGATCAAGCTGAACTTCGAGAACAGCACCCTGCTGGGCGGC
TGGGATCTGAATAAGGAGACAGACAACACAGCCATCATCCTGAGGAAGGATAACCTGTACTATCTGGGCATCATGGA
CAAGAGGCACAATCGCATCTTTCGGAACGTGCCCAAGGCCGATAAGAAGGACTTCTGCTACGAGAAGATGGTGTATA
AGCTGCTGCCTGGCGCCAACAAGATGCTGCCAAAGGTGTTCTTTTCTCAGAGCAGAATCCAGGAGTTTACCCCTTCC
GCCAAGCTGCTGGAGAACTACGCCAATGAGACACACAAGAAGGGCGATAATTTCAACCTGAATCACTGTCACAAGCT
GATCGATTTCTTTAAGGACTCTATCAACAAGCACGAGGATTGGAAGAATTTCGACTTTAGGTTCAGCGCCACCTCCA
CCTACGCCGACCTGAGCGGCTTTTACCACGAGGTGGAGCACCAGGGCTACAAGATCTCTTTTCAGAGCGTGGCCGAT
TCCTTCATCGACGATCTGGTGAACGAGGGCAAGCTGTACCTGTTCCAGATCTATAATAAGGACTTTTCCCCATTCTC
TAAGGGCAAGCCCAACCTGCACACCCTGTACTGGAAGATGCTGTTTGATGAGAACAATCTGAAGGACGTGGTGTATA
AGCTGAATGGCGAGGCCGAGGTGTTCTACCGCAAGAAGAGCATTGCCGAGAAGAACACCACAATCCACAAGGCCAAT
GAGTCCATCATCAACAAGAATCCTGATAACCCAAAGGCCACCAGCACCTTCAACTATGATATCGTGAAGGACAAGAG
ATACACCATCGACAAGTTTCAGTTCCACATCCCAATCACAATGAACTTTAAGGCCGAGGGCATCTTCAACATGAATC
AGAGGGTGAATCAGTTCCTGAAGGCCAATCCCGATATCAACATCATCGGCATCGACAGAGGCGAGAGGCACCTGCTG
TACTATGCCCTGATCAACCAGAAGGGCAAGATCCTGAAGCAGGATACCCTGAATGTGATCGCCAACGAGAAGCAGAA
GGTGGACTACCACAATCTGCTGGATAAGAAGGAGGGCGACCGCGCAACCGCAAGGCAGGAGTGGGGCGTGATCGAGA
CAATCAAGGAGCTGAAGGAGGGCTATCTGTCCCAGGTCATCCACAAGCTGACCGATCTGATGATCGAGAACAATGCC
ATCATCGTGATGGAGGACCTGAACTTTGGCTTCAAGCGGGGCAGACAGAAGGTGGAGAAGCAGGTGTATCAGAAGTT
TGAGAAGATGCTGATCGATAAGCTGAATTACCTGGTGGACAAGAATAAGAAGGCAAACGAGCTGGGAGGCCTGCTGA
ACGCATTCCAGCTGGCCAATAAGTTTGAGTCCTTCCAGAAGATGGGCAAGCAGAACGGCTTTATCTTCTACGTGCCC
GCCTGGAATACCTCTAAGACAGATCCTGCCACCGGCTTTATCGACTTCCTGAAGCCCCGCTATGAGAACCT
Claims (9)
1. a kind of gRNA combination based on CRISPR/Cas12a detection beta Thalassemia gene mutation site, which is characterized in that
GRNA combination include SEQ ID NO.4-8 any one or appoint it is several shown in gRNA.
2. a kind of beta Thalassemia gene mutation site detection method based on CRISPR/Cas12a, which is characterized in that utilize
Cas12a albumen and gRNA carry out CRISPR detection, detect target site nucleotide sequence such as SEQ ID NO.1-3 any one
Or appoint several shown.
3. method according to claim 2, which is characterized in that shown in the sequence of the gRNA such as SEQ ID NO.4-8 is any.
4. method according to claim 2, which is characterized in that the Cas12a albumen be LbCas12a, SsCas12a,
ScCas12a, FnCas12a or AsCas12a.
5. method according to claim 2, which is characterized in that detection architecture includes: 2 μ l RPA products, 45nM Cas12a,
The reporter dna chain of 22.5nM gRNA, the 100nM capable of emitting fluorescence when Cas12a is cut, 0.5 μ l RNase inhibitor and nucleic acid
Enzyme detects buffer.
6. method according to claim 2, which is characterized in that detection program are as follows: reacted 1.5 hours at 37 DEG C, fluorescence is dynamic
Power detects 5 minutes once.
7. a kind of beta Thalassemia gene parting detecting reagent based on CRISPR/Cas12a, which is characterized in that including
Cas12a albumen and gRNA, the sequence of the gRNA such as SEQ ID NO.4-8 are any shown.
8. detection kit according to claim 7, which is characterized in that the Cas12a albumen be LbCas12a,
SsCas12a, ScCas12a, FnCas12a or AsCas12a.
9. detection kit according to claim 7, which is characterized in that further include non-specific single stranded DNA fluorescence probe,
RNase inhibitor and nuclease detect buffer.
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Cited By (1)
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|---|---|---|---|---|
| CN113174433A (en) * | 2021-04-22 | 2021-07-27 | 中南大学 | Cas protein-based detection method |
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