Method for detecting nucleic acid based on dCas9 engineering modified protein and biological membrane layer interference technology
A biotin-dcas9-halo, protein technology, applied in the field of bioengineering, can solve the problems of weakened detection specificity, complex and time-consuming detection system, etc., and achieve the effect of reducing non-specific binding and improving specificity
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Embodiment 1
[0079] Example 1: Preparation of (E.coli) functionalized biotin-dCas9-Halo protein
[0080] (1) Construction of dCas9-Halo expression vector
[0081] The dCas9 gene fragment was obtained by gene synthesis, codon optimization and PCR, and the XbaI restriction site (TCTAGA) was introduced at the 5' end, and the CCACCGAGGTCTAGA sequence was introduced at the 3' end; the Halo gene fragment was obtained from a commercial plasmid by PCR, and at the 5' end The GGTGGCTCCAGATCT sequence was introduced at the end, and the XhoI restriction site (CTCGAG) was introduced at the 3' end. After PCR and XbaI and XhoI double digestion, the dCas9-Halo recombinant gene (sequence shown in SEQ ID NO.3) was cloned into the pET16b plasmid , Obtain the recombinant vector pET16b-dCas9-Halo, and verify it by sequencing.
[0082] The enzyme digestion system is as follows (20μL):
[0083]
[0084] The connection system is as follows (20μL):
[0085]
[0086] (2) Expression and purification of dCas...
Embodiment 2
[0093] Example 2: Features of biotin-dCas9-Halo functional protein
[0094] (1) Specific recognition of biotin-dCas9-Halo functional protein
[0095] Functionalized biotin-dCas9-Halo can bind to sequence-specific double-stranded DNA fragments under the guidance of sgRNA. The present invention verifies the specific binding of biotin-dCas9-Halo to DNA (the DNA sequence is shown in SEQ ID NO.5) by designing and synthesizing the guide sgRNA (sequence shown in SEQ ID NO.6), and we use gel retardation electrophoresis The specific recognition of functional proteins has been verified. When biotin-dCas9-Halo-sgRNA specifically binds to DNA1 to form a complex, its mobility is lower than that of free DNA1 under non-denaturing electrophoresis, which shows that the DNA bands in the complex are more than those not formed. The DNA band of the complex is upper.
[0096] The gel retardation electrophoresis method is as follows
[0097] 1) Configuration of 5% non-denaturing PAGE glue
[0098]...
Embodiment 3
[0104] Example 3: Quantitative nucleic acid detection using CRISPR-BLI detection chip
[0105] 1) Configure 2xPBS buffer: 0.2M PBS (pH6.8~8.0), which contains heparin sodium (10-100μg / ml), BSA (0.2~0.02%), Triton X-100 (0.02~0.1%), Tween -20(0.02~0.1%), NaCl(0.2~1.0mol / L), MgCl 2 (2 ~ 10mmol / L), EDTA (2 ~ 10mmol / ml), denatured salmon sperm DNA (0.2 ~ 0.02%); dilute 2 times at the same time to prepare 1xPBS.
[0106] 2) Prepare CRISPR probe solution: the CRISPR probe compounded with biotin-dCas9-Halo protein and sgRNA is diluted with 1xPBS, and the final concentration is 10-50 μg / mL.
[0107] 3) Prepare DNA standards: DNA standards (S1-S6) are formed by mixing DNA solution and 2xPBS at a volume ratio of 1:1. The concentrations of each standard are: S1: 2.5nM, S2: 5nM, S3: 10nM, S4 : 20nM, S5: 40nM, S6: 80nM. The nucleotide sequence of the DNA standard product is shown in SEQ ID NO.5, and the length is 3.6 kbp.
[0108] 4) Add 1xPBS, CRISPR probe solution, 1xPBS, DNA standar...
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