Patent ID: 12252722

DETAILED DESCRIPTION

For further understanding of the method of the present invention, the present invention will be further described in conjunction with examples.

Example 1

In this example, 48 μg of single-stranded DNA with a length of 253 nt was prepared by the present process, with a purity of 91% and a sequence accuracy of 100%:

The test sample was a DNA sequence with a length of 253 nt (SEQ ID NO: 1).

The production process of the single-stranded DNA in this example was as follows.1) Step 1 (sequence analysis process): After analysis by bioinformatics software, the sequence was found to have no BspQI restriction cleavage site, so BspQI was selected as the final cleavage enzyme.2) Step 2 (primer design process): 5′-AACTATACTGCTCTTCA-3′ (SEQ ID NO:4) and 5′-CCTTGAAGAGCAACTATA-3′ (SEQ ID NO: 5) were respectively added to two ends of the target sequence, and subjected to gene synthesis. The primers for PCR were the forward primer Pf: 5′-AACTATACUGCTCTTCA-3′ (SEQ ID NO:6) and the reverse primer Pr: 5′-TATAGTUGCTCTTCAAGG-3′ (SEQ ID NO:7). The primers were synthesized.3) Step 3 (template amplification and production-self-looping): Using primers (Pf, Pr) with uracil modification, the synthesized fragment with terminal aptamers were amplified. The PCR reaction system was:

Reaction system (100 uL):ReagentVolume253 (288 bp)uL5*phusion HF buffer2010 mM dNTPs2100% DMSO3253 UF (50 uM)1253 UR (50 uM)1DNA template1phusion U Hot1Start DNA polymeraseddH2O71
PCR Reaction Procedure

Reaction condition98° C.30s98° C.10s63° C.30s72° C.8s72° C.5min4° C.Long time
30 Cycles

The electropherogram of the PCR product is shown inFIG.1.

Then the PCR product was recovered, and self-looped. The reaction system for producing gapped circular double-stranded DNA by self-looping using USER enzyme and T4 DNA ligase is:

Total amount ofPCR product:100 nguL253-U0.4810*T4 ligase buffer1USER1T4 ligase0.50ddH2O7.02

The reaction conditions include 30 min at 37° C., 30 min at 20° C., and storage at 4° C.4) Step 4 (rolling circle replication): In a 200 ul PCR tube, 100 ng of the gapped circular DNA sample produced in Step 3 was subjected to rolling circle replication by incubation at 30° C. for 4-8 hrs, and then inactivated at 80° C. for 20 min. The reaction system for rolling circle replication is:

(For example, in 100 ul)

ReagentuLUSER treatment sample1010*phi29 DNA polymerase buffer10dNTP(10 mM)5BSA(2 mg/mL)10phi29 DNA polymerase5ddH2O605) Step 5 (annealing—auto-folding): The product after rolling circle replication was annealed by cooling to form a designed hairpin structure, where the annealing procedure was 5 min at 80° C., cooling to 65° C. at 0.1° C./s, 5 min at 65° C., cooling to 42° C. at 0.1° C./s, 5 min at 42° C., 5 min at 37° C., cooling to 4° C. at 0.1° C./s, and then stored at 4° C.6) Step 6 (restriction endonuclease cleavage-release of the target fragment, that is, the long single-stranded monomer): 15 ul of 10× restriction endonuclease buffer, and 2 ul of the selected type II restriction endonuclease were added to the above reaction product, and ddH2O was added up to a volume of 150 ul. The system was stood at an optimal reaction temperature for the restriction endonuclease for 60 min, and then thermally denatured and inactivated. The electropherogram of the crude reaction product is shown inFIG.2.7) Step 7 (purification and concentration): The cleavage product by restriction endonuclease contains the single-stranded DNA product of the target fragment and the hairpin structure. Since the length of the target sequence is less than 300 nt, magnetic beads are used for efficient purification. The comparison before and after purification is shown inFIG.3. Lane 1 is the crude product before purification, and Lane 2 is the product after magnetic bead purification. The hairpin structure is effectively removed, and the purity is 91%. Next generation sequencing (NGS) was used for sequence verification. The test result of the sequence is shown inFIG.10, and is consistent with SEQ ID NO: 1.

Example 2

In this example, 40 μg of long single-stranded DNA (SEQ ID NO: 2) with a length of 1350 nt was prepared by the present process with a purity of 97% and a sequence accuracy of 100%.

The test sample was a DNA sequence with a length of 1350 nt.

The production process of the long single-stranded DNA in this example was as follows.1) Step 1 (sequence analysis process): After analysis by bioinformatics software, the sequence was found to have no BspQI restriction cleavage site, so BspQI was selected as the final cleavage enzyme.2) Step 2 (primer design process): 5′-AACTATACTGCTCTTCA-3′ (SEQ ID NO:4) and 5′-CCTTGAAGAGCAACTATA-3′ (SEQ ID NO:5) were respectively added to two ends of the target sequence, and subjected to gene synthesis. The primers for PCR were the forward primer Pf: 5′-AACTATACUGCTCTTCA-3′ (SEQ ID NO:6) and the reverse primer Pr: 5′-TATAGTUGCTCTTCAAGG-3′ (SEQ ID NO:7). The primers were synthesized.3) Step 3 (template amplification and production-self-looping): Using primers (Pf, Pr) with uracil modification, the synthesized fragment with terminal aptamers were amplified. The PCR reaction system was:

Reaction system (100 uL):ReagentVolume1350uL5*phusion HF buffer2010 mM dNTPs2100% DMSO31350 UF (50 uM)11350 UR (50 uM)1DNA template1phusion U Hot1Start DNA polymeraseddH2O71
PCR Reaction Procedure

Reaction condition98° C.30s98° C.10s63° C.30s72° C.1min72° C.5min4° C.Long time
30 Cycles

The electropherogram of the PCR product is as shown inFIG.5.

Then the PCR product was recovered, and self-looped. The reaction system for producing gapped circular double-stranded DNA by self-looping using USER enzyme and T4 DNA ligase is:

Total amount ofPCR product:100 nguL253-U0.4810*T4 ligase buffer1USER1T4 ligase0.50ddH2O7.02

The reaction conditions include 30 min at 37° C., 30 min at 20° C., and storage at 4° C.4) Step 4 (rolling circle replication): In a 1.5 ml centrifuge tube, 100 ng of the gapped circular DNA sample produced in Step 3 was subjected to rolling circle replication by incubation at 30° C. for 4-8 hrs, and then inactivated at 80° C. for 20 min. The reaction system for rolling circle replication is (8 ml reaction system):

ReagentuLUSER treatment sample80010*phi29 DNA polymerase buffer800dNTP(10 mM)400BSA(2 mg/mL)800phi29 DNA polymerase200ddH2O48005) Step 5 (annealing—auto-folding): The product after rolling circle replication was annealed by cooling to form a designed hairpin structure, where the annealing procedure was 5 min at 80° C., cooling to 65° C. at 0.1° C./s, 5 min at 65° C., cooling to 42° C. at 0.1° C./s, 5 min at 42° C., 5 min at 37° C., cooling to 4° C. at 0.1° C./s, and then stored at 4° C.6) Step 6 (restriction endonuclease cleavage-release of the target fragment, that is, the long single-stranded monomer): 150 ul of 10× restriction endonuclease buffer, and 20 ul of the selected type II restriction endonuclease were added to the above reaction product, and ddH2O was added up to a volume of 1500 ul. The system was stood at an optimal reaction temperature for the restriction endonuclease for 60 min, and then thermally denatured and inactivated. The electropherogram of the crude reaction product is shown inFIG.5.7) Step 7 (purification and concentration): The cleavage product by restriction endonuclease contains the single-stranded DNA product of the target fragment and the hairpin structure. Since the length of the target sequence is >300 nt, agarose gel electrophoresis is used for extraction and purification. The electropherogram of the purified product is shown inFIG.6, where Lane 1 is the purified product. The hairpin structure and the double-stranded DNA contaminant are effectively removed, the purity is 97%, and the sequence accuracy is 100%. Next generation sequencing (NGS) was used for sequence verification. The test result of the sequence is shown inFIG.11, and is consistent with SEQ ID NO: 2.

Example 3

In this example, 10 μg of long single-stranded DNA (SEQ ID NO: 3) with a length of 2350 nt was prepared by the present process with a purity of 93% and a sequence accuracy of 100%.

The test sample was a DNA sequence with a length of 2350 nt.

The production process of the long single-stranded DNA in this example was as follows.1) Step 1 (sequence analysis process): After analysis by bioinformatics software, the sequence was found to have no BspQI restriction cleavage site, so BspQI was selected as the final cleavage enzyme.2) Step 2 (primer design process): 5′-AACTATACTGCTCTTCA-3′ (SEQ ID NO:4) and 5′-CCTTGAAGAGCAACTATA-3′ (SEQ ID NO:5) were respectively added to two ends of the target sequence, and subjected to gene synthesis. The primers for PCR were the forward primer Pf: 5′-AACTATACUGCTCTTCA-3′ (SEQ ID NO:6) and the reverse primer Pr: 5′-TATAGTUGCTCTTCAAGG-3′ (SEQ ID NO:7). The primers were synthesized.3) Step 3 (template amplification and production-self-looping): Using primers (Pf, Pr) with uracil modification, the synthesized fragment with terminal aptamers were amplified. The PCR reaction system was:

Reaction system (100 uL):ReagentVolume1350uL5*phusion HF buffer2010 mM dNTPs2100% DMSO31350 UF (50 uM)11350 UR (50 uM)1DNA template1phusion U Hot1Start DNA polymeraseddH2O71
PCR Reaction Procedure

Reaction condition98° C.30s98° C.10s63° C.30s72° C.2min72° C.5min4° C.Long time
30 Cycles

The electropherogram of the PCR product is shown inFIG.7.

Then the PCR product was recovered, and self-looped. The reaction system for producing gapped circular double-stranded DNA by self-looping using USER enzyme and T4 DNA ligase is:

Total amount ofPCR product:100 nguL253-U0.4810*T4 ligase buffer1USER1T4 ligase0.50ddH2O7.02

The reaction conditions include 30 min at 37° C., 30 min at 20° C., and storage at 4° C.4) Step 4 (rolling circle replication): In a 1.5 ml centrifuge tube, 100 ng of the gapped circular DNA sample produced in Step 3 was subjected to rolling circle replication by incubation at 30° C. for 4-8 hrs, and then inactivated at 80° C. for 20 min. The reaction system for rolling circle replication is (the final reaction system is 4 ml, with 1 ml/tube of reaction):

ReagentuLUSER treatment sample40010*phi29 DNA polymerase buffer400dNTP(10 mM)200BSA(2 mg/mL)400phi29 DNA polymerase200ddH2O24005) Step 5 (annealing—auto-folding): The product after rolling circle replication was annealed by cooling to form a designed hairpin structure, where the annealing procedure was 5 min at 80° C., cooling to 65° C. at 0.1° C./s, 5 min at 65° C., cooling to 42° C. at 0.1° C./s, 5 min at 42° C., 5 min at 37° C., cooling to 4° C. at 0.1° C./s, and then stored at 4° C.6) Step 6 (restriction endonuclease cleavage-release of the target fragment, that is, the long single-stranded monomer): 150 ul of 10× restriction endonuclease buffer, and 20 ul of the selected type II restriction endonuclease were added to the above reaction product, and ddH2O was added up to a volume of 1500 ul. The system was stood at an optimal reaction temperature for the restriction endonuclease for 60 min, and then thermally denatured and inactivated. The electropherogram of the crude reaction product is shown inFIG.9.7) Step 7 (purification and concentration): The cleavage product by restriction endonuclease contains the single-stranded DNA product of the target fragment and the hairpin structure. Since the length of the target sequence is >300 nt, agarose gel electrophoresis is used for extraction and purification. The electropherogram of the end product after purification is shown inFIG.9, where Lane 1 is the crude product before purification, and Lane 2 is the purified product. The hairpin structure and the double-stranded DNA contaminant are effectively removed, the purity is 95%, and the sequence accuracy is 100%. Next generation sequencing (NGS) was used for sequence verification. The test result of the sequence is shown inFIG.12, and is consistent with SEQ ID NO: 3.

As shown in the above three examples, the present method can be used to produce long single-stranded DNA with a length of 150-2500 nt. This method is applicable to sequences of various lengths, has low requirements on equipment and is easy to be scaled up for production. The purified long single-stranded DNA has a high purity and a sequence fidelity of 100%, thus being suitable for use as an efficient gene knock-in template for CRISPR gene editing.