METHOD FOR SYNTHESIZING SINGLE-STRANDED DNA

Abstract

A method for synthesizing single-stranded DNA, specifically a process for producing single-stranded DNA without base mutations, is provided, by which single-stranded DNA is produced by uracil-specific excision reagent (USER)-mediated self-looping of double-stranded DNA combined with rolling circle replication.

Claims

1. A method for producing a target single-stranded DNA, comprising: (1) obtaining a template double-stranded DNA molecule consisting of a first strand and a reverse complementary second strand, wherein the sequence structure of the first strand is as shown in Formula (I):
5′ left aptamer sequence-target single-stranded DNA sequence-right aptamer sequence 3′  (I) wherein: the left aptamer sequence has a sequence structure of Formula: X.sub.nTX.sub.qX.sub.A; and the right aptamer sequence has a sequence structure of Formula: X.sub.BX.sub.q′X.sub.n-m, in which X.sub.n is a nucleotide sequence consisting of n nucleotides, where the 5′-terminal nucleotide is A and n is any integer of at least 4; X.sub.q is the sequence of a type II restriction endonuclease recognition site, and X.sub.q is not present in the target single-stranded DNA sequence; X.sub.q′ is a reverse complementary sequence of X.sub.q; X.sub.A and X.sub.B are optionally of 0 to several nucleotides, so that X.sub.qX.sub.A and X.sub.BX.sub.q′ constitute the cleavage site of the type II restriction endonuclease respectively, to allow the type II restriction endonuclease to cleave at the 3′ terminus of the X.sub.qX.sub.A sequence and the 5′ terminus of the X.sub.B X.sub.q′ sequence; X.sub.n-m is a sequence of n-m nucleotides from the 5′ terminus of X.sub.n, and m is an integer of 1-3; and A represents the adenine nucleotide; and T represents the thymine nucleotide; (2) with the template double-stranded DNA molecule as a template, performing PCR amplification using a forward primer and a reverse primer to obtain a product double-stranded DNA molecule comprising a first strand containing the target single-stranded DNA sequence and a reverse complementary second strand thereof; wherein the forward primer comprises a sequence of X.sub.nUX.sub.qX.sub.A, and the reverse primer comprises a sequence of X.sub.n-m′X.sub.qX.sub.B′, in which X.sub.n, X.sub.q, X.sub.A, and X.sub.B are the same as in (1); X.sub.n-m′ is a reverse complementary sequence of X.sub.n-m, with the T at the 3′ terminus being replaced by U; X.sub.B′ is a reverse complementary sequence of X.sub.B; and U is the uracil nucleotide; (3) cleaving the product double-stranded DNA molecule at U contained in the two strands with a uracil-specific excision reagent to produce cohesive terminuses at the two ends of the product double-stranded DNA molecule; (4) ligating the two cohesive terminuses of the product double-stranded DNA molecule in the presence of a ligase, to form a circular double-stranded DNA having a gap in the first strand; (5) subjecting the gapped circular double-stranded DNA obtained in Step (4) to rolling circle replication, where the replication starts with the gap in the sense first strand of the product double-stranded DNA molecule and uses the second strand as the template, to obtain a replicon comprising multiple sequence structures as shown in the following formula (II) in tandem:
5′-X.sub.nTX.sub.qX.sub.A-target single-stranded DNA sequence-X.sub.BX.sub.q′-3′  (II) (6) annealing the replicon, so that a hairpin structure is formed between two adjacent target single-stranded DNA sequences in the replicon, wherein preferably the hairpin structure consists of a sequence of X.sub.q′X.sub.nTX.sub.q; (7) treating the replicon with the type II restriction endonuclease, and cleaving at the 5′ terminus and 3 terminus of the X.sub.BX.sub.q′X.sub.nTX.sub.qX.sub.A sequence between adjacent target single-stranded DNA sequences to release multiple target single-stranded DNA sequences.

2. The method according to claim 1, wherein the type II restriction endonuclease is selected from AlwI, BbsI, BbvI, BceAI, BCIVI, BfuAI, BmrI, BpmI, BpuEI, BsaI, BseRI, BsgI, BsmAI, BsmBI, BsmFI, BspMI, BspQI, HphI, HpyAV, FokI, FauI, and HgaI.

3. The method according to claim 1, wherein the uracil-specific excision reagent is a uracil-specific excision enzyme (USER™).

4. The method according to claim 1, wherein the ligase is T4 DNA ligase.

5. The method according to claim 1, wherein steps (3) and (4) occur in the same reaction system in the presence of the uracil-specific excision reagent and the ligase.

6. The method according to claim 1, wherein the rolling circle replication is carried out using a DNA polymerase capable of continuous replication, preferably phi29 DNA polymerase.

7. The method according to claim 1, wherein the type II restriction endonuclease is BspQI.

8. The method according to claim 7, wherein X.sub.qX.sub.A has a sequence of GCTCTTCN, in which N is A, T, C or G, and preferably A; X.sub.BX.sub.q′ has a sequence of N.sub.1N.sub.2N.sub.3N.sub.4GAAGAGC, in which N.sub.1, N.sub.2, N.sub.3, and N.sub.4 are independently selected from A, T, C or G; and more preferably, X.sub.BX.sub.q′ has a sequence of CCTTGAAGAGC.

9. The method according to claim 1, wherein n is 6-10.

10. The method according to claim 1, wherein n is 8.

11. The method according to claim 1, wherein m is 1.

12. The method according to claim 1, wherein X.sub.n has a sequence of AACTATAC, and X.sub.n-m has a sequence of AACTATA.

13. The method according to claim 1, where the target single-stranded DNA has a length of 150-2500 nt.

14. The method according to claim 1, comprising: performing sequence analysis of the target single-stranded DNA sequence before the template double-stranded DNA molecule is produced, and selecting a type II restriction endonuclease that has no cleavage site in the target single-stranded DNA sequence.

15. A kit for amplifying a target DNA sequence, comprising: a left aptamer, having a sequence as shown in a formula of X.sub.nTX.sub.qX.sub.A; and a right aptamer, having a sequence as shown in a formula of X.sub.BX.sub.q′X.sub.n-m, wherein X.sub.n is a nucleotide sequence consisting of n nucleotides, where the 5′-terminal nucleotide is A and n is any integer of at least 4, preferably 6-8, and more preferably 8; X.sub.q is the sequence of a type II restriction endonuclease recognition site, and X.sub.q is not present in the target single-stranded DNA sequence; X.sub.q′ is a reverse complementary sequence of X.sub.q; X.sub.A and X.sub.B are optionally of 0 to several nucleotides, so that X.sub.qX.sub.A and X.sub.BX.sub.q′ constitute a cleavage site of the type II restriction endonuclease respectively, to allow the type II restriction endonuclease to cleave at the 3′ terminus of X.sub.A and the 5′ terminus of the X.sub.B; X.sub.n-m is a sub-sequence of n-m nucleotides from the 5′ terminus of X.sub.n, and m is an integer of 1-3; and A represents the adenine nucleotide; and T represents the thymine nucleotide.

16. The kit according to claim 15, further comprising: a forward primer having a sequence of X.sub.nUX.sub.qX.sub.A, and a reverse primer having a sequence of X.sub.n-m′X.sub.qX.sub.B′, wherein X.sub.n, X.sub.q, X.sub.A, and X.sub.B are the same as in the aptamer; and X.sub.n-m′ is a reverse complementary sequence of X.sub.n-m, with T at the 3′ terminus being replaced by U; X.sub.B′ is a reverse complementary sequence of X.sub.B; and U is the uracil nucleotide; and wherein the forward primer and the reverse primer allow PCR amplification using the target DNA sequence as a template.

17. A kit for producing single-stranded DNA, comprising: a DNA polymerase suitable for polymerase chain reaction; a DNA ligase, preferably T4 DNA ligase; a uracil specific excision reagent, preferably uracil specific excision enzyme; a DNA polymerase suitable for rolling circle amplification, preferably phi29 DNA polymerase; and a type II restriction endonuclease.

18. The kit according to claim 17, further comprising a left aptamer having a sequence as shown in a formula of X.sub.nTX.sub.qX.sub.A, and a right aptamer having a sequence as shown in a formula of X.sub.BX.sub.q′X.sub.n-m, a forward primer having a sequence of X.sub.nUX.sub.qX.sub.A, and a reverse primer having a sequence of X.sub.n-m′X.sub.qX.sub.B′.

19. The kit according to 17, wherein the DNA ligase and the uracil-specific excision reagent are placed in the same container.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0065] FIG. 1 shows the result of agarose gel electrophoresis of the product obtained by PCR amplification of the synthesized fragment with terminal aptamers using primers modified with uracil in Example 1.

[0066] FIG. 2 shows the result of agarose gel electrophoresis of the target fragment that is a long single-stranded monomer released after the hairpin structure is cleaved by BspQI restriction endonuclease in Example 1.

[0067] FIG. 3 shows the result of agarose gel electrophoresis before and after magnetic bead purification of the product obtained after the hairpin structure is cleaved by BspQI restriction endonuclease in Example 1, where Lane 1 is the crude product before purification, and Lane 2 is the product after magnetic bead purification.

[0068] FIG. 4 shows the result of agarose gel electrophoresis of the product obtained by PCR amplification of the synthesized fragment with terminal aptamers using primers modified with uracil in Example 2.

[0069] FIG. 5 shows the result of agarose gel electrophoresis of the target fragment that is a long single-stranded monomer released after the hairpin structure is cleaved by BspQI restriction endonuclease in Example 2.

[0070] FIG. 6 shows the result of agarose gel electrophoresis after magnetic bead purification of the product obtained after the hairpin structure is cleaved by BspQI restriction endonuclease in Example 2.

[0071] FIG. 7 shows the result of agarose gel electrophoresis of the product obtained by PCR amplification of the synthesized fragment with terminal aptamers using primers modified with uracil in Example 3.

[0072] FIG. 8 shows the result of agarose gel electrophoresis of the target fragment that is a long single-stranded monomer released after the hairpin structure is cleaved by BspQI restriction endonuclease in Example 3.

[0073] FIG. 9 shows the result of agarose gel electrophoresis after magnetic bead purification of the product obtained after the hairpin structure is cleaved by BspQI restriction endonuclease in Example 3.

[0074] FIG. 10 shows the sequence of the product prepared in Example 1 determined by next generation sequencing (NGS).

[0075] FIG. 11 shows the sequence of the product prepared in Example 2 determined by next generation sequencing (NGS).

[0076] FIG. 12 shows the sequence of the product prepared in Example 3 determined by next generation sequencing (NGS).

DETAILED DESCRIPTION

[0077] For further understanding of the method of the present invention, the present invention will be further described in conjunction with examples.

Example 1

[0078] In this example, 48 ug 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).

[0079] The production process of the single-stranded DNA in this example was as follows.

[0080] 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.

[0081] 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.

[0082] 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:

TABLE-US-00001 Reaction system (100 uL): Reagent Volume 253 (288 bp) uL 5*phusion HF buffer 20 10 mM dNTPs 2 100% DMSO 3 253 UF (50 uM) 1 253 UR (50 uM) 1 DNA template 1 phusion U Hot 1 Start DNA polymerase ddH2O 71

[0083] PCR Reaction Procedure

TABLE-US-00002 Reaction condition 98° C. 30 s 98° C. 10 s 63° C. 30 s 72° C. 8 s 72° C. 5 min  4° C. Long time

[0084] 30 Cycles

[0085] The electropherogram of the PCR product is shown in FIG. 1.

[0086] 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:

TABLE-US-00003 Total amount of PCR product: 100 ng uL 253-U 0.48 10*T4 ligase buffer 1 USER 1 T4 ligase 0.50 ddH2O 7.02

[0087] The reaction conditions include 30 min at 37° C., 30 min at 20° C., and storage at 4° C.

[0088] 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:

[0089] (For example, in 100 ul)

TABLE-US-00004 Reagent uL USER treatment sample 10 10*phi29 DNA polymerase buffer 10 dNTP(10 mM) 5 BSA(2 mg/mL) 10 phi29 DNA polymerase 5 ddH2O 60

[0090] 5) 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.

[0091] 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 ddH.sub.2O 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 in FIG. 2.

[0092] 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 in FIG. 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 in FIG. 10, and is consistent with SEQ ID NO: 1.

Example 2

[0093] In this example, 40 ug 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%.

[0094] The test sample was a DNA sequence with a length of 1350 nt.

[0095] The production process of the long single-stranded DNA in this example was as follows.

[0096] 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.

[0097] 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.

[0098] 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:

TABLE-US-00005 Reaction system (100 uL): Reagent Volume 1350 uL 5*phusion HF buffer 20 10 mM dNTPs 2 100% DMSO 3 1350 UF (50 uM) 1 1350 UR (50 uM) 1 DNA template 1 phusion U Hot 1 Start DNA polymerase ddH2O 71

[0099] PCR Reaction Procedure

TABLE-US-00006 Reaction condition 98° C. 30 s 98° C. 10 s 63° C. 30 s 72° C. 1 min 72° C. 5 min  4° C. Long time

[0100] 30 Cycles

[0101] The electropherogram of the PCR product is as shown in FIG. 5.

[0102] 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:

TABLE-US-00007 Total amount of PCR product: 100 ng uL 253-U 0.48 10*T4 ligase buffer 1 USER 1 T4 ligase 0.50 ddH2O 7.02

[0103] The reaction conditions include 30 min at 37° C., 30 min at 20° C., and storage at 4° C.

[0104] 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):

TABLE-US-00008 Reagent uL USER treatment sample 800 10*phi29 DNA polymerase buffer 800 dNTP(10 mM) 400 BSA(2 mg/mL) 800 phi29 DNA polymerase 200 ddH2O 4800

[0105] 5) 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.

[0106] 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 ddH.sub.2O 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 in FIG. 5.

[0107] 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 in FIG. 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 in FIG. 11, and is consistent with SEQ ID NO: 2.

Example 3

[0108] In this example, 10 ug 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%.

[0109] The test sample was a DNA sequence with a length of 2350 nt.

[0110] The production process of the long single-stranded DNA in this example was as follows.

[0111] 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.

[0112] 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.

[0113] 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:

TABLE-US-00009 Reaction system (100 uL): Reagent Volume 1350 uL 5*phusion HF buffer 20 10 mM dNTPs 2 100% DMSO 3 1350 UF (50 uM) 1 1350 UR (50 uM) 1 DNA template 1 phusion U Hot 1 Start DNA polymerase ddH2O 71

[0114] PCR Reaction Procedure

TABLE-US-00010 Reaction condition 98° C. 30 s 98° C. 10 s 63° C. 30 s 72° C. 2 min 72° C. 5 min  4° C. Long time

[0115] 30 Cycles

[0116] The electropherogram of the PCR product is shown in FIG. 7.

[0117] 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:

TABLE-US-00011 Total amount of PCR product: 100 ng uL 253-U 0.48 10*T4 ligase buffer 1 USER 1 T4 ligase 0.50 ddH2O 7.02

[0118] The reaction conditions include 30 min at 37° C., 30 min at 20° C., and storage at 4° C.

[0119] 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):

TABLE-US-00012 Reagent uL USER treatment sample 400 10*phi29 DNA polymerase buffer 400 dNTP(10 mM) 200 BSA(2 mg/mL) 400 phi29 DNA polymerase 200 ddH2O 2400

[0120] 5) 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.

[0121] 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 ddH.sub.2O 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 in FIG. 9.

[0122] 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 in FIG. 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 in FIG. 12, and is consistent with SEQ ID NO: 3.

[0123] 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.