METHOD AND REAGENT FOR CONSTRUCTING NUCLEIC ACID DOUBLE-LINKER SINGLE-STRAND CYCLICAL LIBRARY

Abstract

A method and reagent for constructing a nucleic acid double-joint single-strand cyclical library. The method comprises: breaking a nucleic acid into nucleic acid fragments; connecting a first linker sequence; producing by amplification a first product provided with the first linker sequence at either end, where a U nucleobase is provided on a primer sequence; using USER enzyme to cleave the first product and cyclizing to produce a gap; or, a nicking enzyme recognition sequence is also provided on the primer sequence, using the USER enzyme to cleave the first product, cyclizing and using a nicking enzyme for nicking to produce a nick; performing a restrictive nick/gap translation reaction from the nick or the gap; removing by digestion any portion that did not undergo the restrictive nick/gap translation reaction; connecting a second linker sequence; producing by amplification a second product provided with the second linker sequence at either end; denaturing the second product, and using a mediated sequence for cyclization of a single-strand nucleic acid molecule. The method allows an increase in the length of library insert fragments and obviates the need for gel extraction; the single-strand nucleic acid molecule can be cyclized directly when denatured with heat.

Claims

1. A method for constructing a library of single-stranded cyclic nucleic acid fragments having double adaptors, comprising the following steps: disrupting a nucleic acid into nucleic acid fragments for library construction; ligating a first adaptor sequence to both ends of the nucleic acid fragments; performing a first PCR amplification to obtain first products having the first adaptor sequence at both ends, wherein the primer sequences used in the first PCR have a U base site; digesting the first products with a USER enzyme to form sticky ends, followed by cyclization to generate a gap; or alternatively, in the case that the primer sequences used in the first PCR also have a nickase recognition sequence, digesting the first products with a USER enzyme to form sticky ends, followed by cyclization, and then followed by digestion of the cyclized products with a nickase to generate a nick; initiating controlled nick/gap translation reaction from the nick or gap by using the cyclic nucleic acid molecules as template; digesting and removing the portion of the respective cyclic nucleic acid molecules that does not undergo the controlled nick/gap translation reaction to obtain linear nucleic acid molecules; ligating a second adaptor sequence to both ends of the linear nucleic acid molecules; performing a second PCR amplification to obtain second products having the second adaptor sequence at both ends; and denaturing the second products to obtain single-stranded nucleic acid molecules, and cyclizing one of the single-stranded nucleic acid molecules with a mediating sequence complementary to both ends of the single-stranded nucleic acid molecule to obtain the library of single-stranded cyclic nucleic acid fragments having double adaptors.

2. The method according to claim 1, wherein the first adaptor sequence comprises a first 5′ adaptor sequence and a first 3′ L-type adaptor sequence that respectively ligate to the 3′ end and the 5′ end of each strand of the fragments; the first 5′ adaptor sequence comprises a 5′-end-phosphorylated long strand and a complementary short strand, the long strand having a tag sequence in the middle, the short strand having dideoxy modification at the 3′ end, and the short strand comprising a U base site; and a portion of the first 3′ L-type adaptor sequence which is adjacent to the ligated fragment is complementary to part of the bases of the first 5′ adaptor sequence; and said ligating a first adaptor sequence to both ends of the nucleic acid fragments specifically comprises: dephosphorylating the nucleic acid fragments; subjecting the dephosphorylated nucleic acid fragments to end repairing; ligating the first 5′ adaptor sequence to the 3′ end of each strand of the nucleic acid fragments; digesting the U base site of the short strand of the first 5′ adaptor sequence with a USER enzyme; phosphorylating the USER enzyme-digested nucleic acid fragments; and ligating the first 3′ L-type adaptor sequence to the 5′ end of each strand of the phosphorylated nucleic acid fragments.

3. The method according to claim 1, wherein each of the primer sequences used in the first PCR has a nickase recognition sequence and a U base site; and after digesting the U base site with the USER enzyme, sticky ends are formed at both ends of the nucleic acid fragments, and cyclization occurs due to the complementarity of the sticky ends, generating cyclized nucleic acid molecules.

4. The method according to claim 1, wherein one of the primer sequences used in the first PCR has two U base sites, while the other primer sequence has a U base site; and after digesting the U base sites with the USER enzyme, sticky ends are formed at both ends of the nucleic acid fragments, and cyclization occurs due to the complementarity of the sticky ends, generating cyclized nucleic acid molecules.

5. The method according to claim 1, wherein following cyclizing the digested first products, the method further comprises:digesting the nucleic acid molecules that are not cyclized.

6. The method according to claim 1, wherein one of the primer sequences used in the first PCR harbors a biotin label; and prior to the controlled nick/gap translation reaction, streptavidin-labeled magnetic beads are used to bind the products from the first PCR, such that subsequent reactions are performed on the magnetic beads.

7. The method according to claim 1, wherein in the controlled nick/gap translation reaction, the length of the gap translation fragments generated is controlled by controlling at least one factor selected from the group consisting of the molar ratio of dNTPs to the nucleic acid molecules as template, the enzymatic reaction temperature and the enzymatic reaction time.

8. The method according to claim 1, wherein said digesting and removing the portion of the respective cyclic nucleic acid molecules that does not undergo the controlled nick/gap translation reaction specifically comprises:first degrading the cyclic nucleic acid molecules with an enzyme having 5′-3′ exonuclease activity until the gaps at both ends meet; and then degrading the resulting single strands with an enzyme having 3′-5′ exonuclease activity or a single strand exonuclease.

9. The method according to claim 1, wherein the second adaptor sequence comprises a second 5′ adaptor sequence and a second 3′ L-type adaptor sequence that respectively ligate to the 3′ end and the 5′ end of each strand of the linear nucleic acid molecules; the second 5′ adaptor sequence comprises a 5-end-phosphorylated long strand and a complementary short strand, the short strand having dideoxy modification at the 3′ end, and the short strand comprising a U base site; and a portion of the second 3′ L-type adaptor sequence which is adjacent to the ligated fragment is complementary to part of the bases of the second 5′ adaptor sequence; and said ligating a second adaptor sequence to both ends of the linear nucleic acid molecules specifically comprises: dephosphorylating the linear nucleic acid molecules; subjecting the dephosphorylated linear nucleic acid molecules to end repairing; ligating the second 5′ adaptor sequence to the 3′ end of each strand of the linear nucleic acid molecules; digesting the U base site of the short strand of the second 5′ adaptor sequence with a USER enzyme; phosphorylating the USER-enzyme digested fragments; and ligating the second 3′ L-type adaptor sequence to the 5′ end of each strand of the phosphorylated linear nucleic acid molecules.

10. The method according to claim 1, wherein following cyclizing the single-stranded nucleic acid molecules, the method further comprises: digesting the single-stranded nucleic acid molecules that are not cyclized.

11. A reagent for constructing a library of single-stranded cyclic nucleic acid fragments having double adaptors, comprising the following components: a first adaptor sequence, which comprises a first 5′ adaptor sequence and a first 3′ L-type adaptor sequence that respectively ligate to the 3′ end and the 5′ end of each strand of the fragments, wherein the first 5′ adaptor sequence comprises a 5′-end-phosphorylated long strand and a complementary short strand, the long strand having a tag sequence in the middle, the short strand having dideoxy modification at the 3′ end, and the short strand comprising a U base site; and a portion of the first 3′ L-type adaptor sequence which is adjacent to the ligated fragment is complementary to part of the bases of the first 5′ adaptor sequence; primers for a first PCR, which have U base sites or have a nickase recognition sequence and a U base site, and which are used to obtain first products having the first adaptor sequence at both ends by the first PCR amplification; a nickase, which is used for digesting the first products to generate a nick; a USER enzyme, which is used for digesting the first products to generate sticky ends and gaps for cyclization; components for gap translation reaction, which are used for initiating controlled nick/gap translation reaction from the nick or gap by using the cyclic nucleic acid molecules as template; a digestive enzyme, which is used for digesting and removing the portion of the respective cyclic nucleic acid molecules that does not undergo the controlled nick/gap translation reaction to obtain linear nucleic acid molecules; a second adaptor sequence, which comprises a second 5′ adaptor sequence and a second 3′ L-type adaptor sequence that respectively ligate to the 3′ end and the 5′ end of each strand of the linear nucleic acid molecules; wherein the second 5′ adaptor sequence comprises a 5-end-phosphorylated long strand and a complementary short strand, the short strand having dideoxy modification at the 3′ end, and the short strand comprising a U base site; and a portion of the second 3′ L-type adaptor sequence which is adjacent to the ligated fragment is complementary to part of the bases of the second 5′ adaptor sequence; primers for a second PCR, which are used for performing a second PCR amplification to obtain second products having the second adaptor sequence at both ends; and a mediating sequence, which is complementary to both ends of one of the single-stranded nucleic acid molecules obtained following denaturation of the second products, and which is used for cyclizing the single-stranded nucleic acid molecule to obtain the library of single-stranded cyclic nucleic acid fragments having double adaptors.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0050] FIG. 1 is the flow-process diagram showing the method for constructing a library of single-stranded cyclic nucleic acid fragments having double adaptors according to an embodiment of the present invention;

[0051] FIG. 2 is a diagram showing the basic principle underlying the generation of CNT gaps according to an embodiment of the present invention;

[0052] FIG. 3 is a diagram showing the basic principle underlying the generation of CNT nicks according to an embodiment of the present invention;

[0053] FIG. 4 is a diagram comparing the principle of formation of insert fragments and separation and cyclization of single strands according to a prior art process and the process of the present invention;

[0054] FIG. 5 is a diagram comparing a directional adaptor ligation process of the prior art and the L-type adaptor ligation process of the present invention;

[0055] FIG. 6 is a schematic depiction of the library construction strategy of controlled nick/gap translation reaction on magnetic beads (CNT on beads) according to an embodiment of the present invention; and

[0056] FIG. 7 presents graphs showing the results of Agilent 2100 detection of the PCR products following adaptor B ligation according to an embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0057] The present invention is described in further detail below by reference to particular embodiments. Unless otherwise stated, the techniques used in the following embodiments are all conventional techniques known to a person skilled in the art, and the instruments, equipments and reagents used are all publicly available, e.g. commercially available, to a person skilled in the art.

[0058] In the present invention, the concepts of “first” and “second” used in any cases should not be construed as conveying the meaning of order or technique, and they serve only to distinguish the objects to which they refer from other objects.

[0059] Reference is made to FIG. 1, in which a method for constructing a library of single-stranded cyclic nucleic acid fragments having double adaptors according to an embodiment of the present invention is shown. The method comprises the following steps: disrupting a genomic DNA to form nucleic acid fragments for constructing the library; subjecting the fragments to dephosphorylation and end repairing reactions; ligating a 5′ adaptor A sequence to the fragments; subjecting the fragments to USER enzyme digestion and phosphorylation treatment; ligating a 3′ L-type adaptor A sequence to the fragments; subjecting the fragments to PCR amplification to obtain products having the 5′ adaptor A sequence and the 3′ L-type adaptor A sequence at both ends, wherein the sequence of the primers used in the PCR comprises a U base site; digesting the U base site with a USER enzyme to generate gaps, and cyclizing the products following USER enzyme digestion to generate cyclic nucleic acid molecules; initiating controlled nick translation (CNT) from the gaps; degrading the cyclic nucleic acid molecules with an enzyme having 5′-3′ exonuclease activity until the gaps at both ends meet, and then degrading the resulting single strands with an enzyme having 3′-5′ exonuclease activity or a single strand exonuclease to obtain linear nucleic acid molecules; subjecting the linear nucleic acid molecules to dephosphorylation and end repairing; ligating a 5′ adaptor B sequence to the nucleic acid molecules, phosphorylating the nucleic acid molecules, and ligating a 3′ L-type adaptor B sequence to the nuclei acid molecules; subjecting the nucleic acid molecules to PCR amplification to obtain products having the 5′ adaptor B sequence and the 3′ L-type adaptor B sequence at both ends; subjecting the products to denaturation treatment to obtain single-stranded nucleic acid molecules, and cyclizing one of the single-stranded nucleic acid molecules with a mediating sequence complementary to both ends of the single-stranded nucleic acid molecule to obtain the library of single-stranded cyclic nucleic acid fragments having double adaptors.

[0060] In the method according to the present invention for constructing a library of single-stranded cyclic nucleic acid fragments having double adaptors, as shown in FIG. 1, a U base site is introduced into the primers for the first PCR, and is digested with a USER enzyme to generate a gap as an initiation point for controlled nick translation reaction. The basic principle underlying the generation of the gap is as shown in FIG. 2: (1) after ligating the 5′ adaptor A sequence and the 3′ L-type adaptor A sequence, the adaptor A ligation products are amplified using primers respectively having two Us and one U; (2) the U bases are digested with a USER enzyme, forming phosphorylated 3′ ends and 5′ ends at the nicks; (3) performing double strand cyclization by means of the sticky ends generated in the enzyme digestion, wherein after cyclization, the gap on one strand (gap 1, formed by USER enzyme digestion) involves a phosphorylated 3′ end and a phosphorylated 5′ end, and the gap on the other strand (gap 2, formed due to lack of a matching base at the site after cyclization) involves a dephosphorylated 3 ′ end and a phosphorylated 5′end; and (4) performing dephosphorylation treatment to dephosphorylate the 3′ end of gap 1, so as to provide an effective initiation site for CNT.

[0061] In the present invention, nicks can also be generated by employing the principle as shown in FIG. 3 and serve as the initiation site of controlled nick translation reaction. In a prior art process, a III class endonuclease recognition sequence is introduced into the adaptor A sequence, and following adaptor A sequence ligation and cyclization, the double strands are digested with a III class endonuclease to generate linear double-stranded DNA; while in a process of the present invention, nickase recognition sequences are introduced into the adaptor A sequence, and following adaptor A sequence ligation and cyclization, each single strand of the cyclized DNA is digested with a nickase (such as Nb.BsrDI, Nb.BsmI, Nt.BbvCI, Nb.Bbv.Nb.BtsI or Nt.BstNBI, etc.) to generate a nick on each single strand, so as to provide an effective initiation site for CNT.

[0062] In the present invention, the reaction initiated from the nick or gap is referred to as “controlled nick/gap translation reaction”, because the length of the target fragments generated by the reaction can be controlled in a certain range by controlling the usage amount of dNTPs, the usage amount of the nucleic acid molecules as template, the enzyme reaction temperature and time, among other factors. Nucleic acid fragments having a length in a certain range are suitable for a particular sequencing platform. In general, the length of the target fragments in the present invention is preferably controlled in the range of 50-250 bp. Such a length is several times longer than the length of the target fragments obtained by a conventional library construction protocol for a CG sequencing platform. Moreover, the CNT technique of the present application allows for controlling the library insert fragments in a very narrow range without performing gel recovery, which effectively enhances the operability of the gap translation technique.

[0063] Reference is now made to FIG. 4, in which a prior art process and a process of the present invention are compared. The prior art process takes advantage of the cleaving property of a III class endonuclease, which digests the genomic DNA 25-27 bp from both sides of the adaptor A, forming a target DNA fragment of about 104 bp. Subsequently, the DNA fragments of more than 200 bp not having the adaptor A are removed by means of a two-step magnetic bead purification process. Following this sequence selection with magnetic beads, the digestion products obtained are still mingled with some non-target DNA fragments having main bands of 100-200 bp. Following ligation of the adaptor B, the DNA fragments ligated with the adaptor B are amplified with primers having a biotin-labeled base on one of the primers, wherein the single strands amplified with the primer having a biotin-labeled base thereon are non-target single-stranded nucleic acids. Subsequently, DNA fragments ligated with the adaptor B are enriched using streptavidin magnetic beads, and further, DNA fragments ligated with the adaptor A are enriched by capturing via specific sequence hybridization. Finally, the double-stranded DNA is unwound by means of alkali denaturation to elute the target single-stranded nucleic acids from the streptavidin magnetic beads, and then the target single-stranded nucleic acids are cyclized using a mediating sequence. The whole procedure of the prior art process not only involves tedious steps and lengthy operations, but also consumes expensive reagents (mainly streptavidin magnetic beads). In the process of the present invention, a nick is made using a nickase on the two strands of adaptor A respectively. Then, by taking advantage of the nick translation function of a polymerase in the presence of dNTPs, the nicks are moved from the adaptor A region to either side of the adaptor A. The length through which the nicks are moved can be flexibly controlled by controlling the molar ratio of the dNTPs to the template DNA, the reaction temperature and the reaction time, among other conditions, and the size of the main band of the fragments through which the nicks are moved can be controlled in the range of 50-250 bp. Subsequently, the non-target DNA fragments not having adaptor A are digested by a two-step exonuclease digestion reaction, the remaining fragments being the target DNA fragments having adaptor A, which are subjected to ligation with adaptor B and PCR amplification using primers not having a biotin label. The resulting double-stranded DNAs are unwound simply by high temperature denaturation, and the resulting target single strands are cyclized by means of a mediating sequence, thus separating and cyclizing the target single-stranded DNAs. It can be seen that the single strand cyclization process of the present invention only entails denaturation by heat and hybridization with a mediating sequence for successful separation and cyclization of the target single-stranded nucleic acids. Hence, the process of the present invention not only involves simple steps and easy operations, but also eliminates the need to consume large amounts of expensive reagents, leading to decreased cost in library construction.

[0064] In a preferred embodiment of the present invention, ligation is performed using an L-type adaptor instead of a conventional adaptor. Reference is now made to FIG. 5, in which a prior art adaptor ligation process and the adaptor ligation process of the present invention are compared. The prior art process employs a directional adaptor ligation process, whereby directional ligation of the adaptors is ensured and at the same time the problem of inter-ligation of DNA fragments is minimized. The prior art process involves separately designing and stepwise ligating the 5′ adaptor and the 3′ adaptor. The addition of an adaptor on either end entails synergy of an adaptor sequence, a blocking sequence and a primer sequence. The whole procedure entails 6 enzymatic reaction steps, i.e., dephosphorylation, end repairing, adding 5′ adaptor, primer extension, adding 3′ adaptor, nick translation and ligation, as well as 5 purification operations, in order to directionally add the sequence of adaptor A to both ends of the target DNA. Such a prior art process suffers from tedious steps, high cost and long period in library construction (the cost of sequences, the cost of enzymatic reaction reagents, and the cost of purification) and high consumption of samples, which does not comply with the requirement of high efficiency and convenience for library construction. On the contrary, the L-type adaptor ligation process of the present invention can increase library construction efficiency and decrease library construction cost while ensuring directional ligation of the adaptors. Although the L-type adaptor ligation process also employs stepwise ligation, the steps involved are simple in comparison to the prior art process. First, a 5′ adaptor having a blocking sequence is added. The blocking sequence is 12 bp long and completely complementary to the 5′ adaptor, forming a partially complementary double-stranded structure to facilitate ligation of DNA fragments with the 5′ adaptor. The blocking sequence has a dideoxy modification at the 3′ end and a dephosphorylized base at the 5′ end. This not only ensures that the 5′ end adaptor is directionally ligated to the 3′ end of DNA fragments, but also that the blocking sequence would not ligate to the 5′ end of DNA fragments. The blocking sequence has a U base in the middle, such that upon treatment with a USER enzyme, the blocking sequence is “degraded” into two single-stranded DNA fragments of smaller than 8 bp and thus is unwound and detached from the 5′ adaptor. Then, an “L” type single-stranded 3′ adaptor is added via a process of hybridization followed by ligation. Prior to addition of the L-type adaptor, the 5′ end of the DNA fragments needs to be phosphorylated for deblocking. Experiments demonstrated that USER enzyme treatment can be performed concurrently with phosphorylation reaction. Following reaction, purification is conducted with magnetic beads, and the magnetic beads having been washed can be directly resuspended in a ligation reaction buffer for the next step. The L-type adaptor is so ingeniously designed that the last 8 bases at the 3′ end thereof are complementary to the last 8 bases at the 5′ end of the 5′ adaptor, such that the L-type adaptor can directly hybridize to the 5′ adaptor. Then, by using a ligase to block the nick, the L-type 3′ adaptor is ligated to the 5′ end of the DNA fragments. As some of the bases of the L-type adaptor are complementary to some of the bases at the 5′ end of the 5′ adaptor, while the other bases are not complementary to each other, the adaptor appears an L form, hence called L-type adaptor. After the reaction ends, the ligation products having added with the adaptors can be purified and recovered by further adding a suitable amount of a magnetic bead binding buffer into the magnetic beads. The sequence of adaptor A can be directionally added to both ends of the target DNA relatively fast only through five enzymatic reaction steps, i.e., dephosphorylation, end repairing, adding 5′ adaptor, one-step reaction of USER digestion and phosphorylation, adding 5′ L type adaptor, as well as three purification operations in the whole procedure. Thus, the steps are simple, the cost of library construction is lowered, and the period of library construction shortened.

[0065] In a preferred embodiment of the present invention, one of the primer sequences used in the first PCR amplification harbors a biotin label. Prior to the controlled nick/gap translation reaction, streptavidin-labeled magnetic beads are used to bind the products from the first PCR amplification, such that subsequent reactions are performed on the magnetic beads. Reference is now made to FIG. 6, in which the on-beads reactions in the present invention are illustrated. Specifically, one of the strands of the products from the first PCR amplification harbors a biotin label, such that the products having been cyclized can bind with streptavidin-labeled magnetic beads. From step 8 to step 14, the enzymatic reaction in each step is performed on the streptavidin-labeled magnetic beads. After reaction, the magnetic beads only need to be adsorbed onto a magnetic rack to remove the reaction solution and then washed to remove the impurities, in order to be ready for the enzymatic reaction in the next step. This eliminates the need for a purification operation following enzymatic reaction in each step of a conventional library construction process, thus saving the reagents for purification. Moreover, the operation time of the magnetic bead washing process is shorter than that of a conventional purification process.

[0066] The above analysis shows that, in a preferred embodiment of the present invention, by using the unique CNT technique, L-type adaptor ligation technique, and direct single strand cyclization technique following simple thermal denaturation, improvement and optimization are successfully achieved on the library contruction procedure of the double adaptor library construction process with a CG sequencing platform, such that the size of the library insert fragments is increased 2-10 fold compared with the original size, the whole library construction period and cost are decreased by about 40%, and the initial amount required for library construction is decreased from 3 μg to 500 ng.

[0067] The present invention is illustrated in detail with reference to the following example.

[0068] 1. Disruption of genomic DNA:Genomic DNA can be disrupted in a number of methods, and no matter they are a physical ultrasonic method or an enzymatic reaction method, well-established protocols are commercially available. In this example, the physical ultrasonic method was employed for disruption.

[0069] A 96-well PCR plate was added with a polytetrafluoroethylene line. 1 μg of genomic DNA was added, then TE buffer solution or enzyme-free pure water was added to make up to 100 μL. The plate was sealed with a membrane and then placed in an E220 ultrasonic disruptor to conduct ultrasonic disruption. The conditions for disruption were shown in Table 1.

TABLE-US-00001 TABLE 1 Parameter Value Filling factor 21% Pressure (PIP) 500 Pulse factor 500 Disruption time 20 s, twice

[0070] 2. Selection of fragments following disruption: A magnetic bead purification method or gel recovery method can be employed. In this example, the magnetic bead purification method was used.

[0071] The disrupted DNA was added with 45 μL of Ampure XP magnetic beads, and the mixture was mixed well and stood for 7-15 min. The resulting mixture was placed on a magnetic rack, and supernatant was collected. The supernatant was added with 18 μL of Ampure XP magnetic beads, and the mixture was mixed well and stood for 7-15 min. The resulting mixture was placed on a magnetic rack, and supernatant was aspirated off. The magnetic beads were washed twice with 75% ethanol and air dried. Then 30 μL of TE solution was added, and the mixture was mixed well and stood for 7-15 min to allow the recovered products to dissolve.

[0072] 3. Dephosphorylation reaction of the fragments: The recovered products from the previous step were used, and a system was formulated according to Table 2.

TABLE-US-00002 TABLE 2 Reaction component Volume (μL) 10× NEB buffer 2 3.6 Shrimp alkaline phosphatase (1 U/μL) 3.6 Total 7.2

[0073] 7.2 μL of the reaction solution was added to the recovered products from the previous step. The mixture was mixed well and incubated at 37° C. for 45 min and at 65° C. for 10 min. Then the temperature was ramped down to 4° C. at a rate of 0.1° C. per second.

[0074] 4. End repairing of the fragments. A system was formulated according to Table 3.

TABLE-US-00003 TABLE 3 Reaction component Volume (μL) Enzyme-free water 7.32 10× NEB buffer 2 1.08 0.1M adenosine triphosphate 0.48 25 mM deoxyribonucleoside triphosphate 0.48 Bovine serum albumin (10 mg/ml) 0.24 T4 deoxyribonucleic acid polymerase (3 U/μL) 1.2 Total 10.8

[0075] The system was mixed well and the products from the previous step were added. The mixture was mixed well and incubated at 12° C. for 20 min. The reaction was purified with 48 μL of Ampure XP magnetic beads, and the recovered products were dissolved using 40 μL of TE buffer solution.

[0076] 5. Ligation of 5′ adaptor A sequence : The 5′ adaptor A sequence used in this example was as follows (in this example, the sequences are shown in the direction of from 5′ end to 3′ end from left to right, “//” represents modification group, “phos” represents phosphorylation, “dd” represents dideoxy, “bio” represents biotin, and the characters in bold represent a tag sequence).

TABLE-US-00004 5′ adaptor A sequence: (SEQ ID NO: 1) /5phos/ACTGCTGACGTACTGTGTCATAAATAGCACGAGACGTTCTCGA CT/3ddC/; 5′ blocking sequence: (SEQ ID NO: 2) TACGUCAGCAG/3ddT/.

[0077] A mixed solution of 5′ adaptor A (10 μM) was formulated according to Table 4.

TABLE-US-00005 TABLE 4 Reaction component Volume (μL) 5′ adaptor A sequence (100 μM) 12 5′ blocking sequence (100 μM) 10 TE buffer 78 Total 100

[0078] 4.5 μL of the mixed solution of 5′ adaptor A formulated (10 μM) was added into the products of the previous step, and the mixture was sufficiently mixed.

[0079] A ligation reaction system was formulated according to Table 5.

TABLE-US-00006 TABLE 5 Reaction component Volume (μL) Enzyme-free pure water 13.1 2× ligation buffer 1 60 T4 DNA ligase (fast) (600 U/μL) 2.4 Total 75.5

[0080] The 2× ligation buffer 1 used in this example was formulated according to Table 6.

TABLE-US-00007 TABLE 6 Reaction component Volume (μL) 1M tromethamine 37.5 1M citric acid 9.6 1M magnesium chloride 35 1M trisodium citrate 20 100% glycerol 50 10% Tween-80 1 30% polyethylene glycol 8000 333 0.1M adenosine triphosphate 10 0.5M trichloroethyl phosphate (pH 7.0) 2 Enzyme-free pure water 1.9 Total 500

[0081] A mixed solution of the ligation reaction system with the adaptor and the products was mixed well, and the mixture was incubated at 25° C. for 30 min and at 65° C. for 10 min, followed by decreasing the temperature to 4° C.

[0082] 6. One-step reaction of USER enzyme digestion and phosphorylation: Into the reaction solution from the previous step were added 1.2 μL of USER enzyme (1 U/μL) and 1.2 μL of T4 polynueleotide kinase (10 U/μL). The mixture was mixed well and incubated at 37° C. for 20 min. The reaction was purified with 108 μL of Ampure XP magnetic beads. The beads were rinsed with 70% ethanol twice, and with the rinsing liquid having been blot up, were air dried at room temperature for 2 min. Then the beads were resuspended in 48 μL of a 3′ L-type adaptor reaction system.

[0083] 7. Ligation of 3′ L-type adaptor A sequence:The 3′ L-type adaptor A sequence used in this example was as shown below: CGTTCTCGACUCAGCAGT (SEQ ID NO: 3).

[0084] The 3′ L-type adaptor reaction system was formulated according to Table 7:

TABLE-US-00008 TABLE 7 Reaction component Volume (μL) Enzyme-free pure water 28.98 3× ligation buffer 2 16.02 L-type adaptor sequence (100 μM) 1.8 T4 DNA ligase (fast) (600 U/μL) 1.2 Total 48

[0085] The Ampure XP magnetic beads resuspended in 48 μL of the 3′ L-type adaptor reaction system was incubated in an incubator at a rotation speed of 300 rpm at 25° C. for 30 min. After reaction was complete, 43.2 μL of Ampure XP magnetic bead binding buffer was added to conduct incubation at room temperature for 10 min. The supernatant was removed and the beads were washed with 70% ethanol twice, followed by air dried at room temperature for 5-10 min. The recovered products were dissolved with 30 μL of TE buffer solution.

[0086] This step achieved the ligation of the target nucleic acid fragments with the adaptor A. The total amount and yield of the products before and after ligation were as shown in Table 8.

TABLE-US-00009 TABLE 8 Concentration Total amount (ng/μL) (ng) Yield Product 1 5.95 178.5 59.5% Product 2 6.11 183.3 61.1% Product 3 5.92 177.6 59.2%

[0087] 8. Polymerase chain reaction:

[0088] The sequence of primer 1 was as follows:

TABLE-US-00010 (SEQ ID NO: 4) GTCGAGAACGUCTCG/iBiodT/GCTATTTATGACACAGTACGUCAGCA G;

[0089] The sequence of primer 2 was as follows:

TABLE-US-00011 (SEQ ID NO: 5) ACGTTCTCGACUCAGCAG.

[0090] A PCR system was formulated according to Table 9.

TABLE-US-00012 TABLE 9 Reaction component Volume (μL) Enzyme-free pure water 186.5 2× PfuTurbo Cx buffer 275 PfuTurbo Cx heat-activated nucleic acid 11 polymerase (2.5 U/μL) 20 μM primer 1 13.75 20 μM primer 2 13.75 Total volume 500

[0091] Into the above system was added 50 μL (180 ng) of the recovered products from the previous step. The mixture was mixed well and reaction was allowed under the conditions set out in Table 10.

TABLE-US-00013 TABLE 10 Temperature Time Cycle 95° C. 3 min 1 95° C. 30 s  .sup.  7 56° C. 30 s  .sup.  72° C. 4 min 68° C. 10 min  1 Ramping the temperature down to 4° C. at a rate of 0.1° C./s  4° C. Holding the temperature —

[0092] After reaction was complete, purification was conducted using 550 μL of Ampure XP magnetic beads, and the recovered products were dissolved with 80 μL of TE buffer. 1 μL of the recovered products was assayed with a Qubit dsDNA HS assay kit (Invitrogen Corp.) to quantitate the concentration of the products (Table 11). 2 μL of the products was used for reaction at the next step.

TABLE-US-00014 TABLE 11 Concentration Total amount PCR amplification (ng/μL) (ng) efficiency Product 1 57.4 4592 1.59 Product 2 52 4160 1.57

[0093] 9. Removal of uracil: A reaction solution as shown in Table 12 below was formulated.

TABLE-US-00015 TABLE 12 Reaction component Volume (μL) Enzyme-free pure water 25.8 10× Taq buffer 11 USER enzyme (1  U/μL) 13.2 Total volume 50 indicates data missing or illegible when filed

[0094] The above reaction solution was added into 60 μL (2 μg) of the reaction products from the previous step, and the mixture was mixed well and incubated at 37° C. for 1 h.

[0095] 10. Double strand cyclization:Reaction system 1 as shown in Table 13 below was formulated.

TABLE-US-00016 TABLE 13 Reaction component Volume (μL) Enzyme-free pure water 1520 10× TA buffer 180 Total volume 1700

[0096] The reaction products from the previous step were added into reaction system 1. The mixture was mixed well and evenly dispensed into 4 tubes. The tubes were placed in a water bath at 50° C. for reaction for 15 min. After the reaction was complete, the tubes were placed in a water bath at ambient temperature for reaction for 15 min.

[0097] Reaction system 2 as shown in Table 14 below was formulated.

TABLE-US-00017 TABLE 14 Reaction component Volume (μL) Enzyme-free pure water 98 20× Circ buffer 100 T4 DNA ligase (fast) (600 U/μL) 2 Total volume 200

[0098] Into each of the 4 tubes of reaction system 1 was added 50 μL of reaction system 2, and the tubes were incubated at room temperature for 1 h.

[0099] 330 μL of Ampure XP magnetic beads was added into the reaction products of each tube (500 μL). The mixture in each tube was mixed well and stood for 7-15 min. After placing the tubes on a magnetic rack, supernatant was collected. The supernatant was added with 170 μL of Ampure XP magnetic beads, and the mixture was mixed well and stood for 7-15 min. After placing the tubes on a magnetic rack, supernatant was aspirated off, and the magnetic beads were washed with 75% ethanol twice. After air drying the magnetic beads, 65 μL of TE buffer was added to each of the 4 tubes to dissolve the purified products.

[0100] 11. Linear digestion: A reaction system as shown in Table 15 below was formulated.

TABLE-US-00018 TABLE 15 Reaction component Volume (μL) Enzyme-free pure water 0.7 Plasmid-Safe 9X reaction buffer 8.9 Plasmid-Safe ATP-dependent DNase 10.4 Total volume 20

[0101] The products from the previous step were added into the reaction system, and the mixture was mixed well and incubated at 37° C. for 1 h.

[0102] Purification was conducted using 80 μL of Ampure XP magnetic beads, and the recovered products were dissolved using 82 μL of TE buffer. 1 μL of the recovered products were assayed with a Qubit dsDNA HS assay kit (Invitrogen Corp.) to quantitate the concentration of the products. 700 ng of the products was used for reaction at the next step. The initiation site for CNT reaction on the double-strand cyclized DNA formed in this example is in the form of gap.

[0103] 12. Dephosphorylation:A reaction system as shown in Table 16 below was formulated.

TABLE-US-00019 TABLE 16 Reaction component Volume (μL) Enzyme-free pure water 39 10× NEBuffer 3 14 Endo IV (10 U/μL) 7 Total volume 60

[0104] 80 μL (700 ng) of the products from the previous step was added into the reaction system, and the mixture was mixed well and incubated at 37° C. for 1 h.

[0105] Purification was conducted using 210 μL of Ampure XP magnetic beads, and the recovered products were dissolved using 55 μL of TE buffer. The concentration of the products was quantitated using a Qubit dsDNA HS assay kit (Invitrogen Corp.). 500 ng of the products were used for reaction at the next step.

[0106] 13. Binding to streptavidin magnetic beads:Magnetic bead binding buffer and rinsing buffers were formulated according to the systems as shown in Tables 17-19.

TABLE-US-00020 TABLE 17 2× LSBB (Low Salt Bead Binding Buffer) Component Content Trihydroxymethyl aminomethane (pH 7.5) 100 mM Sodium chloride 300 mM Tween-20 0.005%

TABLE-US-00021 TABLE 18 LSWB (Low Salt Bead Wash Buffer) Component Content Trihydroxymethyl aminomethane  50 mM (pH 7.5) Sodium chloride 150 mM Tween-20 0.05%

TABLE-US-00022 TABLE 19 HSWB (High Salt Bead Wash Buffer) Component Content Trihydroxymethyl aminomethane  50 mM (pH 7.5) Sodium chloride 500 mM Tween-20 0.05%

[0107] 75 μL of MyOne Streptavidin C1 magnetic beads was used, and the supernatant was aspirated off. The beads were rinsed with 350 μL of 1×LSBB, then resuspended in 100 μL of 2×LSBB. 100 μL of dephosphorylated cyclized DNA (570 ng) diluted with enzyme-free pure water was added. The mixed solution of the beads and the DNA was incubated at 30 rpm at room temperature for 1 h.

[0108] The beads were rinsed with 350 μL of HSWB and 350 μL of LSWB respectively once, and equilibrated with 200 μL of 1×NEBuffer2 (containing 0.025% Tween-20). After each rinsing, the supernatant should be aspirated off, and after the last equilibration, the supernatant should be aspirated off thoroughly. The beads were placed on ice to precool for at least 10 min.

[0109] 14. On-beads reaction for CNT: A reaction system as shown in Table 20 below was formulated.

TABLE-US-00023 TABLE 20 Volume Reaction component (μL) Enzyme-free pure water 51.2 10 × NEBuffer2 6 2.5 mM dNTP (1:175) 2 PolI DNA polymerase (10 U/μL) 0.8 Total volume 60

[0110] 60 μL of the reaction system was quickly added into the precooled magnetic beads, and the mixture was rapidly mixed well and incubated at 8° C. for 15 min.

[0111] 1.5 μL of 0.5 M EDTA was added, and the mixture was mixed well. The beads were rinsed with 350 μL of HSWB and 350 μL of LSWB respectively once, and equilibrated with 200 μL of 1×NEBuffer4 (containing 0.025% Tween-20). The supernatant was aspirated off thoroughly.

[0112] 15. On-beads reaction for 3′-5′ exonuclease digestion:A reaction system as shown in Table 21 below was formulated.

TABLE-US-00024 TABLE 21 Reaction component Volume (μL) Enzyme-free pure water 65.5 10 × NEBuffer4 8 T7 Exo (10 U/μL) 6.5 Total volume 80

[0113] 80 μL of the reaction system was quickly added into the magnetic beads from the previous step, and the mixture was rapidly mixed well and incubated at 25° C. for 1 h. 2 μL of 0.5 M EDTA was added, and the mixture was mixed well. The beads were rinsed with 350 μL of HSWB and 350 μL of LSWB respectively once, and equilibrated with 200 μL of 1×Exo VII reaction buffer (containing 0.025% Tween-20). The supernatant was aspirated off thoroughly.

[0114] 16. On-beads reaction for single strand digestion:A reaction system as shown in Table 22 below was formulated.

TABLE-US-00025 TABLE 22 Volume Reaction component (μL) Enzyme-free pure water 27.8 5 × ExoVII reaction buffer 7 Exo VII (10 U/μL) 0.2 Total volume 35

[0115] 35 μL of the reaction system was quickly added into the magnetic beads from the previous step, and the mixture was rapidly mixed well and incubated at 37° C. for 30 min. 0.8 μL of 0.5 M EDTA was added, and the mixture was mixed well. The beads were rinsed with 350 μL of HSWB and 350 μL of LSWB respectively once, and equilibrated with 200 μL of 1×Exo VII reaction buffer (containing 0.025% Tween-20). The supernatant was aspirated off thoroughly.

[0116] 17. On-beads reaction for end repairing:A reaction system as shown in Table 23 below was formulated.

TABLE-US-00026 TABLE 23 Reaction component Volume (μL) Enzyme-free pure water 69.2 10 × NEBuffer2 8 25 mM dNTP 0.8 T4 deoxyribonucleic acid polymerase 2 (3 U/μL) Total volume 80

[0117] 80 μL of the reaction system was quickly added into the magnetic beads from the previous step, and the mixture was rapidly mixed well and incubated at 12° C. for 20 min. 2 μL of 0.5 M EDTA was added, and the mixture was mixed well. The beads were rinsed with 350 μL of HSWB and 350 μL of LSWB respectively once, and equilibrated with 200 μL of 1×NEBuffer2 reaction buffer (containing 0.025% Tween-20). The supernatant was aspirated off thoroughly.

[0118] 18. On-beads reaction for dephosphorylation: A reaction system as shown in Table 24 below was formulated.

TABLE-US-00027 TABLE 24 Reaction component Volume (μL) Enzyme-free pure 48 water 10 × NEBuffer2 6 FastAp (1 U/μL) 6 Total volume 60

[0119] 60 μL of the reaction system was quickly added into the magnetic beads from the previous step, and the mixture was rapidly mixed well and incubated at 37° C. for 45 min. 1.5 μL of 0.5 M EDTA was added, and the mixture was mixed well. The beads were rinsed with 350 μL of HSWB and 350 μL of LSWB respectively once, and equilibrated with 200 μL of 1×NEBuffer2 reaction buffer (containing 0.025% Tween-20). The supernatant was aspirated off thoroughly.

[0120] 19. On-beads reaction for ligation of 5′ adaptor B sequence:The adaptor B sequence used in this example was as follows:

TABLE-US-00028 5′ adaptor B sequence: (SEQ ID NO: 6) /5Phos/AAGTCGGAGGCCAAGCGTGCTTAGGA; 5′ blocking sequence: (SEQ ID NO: 7) GCCUCCGACT/ddT/.

[0121] A mixed solution of 5′ adaptor B (10 μM) was formulated according to the system as shown in Table 25.

TABLE-US-00029 TABLE 25 Reaction component Volume (μL) 5′ adaptor B sequence 12 (100 μM) 5′ blocking sequence 10 (100 μM) TE buffer 78 Total 100

[0122] A ligation reaction system was formulated according to the system as shown in Table 26.

TABLE-US-00030 TABLE 26 Volume Reaction component (μL) Enzyme-free pure water 43 3 × ligation buffer 2 27 10 μM of the mixed solution 8 of 5′ adaptor B Total 78

[0123] 78 μL of the reaction system was added into the magnetic beads from the previous step, and the mixture was mixed well. 2 μL of T4 DNA ligase (fast) (600 U/μL) were added rapidly, and the mixture was mixed well and incubated at 25° C. for 60 min and at 65° C. for 10 min, followed by cooling to 4° C. 1.5 μL of T4 polynueleotide kinase (10 U/μL) was added, and the mixture was mixed well and incubated at 37° C. for 20 min. 2 μL of 0.5 M EDTA was added, and the mixture was mixed well. The beads were rinsed with 350 μL of HSWB and 350 μL of LSWB respectively once, and equilibrated with 200 μL of 1×NEBuffer2 reaction buffer (containing 0.025% Tween-20). The supernatant was aspirated off thoroughly.

[0124] 20. On-beads reaction for ligation of 3′ L-type adaptor B sequence:The 3′ L-type adaptor B sequence used in this example was/5Phos/CATGTAGTGTACGATCCGACTT (SEQ ID NO: 8).

[0125] A 3′ L-type adaptor reaction system was formulated according to the system as shown in Table 27:

TABLE-US-00031 TABLE 27 Reaction component Volume (μL) Enzyme-free pure water 44.5 3 × ligation buffer 2 27 L-type adaptor sequence (100 μM) 6.5 T4 DNA ligase (fast) (600 U/μ L) 2 Total 80

[0126] 80 μL of the reaction system was added into the magnetic beads from the previous step, and the mixture was mixed well and incubated at 25° C. for 60 min, followed by decreasing the temperature to 14° C. at a rate of 0.1° C. per second. 2 μL of 0.5 M EDTA was added, and the mixture was mixed well. The beads were rinsed with 350 μL of HSWB and 350 μL of LSWB respectively twice. The supernatant was aspirated off thoroughly.

[0127] 40 μL of 0.1 M sodium hydroxide was added, and the mixture was mixed well and incubated at room temperature for 10 min. The supernatant was aspirated, and 20 μL of 0.3 M acidic buffer was added to neutralize the single-strand products separated, the total volume of the products after neutralization being 60 μL. This step achieved the ligation of the target nucleic acid fragments with adaptor B and obtained the target single-stranded DNA through alkali denaturation and separation.

[0128] 21. Polymerase chain reaction:

TABLE-US-00032 Sequence of primer 1: (SEQ ID NO: 9) /5Phos/CATGTAGTGTACGATCCGACTT; Sequence of primer 2: (SEQ ID NO: 10) TCCTAAGCACGCTTGGCCT.

[0129] A PCR system was formulated according to the system as shown in Table 28.

TABLE-US-00033 TABLE 28 Volume Reaction component (μL) Enzyme-free pure water 160 2 × PfuTurbo Cx buffer 219.8 PfuTurbo Cx heat-activated nucleic acid 8.2 polymerase (2.5 U/μL) 20 μM of primer 1 11 20 μM of primer 2 11 Total volume 410

[0130] Into the above system was added 30 μL of the recovered products from the previous step, and the mixture was mixed well and allowed to react under the conditions as shown in Table 29.

TABLE-US-00034 TABLE 29 Temperature Time Cycle 95° C. 3 min 1 95° C. 30 s 7 56° C. 30 s 72° C. 4 min 68° C. 10 min 1 Ramping the temperature down to 4° C. at a rate of 0.1° C./s  4° C. Holding the temperature —

[0131] After reaction was complete, purification was conducted using 440 μL of Ampure XP magnetic beads, and the recovered product were dissolved with 80 μL of TE buffer. 1 μL of the recovered products was assayed with a Qubit dsDNA HS assay kit (Invitrogen Corp.) to quantitate the concentration of the products. 100 ng of the products was used for reaction at the next step. The size of the PCR product fragments was electrophoretically determined using an Agilent 2100 HS, the results being as shown in FIG. 7.

[0132] 22. Single strand cyclization:

[0133] The mediating fragment has corresponding complementary sequences for ligating both ends of the single strand, the sequence of the mediating fragment being as follows:

TABLE-US-00035 (SEQ ID NO: 11) GTACACTACATGTCCTAAGCACGC.

[0134] 10 μL of the mediating fragment (10 μM) was added into 100 ng of the PCR products from the previous step. The mixture was mixed well and incubated at 95° C. for 3 min, followed by being rapidly placed on ice for cooling. A reaction system as shown in Table 30 below was formulated.

TABLE-US-00036 TABLE 30 Volume Reaction component (μL) Enzyme-free water 36.4 10 × TA buffer (Epicentre Corp.) 12 100 mM adenosine triphosphate 1.2 T4 Ligase (600 U/μL, Enzymatics) 0.4 Total 50

[0135] 50 μL of the reaction system was added into a mixed solution of the PCR products and the mediating fragment. The mixture was mixed well and incubated at 37° C. for 60 min.

[0136] 23. Digestion of linear DNA:A system as shown in Table 31 was formulated.

TABLE-US-00037 TABLE 31 Reaction component Volume (μL) Enzyme-free water 2 10 × TA buffer (Epicentre Corp.) 0.8 Exonuclease 1 (20 U/μL, NEB Corp.) 3.9 Exonuclease 3 (100 U/μL, NEB Corp.) 1.3 Total 8

[0137] 8 μL of the reaction system was added into the ligation reaction solution from the previous step. The mixture was mixed well and incubated at 37° C. for 60 min. 6 μL of 0.5 M EDTA was added and the mixture was mixed well. The products were purified and recovered using 170 μL of PEG32 magnetic beads, and redissolved in 55 μL of TE buffer.

[0138] The concentration and total amount of the final products in this example are as shown in Table 32.

TABLE-US-00038 TABLE 32 Concentration Total amount Molecular weight (ng/μL) (ng) (pmol) Product 1 0.36 19.8 0.27 Product 2 0.34 18.7 0.26

[0139] The results indicated that the concentration and total amount of the respective products met the requirement for subsequent sequencing (molecular weight ≧0.12 pmol). The adaptor B ligation products after PCR and before cyclization were determined using an Agilent 2100 capillary electrophoresis apparatus, the results being as shown in FIG. 7, the abscissa representing the size of base pairs, and the ordinate representing the strength of the fluorescent signal detected. The ordinate value of 0 represents the baseline signal of the detection. The higher the ordinate value, the stronger the fluorescent signal of the sample detected. In the figure, 35 and 1030 correspond to DNA standards of 35 bp and 1030 bp, which were the references for detecting the size of the sample fragments. In the figure, 218 and 219 represent the size of the main bands of product 1 and product 2, i.e., 218 bp and 219 bp respectively. The rectangular box on the right shows the distribution of the bands of the fragments to be detected, as simulated according to the detection signal. The results showed that the target bands obtained (as indicated by the arrows) clustered together, suggesting that the insert fragments could be effectively controlled in a very narrow range by means of CNT. Additionally, according to the size of the main band of the respective PCR products, the size of the adaptor A (54 bp) and the size of the adaptor B (48 bp), the size of the main band of the respective insert fragments could be calculated: 218−54−48=116bp=58bp*2 and 219−54−48=117bp≈59bp*2 respectively, demonstrating that a library of single-stranded cyclic nucleic acid fragments having double adaptors was successfully obtained in this example.

[0140] The disclosure set forth above is intended to describe the present invention in further detail by reference to particular embodiments, and is not to be construed as limiting the practical implementation of the present invention thereto. A number of simple deductions or substitutions could be made by a person of ordinary skill in the art to which the present invention pertains without departing from the concept of the present invention.