阅读说明：本技术 用于产生目标核酸分子的方法和组合物 (Methods and compositions for producing target nucleic acid molecules ) 是由 权圣勋 金廷玟 卢珍星 廉熹兰 柳兑勋 于 2018-02-20 设计创作，主要内容包括：本申请提供一种产生目标核酸分子的方法,包含：(a)提供双链核酸分子,其包括目标序列区域、与目标序列区域的5’端连接且含有一个或多个脱氨碱基的第一侧翼序列区域和与目标序列区域的3’端连接的第二侧翼序列区域；(b)孵育核酸分子和对脱氨碱基具特异性的核酸内切酶,以去除范围从最靠近目标序列区域的5’端的脱氨碱基到核酸分子的5’端的第一侧翼序列区域。本申请还提供用于产生目标核酸分子的组合物,所述目标核酸分子包括双链核酸分子和对脱氨碱基具特异性的核酸内切酶。(The present application provides a method of producing a target nucleic acid molecule comprising: (a) providing a double stranded nucleic acid molecule comprising a target sequence region, a first flanking sequence region linked to the 5 'end of the target sequence region and comprising one or more deaminating bases, and a second flanking sequence region linked to the 3' end of the target sequence region; (b) incubating the nucleic acid molecule with an endonuclease specific for a deaminating base to remove a first flanking sequence region ranging from the deaminating base closest to the 5 'end of the target sequence region to the 5' end of the nucleic acid molecule. The present application also provides compositions for producing a target nucleic acid molecule comprising a double-stranded nucleic acid molecule and an endonuclease specific for a deaminating base.)

1. A method for producing a target nucleic acid molecule, comprising: (a) providing a double stranded nucleic acid molecule comprising a target sequence region, a first flanking sequence region linked to the 5 'end of the target sequence region and comprising one or more deaminating bases, and a second flanking sequence region linked to the 3' end of the target sequence region; and (b) incubating the nucleic acid molecule with an endonuclease specific for a deaminating base to remove a first flanking sequence region ranging from a deaminating base closest to the 5 'end of the target sequence region to the 5' end of the nucleic acid molecule.

2. The method of claim 1, wherein the double-stranded nucleic acid molecule is a product obtained by amplifying a template nucleic acid molecule comprising the target sequence region, a third flanking sequence region attached to the 5' end of the target sequence region, and a fourth flanking sequence region attached to the 3' end of the target sequence region, the 3' end of the target sequence region having a primer set comprising one or more deaminating bases and attached to the fourth flanking sequence region; and the template nucleic acid molecule is prepared by microarray-based synthesis.

3. The method of claim 1, wherein one or more nucleotides are arranged between adjacent deaminating bases.

4. The method of claim 1, wherein the deaminating base is an inosine base or a uracil base.

5. The method of claim 1, wherein the deaminating base is an inosine base and the inosine-specific endonuclease is endonuclease V.

6. The method according to claim 5, wherein the inosine-specific endonuclease is endonuclease V derived from Thermotoga maritima or Escherichia coli.

7. The method of claim 1, wherein the deaminating base is a uracil base and the uracil-specific endonuclease is a uracil-specific excision agent (USER).

8. The method according to claim 1, wherein 3 to 8 nucleotides are arranged between adjacent inosine bases.

9. The method of claim 1, further comprising (c) incubating a nucleic acid molecule free of the first flanking sequence region with 3'→ 5' exonuclease to remove single stranded second flanking sequence region.

10. The method of claim 9, wherein the exonuclease is T4DNA polymerase.

11. The method of claim 9, wherein steps (b) and (c) are performed by a one-step process and the reaction comprising the double-stranded nucleic acid molecule, the deaminating base-specific endonuclease and the exonuclease is incubated at 36 ℃ to 65 ℃ followed by incubation at 20 ℃ to 30 ℃.

12. A composition for producing a target nucleic acid molecule, comprising: a double-stranded nucleic acid molecule comprising a target sequence region, a first flanking sequence region linked to the 5 'end of the target sequence region and comprising one or more deaminating bases, and a second flanking sequence region linked to the 3' end of the target sequence region; and an endonuclease specific for the deaminating base.

13. The composition of claim 12, wherein the double-stranded nucleic acid molecule is a product obtained by amplifying a template nucleic acid molecule comprising the target sequence region, a third flanking sequence region attached to the 5' end of the target sequence region, and a fourth flanking sequence region attached to the 3' end of the target sequence region, the 3' end of the target sequence region having a primer set comprising one or more deaminating bases and attached to the fourth flanking sequence region; and the template nucleic acid molecule is prepared by microarray-based synthesis.

14. The composition of claim 12, wherein one or more nucleotides are arranged between adjacent deaminated bases in the double-stranded nucleic acid molecule.

15. The composition of claim 12, wherein the deaminating base is an inosine base or a uracil base.

16. The composition of claim 12, wherein the deaminating base is an inosine base and the deaminating base-specific endonuclease is endonuclease V.

17. The composition of claim 16, wherein the deaminating base-specific endonuclease is endonuclease V derived from thermatopaus maritima or escherichia coli.

18. The composition of claim 12, wherein the deaminating base is a uracil base and the uracil-specific endonuclease is a uracil-specific excision agent (USER).

19. The composition of claim 12, wherein 3 to 8 nucleotides are arranged between adjacent deaminating bases.

20. The composition of claim 12, further comprising a 3'→ 5' exonuclease.

21. The composition of claim 20, wherein the exonuclease is T4DNA polymerase.

Technical Field

The present application relates to a method and composition for producing a target nucleic acid molecule.

Background

The concentration of products synthesized by current microarray-based gene synthesis techniques is at the femtomolar (femtolar) level. Therefore, it is necessary to increase the concentration of the synthesized product to a higher level by PCR. To meet this requirement, a primer binding region is generally synthesized during gene synthesis, and a restriction enzyme recognition sequence is introduced into the primer binding region. The restriction enzyme recognition sequence is used to remove the primer binding region in subsequent processes.

Restriction enzymes recognize specific nucleotide sequences and cleave DNA in or around the sequence. The enzyme typically recognizes 4 to 8 bases in the sequence. The presence of restriction enzyme recognition sites in a target nucleic acid may be an obstacle to isolation of the complete target nucleic acid. When it is desired to obtain the target nucleic acid in a complete form, it is cumbersome to select an appropriate restriction enzyme depending on the synthetic sequence.

Time and cost problems may arise when special handling of the reaction product with the restriction enzyme is required. The gene synthesis products are assembled into longer nucleic acid molecules by gene assembly. The reaction product with the restriction enzyme may have sticky ends. In this case, an additional process is required to convert the sticky ends into blunt ends (blunteds).

Under these circumstances, the inventors of the present application succeeded in designing a method for producing a target nucleic acid molecule by cleaving a nucleic acid in a sequence-independent manner.

Disclosure of Invention

Problem to be solved by the present application

In one aspect, a method is provided for producing a target nucleic acid molecule from a double stranded nucleic acid molecule comprising a target sequence region, a first flanking sequence region linked to the 5 'end of the target sequence region and comprising one or more deaminating bases, and a second flanking sequence region linked to the 3' end of the target sequence region.

Another aspect provides a composition for producing a target nucleic acid molecule comprising a double-stranded nucleic acid molecule and a deaminating base-specific endonuclease.

Means for solving the problems

In one aspect, there is provided a method for producing a target nucleic acid molecule comprising: (a) providing a double stranded nucleic acid molecule comprising a target sequence region, a first flanking sequence region linked to the 5 'end of the target sequence region and comprising one or more deaminating bases, and a second flanking sequence region linked to the 3' end of the target sequence region; (b) incubating (incubation) the nucleic acid molecule and an endonuclease specific for the deaminating base to remove a first flanking sequence region ranging from the deaminating base closest to the 5 'end of the target sequence region to the 5' end of the nucleic acid molecule.

The first flanking sequence region may have at least 2, at least 3, or at least 4 deaminating bases. In the first flanking sequence region, one or more nucleotides may be arranged between adjacent deaminating bases. The deaminating base can be an inosine base or a uracil base.

The deaminating base can be an inosine base. In this case, 3 to 8 nucleotides may be arranged between adjacent inosine bases. For example, when 3 inosine bases are present in the first flanking sequence region, adjacent inosine bases may be separated by 5 and 8 nucleotides. When 4 inosine bases are present in the first flanking sequence region, adjacent inosine bases may be separated by 4, 3 and 5 nucleotides. Alternatively, the deaminating base can be a uracil base. In this case, one or more nucleotides may be arranged between adjacent uracil bases.

The at least one deaminating base can be located at the first, second, or third nucleotide of the 3' end of the first flanking sequence region. For example, at least one deaminating base can be present in the nucleotide at the 3' end of the first flanking sequence region, a second nucleotide from the 3' end of the first flanking sequence region, or a third nucleotide from the 3' end of the first flanking sequence region

When the base of the deaminating base is an inosine base, the deaminating base-specific endonuclease can be endonuclease V, commonly referred to as a deoxyinosine 3' -endonuclease. Endonuclease V recognizes hypoxanthine (the base of deoxyinosine on single-stranded or double-stranded DNA), and predominantly hydrolyzes the second or third phosphodiester bond that recognizes the 3' end of the base to create a "nick". The inosine-specific endonuclease may be endonuclease V derived from thermatopae maritima (Thermotoga maritima) or escherichia coli (e.

When the deaminating base is a uracil base, the deaminating base-specific endonuclease can be a uracil-specific excision agent (USER). USER is an enzyme that generates a single nucleotide gap at the position of uracil residues. The USER enzyme is a mixture of Uracil DNA Glycosylase (UDG) and DNA glycosylase-lyase endonuclease VIII. UDG catalyzes the cleavage of uracil bases to form abasic sites while leaving the phosphodiester backbone intact. The lyase activity of endonuclease VIII disrupts the phosphodiester backbone on the 3 'and 5' side of the abasic site, thereby releasing abasic deoxyribose.

The double stranded nucleic acid molecule may be a product obtained by amplifying a template nucleic acid molecule comprising a target sequence region, a third flanking sequence region linked to the 5' end of the target sequence region and a fourth flanking sequence region linked to the 3' end of the target sequence region, the 3' end of the target sequence region having a primer set comprising one or more deaminating bases and being attached (annealing) to the fourth flanking sequence region.

The template nucleic acid molecule may be one isolated from an organism, one isolated from a nucleic acid pool, one obtained by genetic engineering modification or combination of isolated nucleic acid fragments, one obtained by chemical synthesis, or a combination thereof. The template nucleic acid molecule may be single-stranded or double-stranded.

alternatively, the template nucleic acid molecule may be prepared by microarray-based synthesis. Microarray-based synthesis refers to a technique for simultaneously synthesizing biochemical molecules of the same, similar or different types in parallel on synthesis sites fixed at intervals in the centimeter or micrometer range on a solid substrate.

The primer set for amplifying the template nucleic acid molecule may be attached to the fourth flanking sequence region of the template nucleic acid molecule and may have at least 2, at least 3, or at least 4 deaminating bases. The deaminating base can be an inosine base or a uracil base.

The deaminating base can be an inosine base. In this case, 3 to 8 nucleotides may be arranged between adjacent inosine bases. For example, when 3 inosine bases are present in the first flanking sequence region, adjacent inosine bases may be separated by 5 and 8 nucleotides. When 4 inosine bases are present in the first flanking sequence region, adjacent inosine bases may be separated by 4, 3 and 5 nucleotides. Alternatively, the deaminating base can be a uracil base. In this case, one or more nucleotides may be arranged between adjacent uracil bases.

The primer set may be a primer having SEQ ID NO: 1 and an oligonucleotide having the nucleotide sequence set forth in SEQ ID NO: 2, a pair of oligonucleotides having the nucleotide sequences shown in SEQ ID NO: 3 and an oligonucleotide having the nucleotide sequence set forth in SEQ ID NO: 4, a pair of oligonucleotides having the nucleotide sequences shown in SEQ ID NO: 5 and an oligonucleotide having the nucleotide sequence set forth in SEQ ID NO: 6, a pair of oligonucleotides having the nucleotide sequences shown in SEQ ID NO: 7 and an oligonucleotide having the nucleotide sequence set forth in SEQ ID NO: 8 or an oligonucleotide having the nucleotide sequence set forth in SEQ ID NO: 15 and an oligonucleotide having the nucleotide sequence set forth in SEQ ID NO: 16, or a pair of oligonucleotides of the nucleotide sequence set forth in seq id no.

The method can further comprise (c) incubating the nucleic acid molecule without the first wing sequence region with a 3'→ 5' exonuclease to remove the single stranded second wing sequence region. The exonuclease may be T4DNA polymerase.

Steps (b) and (c) may be carried out by a one-shot process. According to the one-step method, the reaction comprising the double-stranded nucleic acid molecule, the deaminating base-specific endonuclease and the exonuclease is incubated at a higher temperature (step (b)) and then at a lower temperature (step (c)).

For example, a reaction comprising a double stranded nucleic acid molecule, a deaminating base-specific endonuclease and an exonuclease can be incubated at 36 ℃ to 65 ℃, 38 ℃ to 60 ℃, 40 ℃ to 58 ℃, 40 ℃ to 55 ℃ or 40 ℃ to 50 ℃ for 20 minutes to 40 minutes or 25 minutes to 35 minutes, such as 30 minutes, followed by incubation at 20 ℃ to 30 ℃, 22 ℃ to 28 ℃ or 23.5 ℃ to 26.5 ℃ for 15 minutes to 25 minutes or 18 minutes to 23 minutes, such as 20 minutes.

In another aspect, there is provided a composition for producing a target nucleic acid molecule, comprising: a double-stranded nucleic acid molecule comprising a target sequence region, a first flanking sequence region linked to the 5 'end of the target sequence region and comprising one or more deaminated bases, and a second flanking sequence region linked to the 3' end of the target sequence region; and an endonuclease specific for the deaminating base.

The first flanking sequence region may have at least 2, at least 3, or at least 4 deaminating bases. In the first flanking sequence region, one or more nucleotides may be arranged between adjacent deaminating bases. The deaminating base can be an inosine base or a uracil base. The deaminating base can be an inosine base. In this case, 3 to 8 nucleotides may be arranged between adjacent inosine bases. Alternatively, the deaminating base can be a uracil base. In this case, one or more nucleotides may be arranged between adjacent uracil bases. The at least one deaminating base can be located at the first, second, or third nucleotide of the 3' end of the first flanking sequence region.

When the deaminating base is an inosine base, the deaminating base-specific endonuclease can be endonuclease V. The endonuclease V is the same as described above. When the deaminating base is a uracil base, the deaminating base-specific endonuclease can be a uracil-specific excision agent (USER). The uracil-specific cleavage reagent is the same as described above.

The double stranded nucleic acid molecule may be a product obtained by amplifying a template nucleic acid molecule comprising a target sequence region, a third flanking sequence region linked to the 5' end of the target sequence region, and a fourth flanking sequence region linked to the 3' end of the target sequence region, the 3' end of the target sequence region having a primer set comprising one or more deaminated bases and bound to the fourth flanking sequence region. The template nucleic acid molecule is the same as described above. Alternatively, the template nucleic acid molecule may be prepared by microarray-based synthesis. The primer set for amplifying the template nucleic acid molecule is the same as described above.

The composition may further comprise a 3'→ 5' exonuclease. The exonuclease may be T4DNA polymerase.

Effects of the present application

The methods and compositions for producing a nucleic acid molecule of interest according to aspects can be widely used in the fields of synthetic biology and molecular biology.

Drawings

FIG. 1a shows a schematic diagram of a method for producing a nucleic acid molecule of interest according to one aspect.

FIG. 1b shows a method for preparing a double-stranded nucleic acid molecule in a method for producing a target nucleic acid molecule according to one aspect.

FIG. 2a shows the results of electrophoresis in each step using a primer containing inosine and a restriction enzyme.

FIG. 2b shows the results of electrophoresis in each step using uracil containing primers and restriction enzymes.

FIG. 3 shows the activity of two enzymes at different temperatures.

Fig. 4 shows experimental results of determining whether the purification process can be omitted and a buffer mixture can be used.

FIG. 5a shows a schematic of a one-step reaction according to one aspect.

FIG. 5b shows the results obtained after the next reaction at different temperatures.

Reference numerals:

END represents the result of electrophoresis for blunting the cleavage product by END Repair enzyme

Electrophoresis results of M standard solutions

UP3 represents the result of electrophoresis of the PCR product obtained using the primer set UP3

USER stands for the electrophoresis result of the product cleaved with USER enzyme

Detailed Description

Modes for carrying out the present application

The present application is explained in more detail with reference to the following examples. However, these examples are provided to aid in understanding the present application and are not intended to limit the scope of the present application.

Example 1: cleavage with inosine-containing primers

Preparation of DNA fragments and primer sets and PCR

257 single-stranded DNA fragments of 140bp were prepared from genomic DNA of Mycoplasma genitalium (Mycoplasma genialitium) using a semiconductor-based electrochemical acid production array (CustomARRAY). Each fragment has a common sequence (SEQ ID NOS: 9 and 10) for primer binding, which flanks the target sequence. The 257 fragments were classified into 20 cassettes based on the 80bp overlap region located in the 100bp target sequence.

Primer sets that can be attached to common sequences are prepared. The sequences of the primer sets are identical to the common sequences except that one or more guanine bases in the common sequence are replaced with inosine bases. The primer set was named CP primer set. All primers were custom made by Integrated DNA Technology (Coralville, IA, USA). The CP primer sets are shown in Table 1.

TABLE 1

As shown in Table 1, each of the CP1 primers (SEQ ID NOS: 1 and 2) had an inosine base in front of thymine at the 3' end. Each of the CP2 primer (SEQ ID NOS: 3 and 4) and the CP3 primer (SEQ ID NOS: 5 and 6) had three inosine bases. The adjacent inosine bases in each of the CP2 and CP3 primers were separated by 5 and 8 nucleotides. Unlike the CP2 primer, each CP3 primer has deoxyinosine at the 3' end. Each CP4 primer (SEQ ID NOS: 7 and 8) had four inosine bases. Adjacent inosine bases are separated by 4, 3 and 5 nucleotides.

PCR of the DNA fragments was performed using Taq DNA polymerase (Thermo Scientific) with CP primer set. Specifically, a solution (50. mu.l) containing 700ng of Mycoplasma genitalium genomic DNA and 1pM of each CP primer set was allowed to react at 95 ℃ for 2 minutes. After 10-15 cycles consisting of 95 ℃/30 seconds, annealing temperature/20 seconds and 72 ℃/30 seconds, the reaction was continued for 2 minutes at 72 ℃. The annealing temperature varies depending on the type of primer. The reaction product was purified using QIAGEN MinElute PCR purification kit (QIAGEN, Valencia, CA, USA) and eluted to a final volume of 15 μ Ι.

1.2. Reacting with endonuclease and exonuclease and sequencing

700ng of each DNA-containing purified PCR product from Thermotoga maritima (Tma)) and endonuclease V (Thermo Fisher Scientific, St.Leon-Rot Germany, 5U/. mu.l) were incubated at 65 ℃ for 30 minutes, purified, and eluted to a final volume of 15. mu.l.

Thereafter, the eluate was reacted with T4DNA polymerase (Thermo Scientific, 5U/. mu.l) having 3'→ 5' exonuclease activity at 11 ℃ for 20 minutes or at room temperature for 5 minutes. High resolution electrophoresis was performed in 2.5% agarose gel at 120V for 60-90 min to determine the size and amount of DNA fragments.

For sequencing, the 5 'and 3' phosphate residues were removed by treatment with alkaline phosphatase (Calf interest; New England Biolabs). 1 μ l of TOPO clones of 20 ng/. mu.l DNA from both ends of the phosphate residues were removed using the All in One PCR cloning kit (Biofact) according to the manufacturer's instructions, followed by Sanger sequencing (Macrogen Inc.). To obtain sequence information of a large number of colonies at low cost, all colonies were collected in one tube, cells were cultured in liquid LB medium, and then plasmids were purified using a general express plasmid mini kit. Primers are designed from sequences flanking the cloning site of the plasmid such that the sequence of interest is present in the amplification product. The amplification products were sequenced with illumina miseq. Sequencing results for tens of thousands of templates were obtained.

FIG. 1a shows a schematic diagram of a method for producing a nucleic acid molecule of interest according to one aspect.

FIG. 1b shows a method for preparing a double-stranded nucleic acid molecule in a method for producing a target nucleic acid molecule according to one aspect. As shown in FIG. 1b, the template nucleic acid molecule includes a third flanking sequence region linked to the 5 'end of the target sequence region and a fourth flanking sequence region linked to the 3' end of the target sequence region. A primer set containing a deaminated base was ligated to the fourth flanking sequence region. This binding enables amplification of the template nucleic acid molecule.

FIG. 2a shows the results of electrophoresis in each step using a primer containing inosine and a restriction enzyme. Lanes 1-4 represent PCR products using the universal primer set, CP1 primer set, CP2 primer set, and CP3 primer set, respectively. Lanes 5-8 represent the products obtained by reacting the PCR products in lanes 1-4 with Tma Endo V, respectively. Lanes 9-12 represent the products obtained by reacting the products in lanes 5-8 with T4DNA polymerase, respectively.

As shown in FIG. 2a, for the CP1 primer set (lane 10), cleaved and uncleaved fragments coexist. For the CP2 and CP3 primer sets (lanes 11 and 12), a final product of 100-bp was produced. Sanger sequencing of the final product showed that 73.7% (CP 2) and 93.8% (CP 3) were cleaved.

For the CP4 primer, Sanger sequencing showed 100% cleavage. Illumina Mi-Seq again confirmed the cleavage performance. The results are shown in table 2. In Table 2, F and R represent the forward and reverse primers, respectively, and the Cut (Cut) and Uncut (Uncut) represent the number of Cut and Uncut reads at the inosine base of the primers, respectively. Sample 1 and sample 2 are two parallel experimental groups treated under the same conditions. As a result of Illumina sequencing of sample 1, the F and R primer sites were accurately cleaved in a total of 95365 reads, the target sequence remained only in 94631 reads, the R primer was not cleaved in 732 reads, and the F primer was not cleaved in 2 reads. Illumina sequencing of sample 2 showed that the F and R primers were precisely cleaved and the target sequence remained only in 88649 reads, the R primer was not cleaved in 361 reads, and the F primer was not cleaved in 815 reads. As shown in table 2, 98.97% of the templates were successfully cut. The estimated remainder (1.03%) was due to errors during primer construction. These results conclude that the method of the present application is also effective in large scale experiments.

TABLE 2

Example 2: cleavage with uracil containing primers

2.1. Preparation of primer set and PCR

PCR was performed using a primer set having a sequence shown in Table 3, with the Mycoplasma genitalium-derived DNA fragment described in example 1 as a template. Each primer set includes one or more uracil bases. The primer set was named UP primer set. The UP primer set is shown in Table 3.

TABLE 3

As shown in Table 3, each UP1 primer (SEQ ID NOS: 11 and 12) had one uracil base at the 3 'end, and each UP 2 primer (SEQ ID NOS: 13 and 14) had one uracil base at the fifth or third position of the 3' end. In each UP3 primer (SEQ ID NOS: 15 and 16), 6 or 7 nucleotides are arranged between two uracil bases.

A solution (50. mu.l) containing 1. mu.l of 10. mu.M M. After 11 cycles consisting of 98 ℃/20 sec, 58 ℃/15 sec and 72 ℃/30 sec, the reaction was continued for 2 minutes at 72 ℃. The size of the PCR product was constant at 140 bp.

2.2. Reaction with Endonuclease and exonuclease and sequencing

A solution (100. mu.l) containing 50. mu.l of each PCR product obtained in 2.1, 10. mu.l of 10 XCutSmart buffer and 10. mu.l of USER enzyme (NEB) was incubated at 37 ℃ for 20 minutes, purified, and eluted to a final volume of 12. mu.l. A solution (20. mu.l) containing 10. mu.l of the eluate, 2. mu.l of 10 XEnd Repiair reaction buffer and 1. mu.l of the End Repiair enzyme mixture (NEB) was reacted at 20 ℃ for 30 minutes, purified, and eluted to a final volume of 12. mu.l. High resolution electrophoresis was performed in 2.5% agarose gel at 120V for 60-90 min to determine the size and amount of DNA fragments.

FIG. 2b shows the results of electrophoresis in each step using uracil containing primers and restriction enzymes. In FIG. 2b, UP3 represents the PCR product (140bp) obtained using the uracil-containing primer set UP3, USER represents the product cleaved with USER enzyme, and END represents the blunt-ended cleavage product (100bp) by END Repair enzyme. Sanger sequencing of a total of 83 cleavage products showed that 6 (7.2%) products were 99bp in length and 77 (92.8%) products were 100bp in length, indicating that all nucleic acid fragments were cleaved by uracil containing primers and USER enzyme.

Example 3: extension of enzymatic reaction conditions

3.1. Testing temperature-dependent Activity

The activities of Tma endonuclease V (Tma Endo V) and T4DNA polymerase were tested under various temperature conditions.

FIG. 3 shows the activity of two enzymes at various temperatures.

Tma Endo V was incubated at various temperatures and at the recommended incubation temperature (65 ℃). Lanes 1-4 show the results of electrophoresis after each product obtained by incubation at 25 ℃, 35 ℃, 50 ℃ and 65 ℃ was reacted with T4DNA polymerase at 25 ℃ for 20 minutes. As shown in FIG. 3A, Tma Endo V showed no substantial activity at a temperature ≦ 35 deg.C (lanes 1 and 2). Tma Endo V activity was observed at 50 ℃ and 65 ℃.

The recommended incubation conditions for the 3'→ 5' exonuclease activity of the T4DNA polymerase are 11 ℃/20 minutes or room temperature/5 minutes. However, as shown in FIG. 3B, the T4DNA polymerase retains its activity under various temperature conditions, including 25 ℃, 35 ℃, 50 ℃ and 65 ℃, as well as the recommended incubation temperature (lanes 5-8).

3.2. Testing to determine if the purification process can be omitted and a buffer mixture can be used

The PCR product is purified by removing unnecessary components including salts, nucleotides, enzymes and primers. Subsequent enzymatic treatment also requires cleaning of the DNA sample. However, purification can be costly and difficult to fully automate in large scale experiments. Thus, the omission of purification contributes to saving time and cost, and is therefore advantageous for the operator.

Fig. 4 shows experimental results of determining whether the purification process can be omitted and a buffer mixture can be used. The 140-bp amplification product was obtained using the CP3 primer set, treated with Tma Endo V (lane 2), purified (lane 4) or not (lane 3), and treated with T4DNA polymerase. For lanes 5-8, the resulting reaction products were compared after incubation in a Buffer Mix (BM) of Tma Endo V buffer and T4DNA polymerase buffer. B + in FIG. 4 represents a single addition of T4DNA polymerase buffer.

As shown in FIG. 4, a comparison of lanes 3 and 4 demonstrates that omitting purification prior to addition of T4DNA polymerase does not affect cleavage. It was also demonstrated that the use of a buffer mixture did not inhibit the activity of both enzymes.

3.3. One-step reaction

Considering that omitting the purification or using the buffer mixture had no effect on the activity of the enzymes, a one-step reaction of the two enzymes was carried out in the matrix as demonstrated in 3.2. The optimum temperature and time conditions for this reaction were investigated.

700ng of template substrate, 5 units of Tma Endo V, 1. mu.l of T4DNA polymerase and dNTP, and a buffer mixture were mixed together to prepare 100. mu.l of a solution.

FIG. 5a is a schematic diagram showing a one-step reaction according to one aspect.

FIG. 5b shows the results obtained after the next reaction at different temperatures. Lanes 1-4 represent results obtained after incubation at 50 ℃ for 30 minutes followed by incubation at 25 ℃ for 20 minutes (lane 1), after incubation at 40 ℃ for 30 minutes followed by incubation at 25 ℃ for 20 minutes (lane 2), after incubation at 40 ℃ for 30 minutes followed by incubation at 25 ℃ for 20 minutes (lane 3), and after incubation at 45 ℃ for 30 minutes followed by incubation at 25 ℃ for 20 minutes (lane 4). As shown in FIG. 5b, incubation at 40 ℃ for 30 minutes followed by incubation at 25 ℃ for 20 minutes is the optimal one-step reaction condition.

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<220>

<221> modified_base

<222> (5)

<223> i

<220>

<221> modified_base

<222> (13)

<223> i

<220>

<221> modified_base

<222> (21)

<223> i

<400> 6

cgtgnatgag ganccgcagt n 21

<210> 7

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> CP4_F

<220>

<221> modified_base

<222> (3)

<223> i

<220>

<221> modified_base

<222> (8)

<223> i

<220>

<221> modified_base

<222> (12)

<223> i

<220>

<221> modified_base

<222> (18)

<223> i

<400> 7

gtnccttngc antctcant 19

<210> 8

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> CP4_R

<220>

<221> modified_base

<222> (3)

<223> i

<220>

<221> modified_base

<222> (9)

<223> i

<220>

<221> modified_base

<222> (14)

<223> i

<220>

<221> modified_base

<222> (19)

<223> i

<400> 8

tgnatgagna gccncagtnt 20

<210> 9

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> common_F

<400> 9

gtgccttggc agtctcagt 19

<210> 10

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> common_R

<400> 10

acactgcggg ctcctcatcc acg 23

<210> 11

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> UP1_F

<400> 11

gtgccttggc agtctcagu 19

<210> 12

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> UP1_R

<400> 12

cgtggatgag gagccgcagt gu 22

<210> 13

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> UP2_F

<400> 13

gtgccttggc agtcucagt 19

<210> 14

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> UP2_R

<400> 14

cgtggatgag gagccgcagu gt 22

<210> 15

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> UP3_F

<400> 15

gtgccutggc aguctcag 18

<210> 16

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> UP3_R

<400> 16

cgtggaugag gagcugcagt g 21