Thermally stable single-stranded DNA / RNA ligase and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a thermal-stability single-chain DNA / RNA ligase mutant and application thereof. Compared with the amino acid sequence (pfumlinase) of SEQ ID NO: 1, the DNA / RNA ligase with the mutated lysine provided by the invention has the advantages that the 372 site is subjected to substitution mutation; the amino acid sequence of the nucleic acid ligase is as shown in SEQ ID NO: 4. According to the present invention, the potential connection active site is screened, and the gene mutation modification is performed so as to obtain the Pfumrigase DNA / RNA ligase with the new mutation site; compared with the original Pfumrigase DNA / RNA ligase, the modified Pfumrigase DNA / RNA ligase has the advantage that the modified Pfumrigase DNA / RNA ligase has higher catalytic activity.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a thermostable single-stranded DNA / RNA ligase mutant and a composition, a kit, a ligation method and an application thereof. Background Art

[0002] Nucleic acid ligases belong to the nucleotidyltransferase superfamily. Their function, assisted by metal ions, is to promote the formation of phosphodiester bonds between the 3' and 5' terminal bases of DNA or RNA, thereby achieving self-cyclization of nucleic acid molecules or linear ligation of molecules. Nucleic acid ligases can be divided into two major categories based on the type of substrate they act on: DNA ligases and RNA ligases. Some ligases possess the ability to connect DNA and RNA. These natural nucleic acid ligases, derived from diverse species, exhibit a variety of unique properties, including substrate specificity, sequence preference, thermal stability, salt tolerance, and tolerance to pH changes.

[0003] Currently, the ligation mechanism of nucleic acid ligase is generally believed to catalyze the ligation of bases from the 3' end to the 5' end through a three-step ligation reaction. [1] . The first step is to form an enzyme-AMP complex, the auxiliary factors ATP and NAD+ are cleaved, and the adenylate AMP is transferred to the highly conserved lysine residue in the active region of the nucleic acid ligase to form a covalent enzyme-AMP intermediate complex. The second step is that the enzyme-AMP intermediate complex transfers AMP to the 5' phosphate end of the nucleic acid substrate, thereby forming a 5' adenylylated intermediate product. In the third step, in the active pocket catalyzed by the ligase, the 3' hydroxyl end of the base binds to the enzyme active site, attacks the 5' adenylylated end, and completes the ligation reaction while releasing AMP. The first and second step ligation reactions are both reversible reactions. Conventional nucleic acid ligases complete the connection of the 3' hydroxyl end substrate and the 5' phosphate end substrate through these three steps under the action of auxiliary factors.

[0004] Nowadays, nucleic acid ligase plays a key role in maintaining the stability and integrity of the genome in organisms. At the same time, in in vitro experiments, nucleic acid ligase, as a key tool enzyme in molecular biology research, has played an important role in promoting the advancement of gene synthesis, high-throughput sequencing technology and molecular cloning technology. According to the difference in reaction temperature, nucleic acid ligase can be classified into two types: thermostable and non-thermostable. Although there are many types of nucleic acid ligases, the choice of single-stranded DNA ligases with high thermal stability (>60°C) is relatively limited. These nucleic acid ligases that can directly achieve single-stranded DNA ligation play a vital role in specific application fields, such as single-stranded DNA library construction technology, circularized single-stranded DNA for rolling circle amplification sequencing, and labeling and modification of single-stranded DNA. [2,3] .

[0005] Among the thermostable single-stranded DNA ligases reported so far, Circligase derived from TS2126 phage can catalyze the ligation of 3' hydroxyl termini to 5' phosphate termini at 60°C. [4] . However, its terminal base preference can lead to selective substrate ligation, which in turn causes deviation in the results. In addition, the enzyme is inactivated within 10 minutes at 80°C, and its heat resistance is still insufficient. Although the Mth Rnl mutant (trade name: Thermostable 5'App DNA & RNA Ligase) has good heat resistance, its efficiency in single-stranded DNA ligation is low, and sometimes it cannot meet the needs of related experiments. [5] Taq DNA ligase is a heat-resistant ligase that also exhibits efficient ligation of single-stranded DNA. However, its ligation process requires the presence of a target DNA chain that is completely complementary to the substrate ligation end sequence as a guide, which has limitations. [6] Recently, researchers developed a ligase from Thermomyces butyricum and genetically engineered it, resulting in a heat-resistant single-stranded DNA ligase with an optimal reaction temperature of 65°C, the Hyperligase K370P mutant. This enzyme can directly catalyze the ligation of the 3' hydroxyl terminus to the 5' adenylylated terminus without relying on ATP, and has no obvious terminal base preference. However, during the preparation of the enzyme, the researchers found that its protein solubility was poor, and most of the expressed protein formed inclusion bodies and existed in the precipitate.

[0006] Enzyme modification can be achieved through gene mutation or fusion, chemical or antibody modification, and nucleic acid aptamer modification. Gene mutation modification of certain nucleic acid ligases can help change their properties, enhance their thermal stability or ligation efficiency, or improve their salt and acid resistance. Among them, Mth RNA ligase with a mutation at the core lysine site in the Motif I region can use pre-adenylated single-stranded DNA as a substrate for the ligation reaction, thereby improving its ligation efficiency. [5] The researchers carried out site-directed mutagenesis of the core lysine in the Motif I region of the protein encoded by the Pyrfu_1465 gene in Pyrolobus fumarii, thereby enabling the protein to connect to single-stranded DNA / RNA and belonging to the RNA Ligase 2 family (named Pfum-Thermostable Lysine-MutantssDNA / RNA Ligase, hereinafter referred to as Pfumligase, WO2017160788A3). Pfumligase originates from Pyrolobus fumarii, which has the strongest heat resistance in nature. [7], has extremely high thermal stability and can catalyze the ligation of single-stranded substrates in reaction environments of 75°C and above, however, its ligation efficiency is still insufficient. At the same time, based on the results of active site modification studies of other nucleic acid ligases in the RNA Ligase 2 family, it is suggested that there is still significant potential for improving the ligation efficiency of Pfumligase.

[0007] The references mentioned above are as follows:

[0008] [1] SHUMAN S, LIMAC D. The polynucleotide ligase and RNA capping enzyme superfamily of covalent nucleotidyltransferases[J]. Current opinion instructural biology, 2004, 14(6):757-64;

[0009] [2]MEYER M,KIRCHER M,GANSAUGE MT,et al.A high-coverage genome sequence from an archaic Denisovan individual[J].Science(New York, NY),2012,338(6104):222-6;

[0010] [3]LIQ,ZHANG S,LI W,et al.Programming CircLigase Catalysis for DNARings and Topologies[J].Analytical chemistry,2021,93(3):1801-10;

[0011] [4] BLONDAL T, THORISDOTTIR A, UNNSTEINSDOTTIR U, et al. Isolation and characterization of a thermostable RNA ligase 1 from a Thermus scotoductusbacteriophage TS2126 with good single-stranded DNA ligation properties [J]. Nucleic acids research, 2005, 33(1): 135-42;

[0012] [5]ZHELKOVSKY AM,MCREYNOLDS L A.Structure-function analysis ofMethanobacterium thermoautotrophicum RNA ligase-engineering a thermostableATP independent enzyme[J].BMC molecular biology, 2012,13:24;

[0013] [6]LOHMAN GJ, TABOR S, NICHOLS N M.DNA ligases[J].Current protocols inmolecular biology, 2011, Chapter 3:Unit3.14;

[0014] [7] E,RACHEL R,BURGGRAF S,et al.Pyrolobus fumarii,gen.andsp.nov.,represents a novel group of archaea,extending the upper temperaturelimit for life to 113degrees C[J].Extremophiles:life under extreme conditions,1997,1(1):14-21. Summary of the Invention

[0015] The technical problem to be solved by the present invention is to provide a DNA / RNA ligase with higher catalytic activity and its application. To solve the above technical problem, the present invention provides a DNA / RNA ligase with a lysine mutation, which has a substitution mutation at position 372 compared to the amino acid sequence of SEQ ID NO: 1 (pfumligase); the amino acid sequence of the nucleic acid ligase is as described in SEQ ID NO: 4.

[0016] As an improvement of the DNA / RNA ligase of the present invention, the mutation at position 372 is the replacement of Lys with Glu.

[0017] The present invention also provides a nucleic acid molecule encoding the above-mentioned lysine-mutated DNA / RNA ligase.

[0018] The present invention also provides a vector comprising the nucleic acid molecule described above.

[0019] The present invention also provides a recombinant cell into which the nucleic acid or vector described above is introduced.

[0020] The present invention also provides a composition or kit for ligating single-stranded DNA and / or RNA, comprising the lysine-mutated DNA / RNA ligase as described above.

[0021] The present invention also provides use of the lysine-mutated DNA / RNA ligase described above in preparing a product for ligating single-stranded DNA and / or RNA.

[0022] As an improvement of the use of the present invention: the ligation reaction system is pH = 7.5, 70mM final concentration of Tris-HCl buffer system, 1:1 enzyme / substrate ratio, 10mM Mn 2+ The final concentration was 20% PEG8000; the connection reaction conditions were 95°C for 1 min and 75°C for 2 h.

[0023] As a further improvement of the use of the present invention: the Pfumligase K372E mutant is used for the construction of a single-stranded DNA library.

[0024] As a further improvement of the use of the present invention: the nucleic acid to be tested is ligated to the linker in a first round and then linearly extended, and the linear extension product is ligated to the linker in a second round and then amplified by library PCR.

[0025] The present invention obtains Pfumligase DNA / RNA ligase with new mutation sites by screening potential ligation active sites and implementing genetic mutation modification. Compared with the original Pfumligase DNA / RNA ligase, the partially modified Pfumligase DNA / RNA ligase exhibits higher catalytic activity. In addition, based on the Pfumligase DNA / RNA ligase with significantly enhanced catalytic activity, a single-stranded DNA library construction technology was developed and applied to high-throughput sequencing studies of free DNA samples from human plasma. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The specific embodiments of the present invention are further described in detail below with reference to the accompanying drawings.

[0027] Figure 1 The expression vector construction and electrophoresis identification results of Pfumligase mutant;

[0028] Figure 1 In Chinese: K248A represents mutant Lys248, K370P represents mutant Lys370, K372E represents mutant Lys372, and the same below.

[0029] Figure 2 This is the comparison and verification of the first-generation sequencing results of the Pfumligase mutant nucleic acid sequence; the blue background part is the mutation site peak diagram.

[0030] Figure 3 Figure 3 is the purification and SDS-PAGE electrophoresis identification of Pfumligase and mutant proteins; M is the protein molecular weight standard, F1 is the flow-through collected during Ni column purification injection, Ni and heparin are the Ni column purification product and heparin column purification product, respectively.

[0031] Figure 4 This was a preliminary test of the ligation activity of Pfumligase and its mutants.

[0032] Figure 5 It is a time gradient reaction test for the ligation activity of Pfumligase and mutants;

[0033] Figure 5 Middle: The left figure shows the electrophoresis results of the time-gradient ligation reaction products of Pfumligase and its mutants. The right figure shows the comparison of the 1-h ligation efficiency of Pfumligase and its mutants. The 1-h ligation efficiency in the comparison results is the average of three independent repeated experiments. One-way ANOVA, *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.

[0034] Figure 6 To test the activity of Pfumligase K372E in different buffer systems and pH conditions;

[0035] Figure 6 Middle: The left picture shows the electrophoresis results of the Pfumligase K372E ligation reaction buffer system and pH condition test. The upper right picture shows the comparison of ligation efficiency results of the Pfumligase K372E ligation reaction buffer system and pH condition test. The lower right picture shows the reaction system results after the Pfumligase K372E ligation reaction buffer system and pH condition test.

[0036] Figure 7 Activity test of Pfumligase K372E under different enzyme / substrate ratios;

[0037] Figure 7 Middle: The left figure shows the electrophoresis results of the Pfumligase K372E ligase reaction enzyme / substrate ratio test conditions. The right figure shows the comparison results of the Pfumligase K372E ligase reaction enzyme / substrate ratio test conditions.

[0038] Figure 8To test the activity of Pfumligase K372E under different metal ion conditions;

[0039] Figure 8 Middle: The left figure shows the electrophoresis results of the metal ion type condition test of the Pfumligase K372E ligation reaction. The right figure shows the comparison results of the ligation efficiency of the metal ion type condition test of the Pfumligase K372E ligation reaction.

[0040] Figure 9 Pfumligase K372E in different Mn 2+ Activity test under final concentration conditions;

[0041] Figure 9 Middle: The left picture shows the Pfumligase K372E ligation reaction Mn 2+ Final concentration condition test electrophoresis results, the right figure is Pfumligase K372E ligation reaction Mn 2+ Comparison of ligation efficiency results under final concentration conditions.

[0042] Figure 10 To test the activity of Pfumligase K372E at different final PEG8000 concentrations;

[0043] Figure 10 Middle: The left figure shows the electrophoresis results of the Pfumligase K372E ligation reaction under the conditions of PEG8000 final concentration. The right figure shows the comparison results of the ligation efficiency test under the conditions of PEG8000 final concentration of the Pfumligase K372E ligation reaction.

[0044] Figure 11 To test the activity of Pfumligase K372E under different reaction temperature conditions;

[0045] Figure 11 Middle: The left figure shows the electrophoresis results of the Pfumligase K372E ligation reaction temperature test, and the right figure shows the comparison results of the ligation efficiency of the Pfumligase K372E ligation reaction temperature test.

[0046] Figure 12 To test the activity of Pfumligase K372E under different reaction time conditions;

[0047] Figure 12 Middle: The left figure shows the electrophoresis results of the Pfumligase K372E ligation reaction time condition test, and the right figure shows the comparison results of the ligation efficiency of the Pfumligase K372E ligation reaction time condition test.

[0048] Figure 13Pfumligase K372E is used for ligation reaction of human plasma free DNA (cfDNA);

[0049] The arrows point to the cfDNA fragments connected to the adapter.

[0050] Figure 14 This is a single-strand library construction process based on Pfumligase K372E.

[0051] Figure 15 Library yield and fragment size distribution of Pfum-Pfum library construction technology.

[0052] Figure 16 This is the distribution of starting fragments in sequencing data using the Pfum-Pfum library construction technology. DETAILED DESCRIPTION

[0053] The present invention is further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto:

[0054] Example 1. Screening of potential mutation sites of Pfumligase ligase and construction of expression vector

[0055] The amino acid sequence of pfumligase is shown in SEQ ID NO: 1;

[0056] Mth RNA ligase, a member of the same family as Pfumligase, has been extensively studied, its structure has been elucidated, and its mechanism of catalyzing the ligation reaction is relatively well-defined. Modification of the Lys246 active site in Mth RNA ligase can enhance the ligase's ligation activity. The present invention further identified the corresponding site on Pfumligase, Lys248, and also selected Lys370 and Lys372 as potential mutation sites.

[0057] Based on the selected mutation sites and the corresponding amino acids to be modified, the required PCR primers were designed using Snapgene (v6.2.1) software. After synthesizing the corresponding primers, the wild-type Pfumligase protein nucleic acid sequence (SEQ ID NO: 1) was used as a template for inverse PCR reaction. The product was identified by electrophoresis and was consistent with the expected length ( Figure 1 The purified product was phosphorylated at the 3' end and self-cyclized to form a plasmid, which was then transformed into BL21 (DE3) competent cells. After culture on antibiotic plates, the resistant colonies were selected for first-generation sequencing to confirm that the site mutation was correct ( Figure 2 ) to obtain the protein expression vector and bacterial solution of the Pfumligase mutant. The above experimental procedures all used commercial reagents and strictly followed the operating instructions provided by the kit.

[0058] Lys248 is: compared with the amino acid sequence of SEQ ID NO: 1 (pfumligase), a substitution mutation is present at position 248; Lys is replaced by Ala; the amino acid sequence is as described in SEQ ID NO: 2;

[0059] Lys370 is: compared with the amino acid sequence of SEQ ID NO: 1, a substitution mutation is present at position 370; Lys is replaced by Pro; the amino acid sequence is as described in SEQ ID NO: 3;

[0060] Lys372 is: compared with the amino acid sequence of SEQ ID NO: 1, there is a substitution mutation at position 372; Lys is replaced by Glu; the amino acid sequence is as described in SEQ ID NO: 4.

[0061] Example 2. Protein Induction Expression, Purification and Identification of Pfumligase and Its Mutants

[0062] The low-temperature stored bacterial solution containing the expression plasmid of Pfumligase and its mutant proteins was inoculated into LB medium for recovery. After the recovered bacterial solution was expanded and cultured, it was transferred to 1L LB medium. Cultivate until the OD600 value reaches 0.6-0.8 to ensure that the bacterial solution is in the logarithmic growth phase, add IPTG inducer with a final concentration of 1mmol / L, and culture overnight at 16°C to induce protein expression. Collect and high-pressure crush the bacteria, and purify the products after high-pressure crushing. Protein purification uses Ni column and heparin column, and is carried out according to the instrument operation guide. The concentration of the purified protein product is measured, and the purity is identified by SDS-PAGE ( Figure 3 ),according to Figure 3 , it was found that the content of Pfumligase ligase and its mutants in the supernatant was much higher than that in the precipitate, demonstrating the protein's high solubility and favorable production. Furthermore, the flowthroughs F1 and F2, obtained after the supernatant passed through the Ni column, contained significantly lower levels of the target protein than the supernatant, reflecting the protein's strong affinity for the Ni column and ease of purification. Finally, through a combined purification step using a Ni column and a heparin column, the purity of the obtained protein was significantly improved compared to the supernatant, meeting the requirements for subsequent Pfumligase ligase and its mutants in ligation reactions. The purified Pfumligase and its mutant proteins were mixed with an equal volume of 100% glycerol and stored at -20°C for long-term storage.

[0063] Example 3. Ligation activity test of protein purification products of Pfumligase and its mutants

[0064] (1) Preliminary test of ligation activity of purified products of Pfumligase and its mutant proteins

[0065] The activity of the purified products of Pfumligase and its mutant proteins was evaluated by intermolecular linear ligation reaction. The ligation reaction system is as follows:

[0066] Table 1. Pfumligase ligase and its mutant ligation activity test system

[0067]

[0068]

[0069] illustrate:

[0070] The App-Adapter sequence refers to the universal adapter sequence in second-generation sequencing, with a length of 35 nt. The specific sequence information is: / 5'App / NNNNNNNNAGATCGGAAGAGCGTCGTGTAGGGAAA / iSpC3 / / iSpC3 / / 3'ddC / (5'App is adenylylated, / iSpC3 / / iSpC3 / / 3'ddC / is a terminal modification, and N is a random base).

[0071] The OH-con sequence is the control oligo sequence in the Lucigen circliagse kit.

[0072] The ligation reaction conditions were 95°C for 1 minute and 75°C for 3 hours. After the reaction, the activity of the purified products of Pfumligase and its mutant proteins was compared by Urea-PAGE electrophoresis. The electrophoresis results showed that the enzyme activity was significantly reduced after the K248A mutation, while the enzyme activity of Pfumligase and its K370P and K372E mutants was able to effectively ligate the substrate under the 3-hour reaction conditions ( Figure 4 ).

[0073] (2) Time gradient reaction test of the ligation activity of Pfumligase and its mutant protein purification products

[0074] The ligation activity of Pfumligase and its K370P and K372E mutants was evaluated using different reaction times. The reaction system was the same as in the table, and the ligation reaction time was set to 0.25h, 1h, and 3h. After the reaction, the ligation products were collected and identified by Urea-PAGE electrophoresis. The results of band gray value analysis showed that the enzyme activity of the K370P mutant was significantly decreased compared to Pfumligase after 1h of reaction, while the enzyme activity of the K372E mutant was significantly increased compared to before the mutation ( Figure 5In summary, among the mutants of Pfumligase ligase, the K372E mutant protein exhibited higher catalytic activity, while the K248A and K370P mutations could not enhance the ligation activity of Pfumligase.

[0075] Example 4. Optimization of ligation reaction conditions for the Pfumligase K372E mutant

[0076] The ligase activity of Pfumligase K372E was further tested by varying the buffer system and pH, enzyme / substrate ratio, metal ion concentration, PEG8000 concentration, reaction temperature, reaction time, etc. The results showed that Pfumligase K372E had a high ligase activity in Tris-HCl buffer at pH 7.5, an enzyme-to-substrate ratio of 0.5-1.5 / 1, a reaction temperature of 70 to 85°C, and a Mn concentration of 5 mM or higher. 2+ Under the conditions of a final concentration of 15% or more PEG8000 and a reaction time of more than 1.5 h, the ligation reaction is relatively complete (see Figure 6-12 ).

[0077] Taking into account the degradation effect of high temperature on the adenylylated termini and other factors, the optimal ligation reaction conditions for Pfumligase K372E were established as Tris-HCl, pH = 7.5 buffer system, enzyme / substrate ratio of 1:1, Mn 2+ The final concentration was 10 mM, the final concentration of PEG8000 was 20%, the reaction temperature was 75°C, and the reaction time was 2 hours. Compared to wild-type Pfumligase, replacing the HEPES buffer with Tris shortened the reaction time from 3 hours to 2 hours, demonstrating that the Pfumligase K372E of the present invention has a technical advantage over the original Pfumligase in terms of higher ligation efficiency.

[0078] In summary, we can know that:

[0079] Table 2. Optimal ligation system for Pfumligase K372E

[0080]

[0081] The App-Adapter sequence refers to the universal adapter sequence in second-generation sequencing, with a length of 42 nt. The specific sequence information is: / 5'App / NNNNNNNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC / iSpC3 / / iSpC3 / / 3'ddC / (5'App is adenylylated, / iSpC3 / / iSpC3 / / 3'ddC / is a terminal modification, and N is a random base).

[0082] The ligation reaction conditions were 95°C for 1 min and 75°C for 2 h.

[0083] Example 5. Construction of a single-stranded DNA library using the Pfumligase K372E mutant

[0084] Cell-free DNA (cfDNA) in plasma is a non-invasive biomarker, typically detected using high-throughput sequencing (NGS). Its role in the diagnosis and prognosis of numerous diseases is gaining increasing attention. cfDNA concentrations in plasma are extremely low, and it contains a high proportion of single-stranded DNA. Traditional double-stranded DNA library construction is inefficient, hindering research in areas such as NGS.

[0085] Pfumligase K372E mutant ligase, a highly thermostable single-stranded DNA ligase, has promising applications in single-stranded DNA library construction. Therefore, a single-stranded DNA library construction technique based on the Pfumligase K372E mutant was developed and applied to the detection of cell-free DNA in human plasma.

[0086] (1) Pfumligase K372E ligase is used for ligation of human plasma cfDNA

[0087] Human plasma cfDNA was used as the nucleic acid sample, and the ligation effect was evaluated using Pfumligase K372E ligase. The reaction system was prepared according to the following table:

[0088] Table 3. Pfumligase K372E ligase human plasma cfDNA reaction system

[0089]

[0090] The ligation reaction conditions were 95°C for 1 min and 75°C for 2 h.

[0091] After the reaction, the connection of cfDNA was identified by Urea-PAGE electrophoresis. The results showed that Pfumligase K372E ligase can effectively connect cfDNA to the adapter, preliminarily meeting the requirements of cfDNA single-strand library construction technology ( Figure 13 ).

[0092] (2) Establishment of single-strand library technology based on Pfumligase K372E ligase

[0093] Based on the advantage of Pfumligase K372E ligase in effectively connecting nucleic acid templates and adapters, the effective connection methods of adapters in the first and second rounds of library construction were expanded, and a new single-stranded library construction technology was developed.

[0094] The library construction scheme is named "Pfum-Pfum library construction". The main process includes: the nucleic acid to be tested is connected to the adapter in the first round, followed by linear extension, and the linear extension product is connected to the adapter in the second round and then amplified by library PCR ( Figure 14 ). Using 10ng cfDNA as the starting sample, the process was followed, the number of library PCR cycles was 8, and the library yields of three repeated library constructions were 174.8ng, 160ng, and 178ng, respectively. The results of library product analysis showed that the DNA library fragments were mainly concentrated around 320bp, with a single peak distribution, which was consistent with the length of the cfDNA fragments after ligation with the adapter sequence. This shows that the library construction technology meets the requirements of cfDNA library construction and can be used for subsequent NGS sequencing ( Figure 15 ).

[0095] (3) Sequencing data analysis based on single-strand library construction technology based on Pfumligase K372E ligase

[0096] The three constructed libraries were subjected to NGS sequencing and bioinformatics analysis (Table 4). The sequencing results showed that the sequencing data of the three libraries had a high number of effective reads, a high genome-wide correct mapping ratio (rightmapped ratio) and a low duplication rate (duplicate rate), and could effectively retain a large amount of template DNA fragment information under 150bp, which is conducive to the bioinformatics analysis of shorter cfDNA fragments derived from tumors ( Figure 16 ).

[0097] Table 4. Analysis results of single-stranded library sequencing data based on Pfumligase K372E ligase

[0098]

[0099]

[0100] Finally, it should be noted that the above examples are merely specific embodiments of the present invention. Obviously, the present invention is not limited to the above examples and is subject to numerous variations. All variations that can be directly derived or conceived by a person of ordinary skill in the art from the disclosure of the present invention are considered to be within the scope of protection of the present invention.

Claims

1. A lysine-mutated DNA / RNA ligase, characterized in that: Compared with the amino acid sequence of SEQ ID NO: 1, there is a substitution mutation at position 372; the amino acid sequence of the nucleic acid ligase is as shown in SEQ ID NO:

4.

2. The lysine-mutated DNA / RNA ligase according to claim 1, characterized in that: The mutation at position 372 is a Lys to Glu substitution.

3. A nucleic acid molecule, characterized in that: A DNA / RNA ligase encoding the lysine-mutated DNA / RNA ligase according to claim 1 or 2.

4. A carrier, characterized in that: Comprising the nucleic acid molecule of claim 3.

5. A recombinant cell, characterized in that: The nucleic acid according to claim 3 or the vector according to claim 4 is introduced.

6. A composition or kit for ligating single-stranded DNA and / or RNA, characterized in that: A DNA / RNA ligase comprising the lysine mutation according to claim 1 or 2.

7. Use of the lysine-mutated DNA / RNA ligase according to claim 1 or 2 in the preparation of a product for ligating single-stranded DNA and / or RNA.

8. The method according to claim 7, wherein: The ligation reaction system was pH = 7.5, 70mM final concentration of Tris-HCl buffer system, 1:1 enzyme / substrate ratio, 10mM Mn 2+ final concentration, PEG8000 final concentration of 20%; The ligation reaction conditions were 95°C for 1 min and 75°C for 2 h.

9. The method according to claim 7, wherein: The Pfumligase K372E mutant was used to construct a single-stranded DNA library.

10. The method according to claim 9, wherein: The nucleic acid to be tested is ligated to the adapter in the first round and then linearly extended. The linear extension product is ligated to the adapter in the second round and then amplified by library PCR.

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