CN117946985B - Mutant T4 DNA ligase, kit and application thereof in library construction - Google Patents
Mutant T4 DNA ligase, kit and application thereof in library construction Download PDFInfo
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Abstract
The invention provides a mutant T4 DNA ligase, which is characterized by at least the following six amino acid site mutations based on wild T4 DNA ligase: S14D, Q19I, S40Y, V108F, Q133M, N357P, and 0-3 mutation sites in A85P, V169I, D480M, wherein the amino acid sequence of the wild type T4 DNA ligase is shown as SEQ ID No. 10. The invention also relates to the use of mutant T4 DNA ligases in library construction. Compared with the conventional kit containing the T4 DNA ligase, the kit containing the salt-tolerant heat-stable mutant T4 DNA ligase has the advantages that the heat stability of the ligase is obviously improved, the activity of the ligase in a high-salt environment is obviously improved, the kit is more resistant to storage, the connection efficiency is obviously improved, the self-linking rate is lower, the kit components are fewer, the operation is simpler, and the requirements of automatic platform library establishment can be met.
Description
Technical Field
The invention relates to mutant T4 DNA ligase, coding DNA thereof, a kit and application thereof in library construction, and belongs to the technical field of biology.
Background
Along with the development of life science, we need to analyze genetic information of multiple species, NGS is also called high throughput sequencing, and can read sequences of hundreds of thousands to millions of DNA molecules at a time, and can provide abundant genetic information. Whereas the first step in NGS is library preparation, library preparation is critical to NGS workflow. This step will produce a sequencer compatible DNA or RNA sample. In general library construction, DNA is usually fragmented, the ends of the fragmented DNA are filled up, a is added to the 3 'end and 5' end of the filled up DNA fragments for phosphorylation, and specific linkers are added to the two ends of the DNA fragments to construct a sequencing library.
The linker and the DNA fragment are initially paired by A/T base complementation, and then a phosphodiester bond is formed between the DNA fragment and the 5 '-phosphate end and the 3' -hydroxyl end of the linker under the catalysis of ligase to achieve stable ligation. Therefore, stable and efficient ligase is particularly important for NGS sequencing.
T4 DNA ligase (T4 DNA LIGASE) is a double-stranded DNA ligase which is encoded by the gene of T4 phage 30 and can catalyze the reaction of forming phosphodiester bonds at the 5 '-phosphate end and the 3' -hydroxyl end of adjacent DNA chains, and by utilizing the characteristic, the T4 DNA ligase is widely used in various fields of genetic engineering, including gene cloning, high-throughput sequencing, high-throughput screening and the like.
Since the reaction substrate of T4 DNA ligase is usually subjected to cleavage treatment, it is inevitable that ions of the previous reaction remain in the ligation reaction, but in general, the wild-type (WT) T4 DNA ligase has mild reaction conditions, is heat-sensitive, has low stability, and is easily inactivated. High salt and high temperature can inhibit the enzyme activity and even cause irreversible inactivation. Therefore, the T4 DNA ligase with high efficiency and high stability has important commercial value and scientific research value.
Disclosure of Invention
The invention aims to provide a mutant T4 DNA ligase with high efficiency for library construction, which has higher ligation efficiency.
The invention adopts the technical scheme that: a mutant T4 DNA ligase, which comprises at least the following six amino acid site mutations based on the wild type T4 DNA ligase: S14D (serine at position 14 is mutated to aspartic acid), Q19I (glutamine at position 19 is mutated to isoleucine), S40Y (serine at position 40 is mutated to tyrosine), V108F (valine at position 108 is mutated to phenylalanine), Q133M (glutamine at position 133 is mutated to methionine), N357P (asparagine at position 357 is mutated to proline), and 0 to 3 of the following three mutation sites: a85P (alanine at position 85 mutated to proline), V169I (valine at position 169 mutated to isoleucine), D480M (aspartic acid at position 480 mutated to methionine); wherein the amino acid sequence of the wild type T4 DNA ligase is shown as SEQ ID No. 10. The amino acid sequence of the mutant T4 DNA ligase is shown in any one of SEQ ID No. 1-8.
Among the best-performing mutant T4 DNA ligases are: based on the wild type T4 DNA ligase, the following amino acid site mutations are provided: S14D, Q19I, S40Y, A85P, V F, Q133M, V169I, N357P, D M with an amino acid sequence as shown in SEQ ID NO:8, the coding DNA sequence is shown as SEQ ID NO: shown at 9.
The reaction temperature of the mutant T4 DNA ligase is 15-45 ℃, and the ion concentration in a reaction Buffer of the mutant T4 DNA ligase is 50-350 mM.
The invention also discloses application of the mutant T4 DNA ligase in DNA fragment ligation and library construction.
The invention also discloses a DNA (deoxyribonucleic acid) connecting kit which comprises the mutant T4 DNA ligase.
The invention also discloses a nucleic acid library construction kit which comprises the mutant T4 DNA ligase.
The invention also discloses a rapid DNA library construction kit, which comprises:
Adding reagent A into the end of fragmentation;
A linker ligating reagent, wherein the linker ligating reagent comprises the mutant T4 DNA ligase described above;
library amplification reagents.
Preferably, the end-of-fragmentation reagent A comprises a mixture of enzymes and reaction buffers thereof, wherein the enzymes comprise a fragmenting enzyme, an end-repair enzyme, a phosphokinase, and a DNA polymerase.
Preferably, the reaction temperature of the reagent A for the end-fragmentation is 30-40 ℃ and the reaction time is 3-30 min.
Preferably, the fragmentation end repair A reagent comprises NaCl and MgCl 2, and the concentration of each reagent is 50-350 mM.
Preferably, the concentration of MgCl 2 in the end-of-fragmentation modification A reagent is 300mM and the concentration of NaCl is 200mM.
Preferably, the adaptor-ligation reagent is a mixture of mutant T4 DNA ligase and a reaction buffer therefor.
Preferably, the concentration of salt ions in the adaptor-ligation reagent is 50-350 mM.
Preferably, the reaction temperature of the connector connecting reagent is 15-45 ℃.
Preferably, the mutant T4 DNA ligase is used in an amount of 1000U to 5000U.
Preferably, the library amplification reagent is a mixture of high fidelity DNA polymerase and its reaction buffer.
The rapid and stable nucleic acid library construction kit comprises a fragmentation reagent and a library amplification reagent, wherein the fragmentation reagent and the library amplification reagent can be DNA library construction kit components by a Hieff NGS ® OnePot Pro DNA Library Prep Kit V (Cat.12195) one-step method of the following holy organisms.
The mutant T4 DNA ligase has higher ligation efficiency, and can improve library yield when being used for library construction. The mutant T4 DNA ligase 8 has the characteristics of salt tolerance and thermal stability, has low sensitivity to salt concentration, and can directly accept various enzyme digestion or end repair reactions without purification. The mutant T4 DNA ligase 8 has low temperature sensitivity, can be stored for 4 weeks at room temperature, has no obvious reduction of activity, and ensures that the reaction and storage conditions are more loose and stable. The rapid and stable nucleic acid library construction kit provided by the invention has the advantages that the kit is only three components, the use is more convenient, the suitability of an automatic instrument is high, the library construction can be realized in any input amount of 0.1 ng-2 ug in 2.5 hours, and the requirements of various high-throughput sequencing library construction are met.
Drawings
FIG. 1 shows a library construction process of the present invention.
FIG. 2 comparison of ligation efficiencies of mutant T4 DNA ligase 8 and wild-type T4 DNA ligase.
FIG. 3 comparison of ligation efficiencies of mutant T4 DNA ligase 8 and wild-type T4 DNA ligase in different salt concentration reaction buffers.
FIG. 4 comparison of ligation efficiencies of mutant T4 DNA ligase 8 and wild-type T4 DNA ligase at different reaction temperatures.
FIG. 5 comparison of the time of storage of mutant T4 DNA ligase 8 with wild-type T4 DNA ligase at room temperature.
FIG. 6 mutant T4 DNA ligase 8 and kit therefor, were left at 30℃for 7 days to establish a library.
FIG. 7 mutant T4 DNA ligase 8 and kit therefor were left at 25℃for 4 weeks and library test results were obtained.
FIG. 8 mutant T4 DNA ligase 8 and kit therefor were left at 4℃for 4 weeks and library test results were obtained.
Detailed Description
The following description of the embodiments of the invention is further provided in conjunction with the accompanying drawings, and the specific embodiments described herein are meant to be illustrative of the invention.
Example 1: obtaining mutant T4 DNA ligase
Wild-type T4 DNA ligase has mild reaction conditions, is sensitive to temperature and salt concentration, is extremely easy to inactivate, is unstable and is not shelf stable. Therefore, we entrusted to the following san ZymeEditor platform, to carry out directional modification on wild type T4 DNA ligase, and on the basis of the wild type T4 DNA ligase, to carry out mutation of the following six sites: S14D, Q19I, S40Y, V108F, Q133M, N357P, and 0-3 mutations in three mutation points of A85P, V169I, D M, wherein the amino acid sequence of the wild type T4 DNA ligase is as shown in SEQ ID NO: shown at 10. And the encoding DNA of the T4 DNA ligase mutants 1 to 8 and the wild type T4 DNA ligase shown in Table 1 were synthesized, the encoding genes of the mutant T4 DNA ligases 1 to 8 and the wild type T4 DNA ligase were subjected to protein expression to obtain mutant T4 DNA ligases 1 to 8 and wild type T4 DNA ligases, 100ng was performed by Human Male Genomic (Promega; cat. G1471), and 6 cycles were amplified to construct libraries. The DNA banking process used in the present invention is described with reference to the next holy organism Hieff NGS ® OnePot Pro DNA Library Prep Kit V2 (Cat.12195). Referring to FIG. 1, a one-step DNA library construction procedure was followed. The results are shown in Table 1: compared with the wild type T4 DNA ligase, the library construction yield of the mutant T4 DNA ligase 1-8 is improved, which means that the improvement of the ligation efficiency leads to the increase of the fragment ratio of the ligating linker (high library conversion rate) so as to improve the library yield after amplification, wherein the yield of the mutant T4 DNA ligase 8 is obviously higher than that of the mutant T4 DNA ligase 1-7, and the library construction and performance analysis of the mutant T4 DNA ligase 8 are carried out later.
Table 1: mutant and wild type T4 DNA ligase mutant sites and library construction yield of different T4 DNA ligase
Example 2: mutant T4 DNA ligase 8 and wild-type T4 DNA ligase ligation efficiency test
The 300bp blunt end product (hereinafter referred to as 300A) used in this example was prepared using Lambda DNA (Promega; cat.D152A) as a template and using an end repair and A addition module in the following Saint organism Hieff NGS ® DNA Library Prep Kit (Cat.13577). Then, a ligation reaction was performed in the same ligation Buffer (Cat.13577) using the mutant T4 DNA ligase 8 and the wild-type T4 DNA ligase of example 1:
1) 300ng Lambda DNA (Promega; cat.d152 a) for end repair, add a treatment:
table 2:300A preparation conditions
2) And (3) joint connection:
Table 3: joint connection reaction system and reaction conditions
DNA ADAPTER (Cat, 12312) at a concentration of 15uM
3) After ligation was completed, 1.2 Xbead purification (Yeasen, 12601) was performed, eluting with 20. Mu.L ddH 2 O and 20. Mu.L was taken for quality testing as shown in FIG. 2. Under the same conditions, the adaptor ligation efficiency of mutant T4 DNA ligase 8 was better than that of wild-type T4 DNA ligase.
Example 3: comparison of ligation efficiency of mutant T4 DNA ligase 8 and wild-type T4 DNA ligase in reaction Buffer at different salt concentrations
The 300bp blunt end product (hereinafter referred to as 300A) used in this example was prepared using Lambda DNA (Promega; cat.D152A) as a template and using an end repair and A addition module in the following Saint organism Hieff NGS ® DNA Library Prep Kit (Cat.13577). The ligation reaction was performed in ligation buffers 1 to 8 using mutant T4 DNA ligase 8 and wild-type T4 DNA ligase of example 1:
1) 300ng Lambda DNA (Promega; cat.d152 a) for end repair, add a treatment:
Table 4:300A preparation conditions
2) In order that the ligation reaction was not interfered with by the salt ion in the reaction system of end repair and addition A, 1.2 Xbead purification (Yeasen, 12601) was performed after the completion of this step, and the ligation reaction was performed using 16. Mu.L of ddH 2 O, taking 15. Mu.L.
3) And (3) joint connection:
Table 5: connection reaction Buffer with different salt concentrations
Table 6: joint connection reaction system and reaction conditions
DNA ADAPTER (Cat, 13520) at a concentration of 15uM
4) After ligation was completed, 1.2 Xbead purification (Yeasen, 12601) was performed, eluting with 20. Mu.L ddH 2 O and 20. Mu.L was taken for quality testing as shown in FIG. 3. In ligation reaction Buffer with the same NaCl concentration, the adaptor ligation efficiency of mutant T4 DNA ligase 8 is better than that of wild type T4 DNA ligase, the wild type T4 DNA ligase basically loses the ligase activity at the NaCl concentration of 250mM, and the mutant T4 DNA ligase 8 still has the ligase activity at the NaCl concentration of 350 mM.
Example 4: different ligation temperature test of mutant T4 DNA ligase 8 and wild type T4 DNA ligase
The 300bp blunt end product (hereinafter referred to as 300A) used in this example was prepared using Lambda DNA (Promega; cat.D152A) as a template and using an end repair and A addition module in the following Saint organism Hieff NGS ® DNA Library Prep Kit (Cat.13577). Then, using the mutant T4 DNA ligase 8 and the wild-type T4 DNA ligase of example 1, the test of different ligation temperatures was performed in the same ligation Buffer (Cat.13577):
1) 300ng Lambda DNA (Promega; cat.d152 a) for end repair, add a treatment:
Table 7:300A preparation conditions
2) And (3) joint connection:
Table 8: joint connection reaction system and reaction conditions
DNA ADAPTER (Cat, 13520) at a concentration of 15uM
3) After ligation was completed, 1.2 Xbead purification (Yeasen, 12601) was performed, eluting with 20. Mu.L ddH 2 O and 20. Mu.L was taken for quality testing as shown in FIG. 4. Under the same reaction temperature, the adaptor connection efficiency of the mutant T4 DNA ligase 8 is better than that of the wild type T4 DNA ligase, the optimal reaction temperature of the mutant T4 DNA ligase 8 and the wild type T4 DNA ligase is 15-20 ℃, the wild type T4 DNA ligase almost loses the connection activity at 35 ℃, and the mutant T4 DNA ligase 8 still has the ligase activity at 45 ℃.
Example 5: mutant T4 DNA ligase 8 vs wild-type T4 DNA ligase storage time at RT
The 300bp blunt end product (hereinafter referred to as 300A) used in this example was prepared using Lambda DNA (Promega; cat.D152A) as a template and using an end repair and A addition module in the following Saint organism Hieff NGS ® DNA Library Prep Kit (Cat.13577). Then, using mutant T4 DNA ligase 8 and wild-type T4 DNA ligase stored at room temperature for different times, different ligation temperatures were tested in the same ligation Buffer (Cat. 13577):
1) 300ng Lambda DNA (Promega; cat.d152 a) for end repair, add a treatment:
Table 9:300A preparation conditions
2) And (3) joint connection:
table 10: joint connection reaction system and reaction conditions
DNA ADAPTER (Cat, 12312) at a concentration of 15uM
3) After ligation was completed, 1.2 Xbead purification (Yeasen, 12601) was performed, eluting with 20. Mu.L ddH 2 O and 20. Mu.L was taken for quality testing as shown in FIG. 5. It can be seen that the adaptor ligation efficiency of mutant T4 DNA ligase 8 was better than that of wild-type T4 DNA ligase, and that wild-type T4 DNA ligase lost almost ligation activity when stored at room temperature for 4 days, and mutant T4 DNA ligase 8 remained active when stored at room temperature for 4 weeks.
Example 6: mutant T4 DNA ligase 8 and kit library construction test thereof
The present embodiment was analyzed by using Human Male Genomic (Promega; cat. G1471) as a library scheme as shown below. The DNA banking process used in the present invention is performed with reference to the banking process (FIG. 1) of the next holy organism Hieff NGS ® OnePot Pro DNA Library Prep Kit V (Cat.12195). Several different forms of restriction enzyme library kits were combined using the salt tolerant, thermostable mutant T4 DNA ligase of example 1. The first cleavage library kit, similar in component format to Hieff NGS ® OnePot Pro DNA Library Prep Kit V2 (Cat.12195), contains A, B, C, D, E five components, in which the ligation module containing mutant T4 DNA ligase 8 is separated from the reaction Buffer and the enzyme and Buffer of the cleavage module are also separated. The second kit for enzyme digestion and library construction comprises A, B, C, D components, wherein the enzyme and Buffer of the enzyme digestion module are separated, and the ligation module containing mutant T4 DNA ligase 8 is prepared by premixing the ligase and the reaction Buffer. The third kit for enzyme digestion and library construction comprises A, B, C, D components, wherein the enzyme and Buffer of the enzyme digestion module are premixed together, and the ligation module containing mutant T4 DNA ligase 8 is a ligase and a reaction Buffer are separated. The fourth kit for enzyme digestion and library construction is fully premixed and comprises A, B, C components, wherein the enzyme and Buffer of the enzyme digestion module are premixed together, and the ligation module containing the mutant T4 DNA ligase 8 is prepared by premixing the ligase and the reaction Buffer. Wherein all the pre-mixed reagents are placed at the temperature of-20 ℃ for more than 3 months after pre-mixing.
Library construction comparison was performed using these four cleavage library construction kits:
1) Fragmentation, end repair, and A-addition were performed at 0.1ng, 1ng, 10ng, 100ng, 500ng, 1000ng, 2000ng Human Male Genomic:
Table 11: fragmentation, end repair, and A-addition treatment conditions
2) Linker ligation using a salt tolerant thermostable mutant T4 DNA ligase:
table 12: joint connection reaction system and reaction conditions
DNA ADAPTER is used at a concentration of 0.1ng/2uM, 1ng/2uM, 10ng/5uM, 100ng/7.5uM, 500ng to 2000ng/15uM.
3) Ligation product purification step reference Hieff NGS ® OnePot Pro DNA Library Prep Kit V2 (cat.12195), purification using 0.8 x magnetic beads (Yeasen, 12601); finally, 22. Mu.L of ddH 2 O was used for elution, and finally 20. Mu.L was used for library amplification.
4) Library amplification:
Table 13: library amplification reaction system and reaction conditions
5) Amplification was completed by 1.0 Xbead purification (Yeasen, 12601), elution was performed with 50. Mu.L ddH 2 O, and library construction results are shown in Table 14. It can be seen that the components of the kit are premixed and not premixed, and the difference of the warehouse building output is not large.
Table 14: DNA inventory production with different input
Example 7: mutant T4 DNA ligase 8 and kit database construction stability test thereof
The present embodiment was analyzed by using Human Male Genomic (Promega; cat. G1471) as a library scheme as shown below. The DNA banking process used in the present invention is performed with reference to the banking process (FIG. 1) of the next holy organism Hieff NGS ® OnePot Pro DNA Library Prep Kit V (Cat.12195). The salt-tolerant, thermostable mutant T4 DNA ligase and kit thereof of example 1 were used to test the shelf life at 4℃and 25℃and 30 ℃.
1) Fragmentation, end repair, and a-addition treatment were performed with 30ng, 200ng, 800ng Human Male Genomic:
table 15: fragmentation, end repair, and A-addition treatment conditions
2) Linker ligation using a salt tolerant thermostable mutant T4 DNA ligase:
table 16: joint connection reaction system and reaction conditions
DNA ADAPTER was used at a concentration of 30ng/5uM, 200ng/10uM, 800ng/15uM.
3) Ligation product purification step reference Hieff NGS ® OnePot Pro DNA Library Prep Kit V2 (cat.12195), purification using 0.8 x magnetic beads (Yeasen, 12601); finally, 22. Mu.L of ddH 2 O was used for elution, and finally 20. Mu.L was used for library amplification.
4) Library amplification:
Table 17: library amplification reaction system and reaction conditions
5) After completion of amplification, 1.0 Xbead purification (Yeasen, 12601) was performed, elution was performed using 50. Mu.L of ddH 2 O, the library results were shown in tables 18, 19 and 20, and agarose gel electrophoresis results were shown in FIGS. 6, 7 and 8. As can be seen, the component premix kit showed no significant drop in yield after either 7d at 30℃for 4 weeks, or 4 weeks at 4℃or 25 ℃.
Table 18: warehouse-building yield after 7d of standing at 30 DEG C
Table 19: warehouse-out yield after 4 weeks at 25 DEG C
Table 20: warehouse-out yield after 4 weeks at 4 DEG C
。
Claims (13)
1. A mutant T4 DNA ligase characterized by: based on the wild-type T4 DNA ligase, there are six amino acid site mutations: S14D, Q19I, S40Y, V108F, Q M and N357P, and 0-3 mutation sites in A85P, V169I, D M, wherein the amino acid sequence of the wild type T4 DNA ligase is shown as SEQ ID No. 10.
2. The mutant T4 DNA ligase of claim 1 wherein: the amino acid sequence of the mutant T4 DNA ligase is shown in any one of SEQ ID No. 1-8.
3. The mutant T4 DNA ligase according to claim 1 or 2, wherein the reaction temperature of the mutant T4 DNA ligase is 15-45 ℃, and the ion concentration in the reaction Buffer of the mutant T4 DNA ligase is 50-350 mM.
4. A DNA encoding the mutant T4 DNA ligase of any one of claims 1-3.
5. The coding DNA according to claim 4, wherein the sequence is shown in SEQ ID No. 9.
6. Use of a mutant T4 DNA ligase according to any one of claims 1 to 3 for ligating DNA fragments.
7. Use of a mutant T4 DNA ligase according to any one of claims 1 to 3 in library construction.
8. A DNA ligation kit comprising the mutant T4 DNA ligase of any one of claims 1 to 3.
9. A nucleic acid library kit comprising the mutant T4 DNA ligase of any one of claims 1-3.
10. A rapid DNA pooling kit, characterized in that the kit comprises:
Adding reagent A into the end of fragmentation;
A linker ligating reagent, wherein the linker ligating reagent comprises the mutant T4 DNA ligase of any one of claims 1-3;
library amplification reagents.
11. The rapid DNA pooling kit of claim 10, wherein the end-of-fragmentation repair a reagent comprises a mixture of enzymes and reaction buffers thereof, wherein the enzymes comprise a fragmenting enzyme, an end-repair enzyme, a phosphokinase, and a DNA polymerase.
12. The rapid DNA pooling kit of claim 10, wherein the adaptor-ligation reagent is a mixture of mutant T4 DNA ligase and reaction buffer.
13. The rapid DNA pooling kit of claim 10 wherein the library amplification reagent is a mixture of a high fidelity DNA polymerase and its reaction buffer.
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| CN110914415A (en) * | 2017-05-08 | 2020-03-24 | 科德克希思公司 | Engineered ligase variants |
| US10837009B1 (en) * | 2017-12-22 | 2020-11-17 | New England Biolabs, Inc. | DNA ligase variants |
| CN113774032A (en) * | 2021-11-12 | 2021-12-10 | 翌圣生物科技(上海)股份有限公司 | Recombinant T4 ligase mutant, coding DNA and NGS library construction method |
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| US10837009B1 (en) * | 2017-12-22 | 2020-11-17 | New England Biolabs, Inc. | DNA ligase variants |
| CN113774032A (en) * | 2021-11-12 | 2021-12-10 | 翌圣生物科技(上海)股份有限公司 | Recombinant T4 ligase mutant, coding DNA and NGS library construction method |
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