CN117802211A - Automatic non-natural nucleic acid Sanger sequencing method based on chain termination principle - Google Patents
Automatic non-natural nucleic acid Sanger sequencing method based on chain termination principle Download PDFInfo
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Abstract
The invention discloses an automatic non-natural nucleic acid Sanger sequencing method based on a chain termination principle. The method of the invention obtains the XNA sequencing tool enzyme (Bst F710Y) which can be compatible with the XNA to-be-sequenced chain and ddNTP simultaneously and efficiently by introducing the beneficial mutation in the DNA sequencing enzyme into the active pocket of the homologous XNA reverse transcriptase; compared with a wild type, the apparent primary extension rate of ddNTP by the XNA sequencing tool enzyme is improved by two orders of magnitude, and the same doping rate as that of natural dNTP is achieved; by using the protease as an XNA sequencing tool enzyme, a Sanger sequencing method of the XNA is established, and the continuous sequencing length of the XNA is broken through by 50 bases; the XNA sequencing method is compatible with BigDye modified ddNTP, and the sequencing product can be automatically analyzed and read by a DNA sequencer. The invention lays a foundation for screening and characterizing future functional XNA and developing a data storage system based on XNA.
Description
Technical Field
The invention relates to an automatic non-natural nucleic acid Sanger sequencing method based on a chain termination principle, and belongs to the field of gene sequencing.
Background
Non-natural nucleic acid (XNA) is structurally different from natural deoxyribonucleic acid (deoxyribonucleic acid, DNA) or ribonucleic acid (RNA), and is usually obtained through an organic synthesis manner, so that chemical diversity of nucleic acid can be enriched, performance of nucleic acid can be enhanced, and the non-natural nucleic acid plays a key role in practical applications such as oligonucleotide therapy. For example, sugar or phosphate backbone modified XNAs generally exhibit resistance to nuclease degradation, enhanced biostability, and are widely used in aptamers, ribozymes, and oligonucleotides. Most FDA-approved nucleic acid drugs contain sugar ring or phosphate backbone chemical modifications that enhance the efficacy, metabolic stability, and safety of the nucleic acid drug.
In the screening of functional XNA and the data storage of XNA as information carrier, there is an urgent need to develop a direct sequencing method of XNA. Current DNA sequencing techniques (e.g., sanger and next-generation sequencing technology, NGS) are not compatible with XNA, which can only be read by converting XNA to DNA in a reverse transcription-like manner, and sequencing the DNA. This process is cumbersome and easily results in loss of sequence information. Nanopore sequencing technology can directly read XNA sequences, but its read length is very limited, the root cause being that the motor protein is not compatible with the XNA strand to be sequenced. Thus, the present study is directed to the establishment of a simple and efficient sequencing platform for XNA.
Disclosure of Invention
The invention aims to: it is an object of the present invention to provide a method for Sanger sequencing of unnatural nucleic acids using a variant of XNA reverse transcriptase, which breaks through the continuous sequencing length of XNA by 50 bases.
The technical scheme is as follows: the invention discloses an automatic non-natural nucleic acid Sanger sequencing method based on a chain termination principle, which comprises the following steps of:
(1) Preparation of unnatural nucleic acid sequencing enzymes: introducing a beneficial mutation in the DNA sequencing enzyme into an XNA reverse transcriptase homologous site to obtain an XNA reverse transcriptase variant; chemically characterizing an XNA reverse transcriptase variant, evaluating the compatible effect of the XNA reverse transcriptase variant on four BigDye modified ddNTPs, and determining that the XNA reverse transcriptase variant can be used as an XNA sequencing enzyme if the extension rate of the XNA reverse transcriptase variant on the ddNTPs is consistent with that of the natural dNTPs, the ddNTPs can be randomly and highly-truly doped in the reverse transcription process of the XNA to cause extension termination, and all the four BigDye modified ddNTPs can be connected to a primer within one hour;
(2) Preparing a Sanger sequencing reaction system of the non-natural nucleic acid by using a to-be-sequenced chain of the non-natural nucleic acid and an XNA sequencing enzyme, uniformly mixing, gradient annealing, incubating at a constant temperature, and sequencing by using a denaturing polyacrylamide gel electrophoresis or DNA sequencer after the reaction is finished.
Wherein the DNA sequencing enzyme in step (1) is AmpliTaq TM The beneficial mutation is that 667 phenylalanine is mutated into tyrosine; the XNA reverse transcriptase is Bacillus stearothermophilus DNApolymerase I large fragment, the homologous site is 710 phenylalanine, and the phenylalanine is mutated into tyrosine. The specific operation method of the step (1) comprises the following steps:
1) Performing homology alignment on DNA sequencing enzyme and XNA reverse transcriptase by using Clustal W software, and determining key amino acid sites related to ddNTP recognition; the amino acid sequence of the Bst DNA polymerase is shown as SEQ ID No. 1;
2) The method comprises the steps of fully synthesizing an XNA reverse transcriptase gene, introducing mutation at key amino acid sites, constructing a protein expression vector, transforming into escherichia coli, inducing the expression of an XNA reverse transcriptase variant, and purifying by using a nickel column chromatography.
Wherein the chemically characterizing the XNA reverse transcriptase variant in step (1) comprises the percentage incorporation of the XNA reverse transcriptase variant into ddNTPs at each site, and comparing apparent first order extension rate constants of the wild-type XNA reverse transcriptase and variants thereof for four ddNTPs under more inversion conditions, the XNA reverse transcriptase variant being useful as an ideal XNA sequencing tool enzyme when the extent of its acceptance of ddNTPs is consistent with that of natural dNTPs.
Specifically, the characterization includes the following steps:
1) Mixing an XNA to-be-sequenced chain with a fluorescence modified sequencing primer according to a concentration of 1:1, carrying out denaturation annealing, dividing the mixture into four groups, adding a sequencing buffer, an XNA reverse transcriptase variant, four dNTPs and one ddNTP into each group, wherein the concentration ratio of the dNTPs to the ddNTP is 1:1, and testing the doping percentage of the XNA reverse transcriptase variant to the ddNTP at each site;
2) Mixing an XNA sequence to be detected with a fluorescence modified sequencing primer according to a concentration of 1:1, carrying out gradient annealing, wherein template bases at each group of positions to be extended are A, T, C, G respectively, comparing apparent first-order extension rate constants of wild XNA reverse transcriptase and variants thereof to four ddNTPs under a plurality of turnover conditions, and determining an XNA sequencing tool enzyme.
Wherein, in step (2), when using denaturing polyacrylamide gel electrophoresis, the Sanger sequencing reaction system of the unnatural nucleic acid is configured to: the non-natural nucleic acid strand to be sequenced is annealed with the fluorescent modified sequencing primer and is divided into 4 groups, and each group is added with non-natural nucleic acid sequencing enzyme, four dNTPs, one ddNTP and sequencing buffer.
Specifically, in the step (2), when the denaturing polyacrylamide gel electrophoresis is used, the method comprises the following steps: 1) Mixing an XNA sequence to be detected with a fluorescence modified sequencing primer according to the concentration of 2:1, carrying out gradient annealing, dividing the mixture into four groups, adding a sequencing buffer solution, an XNA sequencing tool enzyme, four dNTPs and one ddNTP into each group, uniformly mixing, and incubating at a constant temperature. 2) At the end of the reaction, the reaction was terminated by adding an equal volume of 8M urea and the sequencing products were read by denaturing PAGE.
Wherein, in step (2), when using a DNA sequencer for sequencing, the configuration of the Sanger sequencing reaction system for non-natural nucleic acids is: annealing the non-natural nucleic acid strand to be sequenced with a sequencing primer, adding a non-natural nucleic acid sequencing enzyme, four dNTPs, four BigDye modified ddNTPs and a sequencing buffer.
Wherein the four BigDye modified ddNTPs are ddTTP-dTMR, ddCTP-dROX, ddATP-dR6G and ddGTP-dR110.
Wherein, in the step (2), when the DNA sequencer is used for sequencing, the method further comprises a purification step after the reaction is finished, and the purification comprises a UNIQ-10 column type oligonucleotide purification method and an ethanol precipitation method.
Specifically, in step (2), when sequencing using a DNA sequencer, the steps include: 1) Mixing an XNA strand to be sequenced and a sequencing primer according to a concentration of 2:1, carrying out gradient annealing, adding a sequencing buffer, an XNA sequencing tool enzyme, four dNTPs and four BigDye modified ddNTPs into each group, uniformly mixing, and incubating at a constant temperature;
2) At the end of the reaction, sequencing product purification was performed using UNIQ-10 oligonucleotide purification kit;
3) Purifying the sequencing product by ethanol precipitation method, volatilizing ethanol at room temperature in dark place, adding deionized formamide (Hi-Di) TM Formamide) dissolving the sequencing sample, and storing at 4 ℃;
4) And (3) placing the sequencing sample into a DNA sequencer for automatic sequencing, and analyzing the XNA sequencing data through Sequencing Analysis analysis software after the data are taken off.
Wherein the length of the non-natural nucleic acid to be sequenced in the step (2) is 12-91 nucleotides.
Wherein the non-natural nucleic acid in step (2) comprises a 2' -fluoroarabinonucleic acid or a threose nucleic acid.
The beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages: according to the method, beneficial mutation in DNA sequencing enzyme is introduced into an active pocket of homologous XNA reverse transcriptase, so that XNA sequencing tool enzyme (Bst F710Y) which can be compatible with XNA to-be-sequenced chains with different skeletons and different lengths and ddNTP simultaneously and efficiently is obtained; compared with a wild type, the apparent primary extension rate of ddNTP by the XNA sequencing tool enzyme is improved by two orders of magnitude, and the same doping rate as that of natural dNTP is achieved; by using the protease as an XNA sequencing tool enzyme, a Sanger sequencing method of the XNA is established, and the continuous sequencing length of the XNA is broken through by 50 bases; the XNA sequencing method is compatible with BigDye modified ddNTP, and the sequencing product can be automatically analyzed and read by a DNA sequencer. The invention also has the advantages of simple system, easily available raw materials, easy reading of sequencing results and the like. The invention lays a foundation for screening and characterizing future functional XNA and developing a data storage system based on XNA.
Drawings
FIG. 1 is a schematic diagram of a method for Sanger sequencing of unnatural nucleic acids according to the present invention;
FIG. 2 shows the introduction of the mutation site of XNA reverse transcriptase and the effect display in the present invention;
FIG. 3 is a representation of the system of XNA sequencers of the present invention;
FIG. 4 shows the sequencing effect of the non-natural nucleic acid Sanger sequencing method established in the invention on the strands to be sequenced of different frameworks XNA;
FIG. 5 is a graphical representation of the results of the non-natural nucleic acid Sanger sequencing method of the present invention on the sequencing of XNA long-chain to be tested;
FIG. 6 is a graphical representation of the results of the automated sequencing of XNA achieved by the non-native nucleic acid Sanger sequencing method established in connection with the DNA generation sequencing platform of the present invention.
Detailed Description
The technical scheme of the invention is further described below with reference to the accompanying drawings.
Example 1 early verification
The verification content mainly comprises:
1) Obtaining the XNA strand to be sequenced 2' -fluoroarabinonucleic acid (fluoroarabinose nucleic acid, FANA) and threose nucleic acid (threose nucleic acid, TNA) by chemical solid phase synthesis or enzymatic synthesis, downstream of which comprises a sequencing primer binding region;
2) Using fluorescent modified sequencing primers, XNA reverse transcriptase Bst and four ddntps, verifying XNA Sanger sequencing feasibility;
the detailed preparation and operation processes are as follows:
1) Designing and synthesizing FANA single chain and TNA single chain to be detected:
a. FANA sequences were synthesized solid phase by four FANA phosphoramidite monomers (2 '-F-2' -ara-Bz-dA phosphoramidite,2'-F-2' -ara-N4-Bz-dC phosphoramidite,2'-F-2' -ara-N2-ibu-dG phosphoramidite,2'-F-2' -ara-dU phosphoramidite): 6mg of solid phase carrier (Ji Ma gene, 30-1100-XX) was weighed, put into a synthesis column, and four FANA phosphoramidite monomers, 0.25g each, were each dissolved in 2.5mL of acetonitrile, and put into four monomer bottles in sequence, and FANA solid phase synthesis was started. After the reaction, transferring the solid phase carrier into an EP tube, adding concentrated ammonia water, reacting for 16 hours at 55 ℃, centrifuging at 12000rpm to obtain a supernatant, adding one tenth volume of 3M sodium acetate solution, three times volume of absolute ethyl alcohol, shaking and uniformly mixing, freezing at-80 ℃ for 1 hour, centrifuging at 12000rpm for 10 minutes, discarding the supernatant, adding 75% of ethyl alcohol, and washing the precipitate twice. 200. Mu.L of 8M urea solution was addedDissolving the precipitate, cutting gel by 12% denaturing PAGE to purify target FANA sequence, placing gel block into ultrapure water, oscillating at 80 ℃ for 2 hours, sucking supernatant, desalting by a 3kDa ultrafiltration tube, and concentrating the product. Finally 2 FANA single chains were synthesized, respectively (The underlined portion is the primer binding region, bold is the region to be measured)。
b. Four TNA triphosphate monomers (tTTP, tATP, tCTP, tGTP, collectively tNTPs) (Curr Protoc NucleicAcid chem.2013, chapter 4:4.54.1-4.54.17) were synthesized by organic synthesis methods reported in the literature as substrates for TNA enzymatic synthesis; purified Kod-RSGA polymerase (ACS Synth biol.2020,9, 1873-1881) was expressed by methods reported in the literature; the TNA test sequence is synthesized by an enzymatic synthesis method: 1mL of the DNA template (10. Mu.M) was mixed uniformly with 1mL of the primer P5 (10. Mu.M) (see Table 1 in detail), gradient annealed (heating at 95℃for 5 minutes followed by gradual cooling to 4℃at a rate of 10℃C/min), followed by adding 100. Mu.M of tNTPs, 1x Thermopol buffer (20 mM Tris-HCl,10mM (NH) to the annealing reaction system 4 ) 2 SO 4 ,10mM KCl,2mM MgSO 4 0.1% Triton X-100, pH 8.8) and 1mg/mL Kod-RSGA polymerase, total volume 10mL, were mixed well and reacted in a water bath at 55℃for 4 hours. After the reaction was completed, the reaction products were characterized by 12% denaturing PAGE, 100W electrophoresis was performed for 1 hour, 3 XGelRed shake-stained gel, and an ultraviolet gel imaging system was used to image. The DNA-TNA chimeric sequence was purified by ethanol precipitation and gel cutting in the same manner as described in example 1, 1) a. The DNA-TNA chimeric sequence was treated with 1U/mL venom phosphodiesterase at 37℃for 16 hours to remove the DNA primer. After the reaction was completed, ethanol precipitation, gel cutting and TNA sequence purification were performed in the same manner as described in example 1, 1) a, nanodrop concentration was measured, and the mixture was stored in a refrigerator at-20 ℃.
TABLE 1 DNA templates, DNA primers and synthetic TNA Single-stranded sequences
Note that: the underlined part is the primer binding region, bold is the region to be detected, and pG is the residual guanine DNA monophosphate.
2) XNA sequencing Effect study of wild-type reverse transcriptase Bst:
single-stranded T1 (2. Mu.M) of 2. Mu.LTNA, 2. Mu.L of Cy5.5 modified sequencing primer P1 (2. Mu.M) (see Table 2 in particular), 2. Mu.L of 10x Thermopol buffer (200 mM Tris-HCl,100mM (NH) 4 ) 2 SO 4 ,100mM KCl,20mM MgSO 4 1% Triton X-100, pH 8.8) and 6. Mu.L of ultrapure water were mixed uniformly, and subjected to gradient annealing (heating at 95℃for 5 minutes, followed by gradual cooling to 4℃at a rate of 10℃per minute) in a PCR instrument. After completion of annealing, four sets of four dNTPs (except for the final concentration of dNTPs of the same base as ddNTPs of 2.5. Mu.m, the other three were 25. Mu.m), one ddNTP (final concentration of ddATP of 200. Mu.m, ddCTP of 300. Mu.m, ddTTP of 400. Mu.m, ddGTP of 200. Mu.m) and 2. Mu.L of Bst 2.0DNA polymerase (3.3 mg/mL) were added in each set, and the total volume was 20. Mu.L. After being evenly mixed, the mixture is placed in a water bath kettle with the temperature of 56 ℃ for reaction for 4 hours. After the reaction was completed, the reaction was terminated by adding an equal volume of 8M urea, analyzed by 12% denaturing PAGE, run at 60W for 2.5h, and imaged using an Odyssey CLx near infrared two-color fluorescence imaging system. As a result, as shown in fig. 2 b), wild-type Bst can randomly incorporate four ddntps during reverse transcription of TNA, but the efficiency of recognition of ddntps is very low; in the case of ddNTPs two orders of magnitude higher than dNTP concentrations, it is still preferable to select dNTPs as substrates, extending the vast majority of primers to full length.
TABLE 2 sequencing primers
PrimerP1 | Cy5.5-GCATCAGATCTCTAACTCAT |
PrimerP2 | Cy5.5-GCATCAGATCTCTAACTCATA |
PrimerP3 | Cy5.5-GCATCAGATCTCTAACTCATAC |
PrimerP4 | Cy5.5-GCATCAGATCTCTAACTCATACT |
PrimerP6 | GCATCAGATCTCTAACTCAT |
Example 2 development of a tool enzyme suitable for XNA Sanger sequencing
The content mainly comprises:
1) The XNA sequencing tool enzyme was designed, expressed and purified, and the results are shown in FIG. 2;
2) Biochemical characterization of XNA sequencing tool enzyme, the results are shown in figure 3;
the detailed preparation and operation processes are as follows:
a. bst DNA polymerase (Bacillus stearothermophilus DNA polymerase I large fragment, bst) amino acid sequence (299-876,SEQ ID No.1:AKMAFTLADRVTEEMLADKAALVVEVVEENYHDAPIVGIAVVNEHGRFFLRPETALADPQFVAWLGDETKKKSMFDSKRAAVALKWKGIELCGVSFDLLLAAYLLDPAQGVDDVAAAAKMKQYEAVRPDEAVYGKGAKRAVPDEPVLAEHLVRKAAAIWALERPFLDELRRNEQDRLLVELEQPLSSILAEMEFAGVKVDTKRLEQMGEELAEQLRTVEQRIYELAGQEFNINSPKQLGVILFEKLQLPVLKKTKTGYSTSADVLEKLAPYHEIVENILHYRQLGKLQSTYIEGLLKVVRPDTKKVHTIFNQALTQTGRLSSTEPNLQNIPIRLEEGRKIRQAFVPSESDWLIFAADYSQIELRVLAHIAEDDNLMEAFRRDLDIHTKTAMDIFQVSEDEVTPNMR) using Clustal WRQAKAVNFGIVYGISDYGLAQNLNISRKEAAEFIERYFESFPGVKRYMENIVQEAKQKGYVTTLLHRRRYLPDITSRNFNVRSFAERMAMNTPIQGSAADIIKKAMIDLNARLKEERLQARLLLQVHDELILEAPKEEMERLCRLVPEVMEQAVTLRVPLKVDYHYGSTWYDAK) and AmpliTaq TM The sequencing enzyme is subjected to homology alignment, and the beneficial mutation is selected as AmpliTaq TM The amino acid 710 of Bst DNA polymerase is mutated from phenylalanine to tyrosine, and the codon optimization and total gene synthesis of the escherichia coli are carried out. The N-terminal His-tagged gene fragment (SEQ ID No.2:5' -ATGAGAGGATCTCACCATCACCATCACCATACGGATCCAAGCGGCCTGGTGCCGCGCGGCAGCATGGCAAAAATGGC)
GTTCACCCTGGCTGACCGCGTGACCGAAGAAATGCTGGCTGATAAAGCGGCGCTC
GTGGTTGAAGTGGTTGAAGAAAACTACCACGACGCGCCGATTGTGGGTATCGCAG
TTGTTAACGAACATGGCCGTTTCTTTCTGCGTCCGGAAACCGCGCTGGCGGACCCG
CAGTTCGTTGCATGGCTGGGTGATGAAACCAAAAAGAAATCTATGTTCGATTCCAA
ACGTGCGGCGGTGGCTCTGAAATGGAAAGGTATCGAACTGTGCGGCGTTTCTTTC
GATCTGCTGCTGGCGGCATATCTGCTGGATCCGGCTCAGGGCGTTGATGATGTTGC
AGCAGCTGCGAAAATGAAACAATATGAAGCGGTGCGCCCGGATGAAGCAGTGTAT
GGTAAAGGCGCTAAACGTGCAGTTCCGGACGAACCGGTTCTGGCGGAACACCTGG
TGCGCAAAGCAGCAGCGATCTGGGCGCTGGAGCGTCCGTTCCTGGATGAACTGCG
TCGTAACGAACAGGATCGTCTGCTGGTTGAACTGGAACAGCCGCTGTCTAGCATCC
TGGCTGAAATGGAATTCGCGGGCGTTAAAGTGGATACTAAACGTCTGGAACAGAT
GGGTGAAGAACTGGCCGAACAGCTGCGTACGGTGGAACAGCGCATCTACGAACT
GGCAGGCCAGGAATTCAACATTAACTCCCCGAAACAGCTGGGTGTGATCCTGTTC
GAAAAACTGCAGCTGCCGGTGCTGAAGAAAACCAAAACTGGTTACAGCACCAGC
GCAGATGTGCTTGAAAAACTGGCCCCGTACCACGAAATCGTTGAAAATATTTTACA
TTACCGTCAGCTGGGTAAACTGCAGTCCACGTACATCGAAGGTCTGCTGAAAGTT
GTTCGTCCGGATACTAAAAAAGTTCACACCATTTTCAACCAGGCGCTGACCCAGAC
CGGTCGTCTGTCCTCTACCGAACCGAACCTGCAGAACATCCCGATCCGCCTGGAA
GAAGGTCGCAAAATCCGTCAGGCCTTCGTGCCGAGCGAATCTGATTGGCTGATCTT
TGCCGCTGATTATAGCCAGATTGAACTGCGTGTTCTGGCTCATATCGCGGAAGATGA
TAACCTGATGGAAGCCTTTCGTCGTGACCTGGATATTCACACCAAAACCGCAATGG
ATATCTTTCAGGTTTCCGAAGATGAAGTAACTCCTAACATGCGTCGTCAGGCAAAA
GCGGTTAACTACGGCATCGTGTATGGCATTAGCGATTATGGCCTGGCACAGAACCT
GAACATTTCTCGTAAAGAAGCAGCTGAATTTATCGAACGTTACTTTGAATCTTTCCC
AGGCGTTAAACGTTACATGGAAAACATTGTTCAGGAAGCGAAACAGAAAGGCTAT
GTGACCACCCTGCTGCACCGTCGTCGTTATCTGCCGGATATTACTTCCCGCAACTTT
AACGTTCGTAGCTTCGCTGAACGTATGGCGATGAACACCCCGATCCAGGGCAGCG
CGGCGGATATCATTAAAAAAGCGATGATTGACCTGAACGCGCGCCTGAAAGAAGA
ACGTCTGCAGGCTCGCCTGCTGCTGCAGGTTCATGATGAACTGATCCTGGAAGCAC
CGAAAGAAGAAATGGAACGTCTGTGCCGTCTGGTTCCGGAAGTTATGGAACAGGC
TGTGACCCTGCGTGTTCCGCTGAAAGTTGATTACCACTACGGTAGCACCTGGTACGACGCTAAATGA-3') was constructed into the protein expression vector pQE-80L (Biobw Co., bio-118267), and sequencing verified. BL21 (DE 3) competent cells were thawed on ice, 100ng of the above recombinant plasmid (1. Mu.L) was transformed into 50. Mu.L competent cells, plated on LB solid medium plates containing ampicillin resistance, and placed in an incubator at 37℃for inversion culture for 16 hours.
b. The monoclonal after sequencing verification is inoculated into 1L of liquid LB culture medium containing ampicillin resistance, the liquid LB culture medium is subjected to expansion culture at 37 ℃, and when the OD value of bacterial liquid reaches 0.6, 100mM isopropyl-beta-D-thiogalactoside (IPTG) is added into the bacterial liquid to induce the expression of Bst F710Y polymerase variant. After 12 hours, the bacterial solution was centrifuged and the supernatant was discarded. The pellet was collected and resuspended in 30mL PBS bufferMiddle (1 mM KH) 2 PO 4 ,3m M Na 2 HPO 4 ,2mM MgCl 2 155mM NaCl,pH 7.4) the bacteria were sonicated using a cytobreaker. The bacterial lysate was placed in a water bath, treated at 60℃for 40 minutes, ultracentrifuged at 8000rpm, and the supernatant was collected and filtered using a 0.22 μm filter membrane. The target protein was purified using Ni-NTA affinity gel resin. SDS-PAGE was used to verify the molecular weight and purity of the target protein. The eluate containing the target protein was collected, the target protein was replaced in Bst DNA polymerase storage buffer (50 mM Tris-HCl,150mM Na Cl,1mM EDTA,10mM. Beta. -ME, pH 7.5) using a 30kDa ultrafiltration tube, the concentration was determined using Nanodrop, and the solution was stored in a refrigerator at-20 ℃.
c. To the EP tube were added 1. Mu.L XNA template (2. Mu.M) (TNA sequencing effect analysis used T1; FANA sequencing effect analysis used F1), 1. Mu. L P1 (2. Mu.M), 1. Mu.L 10x Thermopol buffer (200 mM Tris-HCl,100mM (NH) 4 ) 2 SO 4 ,100mM KCl,20mM MgSO 4 1% Triton X-100, pH 8.8) and 4. Mu.L of ultrapure water were uniformly mixed and then gradient annealed (heated at 95℃for 5 minutes followed by gradual cooling to 4℃at a rate of 10℃per minute) and the mixture was divided into four groups, each of which was added with four dNTPs (each having a final concentration of 125. Mu.M), one ddNTP (having a final concentration of 125. Mu.M) and Bst F710Y polymerase variant prepared in step b (having a final concentration of 0.6M g/mL), and the total volume of the reaction system was 10. Mu.L. After being evenly mixed, the mixture is placed in a water bath kettle with the temperature of 56 ℃ for 4 hours of incubation. After the reaction was completed, the reaction was terminated by adding an equal volume of 8M urea, analyzed by 12% denaturing PAGE, run at 60W for 2.5 hours, and imaged using Odyssey CLx near infrared two-color fluorescence imaging system. The percent incorporation of ddNTP per site was calculated from the fluorescence value of the extension product bands. As shown in the results of figures 3 a) and 3 d), under the competing condition that the concentration of dNTP and ddNTP in the system is equal, the doping rate of the Bst F710Y polymerase variant of the invention to the ddNTP at each site is higher than that of dNTP #>50%) demonstrated that the effect of recognizing ddNTP is better than that of natural dNTP.
d. The extension rate of the wild-type Bst DNA polymerase and the F710Y polymerase variant to ddNTP was determined using XNA mode sequence. mu.L of TNA template (2. Mu.M) (T1 (corresponding to ddATP), T2 (corresponding to ddCTP), T3 (corresponding to ddTTP)) And T4 (corresponding ddGTP)), 5. Mu.L of Cy5.5 modified sequencing primer P1 (2. Mu.M), 5. Mu.L of 10x Thermopol buffer (200 mM Tris-HCl,100mM (NH) 4 ) 2 SO 4 ,100mM KCl,20mM MgSO 4 1% Triton X-100, pH 8.8), 25. Mu.L of ultrapure water and 5. Mu.L of ddNTP (125. Mu.M). Mixing well, placing in a PCR instrument, and performing gradient annealing (heating at 95 ℃ C. For 5 minutes, and then gradually cooling to 4 ℃ C. At a speed of 10 ℃ C./minute). mu.L of 0.6mg/mL Bst F710Y polymerase variant was added, mixed well and incubated in a 56℃water bath. At specific time points (15 s,30s,45s,1min,2min,3min,6min,10min,15min,30 min), 4. Mu.L of the reaction solution was taken out and added to 10. Mu.L of 8M urea, and mixed well on ice. The reaction was characterized by 12% denaturing PAGE, electrophoresed at 60W for 2.5h, imaged using an Odyssey CLx near infrared two-color fluorescence imaging system, and the reaction yield calculated from the fluorescence values of the primer and extension product bands, the apparent first order extension rate constants were calculated. As a result, as shown in FIG. 3 c), 3F), the apparent first order extension rate constant for ddNTP was increased by two orders of magnitude with either T NA (3 c) or FANA (3F) as templates, compared to the wild type (black) Bst F710Y polymerase variant (red).
Example 3 automated Sanger sequencing method to establish XNA
The content mainly comprises:
1) Sanger sequencing of XNA strands to be sequenced of different backbones, different sequences, and different lengths was performed using XNA sequencing-tool enzyme (Bst F710Y polymerase variant), and the results are shown in FIGS. 4 and 5.
2) The results are shown in FIG. 6 using four BigDye modified ddNTPs (ddTTP-dTMR, d dCTP-dROX, ddATP-dR6G, ddGTP-dR 110) (Superyears, 20200204IIIU,20200205IIIC,20200206IIIA,20200210 IIIG) compatible with the DNA generation sequencing platform.
The detailed preparation and operation processes are as follows:
a. mu.L of TNA template (T1, T5, T6 or T9, 6. Mu.M, respectively) or FANA template (F1 and F2, 6. Mu.M, respectively), 2. Mu.L of Cy5.5-labeled sequencing primer P1 (2. Mu.M), 2. Mu.L of 10x Thermop ol buffer (200 mM Tris-HCl,100mM (NH) 4 ) 2 SO 4 ,100mM KCl,20mM MgSO 4 1% Triton X-100, pH 8.8) and 6. Mu.L of ultrapure water were added to the EP tube, mixed well, gradient annealed (heating at 95℃for 5 minutes followed by gradual cooling to 4℃at a rate of 10℃per minute) and divided into four groups each of which was added four dNTPs (each at a final concentration of 125. Mu.M), one ddNTP (final concentration: ddATP 9. Mu.m, ddCTP 12. Mu.m, ddTTP 6. Mu.m, ddGTP 12. Mu.m, and Bst F710Y polymerase variant (final concentration 0.6 mg/mL) in a total volume of 20. Mu.L were mixed and incubated in a 56℃water bath for 5 hours. Analytical methods are the same as in examples 1, 2).
b. mu.L of XNA template (2. Mu.M) (TNA assay using T1 (corresponding ddATP), T2 (corresponding ddCTP), T3 (corresponding ddTTP) and T4 (corresponding ddGTP), respectively, FANA assay using F1 (corresponding to four ddNTPs)), 1. Mu.L of Cy5.5 modified sequencing primer (2. Mu.M) (TNA assay using P1 (corresponding to four ddNTPs), FANA assay using P1 (corresponding to ddATP), P2 (corresponding to ddCTP), P3 (corresponding to ddTTP) and P4 (corresponding to ddGTP), respectively, FANA assay using P1), 1. Mu.L 10x Thermopol buffer (200 mM Tris-HCl,100mM (NH) 4 ) 2 SO 4 ,100mM KCl,20mM MgSO 4 1% Triton X-100, pH 8.8), 5. Mu.L of ultrapure water and 1. Mu.L of BigDye modified ddNTP (50. Mu.M) were added to the EP tube, mixed well, gradient annealed (heating at 95℃for 5 minutes followed by gradual cooling to 4℃at a rate of 10℃per minute), followed by 1. Mu.L of Bst F710Y polymerase variant (6.6 mg/mL), mixed well and incubated in a 56℃water bath for 1 hour. Analytical methods are the same as in examples 1, 2).
c. mu.L of TNA template (4. Mu.M, T7 or T8), 6. Mu.L of unlabeled sequencing primer P6 (2. Mu.M), 6. Mu.L of 10x Thermopol buffer (200 mM Tris-HCl,100mM (NH) 4 ) 2 SO 4 ,100mM KCl,20mM MgSO 4 1% Triton X-100, pH 8.8), 19.2. Mu.L of ultrapure water, 6. Mu.L of dNTPs (125. Mu.M for each of the four types), 1.8. Mu.L of ddATP-dR6G (50. Mu.M), 1.8. Mu.L of ddGTP-dR110 (50. Mu.M), 2.4. Mu.L of ddCTP-dROX (50. Mu.M) and 1.8. Mu.L of ddTTP-dTMR (50. Mu.M) were added to the EP tube, mixed well, gradient annealed (heated at 95℃for 5 minutes, followed by gradual cooling to 4℃at a rate of 10℃per minute), followed by adding 6. Mu.L of Bst F710Y polymerase variant (6.6 mg/mL), and incubating in a 56℃water bath after mixing well for 6 hours. At the end of the reaction, purification reagents were used with UNIQ-10 oligonucleotidesThe cassette (division of biological engineering (Shanghai), cat: B511143) was used for sequencing product purification. Subsequently, the sequencing product was further purified by ethanol precipitation (same as in example 1, 1) a), volatilizing at room temperature in the absence of light to remove ethanol, and adding Hi-Di TM Formalmide dissolves the sequencing sample, places the sequencing sample on a DNA sequencer, sends the DNA sequencer to capillary electrophoresis for automatic sequencing, and analysis is carried out on XNA sequencing data through Sequencing Analysis analysis software after completion.
The Sanger sequencing results of the XNA strand to be sequenced show (FIGS. 4, 5) that termination bands are present at the desired positions, and that the sequence information of the XNA can be easily read from the termination bands. The results of automated sequencing of XNA using a DNA sequencer are shown in fig. 6 d); the sequence information corresponds to the dye peak positions one by one. Bst F710Y proved to be useful as an enzyme for XNA sequencing tools, and the method breaks through the continuous sequencing length of XNA by 50 bases.
Claims (10)
1. An automated non-native nucleic acid Sanger sequencing method based on the chain termination principle, comprising the steps of:
(1) Preparation of unnatural nucleic acid sequencing enzymes: introducing a beneficial mutation in the DNA sequencing enzyme into an XNA reverse transcriptase homologous site to obtain an XNA reverse transcriptase variant; characterizing an XNA reverse transcriptase variant, evaluating the compatible effect of the XNA reverse transcriptase variant on four BigDye modified ddNTPs, and determining that the XNA reverse transcriptase variant can be used as an XNA sequencing enzyme if the extension rate of the XNA reverse transcriptase variant on the ddNTPs is consistent with that of the natural dNTPs, the ddNTPs can be randomly and highly-truly doped in the reverse transcription process of the XNA to cause extension termination, and all the four BigDye modified ddNTPs can be connected to a primer in one hour;
(2) Preparing a Sanger sequencing reaction system of the non-natural nucleic acid by using a to-be-sequenced chain of the non-natural nucleic acid and an XNA sequencing enzyme, uniformly mixing, gradient annealing, incubating at a constant temperature, and sequencing by using a denaturing polyacrylamide gel electrophoresis or DNA sequencer after the reaction is finished.
2. The method of automated non-natural nucleic acid Sanger sequencing based on the chain termination principle according to claim 1, wherein the specific method of operation of step (1) is:
1) Performing homology alignment on DNA sequencing enzyme and XNA reverse transcriptase by using Clustal W software, and determining key amino acid sites related to ddNTP recognition;
2) The method comprises the steps of fully synthesizing an XNA reverse transcriptase gene, introducing mutation at key amino acid sites, constructing a protein expression vector, transforming into escherichia coli, inducing the expression of an XNA reverse transcriptase variant, and purifying by using a nickel column chromatography.
3. The method for automatic non-natural nucleic acid Sanger sequencing based on the chain termination principle according to claim 1, wherein the DNA sequencing enzyme in the step (1) is AmpliTaq TM The beneficial mutation is that 667 phenylalanine is mutated into tyrosine; the XNA reverse transcriptase is Bst DNA polymerase, the homologous site is 710-position phenylalanine, and the phenylalanine is mutated into tyrosine.
4. The automated non-native nucleic acid Sanger sequencing method based on the chain termination principle according to claim 1, wherein the characterizing the XNA reverse transcriptase variant in step (1) comprises the percentage of incorporation of the XNA reverse transcriptase variant into ddntps at each site, and the apparent first order extension rate constants of the wild type XNA reverse transcriptase and its variants to four ddntps under more inverted conditions, when the extent of acceptance of ddntps by the XNA reverse transcriptase variant is consistent with that of native dntps, is used as an ideal XNA sequencing tool enzyme.
5. The automated non-natural nucleic acid Sanger sequencing method based on the chain termination principle according to claim 1, wherein in step (2), when denaturing polyacrylamide gel electrophoresis is used, the configuration of the Sanger sequencing reaction system of the non-natural nucleic acid is as follows: annealing the non-natural nucleic acid to-be-sequenced chain and the fluorescence modified sequencing primer, dividing the non-natural nucleic acid to-be-sequenced chain into four groups, and adding non-natural nucleic acid sequencing enzyme, four dNTPs, one ddNTP and a sequencing buffer into each group.
6. The automated non-natural nucleic acid Sanger sequencing method based on the chain termination principle according to claim 1, wherein in step (2), when sequencing using a DNA sequencer, the configuration of the Sanger sequencing reaction system of the non-natural nucleic acid is as follows: annealing the non-natural nucleic acid strand to be sequenced with a sequencing primer, adding a non-natural nucleic acid sequencing enzyme, four dNTPs, four BigDye modified ddNTPs and a sequencing buffer.
7. The method of automated non-natural nucleic acid Sanger sequencing based on the chain termination principle according to claim 6, wherein the four BigDye-modified ddntps are ddTTP-dTMR, ddCTP-dtrox, ddATP-dR6G, ddGTP-dR110.
8. The method according to claim 1, wherein in the step (2), when the DNA sequencer is used for sequencing, the method further comprises a purification step after the reaction is finished, wherein the purification comprises a UNIQ-10 column oligonucleotide purification method and an ethanol precipitation method.
9. The method for automated non-natural nucleic acid Sanger sequencing based on the chain termination principle according to claim 1, wherein the non-natural nucleic acid to be sequenced in step (2) has a chain length of 12 to 91 nucleotides.
10. The method of automated non-natural nucleic acid Sanger sequencing based on the chain termination principle according to claim 1, characterized in that the non-natural nucleic acid in step (2) comprises 2' -fluoroarabinonucleic acid or threose nucleic acid.
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