Reverse transcription mutants and their applications

Abstract

The present invention belongs to the field of biotechnology, and discloses an MMLV reverse transcription mutant, which is obtained by replacing the RNaseH domain of a wild-type MMLV reverse transcriptase with a domain that enhances binding to an RNA-DNA hybrid chain. The present invention also performs mutation design on the mutant, wherein the preferred mutation combination is E69K, H126Y, N131M, H204R, and T306K. Compared with the wild type, the MMLV reverse transcription mutant of the present invention has a higher amplification efficiency. The present invention also provides the sequence of the MMLV reverse transcriptase mutant of the present invention and its application in a reverse transcription reaction.

Description

[0001] This application is a divisional application of patent application 202210621426.3 entitled “Reverse transcription mutants and their applications” filed by the applicant on June 1, 2022. Technical Field

[0002] The invention belongs to the field of biological technology, and more specifically, relates to a reverse transcription mutant and an application thereof. Background Art

[0003] Reverse transcriptase is a polymerase that uses RNA as a template and incorporates substrate dNTPs to synthesize DNA. It has a wide range of applications in the field of molecular biology. The reverse transcriptase derived from Murine-Moloneyleukemia virus (MMLV) (hereinafter referred to as MMLV reverse transcriptase) is an important tool enzyme widely used in basic scientific research. MMLV reverse transcriptase has high activity in a relatively wide temperature range of 37℃ to 60℃, and is particularly suitable for the amplification of longer fragments and RNA templates with complex secondary structures.

[0004] The wild-type MMLV reverse transcriptase contains a reverse transcription catalytic domain and an RNaseH domain. The RNaseH domain itself has binding sites for small chemical molecules, which may include heparin and bile salts from animals, polysaccharides and polyphenols from plants, and alcohols used in the RNA extraction process. The binding of these small chemical molecules to the RNaseH domain will cause a decrease in the continuous reverse transcription ability of the MMLV reverse transcriptase. In addition, the wild-type MMLV catalytic domain has a weak ability to bind to the template chain RNA, which will also cause the MMLV reverse transcriptase to have a low continuous reverse transcription ability.

[0005] Therefore, there is a need in the art for an improved reverse transcriptase having enhanced amplification efficiency to meet the needs of reverse transcription reaction applications in the art. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention improves the MMLV reverse transcriptase to obtain an MMLV reverse transcriptase mutant with enhanced amplification efficiency, including replacing the RNaseH domain with other domains that enhance the binding to the RNA-DNA hybrid chain, and further mutating the sites near the interaction interface between the catalytic domain and the RNA template to enhance the interaction between the MMLV reverse transcriptase and the RNA template.

[0007] The invention provides a MMLV reverse transcription mutant. Compared with a wild type, the mutant has a higher amplification efficiency.

[0008] The present invention provides an MMLV reverse transcriptase mutant, which is obtained by replacing the RNaseH domain of a wild-type MMLV reverse transcriptase (SEQ ID NO.1) with an sso7d-like family protein from Sulfuris phaeratokodaii, wherein the lysine (K) at position 13 of the sso7d-like family protein is mutated to leucine (L), and the sequence thereof is shown in SEQ ID NO.2.

[0009] In one embodiment, the RNaseH domain replacement is performed after the 474th amino acid residue of the wild-type MMLV reverse transcriptase, preferably, at positions 474 to 495, and more preferably, at position 495. The sequence of the replaced MMLV reverse transcriptase mutant is shown in SEQ ID NO.3.

[0010] In one embodiment, the present invention also carries out mutation design for the MMLV reverse transcriptase with the RNaseH domain replaced, and the sites where the mutation occurs include glutamic acid (H) at position 69, histidine (H) at position 126, asparagine (N) at position 131, aspartic acid (D) at position 200, histidine (H) at position 204, threonine (T) at position 306, proline (P) at position 326, threonine (T) at position 330, proline (P) at position 401, glutamine (Q) at position 374, and glutamine (Q) at position 430.

[0011] In one embodiment, the MMLV reverse transcriptase mutant of the present invention is selected from E69K, P326K, P401K, H204R, T306K, H126Y, N131M, D200K, T330P, P326K, P401K, Q374K, Q430K or a combination thereof; preferably, the MMLV reverse transcriptase mutant comprises an amino acid substitution selected from (1)-(5):

[0012] (1) P326K, P401K, H126Y and N131M;

[0013] (2) E69K, H126Y, N131M, H204R and T306K;

[0014] (3) E69K, D200K and T330P;

[0015] (4) E69K, P326K, P401K, Q374K and Q430K;

[0016] (5) E69K, D200K, P326K, P401K, T330P and T306K.

[0017] The present invention also provides the polynucleotide sequence of the MMLV reverse transcriptase mutant as described above.

[0018] The present invention also provides an expression vector, which comprises the polynucleotide sequence of the MMLV reverse transcriptase mutant.

[0019] The present invention also provides a host cell, which comprises the polynucleotide sequence of the MMLV reverse transcriptase mutant or the expression vector as described above.

[0020] The present invention also provides the use of the MMLV reverse transcriptase mutant of the present invention in a reverse transcription reaction.

[0021] The present invention also provides a kit for performing a reverse transcription reaction, which comprises the MMLV reverse transcriptase mutant as described above. Preferably, the kit further comprises a reverse transcription reaction buffer and dNTP. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : Western blot showing the expression and purification of wild-type MMLV reverse transcriptase and mutants M1 to M5, where WT is the wild type, M1 to M5 are five mutants, and Mw is a protein molecular weight marker.

[0023] Figure 2 : AC are fluorescence quantitative detection diagrams for three different genes (SCD1, Cyp7a1 and GADPH) using RNA from mice, wherein the Y-axis indicates the amplification signal and the X-axis indicates the amplification cycle number.

[0024] Figure 3 : AC are fluorescence quantitative detection graphs for three different genes (60S, E12 and FAD8) using RNA from peanut, wherein the Y-axis indicates the amplification signal and the X-axis indicates the amplification cycle number. DETAILED DESCRIPTION

[0025] Without wishing to be confined to a specific theory, the inventors believe that the RNaseH domain has the activity of digesting RNA in the RNA-DNA hybrid chain, which is unfavorable for the multi-cycle iterative reverse transcription of RNA, and therefore needs to be inactivated. The RNaseH domain itself can bind to the RNA-DNA hybrid chain, and plays an important role in the continuous reverse transcription ability of the MMLV reverse transcriptase. Deleting this domain will cause a decrease in the continuous reverse transcription ability of the MMLV reverse transcriptase. Therefore, the conventional method is to retain the RNaseH domain, and under the condition that it can still bind to the RNA-DNA hybrid chain, mutate the key sites related to the digestive activity in the RNaseH domain.

[0026] The MMLV reverse transcriptase mutant provided by the present invention is obtained by improving the wild-type MMLV reverse transcriptase, wherein the sequence of the wild-type MMLV reverse transcriptase is shown in SEQ ID NO.1.

[0027] The specific scheme of the present invention is as follows:

[0028] (1) Replace the RNaseH domain.

[0029] In one embodiment of the present invention, the RNaseH domain of MMLV reverse transcriptase is replaced with a sso7d-like family protein from Sulfurisphaera tokodaii.

[0030] Since the sso7d-like family protein has RNase activity, in one embodiment of the present invention, the RNaseH domain of the MMLV reverse transcriptase is replaced by the sso7d-like family protein from Sulfurisphaera tokodaii, wherein the lysine (K) at position 13 in the sso7d-like family protein from Sulfurisphaera tokodaii is replaced by leucine (L), and its sequence is shown in SEQ ID NO.2.

[0031] The amino acid residues at positions 362 to 474 of the MMLV reverse transcriptase are the connection domain, which is used to connect the thumb domain located in the range of amino acid residues 276 to 361 and the RNaseH domain located in the range of amino acid residues 474 to 672. The connection domain has an important regulatory function for the overall activity of the MMLV reverse transcriptase, so the replacement of the RNaseH domain in the present invention is performed after the 474th amino acid residue of the MMLV reverse transcriptase, which can be from position 474 to position 495, and preferably, the RNaseH domain is replaced at position 495.

[0032] In one embodiment of the present invention, the sequence of MMLV reverse transcriptase after replacing the RNaseH domain is shown as SEQ ID NO.3.

[0033] (2) Design of MMLV reverse transcriptase mutants.

[0034] In order to further improve the activity of MMLV reverse transcriptase, the inventors analyzed the structure of MMLV and obtained amino acid sites that may affect the template binding ability, including glutamic acid (H) at position 69, histidine (H) at position 126, asparagine (N) at position 131, aspartic acid (D) at position 200, histidine (H) at position 204, threonine (T) at position 306, proline (P) at position 326, threonine (T) at position 330, glutamine (Q) at position 374, proline (P) at position 401, and glutamine (Q) at position 430.

[0035] In the present invention, the following sites can be specifically designed for mutation:

[0036] (1) Glutamic acid at position 69, threonine at position 306, proline at position 326, glutamine at position 374, proline at position 401, and glutamine at position 430. These sites are located near the template chain and can bind to the phosphate backbone, and thus can be mutated to histidine, lysine, or arginine, preferably to lysine (K);

[0037] (2) Histidine at position 126, which faces the phosphate backbone of the template, can be mutated to tyrosine (Y), which is also aromatic and has only one benzene ring side chain, in order to enhance its interaction with the template chain and to ensure that the secondary structure is not affected;

[0038] (3) Asparagine at position 131 is closer to the base and is presumed to bind to the template chain mainly by hydrophobic interaction. Therefore, it can be mutated to methionine, leucine or isoleucine, preferably to methionine (M);

[0039] (4) Aspartic acid at position 200 and histidine at position 204. These two sites are far away from the template chain but close to the β-fold involved in catalysis, which indirectly regulate the interaction between the β-fold and the template chain. These two sites can be mutated to lysine, arginine or asparagine, preferably to lysine (K);

[0040] (5) The threonine at position 330 is adjacent to the α-helix that binds to the template and can affect the interaction between the reverse transcriptase and the template. Preferably, it is mutated to proline (P).

[0041] For the above mutation sites, the specific combinations provided by the present invention are shown in Table 1 below:

[0042] Table 1 Mutation site combinations

[0043]

[0044] (3) Obtaining MMLV reverse transcriptase mutants.

[0045] After designing the nucleic acid sequence of MMLV reverse transcriptase (as shown in SEQ ID NO.10 to SEQ ID NO.14), connect it to the commercial expression vector pET series based on T7 promoter, and use the commercial site-directed mutagenesis kit to obtain mutants. The expression vector used contains an operon sequence that responds to IPTG-induced expression, while ensuring efficient expression of the protein in Escherichia coli. The specific culture temperature of Escherichia coli is 16°C, 25°C, 30°C or 37°C, and the culture time can be 20h, 16h, 12h or 4h. Preferably, the present invention uses 16°C for induced expression, and the induction time is 20h.

[0046] (4) Isolation and purification of MMLV reverse transcriptase mutants.

[0047] After reaching the set induction expression culture time, the cultured bacteria are collected by centrifugation and resuspended by oscillation with a buffer. The pH of the buffer is 7.0-8.0, and it contains 10-50mM Trima, HEPES or Tricine as a pH buffer, and 200mM-500mM NaCl or KCl as a salt. The buffer used for lysis and purification contains 0.5mM-2mM reducing agent, which can prevent the erroneous disulfide bond folding inside the enzyme. The buffer is prepared with nuclease-free water, filtered and sterilized at high temperature and high pressure. The resuspended bacterial suspension is lysed and broken by ultrasound, and then an affinity chromatography method is used to obtain a higher purity MMLV reverse transcriptase mutant. Furthermore, ion exchange chromatography is used to remove more impurities so that the purity of the mutant target protein reaches more than 90%. The mutant target protein is finally dialyzed into the storage buffer.

[0048] (5) Detection of the amplification efficiency of MMLV reverse transcriptase mutants.

[0049] RNA from two different species was used for reverse transcription, with a template amount of 500 ng. The components necessary for reverse transcription and the obtained MMLV reverse transcriptase mutant were added, and primers at different sites were designed to synthesize cDNA fragments, and then the amount of the product was detected by qPCR fluorescence quantification.

[0050] Compared with the prior art, the technical solution of the present invention has the beneficial effect that the mutant of the present invention has a higher amplification efficiency.

[0051] Example

[0052] Example 1: Expression and purification of reverse transcriptase.

[0053] Add wild-type MMLV reverse transcriptase and its mutant plasmids to BL21 (DE3) competent cells, ice bath for 30 minutes, heat shock at 42°C for 90 seconds, and ice bath for 2 minutes. Add 900μL LB, incubate at 37°C / 220rpm / 1h, spread on LB plates containing kanamycin resistance, and invert overnight culture at 37°C. Pick the above single clone and inoculate it in 10mL LB (Kan+), shake at 37°C, 220rpm until turbidity, inoculate the bacterial liquid in 1000mL LB (Kan+), the total initial bacterial volume is 8L, shake at 37°C, 220rpm to OD=0.6, add 0.5mM IPTG 500μL, and induce overnight at 16°C / 120rpm for 18h. The induced bacterial solution was centrifuged at 4°C, 3500rpm for 10min, the bacterial cells were collected, and resuspended in 100mL buffer (20mM Tris-Cl 7.6, 300mM NaCl, 10% glycerol), ultrasonically disrupted for 15min, and then centrifuged at 4°C, 9500rpm for 30min, and the supernatant was collected. The supernatant was added to the affinity column, and after loading, the Ni-NTA chromatography column was equilibrated with affinity buffer for 5 column volumes, and washed with washing buffer (20mM Tris-Cl 7.6, 300mM NaCl, 50mM imidazole, 10% glycerol) for 5 column volumes, and finally eluted with elution buffer (20mM Tris-Cl7.6, 150mM NaCl, 300mM imidazole, 10% glycerol). Then, anion exchange chromatography was used for further purification, and the column was balanced with buffer (20mMTris-Cl7.6, 300mMNaCl, 10% glycerol), and then the sample was directly loaded for purification, and the flow-through sample was collected and analyzed by SDS-PAGE electrophoresis. The protein sample was dialyzed overnight with dialysis buffer (20mMTris-Cl7.6, 300mMNaCl, 1mMDTT, 1mMEDTA, 0.2% IPGAL-CA630, 50% glycerol), and the target protein was collected and stored at -20°C.

[0054] The protein results obtained are as follows Figure 1 As shown, the wild-type MMLV reverse transcriptase and each mutant can be well expressed in a soluble form.

[0055] Example 2: Amplification efficiency detection.

[0056] 1 μg of RNA was used as a template, and the RNA template was derived from mice and peanuts. Three genes at different loci of each species (mouse: SCD1, Cyp7a1 and GADPH; peanut: 60S, E12 and FAD8) were selected for reverse transcription. The specific reaction system is shown in Table 2. The reaction conditions were: incubation at 25°C for 10 min, incubation at 50°C for 5 min, and reaction at 85°C for 5 min.

[0057] Then, fluorescent quantitative PCR was used for detection. After the reverse transcription product was diluted 3 times, 1 μL was taken for qRT-PCR reaction. The reaction system is shown in Table 3, the reaction conditions are shown in Table 4, and the primer sequences used are shown in Table 5.

[0058] Table 2 Reverse transcription reaction system

[0059]

[0060] Table 3 qRT-PCR reaction system

[0061]

[0062]

[0063] Table 4 qRT-PCR reaction conditions

[0064]

[0065] Table 5 Primer sequences

[0066]

[0067] The comparison of Ct values ​​is used to determine the majority of product amounts, and thus the amplification efficiency. In short, the smaller the Ct value, the lower the number of cycles required to reach the threshold of the fluorescent quantitative PCR instrument, that is, the higher the initial reverse transcription product amount, the higher the efficiency of the reverse transcriptase.

[0068] The results are as follows Figure 2 (Mouse gene fluorescence quantitative detection) and Figure 3 (Fluorescence quantitative detection of peanut genes) shows that the amplification efficiency of reverse transcriptase mutants M1 to M5 is better than that of wild-type reverse transcriptase, among which mutant M5 performs better, which can be up to 4 Ct higher than the wild type in mouse gene fluorescence quantitative detection and up to 2 Ct values ​​higher than the wild type in peanut gene fluorescence quantitative detection.

[0069] The sequence is as follows:

[0070] SEQ ID NO.1 (sequence of wild-type MMLV):

[0071] TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLL

[0072] SEQ ID NO.2 (Mutated Sulfurisphaera tokodaii sequence): MVTVKFKYKGEELEVDISKIKKVWRVGKMISFTYDDNGKTGRGAVSEKDAPKELLQM LEKSGKK

[0073] SEQ ID NO.3 (Sequence after replacing the RNaseH domain):

[0074] TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNGTGGGGMVTVKFKYKGEELEVDISKIKKVWRVGKMISFTYDDNGKTGRGAVSEKDAPKELLQMLEKSGKK

[0075] SEQ ID NO.4 (Mutant M1)

[0076] TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIYPTVPMPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYKLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDKVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNGTGGGGMVTVKFKYKGEELEVDISKIKKVWRVGKMISFTYDDNGKTGRGAVSEKDAPKELLQMLEKSGKK

[0077] SEQ ID NO.5 (Mutant M2)

[0078] TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQKARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIYPTVPMPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALRRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNGTGGGGMVTVKFKYKGEELEVDISKIKKVWRVGKMISFTYDDNGKTGRGAVSEKDAPKELLQMLEKSGKK

[0079] SEQ ID NO.6 (Mutant M3)

[0080] TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQKARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFKEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNGTGGGGMVTVKFKYKGEELEVDISKIKKVWRVGKMISFTYDDNGKTGRGAVSEKDAPKELLQMLEKSGKK

[0081] SEQ ID NO.7 (Mutant M4)

[0082] TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQKARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYKLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKKGYAKGVLTQKLGPWRRPVAYLSKKLDKVAAGWPPCLRMVAAIAVLTKDAGKLTMGKPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNGGGTGGGGMVTVKFKYKGEELEVDISKIKKVWRVGKMISFTYDDNGKTGRGAVSEKDAPKELLQMLEKSGKK

[0083] SEQ ID NO.8 (Mutant M5)

[0084] TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQKARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFKEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLWIPGFAEMAAPLYKLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDKVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNGTGGGGMVTVKFKYKGEELEVDISKIKKVWRVGKMISFTYDDNGKTGRGAVSEKDAPKELLQMLEKSGKK

[0085] SEQ ID NO.9 (Nucleic acid sequence after replacing the RNaseH domain)

[0086]

[0087] SEQ ID NO.10 (Mutant M1 nucleic acid sequence)

[0088]

[0089] SEQ ID NO.11 (Mutant M2 nucleic acid sequence)

[0090]

[0091] SEQ ID NO.12 (Mutant M3 nucleic acid sequence)

[0092]

[0093] SEQ ID NO.13 (Mutant M4 nucleic acid sequence)

[0094]

[0095] SEQ ID NO.14 (Mutant M5 nucleic acid sequence)

[0096]

Claims

1. A MMLV reverse transcriptase mutant, characterized in that: The amino acid sequence of the mutant is shown in SEQ ID NO.

5.

2. A polynucleotide sequence encoding the MMLV reverse transcriptase mutant as claimed in claim 1. An expression vector comprising the polynucleotide sequence according to claim 2 .

4. A host cell comprising the polynucleotide sequence of claim 2 or the expression vector of claim 3.

5. Use of the MMLV reverse transcriptase mutant as claimed in claim 1 in a reverse transcription reaction.

6. A kit for performing a reverse transcription reaction, characterized in that: The invention comprises the MMLV reverse transcriptase mutant as claimed in claim 1.

7. The kit for performing a reverse transcription reaction according to claim 6, characterized in that: Also includes: Reverse transcription reaction buffer, dNTPs.

Images