KR101473988B1 - Reverse Transcriptase Having Improved Thermostability - Google Patents

Reverse Transcriptase Having Improved Thermostability Download PDF

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KR101473988B1
KR101473988B1 KR1020130165840A KR20130165840A KR101473988B1 KR 101473988 B1 KR101473988 B1 KR 101473988B1 KR 1020130165840 A KR1020130165840 A KR 1020130165840A KR 20130165840 A KR20130165840 A KR 20130165840A KR 101473988 B1 KR101473988 B1 KR 101473988B1
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박한오
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주성모
황병오
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Abstract

본 발명은 열안정성이 증가된 역전사효소에 관한 것으로서, 보다 상세하게는 서열번호 1로 기재되는 M-MLV 유래 역전사효소의 아미노산 서열의 63번째의 글루타민(Q63), 264번째의 리신(K264), 295번째의 리신(K295), 306번째의 트레오닌(T306), 346번째의 글루탐산(E346), 408번째의 프롤린(P408), 438번째의 히스티딘(H438) 및 454번째의 아스파라긴(N454)으로 이루어진 군으로부터 선택되는 하나 이상의 아미노산이 다른 아미노산으로 치환되어 열안정성이 증가된 돌연변이 역전사효소에 관한 것이다. 본 발명의 돌연변이 역전사효소는 야생형 역전사효소와 비교하여 뛰어난 열안정성을 나타내므로, 고온에서도 안정한 2차 구조를 형성할 수 있는 특정한 RNA의 구조적 방해로 인해 효율적으로 역전사 산물을 얻지 못하는 경우라도 안정적으로 역전사 활성을 나타내어 원하는 cDNA를 얻을 수 있다는 장점이 있다.The present invention relates to a reverse transcriptase having increased thermostability. More particularly, the present invention relates to a reverse transcriptase having an amino acid sequence of 63rd glutamine (Q63), 264th lysine (K264) A group consisting of 295th lysine (K295), 306th threonine (T306), 346th glutamic acid (E346), 408th proline (P408), 438th histidine (H438) and 454th asparagine (N454) Is replaced by another amino acid to thereby increase the thermal stability of the mutant reverse transcriptase. Since the mutant reverse transcriptase of the present invention exhibits excellent thermal stability compared with the wild type reverse transcriptase, even if the reverse transcription product can not be efficiently obtained due to the structural interference of a specific RNA capable of forming a stable secondary structure even at a high temperature, And the desired cDNA can be obtained.

Description

열안정성이 증가된 역전사효소{Reverse Transcriptase Having Improved Thermostability}Reverse Transcriptase Having Improved Thermostability}

본 발명은 역전사효소에 관한 것으로서, 보다 상세하게는 종래 역전사효소를 구성하는 특정 아미노산 잔기를 돌연변이시킴으로써 열안정성을 증가시킨 역전사효소에 관한 것이다.The present invention relates to a reverse transcriptase, and more particularly, to a reverse transcriptase having increased thermal stability by mutating a specific amino acid residue constituting a conventional reverse transcriptase.

RNA 종양 바이러스, 특히 몰로니 설치류 백혈병 바이러스(Moloney-Murine Leukemia Virus, M-MLV)나 인간 후천성 면역결핍 바이러스(HIV), 조류 골수아구증 바이러스(Avian Myeloblastosis Virus, AMV) 유래의 역전사효소(reverse transcriptase)에 관해 많은 연구가 이루어지고, 다양한 기능, 성질이 알려져 오고 있다. RNA을 주형으로 사용하여 이것에 상보적인 DNA(cDNA)를 합성할 수 있다고 하는 특징적인 성질로 인해 역전사효소는 많은 분자생물학적 방법, 예를 들어 cDNA 라이브러리의 구축, 역전사 및 중합효소연쇄반응(polymerase chain reaction, PCR) 등에 사용되고 있다.Reverse transcriptase derived from RNA tumor viruses, especially Moloney-Murine Leukemia Virus (M-MLV), Human Acquired Immunodeficiency Virus (HIV), and Avian Myeloblastosis Virus (AMV). ), many studies have been made, and various functions and properties have been known. Due to its characteristic property that RNA can be used as a template to synthesize DNA (cDNA) complementary to it, reverse transcriptase is used in many molecular biological methods, such as construction of cDNA libraries, reverse transcription and polymerase chain reactions. reaction, PCR), etc.

3가지 프로토타입(prototype) 형태의 레트로바이러스 역전사효소가 광범위하게 연구되고 있다. 몰로니 설치류 백혈병 바이러스의 역전사효소는 RNA 의존성 DNA 중합효소 및 RNase H 활성을 갖는 78 kDa의 단일 서브유닛(subunit)을 함유한다. 상기 효소는 E. coli에서 완전한 활성형으로 클로닝 및 발현되어 있다. HIV의 역전사효소는 p66 및 p51의 서브유닛의 헤테로다이머(hetero-dimer)로서, p51 서브유닛은 p66 서브유닛의 단백질분해성 절단에 의해 생성된다. p66 서브유닛은 RNA 의존성 DNA 중합효소 및 RNase H 도메인(domain) 모두를 갖고 있지만, p51 서브유닛은 DNA 중합효소 도메인만을 갖는다. 활성형 HIV의 p66/p51 역전사효소는 E. coli을 포함하는 다수의 발현 숙주에서 클로닝 및 발현되고 있다. HIV p66/p51 헤테로다이머 내에서, 51 kDa의 서브유닛은 촉매적으로 불활성이고, 66 kDa의 서브유닛은 DNA 중합효소 및 RNase H 활성 모두를 갖는다. 또한, 라우스 육종 바이러스(Rous Sarcoma Virus, RSV)의 역전사효소, 조류 골수아구증 바이러스의 역전사효소, 조류 적아구증 바이러스(Avian Erythroblastosis Virus, AEV) 헬퍼 바이러스 MCAV의 역전사효소, 조류 골수구종증 바이러스(Avian Myelocytomatosis Virus) MC29 헬퍼 바이러스 MCAV의 역전사효소, 조류 세망내피증 바이러스(Avian Reticuloendotheliosis Virus, REV-T) 헬퍼 바이러스 REV-A의 역전사효소, 조류 육종 바이러스 UR2의 헬퍼 바이러스 UR2AV의 역전사효소, 조류 육종 바이러스 Y73의 헬퍼 바이러스 YAV의 역전사효소, 라우스 연관 바이러스(Rous Associated Virus, RAV)의 역전사효소 및 골수아구증 연관 바이러스(Myeloblastosis Associated Virus, MAV)의 역전사효소와 같은 조류 육종 백혈병 바이러스(Avian Sarcoma-Leukosis Virus, ASLV)의 역전사효소도 2가지 서브유닛 알파(대략 62 kDa) 및 베타(대략 94 kDa)의 헤테로다이머이며, 상기 알파는 베타로부터 단백질분해성 절단에 의해 생성된다. ASLV 역전사효소는 알파-베타 및 베타-베타의 2가지 촉매적으로 활성인 구조 형태로 존재할 수 있다. 침강 분석에 의해, 상기 알파-베타 및 베타-베타 구조는 다이머이고, 상기 알파는 모노머 형태와 다이머 형태 사이에서 평형상태로 존재한다는 것이 알려져 있다. ASLV의 알파-베타 및 베타-베타 역전사효소는 동일한 단백질 복합체 내에서 DNA 중합효소 활성, RNase H 활성 및 DNA 엔도뉴클레아제(endonuclease)(integrase) 활성의 3가지 다른 활성을 갖고 있는 종래 공지된 유일한 레트라바이러스 역전사효소이다. 상기 알파 형태는 인테그라제 도메인 및 활성을 갖고 있지 않다.Three prototype types of retroviral reverse transcriptase are being studied extensively. The reverse transcriptase of Moloney rodent leukemia virus contains a single subunit of 78 kDa with RNA-dependent DNA polymerase and RNase H activity. The enzyme has been cloned and expressed in its fully active form in E. coli. The reverse transcriptase of HIV is a heterodimer of the subunits of p66 and p51, and the p51 subunit is produced by proteolytic cleavage of the p66 subunit. The p66 subunit has both an RNA-dependent DNA polymerase and an RNase H domain, whereas the p51 subunit has only a DNA polymerase domain. The p66/p51 reverse transcriptase of active HIV has been cloned and expressed in a number of expression hosts including E. coli. Within the HIV p66/p51 heterodimer, the 51 kDa subunit is catalytically inactive and the 66 kDa subunit has both DNA polymerase and RNase H activities. In addition, reverse transcriptase of Rous Sarcoma Virus (RSV), reverse transcriptase of avian myeloblastosis virus, reverse transcriptase of Avian Erythroblastosis Virus (AEV) helper virus MCAV, and avian myelocytosis virus (Avian Myelocytomatosis Virus) MC29 helper virus MCAV reverse transcriptase, Avian Reticuloendotheliosis Virus (REV-T) helper virus REV-A reverse transcriptase, avian sarcoma virus UR2 helper virus UR2AV reverse transcriptase, avian sarcoma virus Y73 Avian Sarcoma-Leukosis Virus (Avian Sarcoma-Leukosis Virus), such as reverse transcriptase of YAV's helper virus, reverse transcriptase of Rous Associated Virus (RAV) and reverse transcriptase of Myeloblastosis Associated Virus (MAV). ASLV) is also a heterodimer of two subunits alpha (approximately 62 kDa) and beta (approximately 94 kDa), which alpha is produced by proteolytic cleavage from beta. ASLV reverse transcriptase can exist in two catalytically active structural forms, alpha-beta and beta-beta. By sedimentation analysis, it is known that the alpha-beta and beta-beta structures are dimers, and that the alpha exists in equilibrium between the monomer form and the dimer form. The alpha-beta and beta-beta reverse transcriptases of ASLV are the only conventionally known to have three different activities: DNA polymerase activity, RNase H activity and DNA endonuclease (integrase) activity within the same protein complex. It is a retroviral reverse transcriptase. The alpha form has no integrase domain and activity.

역전사효소 매개성 역전사에 의해 mRNA로부터 cDNA로 변환되는 것은 많은 유전자 발현 연구에 있어서 중요하다. 그러나, 역전사를 촉매하기 위해 변형되지 않은 역전사효소를 사용하는 것은 여러 가지 측면에서 바람직하지 않다. 먼저, 역전사효소는 역전사효소에 존재하는 RNase H 활성에 의해 첫 번째 가닥(strand) 반응이 개시되거나 완료되기 전에 RNA 주형을 분해해 버리는 경우가 있다. 또한, mRNA 주형 분자의 미스-프라이밍(mis-priming)으로 인해 cDNA의 첫 번째 가닥에 오류가 발생할 수 있다. 사실, cDNA 합성 과정에서 HIV 역전사효소의 경우에는 3,000 내지 6,000 뉴클레오티드 당 1개 염기, AMV 역전사효소의 경우에는 10,000 뉴클레오티드 당 1개 염기에 오류가 발생하는 것으로 나타났다.The conversion from mRNA to cDNA by reverse transcriptase-mediated reverse transcription is important for many gene expression studies. However, the use of an unmodified reverse transcriptase to catalyze reverse transcription is undesirable in several respects. First, reverse transcriptase may degrade the RNA template before the first strand reaction is initiated or completed by RNase H activity present in reverse transcriptase. In addition, errors may occur in the first strand of cDNA due to mis-priming of the mRNA template molecule. In fact, in the cDNA synthesis process, an error occurs at 1 base per 3,000 to 6,000 nucleotides in the case of HIV reverse transcriptase and 1 base per 10,000 nucleotides in the case of AMV reverse transcriptase.

역전사효소의 효능에 영향을 미치는 다른 인자는 RNA가 2차 구조를 형성할 수 있는지 여부이다. 이와 같은 2차 구조는, 예를 들면, RNA 분자 영역이 교잡하여 이중 가닥 RNA를 형성할 정도로 충분히 상보성이 있을 때 형성될 수 있다. 일반적으로 RNA 2차 구조의 형성은 RNA 분자를 함유하는 용액의 온도를 상승시킴으로써 감소될 수 있다. 따라서, 많은 경우 37℃ 이상의 온도에서 RNA를 역전사하는 것이 바람직하다. 그러나, 본 기술분야에 공지된 역전사효소는 일반적으로 37℃보다 상당히 높은 온도(예를 들면, 50℃)에서 배양해야만 활성을 잃어버린다.Another factor that influences the efficacy of reverse transcriptase is whether RNA can form secondary structures. Such secondary structures can be formed when, for example, regions of RNA molecules are sufficiently complementary to hybridize to form double-stranded RNA. In general, the formation of RNA secondary structures can be reduced by raising the temperature of the solution containing the RNA molecule. Therefore, in many cases, it is desirable to reverse transcription of RNA at a temperature of 37°C or higher. However, reverse transcriptase known in the art generally loses its activity only when incubated at a temperature significantly higher than 37°C (eg, 50°C).

열안정성 역전사효소를 가공하기 위한 다양한 방법이 본 기술분야에 공지되어 있다. 상기 방법으로는 역전사효소 활성을 갖는 열안정성 DNA 중합효소를 이용하는 방법, 열안정성 DNA 중합효소에 변이를 유발시켜 그 역전사효소 활성을 증대시키는 방법, 열 불안정성 역전사효소에 변이를 유발시키는 공정, Taq / Tth DNA 중합효소의 존재하에 Mg2 + 대신에 Mn2 +를 이용하는 방법, 열 불안정성 역전사효소와 함께 트레할로스와 같은 첨가물을 이용하는 방법 등을 포함한다.Various methods are known in the art for processing thermostable reverse transcriptases. The above methods include a method of using a heat-stable DNA polymerase having reverse transcriptase activity, a method of inducing a mutation in a heat-stable DNA polymerase to increase its reverse transcriptase activity, a step of inducing a mutation in a heat-labile reverse transcriptase, Taq / in the presence of the Tth DNA polymerase with the method, heat labile reverse transcriptase using a Mn + 2 in place of Mg 2 +, and the like method using an additive such as trehalose.

또한, 본 기술분야의 숙련된 당업자는 DNA 또는 RNA 주형을 중합할 때의 정확성(fidelity)을 증가시키기 위하여 여러 가지 효소 조성물과 방법을 사용해 오고 있다. 예를 들면, 쉐벨레프 등(Shevelev et al., Nature Rev . Mol . Cell Biol. 3:364 (2002))은 3'-5' 엑소뉴클레아제에 대한 리뷰논문을 제공하고 있다. 페리노 등(Perrino et al., PNAS USA, 86:3085 (1989))은 송아지 흉선 DNA 중합효소 알파의 정확성을 증가시키기 위하여 E. coli DNA 중합효소 Ⅲ의 입실론 서브유닛을 사용하고 있다. 바크하나쉬빌리(Bakhanashvili, Eur. J. Biochem. 268:2047 (2001))는 p53 단백질의 교정(proofreading) 활성을 개시하고 있고, 후앙 등(Huang et al., Oncogene, 17:261 (1998))은 DNA 복제의 정확성을 증강시키기 위하여 p53을 사용하는 것에 대해 개시하고 있다. 또한, 미국 특허공개 제2003/0198944A1호 및 미국 특허 제6,518,019호는 2개 이상의 역전사효소(예컨대, 각각의 역전사효소는 상이한 전사 정지 부위를 가짐) 및 필요에 따라 1개 이상의 DNA 중합효소를 함유하는 효소 혼합물을 개시하고 있다. 미국 특허공개 제2002/0119465A1호는 변이체 열안정성 DNA 중합효소 및 변이체 역전사효소를 포함하는 조성물(예컨대, 변이체 Taq DNA 중합효소 및 변이체 MMLV-RT)을 개시하고 있다. 미국 특허 제6,485,917B1호, 미국 특허공개 제2003/0077762호 및 유럽 특허공개 EP1132470은 역전사효소 활성을 갖는 효소 및 3'-5' 엑소뉴클레아제 활성을 갖는 α형 DNA 중합효소의 존재하에 cDNA를 합성하는 방법을 개시하고 있다.In addition, those skilled in the art have used various enzyme compositions and methods to increase fidelity when polymerizing DNA or RNA templates. For example, Shevelev et al., Nature Rev. Mol . Cell Biol . 3:364 (2002)) provides a review paper on 3'-5' exonucleases. Perrino et al. (PNAS USA, 86:3085 (1989)) used the epsilon subunit of E. coli DNA polymerase III to increase the accuracy of calf thymus DNA polymerase alpha. Bakhanashvili ( Eur. J. Biochem . 268:2047 (2001)) discloses the proofreading activity of p53 protein, Huang et al., Oncogene , 17:261 (1998) ) Discloses the use of p53 to enhance the accuracy of DNA replication. In addition, U.S. Patent Publication No. 2003/0198944A1 and U.S. Patent No. 6,518,019 contain two or more reverse transcriptases (e.g., each reverse transcriptase has a different transcription stop site) and, if necessary, one or more DNA polymerases. Enzyme mixtures are disclosed. US 2002/0119465A1 discloses a composition comprising a variant thermostable DNA polymerase and a variant reverse transcriptase (eg, variant Taq DNA polymerase and variant MMLV-RT). U.S. Patent No. 6,485,917B1, U.S. Patent Publication No. 2003/0077762 and European Patent Publication EP1132470 disclose cDNA in the presence of an enzyme having a reverse transcriptase activity and an α-type DNA polymerase having a 3'-5' exonuclease activity. A method of synthesis is disclosed.

역전사효소의 RNase H 활성을 제거하면 RNA 주형의 RNA 분해 문제를 배제할 수 있고, 또한 역전사 효율을 개선할 수 있다. 그러나, 이러한 역전사효소('RNase H-' 형)는 미스-프라이밍 및 mRNA 2차 구조와 같은 문제는 개선할 수 없다는 단점이 있다. 또한, 종래의 역전사효소는 대체로 낮은 열안정성을 가지며, 이에 따라 역전사 반응이 상대적으로 낮은 온도에서만 진행되고 있으므로, 경우에 따라 65℃ 이상의 높은 온도에서도 안정한 2차 구조를 형성하는 RNA 진행의 구조적 방해로 인해 효율적으로 역전사 산물을 얻지 못한다는 한계를 가지고 있다. 이 같은 한계는 전체 RNA로부터 cDNA를 제작하는 과정뿐만 아니라 RNA 검출 및 프로파일링 등과 같이 역전사 반응이 요구되는 다양한 생화학적 실험에서 주요한 제한 요소로 작용하고 있다. 따라서, 보다 고온에서도 안정하게 역전사 활성을 가지는 신규한 역전사효소의 필요성이 증가되고 있다.If the RNase H activity of the reverse transcriptase is removed, the problem of RNA degradation of the RNA template can be excluded, and the reverse transcription efficiency can be improved. However, this reverse transcriptase ('RNase H-' type) has a disadvantage in that it cannot improve problems such as miss-priming and mRNA secondary structure. In addition, conventional reverse transcriptase generally has low thermal stability, and thus reverse transcription reaction proceeds only at a relatively low temperature. In some cases, it is due to structural interference with RNA progression that forms a stable secondary structure even at a high temperature of 65°C or higher. Therefore, it has a limitation in that it cannot efficiently obtain reverse transcription products. This limitation acts as a major limiting factor in various biochemical experiments that require reverse transcription, such as RNA detection and profiling, as well as the process of producing cDNA from total RNA. Accordingly, there is an increasing need for a novel reverse transcriptase having a stable reverse transcription activity even at a higher temperature.

미국 특허공개 제2003/0198944A1호US Patent Publication No. 2003/0198944A1 미국 특허 제6,518,019호U.S. Patent No. 6,518,019 미국 특허공개 제2002/0119465A1호US Patent Publication No. 2002/0119465A1 미국 특허 제6,485,917B1호U.S. Patent No. 6,485,917B1 미국 특허공개 제2003/0077762호US Patent Publication No. 2003/0077762 유럽 특허공개 EP1132470European Patent Publication EP1132470

Shevelev et al., Nature Rev. Mol. Cell Biol. 3:364 (2002) Shevelev et al., Nature Rev. Mol. Cell Biol. 3:364 (2002) Perrino et al., PNAS USA, 86:3085 (1989) Perrino et al., PNAS USA, 86:3085 (1989) Bakhanashvili, Eur. J. Biochem. 268:2047 (2001) Bakhanashvili, Eur. J. Biochem. 268:2047 (2001) Huang et al., Oncogene, 17:261 (1998) Huang et al., Oncogene, 17:261 (1998)

본 발명은 상기와 같은 종래 역전사효소가 갖고 있는 문제점을 개선하기 위해 도출된 것으로서, M-MLV 유래 역전사효소의 특정 아미노산 잔기를 돌연변이시킴으로써 RNA의 구조변이가 일어날 수 있는 온도 이상의 고온에서도 뛰어난 역전사 활성을 가져 cDNA를 안정적으로 생성할 수 있는 열안정성이 증가된 역전사효소를 제공하는 것을 목적으로 한다.The present invention was derived to improve the problems of the conventional reverse transcriptase as described above, and by mutating a specific amino acid residue of the M-MLV-derived reverse transcriptase, it exhibits excellent reverse transcription activity even at a high temperature above the temperature at which a structural change of RNA can occur. It is an object of the present invention to provide a reverse transcriptase with increased thermal stability that can stably produce cDNA.

본 발명에 기재된 용어, 기술 등은 달리 언급하지 않는 한 본 발명이 속하는 기술분야에서 일반적으로 사용되는 의미로 사용된다. 또한, 본 명세서에 개시된 문헌들은 모두 본 발명을 설명하기 위한 문헌으로 본 명세서 내에 포함된다.Unless otherwise stated, terms, techniques, and the like described in the present invention have the meanings generally used in the technical field to which the present invention belongs. In addition, all of the documents disclosed in the present specification are included in the present specification as documents for explaining the present invention.

본 발명에 있어서, "역전사효소 활성" 또는 "역전사"란 용어는 효소가 RNA 사슬을 주형으로 이용하여 DNA 사슬(즉, 상보적인 DNA 또는 cDNA)을 합성할 수 있는 능력을 말한다. 본 발명에 있어서,"역전사효소"란 본 명세서에서 개시되거나 본 기술분야에서 공지인 임의의 방법에 따라 측정할 때 역전사효소 활성을 나타내는 임의의 효소를 말한다.In the present invention, the term "reverse transcriptase activity" or "reverse transcription" refers to the ability of an enzyme to synthesize a DNA chain (ie, complementary DNA or cDNA) using an RNA chain as a template. In the present invention, "reverse transcriptase" refers to any enzyme that exhibits reverse transcriptase activity when measured according to any method disclosed herein or known in the art.

본 발명에 있어서, "역전사 활성" 또는 "역전사효소 활성"이란 용어는 RNA 사슬을 주형으로 이용하여 DNA 사슬(즉, cDNA)을 합성하는 효소(예를 들면,역전사효소 또는 DNA 중합효소)의 능력을 나타내기 위해 상호 교환가능하게 사용된다.In the present invention, the term "reverse transcriptase activity" or "reverse transcriptase activity" refers to the ability of an enzyme (eg, reverse transcriptase or DNA polymerase) to synthesize a DNA chain (ie, cDNA) using an RNA chain as a template. It is used interchangeably to indicate

본 발명에 있어서, "변이" 또는 "돌연변이"란 용어는 DNA에 의해 코딩되는 아미노산 서열을 변화시키기 위하여 야생형 DNA 서열에 도입되는 변화를 나타낸다. 여기에는 치환, 삽입, 결실, 점 돌연변이 등이 있지만 이에 한정되는 것은 아니다.In the present invention, the term "mutation" or "mutation" refers to a change introduced into a wild-type DNA sequence to change the amino acid sequence encoded by the DNA. These include, but are not limited to, substitutions, insertions, deletions, and point mutations.

본 발명에 있어서, "야생형(wild type)"이란 용어는 유전자 또는 유전자 산물이 천연에 존재하는 공급원으로부터 단리되는 경우와 동일한 특징을 가지는 유전자 또는 유전자 산물을 말한다. 이와 대조적으로, "변형된(modified)" 또는 "변이체(mutant)"란 용어는 야생형의 유전자 또는 유전자 산물과 비교하여 변경되는 특징을 갖고 있는 유전자 또는 유전자 산물을 나타낸다.In the present invention, the term "wild type" refers to a gene or gene product having the same characteristics as when a gene or gene product is isolated from a natural source. In contrast, the term "modified" or "mutant" refers to a gene or gene product that has characteristics that are altered compared to a wild-type gene or gene product.

본 발명에 있어서, "발현 벡터(expression vector)"란 용어는 코딩 서열(coding sequence)과 프로모터(promoter)를 반드시 포함하는 하나 이상의 전사 조절 부위들이 작동 가능하게 연결된 집합체(발현체, expression cassette), 또는 상기의 집합체를 포함하는 벡터를 의미한다.In the present invention, the term "expression vector" refers to a collection (expression cassette) in which one or more transcription control regions necessarily including a coding sequence and a promoter are operably linked, Or it means a vector containing the aggregate.

본 발명에 있어서, "코딩 서열"이란 용어는 특정 아미노산 또는 기능적인 RNA를 암호화하는 DNA 서열을 의미한다.In the present invention, the term "coding sequence" refers to a DNA sequence encoding a specific amino acid or functional RNA.

본 발명에 있어서, "프로모터"란 용어는 코딩 서열의 RNA로의 전사과정을 유도하는 부위로서, 통상적으로 프로모터는 중합효소와 전사인자들이 결합하는 부위를 나타낸다.In the present invention, the term "promoter" refers to a site that induces a transcription process of a coding sequence into RNA, and generally, a promoter refers to a site where a polymerase and transcription factors bind.

본 발명에 있어서, 역전사효소를 구성하는 아미노산 잔기들은 표 1에 나타낸 3개 또는 1개 알파벳의 약어로 표현된다.In the present invention, amino acid residues constituting reverse transcriptase are represented by three or one alphabetic abbreviations shown in Table 1.

알라닌Alanine AA AlaAla 시스테인Cysteine CC CysCys 아스파르트산Aspartic acid DD AspAsp 글루탐산Glutamic acid EE GluGlu 페닐알라닌Phenylalanine FF PhePhe 글리신Glycine GG Gly Gly 히스티딘Histidine HH HisHis 이소루이신Isoleucine II IleIle 리신Lee Sin KK LysLys 루이신Leucine LL Leu Leu 메티오닌Methionine MM MetMet 아스파라긴Asparagine NN Asn Asn 프롤린Proline PP ProPro 글루타민Glutamine QQ Gln Gln 아르기닌Arginine RR Arg Arg 세린Serine SS SerSer 트레오닌Threonine TT Thr Thr 셀레노시스테인Selenocysteine UU SecSec 발린Valine VV ValVal 트립토판Tryptophan WW Trp Trp 티로신Tyrosine YY TyrTyr

이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.

본 발명은 서열번호 1로 기재되는 M-MLV 유래 역전사효소의 아미노산 서열의 63번째의 글루타민(Q63), 264번째의 리신(K264), 295번째의 리신(K295), 306번째의 트레오닌(T306), 346번째의 글루탐산(E346), 408번째의 프롤린(P408), 438번째의 히스티딘(H438) 및 454번째의 아스파라긴(N454)으로 이루어진 군으로부터 선택되는 하나 이상의 아미노산이 다른 아미노산으로 치환되어 열안정성이 증가된 역전사효소를 제공한다.The present invention is the amino acid sequence of the M-MLV derived reverse transcriptase described in SEQ ID NO: 1 at the 63rd glutamine (Q63), the 264th lysine (K264), the 295th lysine (K295), and the 306th threonine (T306). , At least one amino acid selected from the group consisting of glutamic acid at position 346 (E346), proline at position 408 (P408), histidine at position 438 (H438) and asparagine at position 454 (N454) is substituted with another amino acid to provide thermal stability. Provides increased reverse transcriptase.

상기와 같은 아미노산의 치환은 63번째 글루타민에서 루이신으로의 치환(Q63L), 264번째 리신에서 루이신으로의 치환(K264L), 295번째 리신에서 글루타민으로의 치환(K295Q), 306번째 트레오닌에서 루이신으로의 치환(T306L), 346번째 글루탐산에서 메티오닌으로의 치환(E346M), 408번째 프롤린에서 글루탐산으로의 치환(P408E), 438번째 히스티딘에서 티로신으로의 치환(H438Y) 및 454번째 아스파라긴에서 페닐알라닌으로의 치환(N454F)으로 이루어진 군으로부터 선택되는 하나 이상의 아미노산 치환을 포함하지만 이에 한정되는 것은 아니다(도 1 참조). 본 발명의 한 구현예에 따르면, 상기 8군데 위치의 아미노산이 각각 치환된 서열번호 2 내지 서열번호 9로 기재되는 아미노산 서열을 갖는 8가지 돌연변이 역전사효소의 열안정성 효과를 확인하였지만, 2군데 이상의 위치에서 동시에 돌연변이가 일어난 역전사효소도 고온에서의 열안정성을 갖고 있는 한 본 발명의 사상의 범위 내에서 본 발명의 권리범위 내에 포함될 수 있다.Substitution of amino acids as described above is a substitution from glutamine at position 63 to leucine (Q63L), lysine at position 264 to leucine (K264L), lysine at position 295 to glutamine (K295Q), threonine at position 306 to Lewis Substitution with god (T306L), glutamic acid at position 346 to methionine (E346M), proline at position 408 to glutamic acid (P408E), histidine at position 438 to tyrosine (H438Y) and asparagine at position 454 to phenylalanine A substitution of one or more amino acids selected from the group consisting of (N454F) is included, but is not limited thereto (see FIG. 1 ). According to one embodiment of the present invention, the heat stability effect of eight mutant reverse transcriptases having an amino acid sequence represented by SEQ ID NO: 2 to SEQ ID NO: 9 in which the amino acids at the 8 positions are substituted, respectively, was confirmed, but at least two positions Reverse transcriptases in which mutations have occurred at the same time may also be included within the scope of the present invention within the scope of the present invention as long as it has thermal stability at high temperatures.

본 발명의 바람직한 구현예에 따르면, 본 발명의 돌연변이 역전사효소는 야생형 역전사효소에 비해 열안정성을 가지며, 그 중에서도 K295Q, T306L 및 P408E 돌연변이 역전사효소는 뛰어난 열안정성을 나타낸다(도 3 참조). 특히, 가장 뛰어난 열안정성을 나타내는 T306L 돌연변이 역전사효소의 경우, 60℃, 65℃ 및 70℃에서도 야생형 M-MLV 유래 역전사효소보다 뛰어난 열안정성을 나타내어 cDNA를 생성할 수 있다(도 4 참조).According to a preferred embodiment of the present invention, the mutant reverse transcriptase of the present invention has heat stability compared to the wild-type reverse transcriptase, and among them, K295Q, T306L and P408E mutant reverse transcriptases exhibit excellent heat stability (see FIG. 3). In particular, in the case of the T306L mutant reverse transcriptase exhibiting the most excellent thermal stability, cDNA can be generated by exhibiting superior thermal stability than the wild-type M-MLV-derived reverse transcriptase even at 60°C, 65°C, and 70°C (see FIG. 4).

본 기술분야의 숙련된 당업자는 일반적으로 상기와 같이 치환된 아미노산이 상대적으로 유사한 특성의 다른 아미노산(즉, 보존적 아미노산 치환)으로 교체되어도 유사한 생리학적 및 생화학적 특성을 가질 수 있을 것임을 인식할 것이다. 다양한 아미노산의 특성과 단백질 구조 및 기능에 대한 아미노산 치환의 효과는 본 기술분야의 광범위한 연구 및 지식의 대상이 되어 왔다.Those skilled in the art will generally recognize that amino acids substituted as described above may have similar physiological and biochemical properties even if they are replaced with other amino acids of relatively similar properties (i.e., conservative amino acid substitutions). . The effect of amino acid substitution on the properties and protein structure and function of various amino acids has been the subject of extensive research and knowledge in the art.

예를 들면, 아미노산의 히드로패틱(hydropathic) 지수가 고려될 수 있다(Kyte & Doolittle, 1982, J. Mol . Biol., 157:105-132). 아미노산의 상대적인 히드로패틱 특성이 결과물인 단백질의 2차 구조에 기여하며, 이는 다시 상기 단백질과 다른 분자와의 상호작용을 정의하게 된다. 각각의 아미노산은 그 소수성 및 전하 특성에 기초하여 히드로패틱 지수가 할당되어 있으며(Kyte & Doolittle, 1982, J. Mol . Biol., 157:105-132), 이들은 다음과 같다: 이소루이신(+4.5); 발린(+4.2); 루이신(+3.8); 페닐알라닌(+2.8); 시스테인/시스틴(+2.5); 메티오닌(+1.9); 알라닌(+1.8); 글리신(-0.4); 트레오닌(-0.7); 세린(-0.8); 트립토판(-0.9); 티로신(-1.3); 프롤린(-1.6); 히스티딘(-3.2); 글루탐산(-3.5); 글루타민(-3.5); 아스파르트산(-3.5); 아스파라긴(-3.5); 리신(-3.9); 및 아르기닌(-4.5). 보존적 치환을 함에 있어서, 그 히드로패틱 지수가 ±2 이내인 아미노산을 사용하는 것이 바람직하고, ±1 이내가 더욱 바람직하며, ±0.5 이내가 훨씬 더 바람직하다.For example, the hydropathic index of amino acids can be considered (Kyte & Doolittle, 1982, J. Mol . Biol ., 157:105-132). The relative hydropathic properties of amino acids contribute to the secondary structure of the resulting protein, which in turn defines the protein's interaction with other molecules. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge properties (Kyte & Doolittle, 1982, J. Mol . Biol ., 157:105-132), which are as follows: isoleucine (+) 4.5); Valine (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Cysteine/cystine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6); Histidine (-3.2); Glutamic acid (-3.5); Glutamine (-3.5); Aspartic acid (-3.5); Asparagine (-3.5); Lysine (-3.9); And arginine (-4.5). In making conservative substitutions, it is preferable to use an amino acid whose hydropathic index is within ±2, more preferably within ±1, and even more preferably within ±0.5.

아미노산 치환은 또한 아미노산 잔기의 친수성을 고려할 수 있다(예컨대, 미국 특허 제4,554,101호 참조). 친수성 값은 각각의 아미노산 잔기에 대해 할당되어 있으며, 이들은 다음과 같다: 아르기닌(+3.0); 리신(+3.0); 아스파르트산(+3.0); 글루탐산(+3.0); 세린(+0.3); 아스파라긴(+0.2); 글루타민(+0.2); 글리신(0); 트레오닌(-0.4); 프롤린(-0.5 .+-.1); 알라닌(-0.5); 히스티딘(-0.5); 시스테인(-1.0); 메티오닌(-1.3); 발린(-1.5); 루이신(-1.8); 이소루이신(-1.8); 티로신(-2.3); 페닐알라닌(-2.5); 트립토판(-3.4). 아미노산을 유사한 친수성을 갖는 다른 아미노산으로 교체하는 것이 바람직하지만 필수적인 것은 아니다.Amino acid substitutions can also take into account the hydrophilicity of amino acid residues (see, eg, US Pat. No. 4,554,101). Hydrophilicity values are assigned for each amino acid residue, which are as follows: arginine (+3.0); Lysine (+3.0); Aspartic acid (+3.0); Glutamic acid (+3.0); Serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (-0.4); Proline (-0.5 .+-.1); Alanine (-0.5); Histidine (-0.5); Cysteine (-1.0); Methionine (-1.3); Valine (-1.5); Leucine (-1.8); Isoleucine (-1.8); Tyrosine (-2.3); Phenylalanine (-2.5); Tryptophan (-3.4). It is desirable, but not necessary, to replace the amino acid with another amino acid having similar hydrophilicity.

다른 고려사항으로는 아미노산 측쇄의 크기를 고려할 수 있다. 예를 들면, 글리신 또는 세린과 같은 조밀한 측쇄를 갖는 아미노산을 예컨대 트립토판, 티로신과 같은 커다란 측쇄를 갖는 아미노산으로 교체하는 것은 일반적으로 바람직하지 않을 것이다. 단백질 2차 구조에 대한 다양한 아미노산 잔기의 영향도 고려사항이다. 경험적인 연구를 통해, 단백질 도메인이 알파-나선, 베타-시트 또는 리버스 턴(reverse turn) 2차 구조를 채택하는 경향에 대해 상이한 아미노산 잔기가 미치는 영향이 측정되어 왔고, 본 기술분야에 알려져 있다(예컨대, Chou & Fasman, 1974, Biochemistry, 13:222-245; 1978, Ann . Rev . Biochem., 47:251-276; 1979, Biophys. J., 26:367-384 참조).Another consideration may be the size of the amino acid side chains. For example, it would generally not be desirable to replace an amino acid having a dense side chain such as glycine or serine with an amino acid having a large side chain such as tryptophan or tyrosine. The influence of various amino acid residues on protein secondary structure is also a consideration. Through empirical studies, the influence of different amino acid residues on the tendency of protein domains to adopt an alpha-helix, beta-sheet or reverse turn secondary structure has been determined and is known in the art ( For example, Chou & Fasman, 1974, Biochemistry , 13: see 367-384): J, 26 1979, Biophys:;; 251-276. 222-245 1978, Ann Rev Biochem, 47.....

이러한 고려사항 및 광범위한 경험적 연구에 기초하여, 보존적 아미노산 치환의 표가 만들어져 왔고, 본 기술분야에 알려져 있다. 예를 들면, 아르기닌 및 리신; 글루탐산 및 아스파르트산; 세린 및 트레오닌; 글루타민 및 아스파라긴; 및 발린, 루이신 및 이소루이신이다. 다른 한편으로, Ala(A) leu, ile, val; Arg(R) gln, asn, lys; Asn(N) his, asp, lys, arg, gln; Asp(D) asn, glu; Cys(C) ala, ser; Gln(Q) glu, asn; Glu(E) gln, asp; Gly(G) ala; His(H) asn, gln, lys, arg; Ile(I) val, met, ala, phe, leu; Leu(L) val, met, ala, phe, ile; Lys(K) gln, asn, arg; Met(M) phe, ile, leu; Phe(F) leu, val, ile, ala, tyr; Pro(P) ala; Ser(S), thr; Thr(T) ser; Trp(W) phe, tyr; Tyr(Y) trp, phe, thr, ser; Val(V) ile, leu, met, phe, ala 이다.Based on these considerations and extensive empirical studies, tables of conservative amino acid substitutions have been created and are known in the art. For example arginine and lysine; Glutamic acid and aspartic acid; Serine and threonine; Glutamine and asparagine; And valine, leucine and isoleucine. On the other hand, Ala(A) leu, ile, val; Arg(R) gln, asn, lys; Asn(N) his, asp, lys, arg, gln; Asp(D) asn, glu; Cys(C) ala, ser; Gln(Q) glu, asn; Glu(E) gln, asp; Gly(G) ala; His(H) asn, gln, lys, arg; Ile(I) val, met, ala, phe, leu; Leu(L) val, met, ala, phe, ile; Lys(K) gln, asn, arg; Met(M) phe, ile, leu; Phe(F) leu, val, ile, ala, tyr; Pro(P) ala; Ser(S), thr; Thr(T) ser; Trp(W) phe, tyr; Tyr(Y) trp, phe, thr, ser; Val(V) ile, leu, met, phe, ala.

아미노산 치환에 대한 다른 고려사항으로는 그 잔기가 단백질의 내부에 위치해 있는지, 또는 용매에 노출되어 있는지 여부를 포함한다. 내부의 잔기의 경우, 보존적 치환은 다음을 포함할 수 있다: Asp 및 Asn; Ser 및 Thr; Ser 및 Ala; Thr 및 Ala; Ala 및 Gly; Ile 및 Val; Val 및 Leu; Leu 및 Ile; Leu 및 Met; Phe 및 Tyr; Tyr 및 Trp. 용매에 노출된 잔기의 경우, 보존적 치환은 다음을 포함할 수 있다: Asp 및 Asn; Asp 및 Glu; Glu 및 Gln; Glu 및 Ala; Gly 및 Asn; Ala 및 Pro; Ala 및 Gly; Ala 및 Ser; Ala 및 Lys; Ser 및 Thr; Lys 및 Arg; Val 및 Leu; Leu 및 Ile; Ile 및 Val; Phe 및 Tyr.Other considerations for amino acid substitution include whether the residue is located inside the protein or whether it is exposed to a solvent. For internal residues, conservative substitutions may include: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala; Ala and Gly; Ile and Val; Val and Leu; Leu and Ile; Leu and Met; Phe and Tyr; Tyr and Trp. For solvent exposed residues, conservative substitutions may include: Asp and Asn; Asp and Glu; Glu and Gin; Glu and Ala; Gly and Asn; Ala and Pro; Ala and Gly; Ala and Ser; Ala and Lys; Ser and Thr; Lys and Arg; Val and Leu; Leu and Ile; Ile and Val; Phe and Tyr.

아미노산 치환을 결정함에 있어서, 양으로 대전된 잔기(예컨대, His, Arg, Lys)와 음으로 대전된 잔기(예컨대, Asp, Glu) 사이의 이온 결합(염 가교) 또는 인접한 시스테인 잔기 사이의 이황화 결합의 형성과 같은 분자간 또는 분자내 결합의 존재를 고려하는 것이 바람직하다.
In determining amino acid substitution, ionic bonds (salt bridges) between positively charged residues (e.g., His, Arg, Lys) and negatively charged residues (e.g., Asp, Glu) or disulfide bonds between adjacent cysteine residues It is desirable to take into account the presence of intermolecular or intramolecular bonds, such as the formation of.

또한, 본 발명은 상기 열안정성이 증가된 역전사효소를 코딩하는 유전자를 제공한다.In addition, the present invention provides a gene encoding the reverse transcriptase having increased thermal stability.

상기 유전자는 서열번호 1로 기재되는 야생형 역전사효소의 63번째 글루타민에서 루이신으로의 치환(Q63L), 264번째 리신에서 루이신으로의 치환(K264L), 295번째 리신에서 글루타민으로의 치환(K295Q), 306번째 트레오닌에서 루이신으로의 치환(T306L), 346번째 글루탐산에서 메티오닌으로의 치환(E346M), 408번째 프롤린에서 글루탐산으로의 치환(P408E), 438번째 히스티딘에서 티로신으로의 치환(H438Y) 및 454번째 아스파라긴에서 페닐알라닌으로의 치환(N454F)으로 이루어진 군으로부터 선택되는 하나 이상의 아미노산 치환을 포함하는 돌연변이 역전사효소를 코딩하는 유전자이다. 본 발명의 한 구현예에 따르면, 본 발명의 유전자는 상기 8군데 위치의 아미노산이 각각 치환된 서열번호 2 내지 서열번호 9로 기재되는 아미노산 서열을 갖는 8가지 돌연변이 역전사효소를 코딩하는 서열번호 11 내지 서열번호 18로 기재되는 염기서열을 갖는 유전자인 것이 바람직하지만 이에 한정되는 것은 아니다. 예컨대, 서열번호 1로 기재되는 M-MLV 유래 역전사효소의 아미노산 서열의 63번째의 글루타민(Q63), 264번째의 리신(K264), 295번째의 리신(K295), 306번째의 트레오닌(T306), 346번째의 글루탐산(E346), 408번째의 프롤린(P408), 438번째의 히스티딘(H438) 및 454번째의 아스파라긴(N454) 중 2가지 이상이 돌연변이된 역전사효소를 코딩하는 임의의 유전자도 본 발명의 권리범위 내에 포함될 수 있다(도 2a 내지 도 2d 참조).The gene is a wild-type reverse transcriptase represented by SEQ ID NO: 1 at 63 glutamine to leucine substitution (Q63L), 264 lysine to leucine substitution (K264L), 295 lysine to glutamine substitution (K295Q) , Threonine at position 306 to leucine (T306L), glutamic acid at position 346 to methionine (E346M), proline at position 408 to glutamic acid (P408E), histidine at position 438 to tyrosine (H438Y), and It is a gene encoding a mutant reverse transcriptase containing one or more amino acid substitutions selected from the group consisting of substitution of asparagine at position 454 to phenylalanine (N454F). According to one embodiment of the present invention, the gene of the present invention is SEQ ID NO: 11 to encode eight mutant reverse transcriptases having an amino acid sequence represented by SEQ ID NO: 2 to SEQ ID NO: 9 in which the amino acids at the 8 positions are respectively substituted. It is preferably a gene having a nucleotide sequence represented by SEQ ID NO: 18, but is not limited thereto. For example, glutamine at the 63rd position (Q63), lysine at the 264th position (K264), lysine at the 295th position (K295), threonine at the 306th position in the amino acid sequence of the reverse transcriptase derived from M-MLV shown in SEQ ID NO: 1, Any gene encoding a reverse transcriptase in which two or more of glutamic acid at position 346 (E346), proline at position 408 (P408), histidine at position 438 (H438), and asparagine at position 454 (N454) are mutated can also be used in the present invention. It may be included within the scope of rights (see FIGS. 2A to 2D).

또한, 본 발명에서 언급된 상기 유전자들과 실질적으로 동일하고 기능적으로 동등한 폴리뉴클레오티드도 본 발명의 유전자의 범위에 포함된다. '실질적으로 동일하고 기능적으로 동등한 폴리뉴클레오타이드'란 공지된 알고리즘의 컴퓨터화된 실행에 의해서 또는 조사에 의해서 적절히 배열되었을 때 두 개의 폴리뉴클레오타이드가 적어도 70％, 바람직하게는 적어도 80％, 더욱 바람직하게는 적어도 90％, 가장 바람직하게는 적어도 95％ 이상의 서열 상동성을 공유하는 것을 의미한다.In addition, polynucleotides that are substantially identical and functionally equivalent to the genes mentioned in the present invention are also included in the scope of the genes of the present invention. The term'substantially identical and functionally equivalent polynucleotides' means that two polynucleotides are at least 70%, preferably at least 80%, more preferably when properly arranged by computerized execution of known algorithms or by investigation. It is meant to share at least 90%, most preferably at least 95%, sequence homology.

또한, 본 발명은 상기 유전자를 포함하는 발현 벡터를 제공한다.In addition, the present invention provides an expression vector containing the gene.

본 발명의 발현 벡터의 제조에 사용될 수 있는 모 벡터에는 특별한 제한이 없으며, 통상의 원핵생물 또는 진핵생물 형질전환용 벡터가 사용될 수 있다. 본 발명의 바람직한 구현예에서는 서열번호 11 내지 서열번호 18로 기재되는 각각의 돌연변이 유전자를 핵외 유전자 벡터 PET 22b(+)에 삽입시킨 재조합 발현 벡터를 제조하였다.There is no particular limitation on the parent vector that can be used to prepare the expression vector of the present invention, and a vector for transformation of a conventional prokaryote or eukaryote may be used. In a preferred embodiment of the present invention, a recombinant expression vector was prepared in which each mutant gene represented by SEQ ID NO: 11 to SEQ ID NO: 18 was inserted into the extranuclear gene vector PET 22b(+).

또한, 본 발명은 상기 발현 벡터로 형질전환된 형질전환체를 제공한다.In addition, the present invention provides a transformant transformed with the expression vector.

본 발명의 형질전환체는 상기 발현 벡터를 임의의 원핵세포 또는 진핵세포에 도입함으로써 용이하게 제조될 수 있으며, 특정한 벡터를 세포내로 도입시키는 방법은 당업자에게 잘 알려져 있다. 이러한 방법의 예로는 리포펙타민 방법 등이 있다. 본 발명의 바람직한 구현예에서는 상기 돌연변이 유전자가 삽입된 PET 22b(+) 벡터를 대장균 DH5α 숙주 세포에 도입한 형질전환체를 제조하였다.The transformant of the present invention can be easily prepared by introducing the expression vector into any prokaryotic or eukaryotic cell, and a method of introducing a specific vector into a cell is well known to those skilled in the art. Examples of such methods include the lipofectamine method. In a preferred embodiment of the present invention, a transformant was prepared by introducing the mutant gene-inserted PET 22b(+) vector into E. coli DH5α host cells.

또한, 본 발명은 상기 역전사효소를 포함하는 역전사 반응용 키트를 제공한다.In addition, the present invention provides a reverse transcription reaction kit comprising the reverse transcriptase.

본 발명의 역전사 반응용 키트는 상기 역전사효소 이외에도 역전사 반응에 필요한 통상적인 구성성분, 예를 들면, 증폭하기 원하는 표적 유전자 또는 올리고뉴클레오티드에 특이적으로 결합하는 프라이머쌍, dNTP, 반응완충액, 선택적으로는 DNA 중합효소 등을 추가로 포함할 수 있다.In addition to the reverse transcriptase, the kit for reverse transcription of the present invention is a primer pair that specifically binds to a target gene or oligonucleotide that is desired to be amplified, such as a primer pair, dNTP, a reaction buffer, optionally It may further include a DNA polymerase and the like.

본 발명의 돌연변이 역전사효소는 야생형 역전사효소와 비교하여 뛰어난 열안정성을 나타내므로, 고온에서도 안정한 2차 구조를 형성할 수 있는 특정한 RNA의 구조적 방해로 인해 효율적으로 역전사 산물을 얻지 못하는 경우라도 안정적으로 역전사 활성을 나타내어 원하는 cDNA를 얻을 수 있다.The mutant reverse transcriptase of the present invention exhibits excellent thermal stability compared to the wild-type reverse transcriptase, so even when the reverse transcription product cannot be efficiently obtained due to structural interference of a specific RNA that can form a stable secondary structure even at high temperature, it is stable reverse transcription. It shows activity and can obtain a desired cDNA.

도 1은 본 발명의 돌연변이 역전사효소에서 돌연변이된 아미노산의 위치 및 종류를 나타낸 것이다.
도 2a 내지 도 2b는 본 발명의 돌연변이 역전사효소를 코딩하는 유전자에서 돌연변이된 염기의 위치 및 그 서열을 나타낸 것이다.
도 3은 야생형 역전사효소(서열번호 1)와 본 발명의 돌연변이 역전사효소(서열번호 2 내지 서열번호 9)를 이용하여 42, 50, 60, 65 및 70℃에서 역전사 반응시킨 결과를 보여주는 전기영동 사진이다.
도 4는 야생형 역전사효소(서열번호 1)와 본 발명의 T306L 돌연변이 역전사효소(서열번호 5)를 이용하여 37, 42, 50, 60, 65 및 70℃에서 역전사 반응시킨 결과를 보여주는 전기영동 사진이다.1 shows the location and type of amino acids mutated in the mutant reverse transcriptase of the present invention.
2A to 2B show the positions and sequences of mutated bases in the gene encoding the mutant reverse transcriptase of the present invention.
3 is an electrophoretic photograph showing the results of reverse transcription reaction at 42, 50, 60, 65 and 70°C using wild-type reverse transcriptase (SEQ ID NO: 1) and mutant reverse transcriptase of the present invention (SEQ ID NO: 2 to SEQ ID NO: 9) to be.
Figure 4 is an electrophoresis photograph showing the results of reverse transcription reaction at 37, 42, 50, 60, 65 and 70°C using wild-type reverse transcriptase (SEQ ID NO: 1) and T306L mutant reverse transcriptase of the present invention (SEQ ID NO: 5). .

이하, 본 발명을 실시예에 의해 상세히 설명한다.Hereinafter, the present invention will be described in detail by examples.

단, 하기 실시예는 본 발명을 예시하기 위한 것일 뿐, 본 발명의 내용이 하기 실시예에 의해 한정되는 것은 아니다.
However, the following examples are for illustrative purposes only, and the contents of the present invention are not limited by the following examples.

실시예Example 1. 돌연변이 1. mutation 역전사효소를Reverse transcriptase 코팅하는 유전자의 제작 Construction of the gene to coat

서열번호 1로 기재되는 아미노산 서열을 갖는 M-MLV 유래 역전사효소에 돌연변이를 도입하기 위하여, 상기 M-MLV 유래 역전사효소를 코딩하는 유전자 상의 염기서열을 치환하였다. 구체적으로, 서열번호 1로 기재되는 아미노산 서열을 갖는 M-MLV 유래 역전사효소에서 63번째 글루타민에서 루이신으로의 변이(Q63L), 264번째 리신에서 루이신으로의 변이(K264L), 295번째 리신에서 글루타민으로의 변이(K295Q), 306번째 트레오닌에서 루이신으로의 변이(T306L), 346번째 글루탐산에서 메티오닌으로의 변이(E346M), 408번째 프롤린에서 글루탐산으로의 변이(P408E), 438번째 히스티딘에서 티로신으로의 변이(H438Y 변이) 및 454번째 아스파라긴에서 페닐알라닌으로의 변이(N454F)가 도입된 8가지 돌연변이 역전사효소를 제작하였으며(도 1 참조), 이를 위하여 상기 서열번호 1의 아미노산 서열을 갖는 역전사효소를 코딩하는 서열번호 10으로 기재되는 유전자에 돌연변이를 도입하였다(도 2a 내지 도 2d 참조). 상기 돌연변이 유전자는 각각 서열번호 11 내지 서열번호 18로 기재되는 염기서열을 갖는다. 상기 돌연변이 유전자의 제조 과정은 다음과 같다:In order to introduce a mutation into the M-MLV-derived reverse transcriptase having the amino acid sequence shown in SEQ ID NO: 1, the nucleotide sequence on the gene encoding the M-MLV-derived reverse transcriptase was substituted. Specifically, M-MLV-derived reverse transcriptase having the amino acid sequence shown in SEQ ID NO: 1 at the 63rd glutamine to leucine (Q63L), the 264th lysine to leucine (K264L), at the 295th lysine Glutamine mutation (K295Q), threonine to leucine at position 306 (T306L), glutamic acid to methionine at position 346 (E346M), proline to glutamic acid at position 408 (P408E), histidine to tyrosine at position 438 Eight mutant reverse transcriptases in which mutations to (H438Y mutation) and 454th asparagine to phenylalanine (N454F) were introduced were prepared (see FIG. 1), and for this purpose, reverse transcriptase having the amino acid sequence of SEQ ID NO: 1 was used. A mutation was introduced into the gene represented by the coding SEQ ID NO: 10 (see FIGS. 2A to 2D). Each of the mutant genes has a nucleotide sequence represented by SEQ ID NO: 11 to SEQ ID NO: 18. The manufacturing process of the mutant gene is as follows:

대장균(Escherichia coli)에서의 단백질 발현량을 높이기 위하여 서열번호 10으로 기재되는 M-MLV 역전사효소의 유전자 서열을 대장균의 코돈 이용성(codon usage)에 맞추어 최적화시켜 서열번호 19로 기재되는 염기서열을 얻었다. 상기 최적화된 염기서열을 바탕으로 야생형 유전자와 K295Q 돌연변이 유전자를 합성하였다. 먼저, 서열번호 19로 기재되는 야생형 유전자의 염기서열을 얻기 위하여, 전체 염기서열을 바탕으로 센스 가닥과 안티센스 가닥을 각각 어닐링(annealing) 온도가 약 60℃(20 내지 40 염기)가 되도록 설계한 후, 설계된 올리고뉴클레오티드들을 합성하였다(바이오니아, KOREA). 합성된 올리고뉴클레오티드들은 각각 서열번호 20 내지 서열번호 149로 기재되는 염기서열을 갖는다. 상기 합성된 올리고뉴클레오티드들을 사용하여 키네이션(Kination)-LCR법(대한민국 특허출원 제2008-0025050호)으로 40℃와 60℃에서 반복적으로 반응시켜 라이게이션(ligation)시킴으로써 서열번호 19로 기재되는 염기서열을 갖는 이중가닥의 전체 유전자를 합성하였다. 또한, K295Q 돌연변이 유전자는 서열번호 77 및 서열번호 78로 기재되는 염기서열 대신에 서열번호 150 및 서열번호 151로 기재되는 염기서열을 사용한 것을 제외하고는 상기 야생형 유전자와 동일한 방법을 이용하여 합성하였다. 합성이 완료된 유전자를 95℃에서 5분 동안 사전 열처리한 후, 서열번호 152 및 서열번호 153으로 기재되는 프라이머쌍을 이용하여 95℃에서 1분, 65℃에서 1분, 72℃에서 2분30초 반응시켰으며, 이 과정을 30회 반복한 뒤 72℃에서 추가적으로 10분간 반응시켜 상기 유전자를 증량하였다. 상기 증량된 합성 유전자를 pGEM-T easy vector(Promega, USA)에 라이게이션시킨 후, 대장균 DH5a(RBC, USA)에 형질전환시켜 야생형 및 K295Q 돌연변이 유전자의 클론을 다량 확보하였다.In order to increase the protein expression level in Escherichia coli , the gene sequence of the M-MLV reverse transcriptase described in SEQ ID NO: 10 was optimized according to the codon usage of Escherichia coli to obtain the nucleotide sequence shown in SEQ ID NO: 19. . Based on the optimized nucleotide sequence, a wild-type gene and a K295Q mutant gene were synthesized. First, in order to obtain the nucleotide sequence of the wild-type gene represented by SEQ ID NO: 19, the sense strand and the antisense strand were designed to each have an annealing temperature of about 60°C (20 to 40 bases) based on the entire nucleotide sequence. , Designed oligonucleotides were synthesized (Bionia, KOREA). The synthesized oligonucleotides each have a nucleotide sequence represented by SEQ ID NO: 20 to SEQ ID NO: 149. The base described in SEQ ID NO: 19 by repeatedly reacting at 40°C and 60°C using the synthesized oligonucleotides by the Kination-LCR method (Korean Patent Application No. 2008-0025050). The entire double-stranded gene having the sequence was synthesized. In addition, the K295Q mutant gene was synthesized using the same method as the wild-type gene, except that the nucleotide sequences shown in SEQ ID NO: 150 and SEQ ID NO: 151 were used instead of the nucleotide sequences shown in SEQ ID NO: 77 and SEQ ID NO: 78. After pre-heating the synthesized gene at 95°C for 5 minutes, using a primer pair described in SEQ ID NO: 152 and SEQ ID NO: 153 for 1 minute at 95°C, 1 minute at 65°C, 2 minutes 30 seconds at 72°C The reaction was carried out, and after repeating this process 30 times, the gene was increased by reacting for an additional 10 minutes at 72°C. The increased synthetic gene was ligated into a pGEM-T easy vector (Promega, USA), and then transformed into E. coli DH5a (RBC, USA) to obtain a large amount of clones of wild-type and K295Q mutant genes.

이렇게 합성된 야생형 유전자 서열을 주형으로 하여 Q63L, K264L, T306L, E346M, P408E, H438Y 및 N454F를 위치지정 돌연변이법(site-directed mutagenesis)을 이용하여 돌연변이시켰다. 상기 위치지정 돌연변이법은 다음과 같이 진행하였다: 먼저, 상기 돌연변이 유전자에 대하여 서열번호 154 내지 서열번호 167로 기재되는 염기서열을 갖는 올리고뉴클레오티드를 합성하였다. 95℃에서 5분 동안 열처리한 후, 상기 합성된 올리고뉴클레오티드를 프라이머로 pfu DNA 중합효소를 사용하여 95℃에서 1분, 65℃에서 1분, 72℃에서 5분30초 반응시켰으며, 이 과정을 총 30회 반복한 뒤 다음 72℃에서 추가 반응시켜 돌연변이 유전자 산물을 생성하였다. 이렇게 생성한 돌연변이 유전자 산물들을 키네이션-자가 라이게이션(self ligation)시킨 후 대장균 DH5a(RBC, USA)에 형질전환시켜 Q63L, K264L, T306L, E346M, P408E, H438Y 및 N454F 돌연변이 유전자의 클론을 다량 확보하였다.
Using the thus synthesized wild-type gene sequence as a template, Q63L, K264L, T306L, E346M, P408E, H438Y and N454F were mutated using site-directed mutagenesis. The position-directed mutation method was carried out as follows: First, oligonucleotides having nucleotide sequences represented by SEQ ID NOs: 154 to 167 were synthesized for the mutant gene. After heat treatment at 95° C. for 5 minutes, the synthesized oligonucleotide was reacted for 1 minute at 95° C., 1 minute at 65° C., 5 minutes 30 seconds at 72° C. using pfu DNA polymerase as a primer. After repeating a total of 30 times, a mutant gene product was generated by further reacting at the next 72°C. The mutant gene products thus generated were subjected to kination-self ligation and then transformed into E. coli DH5a (RBC, USA) to secure large amounts of clones of the Q63L, K264L, T306L, E346M, P408E, H438Y and N454F mutant genes. I did.

실시예Example 2. 돌연변이 유전자의 형질전환 2. Transformation of mutant gene

핵외유전자 PET 22b(+)(NOVAGEN CO.)를 재조합 벡터로 사용하여 실시예 1에서 제조된 8가지 돌연변이 역전사효소 유전자를 클로닝하였다. 이를 위하여, pGEM-T easy 벡터(Promega)에 삽입되어 합성된 변이 유전자(Q63L, K264L, T306L 및 E346M)를 하기 프라이머쌍을 이용하여 PCR을 진행하였다.Eight mutant reverse transcriptase genes prepared in Example 1 were cloned using the extranuclear gene PET 22b(+) (NOVAGEN CO.) as a recombinant vector. To this end, the mutant genes (Q63L, K264L, T306L and E346M) synthesized by being inserted into the pGEM-T easy vector (Promega) were subjected to PCR using the following primer pairs.

센스 가닥: 5'-GCG CGC CAT ATG CTG AAC ATC GAA GAC GAA CAC CGT CTG CAC GAA AC-3'(Nde Ⅰ)(서열번호 168)Sense strand: 5'-GCG CGC CAT ATG CTG AAC ATC GAA GAC GAA CAC CGT CTG CAC GAA AC-3' (Nde I) (SEQ ID NO: 168)

안티센스 가닥: 5'-GCG CGC GCG GCC GCT TAG ATC AGC AGG GTA GAG GTG TCC GGG GTT TC-3'(Not Ⅰ)(서열번호 169)Antisense strand: 5'-GCG CGC GCG GCC GCT TAG ATC AGC AGG GTA GAG GTG TCC GGG GTT TC-3' (Not I) (SEQ ID NO: 169)

다른 변이 유전자(K295Q, P408E, H438Y, N454F) 경우 하기 프라이머쌍을 이용하여 PCR을 진행하였다.In the case of other mutant genes (K295Q, P408E, H438Y, N454F), PCR was performed using the following primer pairs.

센스 가닥: 5'-GCG CGC CAT ATG CTG AAC ATC GAA GAC GAA CAC CGT CTG CAC GAA AC-3'(Nde Ⅰ)(서열번호 170)Sense strand: 5'-GCG CGC CAT ATG CTG AAC ATC GAA GAC GAA CAC CGT CTG CAC GAA AC-3' (Nde I) (SEQ ID NO: 170)

안티센스 가닥: 5'-GCG CGC GCG GCC GCG ATC AGC AGG GTA GAG GTG TCC GGG GTT TC-3'(Not Ⅰ)(서열번호 171)Antisense strand: 5'-GCG CGC GCG GCC GCG ATC AGC AGG GTA GAG GTG TCC GGG GTT TC-3' (Not I) (SEQ ID NO: 171)

핵외 유전자 벡터 PET 22b(+)와 PCR을 통해 얻은 각 M-MLV 역전사 유전자 단편들에 대하여, 5' 말단은 NdeⅠ, 3' 말단은 NotⅠ을 처리하여 절단한 후 겔 추출 키트(BIONEER CO.)를 이용하여 정제하였다. 이 후 T4 DNA 라이게이즈(ligase)(TAKARA CO.)를 이용하여 상기 절단된 단편들을 16℃에서 2시간 반응시켜 각각의 돌연변이 유전자를 핵외 유전자 벡터 PET 22b(+)에 삽입하였다.For each M-MLV reverse transcription gene fragment obtained through the extranuclear gene vector PET 22b(+) and PCR, the 5'end was treated with Nde I and the 3'end was cut with Not I, and then a gel extraction kit (BIONEER CO.) was used. It was purified using. Thereafter, the cut fragments were reacted at 16° C. for 2 hours using T4 DNA ligase (TAKARA CO.), and each mutant gene was inserted into the extranuclear gene vector PET 22b(+).

상기 돌연변이 유전자가 삽입된 PET 22b(+) 벡터를 대장균(Escherichia coli) DH5α 숙주 세포에 형질전환시킴으로써 재조합 숙주 세포를 생성하였다. 상기 형질전환은 다음과 같은 순서로 진행하였다: -70℃에서 냉동된 상태의 DH5α 숙주 세포를 해동하였다. 해동된 숙주 세포와 상기 DNA 재조합 벡터 1 ㎍/㎕를 섞은 후 30분간 얼음에서 방치하였고, 42℃에서 90초간 열처리를 하였다. 열처리한 숙주 세포를 암피실린(50 ㎎/㎖)이 들어있는 LB 세포배양액 플레이트에 도말한 후 37℃에서 밤새 배양하여 클론을 얻었다. 얻어진 클론은 하기 염기서열을 갖는 프라이머쌍을 이용하여 각 유전자 서열을 확인하였으며, 확인된 클론은 플라스미드를 다시 BL21(DE3) 숙주 세포에 형질 전환시켜 발현 균주를 확보하였다.PET 22b (+) vector into which the mutant gene was inserted was transferred to Escherichia coli) DH5α host cells were transformed to generate recombinant host cells. The transformation was carried out in the following order: DH5α host cells in a frozen state at -70°C were thawed. The thawed host cells and 1 µg/µl of the DNA recombinant vector were mixed and left on ice for 30 minutes, followed by heat treatment at 42°C for 90 seconds. The heat-treated host cells were plated on an LB cell culture medium plate containing ampicillin (50 mg/ml) and cultured overnight at 37°C to obtain clones. The obtained clone was confirmed for each gene sequence using a primer pair having the following nucleotide sequence, and the confirmed clone was transformed into a BL21 (DE3) host cell again to obtain an expression strain.

T7 프로모터 센스 가닥: 5'-TAA TAC GAC TCA CTA TAG GG-3'(서열번호 172)T7 promoter sense strand: 5'-TAA TAC GAC TCA CTA TAG GG-3' (SEQ ID NO: 172)

T7 터미네이터 안티센스 가닥: 5'-GCT AGT TAT TGC TCA GCG G-3'(서열번호 173)
T7 terminator antisense strand: 5'-GCT AGT TAT TGC TCA GCG G-3' (SEQ ID NO: 173)

실시예Example 3. 돌연변이 3. Mutations 역전사효소의Reverse transcriptase 발현 Manifestation

BL21(DE3) 숙주 세포에 형질전환한 발현 균주의 단일 콜로니를 취한 후 앰피시린(50 ㎎/㎖)이 포함된 LB(Luria-Bertani medium) 세포배양액 15 ㎖에 넣고 37℃에서 밤새 배양하여 시드(seed) 균주 세포를 확보하였다. 앰피시린이 포함된 LB 세포배양액 1 ℓ에 상기 시드 균주 세포를 넣고 37℃에서 2시간 동안 배양한 후, 0.5 mM IPTG(Isopropyl β-D-1-thiogalactopyranoside)를 첨가하여 2시간 더 발현시켰다. 배양된 각각의 균주 세포를 원심분리한 후 얻어진 펠렛(pellet)을 -50℃에서 정제하기 전까지 보관하였다.
After taking a single colony of the expression strain transformed into BL21(DE3) host cell, put it in 15 ml of LB (Luria-Bertani medium) cell culture solution containing ampicillin (50 mg/ml) and incubated overnight at 37°C to seed. (seed) strain cells were obtained. The seed strain cells were added to 1 ℓ of the LB cell culture solution containing ampicillin and incubated at 37° C. for 2 hours, and then 0.5 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside) was added and expressed for 2 hours. After centrifuging the cultured cells of each strain, the obtained pellet was stored at -50°C until purification.

실시예Example 4. 돌연변이 4. Mutation 역전사효소의Reverse transcriptase 정제 refine

실시예 3에서 얻어진 동결 세포덩어리 5 g을 파쇄한 후, 4℃에서 교반하에 용균 완충액(25 mM 트리스 염산(Tris-HCl pH 7.6), 1 mM EDTA, 10%(v/v) β-머갑토에탄올, 페닐메틸술포닐 플루오라이드(PMSF) 20 ㎖)에 30분간 현탁 시켰다. 그 다음 세포균질기(fisher sonicator)로 세포를 용균시켰다. 그 다음, 상기 세포 용균물을 4℃에서 1시간 동안 11,000 rpm에서 원심분리시키고, 펠렛을 따라내어 버렸다. 맑은 용균물을 즉시 완충 용액(40 mM 트리스 염산(Tris-HCl pH 7.6), 2 mM EDTA, 28.58 mM β-머갑토에탄올, 100 mM KCl)에 투석하였다.
After crushing 5 g of the frozen cell mass obtained in Example 3, lysis buffer (25 mM Tris hydrochloric acid (Tris-HCl pH 7.6), 1 mM EDTA, 10% (v/v) β-mergatto) under stirring at 4° C. It was suspended in ethanol and phenylmethylsulfonyl fluoride (PMSF) (20 ml) for 30 minutes. Then, the cells were lysed with a fisher sonicator. Then, the cell lysate was centrifuged at 11,000 rpm for 1 hour at 4° C., and the pellet was decanted. The clear lysate was immediately dialyzed against a buffer solution (40 mM Tris hydrochloric acid (Tris-HCl pH 7.6), 2 mM EDTA, 28.58 mM β-mercaptoethanol, 100 mM KCl).

실시예Example 5. 돌연변이 5. Mutation 역전사효소의Reverse transcriptase 크로마토그래피 Chromatography

돌연변이 역전사 효소를 정제하기 위해서 아마시암 FPLC 장치를 사용하여 수행하였다. 1차 칼럼은 아마시암 XK16(1.6 ㎝×30.0 ㎝)을 사용하였다. 칼럼에 이온교환 수지 칼럼 크로마토그래피 DEAE hyper-D(pall co.)를 충진하고 1 M KCl을 함유하는 칼럼 완충액 250 ㎖로 세척한 후 완충용액으로 평형화시킨 60 ㎖의 베드를 얻었다. 그 다음, 투명한 세포 용균물 20 ㎖를 5 ㎖/분의 유속으로 칼럼에 적용하였다. 적용된 용균물 중 칼럼에 흡착되지 않은 용균물을 획득하여 다음 칼럼에 적용하였다. 얻어진 용균물을 2차 크로마토그래피에 적용하였다. 칼럼 아마시암 XK16(1.6 ㎝×30.0 ㎝)을 세척하고 평형화시킨 후, 친화성 크로마토그래피 헤파린 hyper-D(pall co.)를 충진하여 50 ㎖의 베드를 얻었다. 그 다음, 완충용액으로 평형화시켰다. 용균물을 칼럼에 적용한 후, 칼럼 완충액 중 0.2 M∼0.6 M KCl 선형 염 구배 100∼150 ㎖를 사용하여 용출시켰다. 용출시킨 용균물을 칼럼 26X60 S-200 gel filteration(GE healthcare)에 적용하여 고순도의 역전사효소를 얻었다. 칼럼 분획을 SDS-폴리아크릴아미드 겔 전기영동 (SDS-PAGE)에 적용한 후 염색(coomassie brilliant blue staining)을 수행하여 분석하였다. 각 분획에서 취한 10 ㎕ 분획물을 각각의 겔 레인에서 분석하였다. 야생형과 비슷한 분자량(74 kDa)의 단백질을 취하였고, 상기 단백질의 안정성을 확보하기 위하여 보존액(20 mM 트리스-염산(pH 7.6), 0.1 mM EDTA, 150 mM NaCl, 0.1% IGEPAL CA-630(Polyethoxyethanol) 1 mM DTT(Dithiothreitol), 50% 글리세롤(Glycerol)로 투석을 진행하여 돌연변이 효소를 얻어 -20℃에서 보관하였다.
To purify the mutant reverse transcriptase, it was carried out using an Amsiam FPLC apparatus. As the primary column, flaxiam XK16 (1.6 cm x 30.0 cm) was used. The column was filled with ion exchange resin column chromatography DEAE hyper-D (pall co.), washed with 250 ml of a column buffer containing 1 M KCl, and then equilibrated with a buffer solution to obtain a 60 ml bed. Then, 20 ml of clear cell lysate was applied to the column at a flow rate of 5 ml/min. Among the applied lysates, lysates that were not adsorbed on the column were obtained and applied to the next column. The obtained lysate was subjected to secondary chromatography. After washing and equilibrating the column flaxiam XK16 (1.6 cm×30.0 cm), affinity chromatography heparin hyper-D (pall co.) was added to obtain a 50 ml bed. Then, it was equilibrated with a buffer solution. After the lysate was applied to the column, it was eluted using a 0.2 M to 0.6 M KCl linear salt gradient of 100 to 150 ml in a column buffer. The eluted lysate was applied to a column 26X60 S-200 gel filteration (GE healthcare) to obtain a high-purity reverse transcriptase. The column fraction was subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE), followed by staining (coomassie brilliant blue staining) to analyze. The 10 μl fractions taken from each fraction were analyzed in each gel lane. A protein with a molecular weight (74 kDa) similar to that of the wild type was taken, and to secure the stability of the protein, a preservative solution (20 mM Tris-HCl (pH 7.6), 0.1 mM EDTA, 150 mM NaCl, 0.1% IGEPAL CA-630 (Polyethoxyethanol)) ) Dialysis was performed with 1 mM DTT (Dithiothreitol) and 50% glycerol to obtain a mutant enzyme and stored at -20°C.

실시예Example 6. 돌연변이 6. Mutation 역전사효소를Reverse transcriptase 이용한 Used RTRT -- PCRPCR 수행 Perform

상기 분리된 역전사효소의 활성을 확인하기 위하여 역전사 PCR을 수행하였다. 역전사 PCR에 사용한 물질은 다음과 같으며, 전체 RNA는 5 ng/㎕∼500, 50, 5 pg/㎕의 농도를 사용하였다.Reverse transcription PCR was performed to confirm the activity of the isolated reverse transcriptase. Materials used for reverse transcription PCR were as follows, and total RNA was used at concentrations of 5 ng/µl to 500, 50, and 5 pg/µl.

5X 반응 버퍼 4 ㎕(Bioneer co.)4 μl of 5X reaction buffer (Bioneer co.)

10 mM dNTP 2 ㎕10 mM dNTP 2 μl

dT(18) 10 pmol 1 ㎕dT(18) 10 pmol 1 μl

100 mM DTT 2 ㎕100 mM DTT 2 μl

RNase 억제제(50 ng/㎕) 1 ㎕1 μl of RNase inhibitor (50 ng/μl)

증류수 7 ㎕7 µl of distilled water

전체 RNA 2 ㎕2 μl of total RNA

비교예로는 야생형 역전사효소를 이용하였으며, 42℃, 50℃, 60℃, 65℃ 및 70℃(T306L 변이는 37℃에서의 반응을 추가함)에서 1 시간 역전사 PCR을 수행하였다. 상기에서 생성된 cDNA를 이용하여 PCR을 수행하였다. 증폭할 유전자로는 GAPDH를 사용하였으며(유전자 크기 500 bp), 하기 염기서열을 갖는 프라이머쌍을 이용하였다.As a comparative example, wild-type reverse transcriptase was used, and reverse transcription PCR was performed for 1 hour at 42°C, 50°C, 60°C, 65°C and 70°C (T306L mutation added the reaction at 37°C). PCR was performed using the cDNA generated above. GAPDH was used as a gene to be amplified (gene size 500 bp), and a primer pair having the following nucleotide sequence was used.

센스 가닥: 5'-GAAGGTGAAGGTCGGAGTCAACG-3'(서열번호 174)Sense strand: 5'-GAAGGTGAAGGTCGGAGTCAACG-3' (SEQ ID NO: 174)

안티센스 가닥: 5'-AGTCCTTCCACGATACCAAAGTTG-3'(서열번호 175)Antisense strand: 5'-AGTCCTTCCACGATACCAAAGTTG-3' (SEQ ID NO: 175)

조제물로는 하기 조성의 것을 사용하였다:As the preparation, those of the following composition were used:

PCR 프리믹스 타입(바이오니아)PCR premix type (Bionia)

센스 프라이머: 5 p㏖, 1 ㎕Sense primer: 5 p㏖, 1 µl

안티센스 프라이머: 5 p㏖, 1 ㎕Antisense primer: 5 p㏖, 1 µl

증류수 13 ㎕13 µl of distilled water

cDNA 5 ㎕5 µl of cDNA

PCR은 95℃ 30초, 57℃ 30초 및 72℃ 30초의 반응 조건에서 30 사이클 수행하였고, PCR 결과물을 아가로스 겔에 전기영동하여 증폭 산물을 확인하였다.PCR was performed for 30 cycles under reaction conditions of 95°C for 30 seconds, 57°C for 30 seconds, and 72°C for 30 seconds, and the PCR result was subjected to electrophoresis on an agarose gel to confirm the amplification product.

그 결과, 야생형 M-MLV에서 유래된 본 발명의 돌연변이 역전사효소는 야생형 역전사효소에 비해 뛰어난 열안정성을 가지며, 그 중에서도 K295Q, T306L 및 P408E 돌연변이 역전사효소는 특히 뛰어난 열안정성을 나타내었다. 특히, 반응 온도 60℃에서 야생형 M-MLV 역전사효소는 역전사 활성을 갖지 못하지만, K295Q, T306L 및 P408E 돌연변이 역전사효소는 역전사 활성을 유지하였다(도 3). 가장 뛰어난 열안정성을 나타내는 T306L 돌연변이 역전사효소의 경우, 37℃, 42℃ 및 50℃에서는 야생형 M-MLV 유래 역전사효소와 동등한 열안정이 나타내었지만, 60℃, 65℃ 및 70℃에서는 야생형 M-MLV 유래 역전사효소보다 뛰어난 열안정성을 나타내었다(도 4).As a result, the mutant reverse transcriptase of the present invention derived from wild-type M-MLV has superior thermal stability compared to the wild-type reverse transcriptase, and among them, K295Q, T306L and P408E mutant reverse transcriptases exhibited particularly excellent thermal stability. In particular, wild-type M-MLV reverse transcriptase did not have reverse transcription activity at a reaction temperature of 60° C., but K295Q, T306L and P408E mutant reverse transcriptases maintained reverse transcription activity (FIG. 3). In the case of the T306L mutant reverse transcriptase, which exhibits the best thermal stability, at 37°C, 42°C and 50°C, the same thermal stability as the wild-type M-MLV-derived reverse transcriptase was shown, but at 60°C, 65°C and 70°C, the wild-type M-MLV It showed superior thermal stability than the derived reverse transcriptase (FIG. 4).

<110> BIONEER CORPORATION <120> Reverse Transcriptase Having Improved Thermostability <130> 2013-dpa-0708 <160> 175 <170> KopatentIn 2.0 <210> 1 <211> 672 <212> PRT <213> Moloney Murine Leukemia Virus <400> 1 Met Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu 1 5 10 15 Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala 20 25 30 Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu 35 40 45 Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr 50 55 60 Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg 65 70 75 80 Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr 85 90 95 Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val 100 105 110 Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr 115 120 125 Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln 130 135 140 Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu 145 150 155 160 His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu 165 170 175 Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe 180 185 190 Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala 195 200 205 Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp 210 215 220 Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr 225 230 235 240 Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala 245 250 255 Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu 260 265 270 Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val 275 280 285 Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu 290 295 300 Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met 305 310 315 320 Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp 325 330 335 Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu 340 345 350 Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu 355 360 365 Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys 370 375 380 Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp 385 390 395 400 Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile 405 410 415 Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu 420 425 430 Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro 435 440 445 Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu 450 455 460 Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro 465 470 475 480 Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu 485 490 495 Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln 500 505 510 Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asp Gly Ser Ser Leu 515 520 525 Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr 530 535 540 Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg 545 550 555 560 Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys 565 570 575 Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His 580 585 590 Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly 595 600 605 Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu 610 615 620 Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys 625 630 635 640 Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala 645 650 655 Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ile 660 665 670 <210> 2 <211> 672 <212> PRT <213> Moloney Murine Leukemia Virus <220> <221> MUTAGEN <222> (63) <223> mutant reverse transcriptase having leucine residue instead of glutamine residue at 63 position of SEQ. ID. No: 1 <400> 2 Met Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu 1 5 10 15 Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala 20 25 30 Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu 35 40 45 Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Leu Tyr 50 55 60 Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg 65 70 75 80 Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr 85 90 95 Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val 100 105 110 Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr 115 120 125 Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln 130 135 140 Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu 145 150 155 160 His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu 165 170 175 Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe 180 185 190 Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala 195 200 205 Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp 210 215 220 Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr 225 230 235 240 Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala 245 250 255 Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu 260 265 270 Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val 275 280 285 Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu 290 295 300 Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met 305 310 315 320 Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp 325 330 335 Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu 340 345 350 Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu 355 360 365 Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys 370 375 380 Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp 385 390 395 400 Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile 405 410 415 Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu 420 425 430 Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro 435 440 445 Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu 450 455 460 Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro 465 470 475 480 Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu 485 490 495 Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln 500 505 510 Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asp Gly Ser Ser Leu 515 520 525 Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr 530 535 540 Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg 545 550 555 560 Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys 565 570 575 Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His 580 585 590 Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly 595 600 605 Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu 610 615 620 Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys 625 630 635 640 Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala 645 650 655 Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ile 660 665 670 <210> 3 <211> 672 <212> PRT <213> Moloney Murine Leukemia Virus <220> <221> MUTAGEN <222> (264) <223> mutant reverse transcriptase having leucine residue instead of lysine residue at 264 position of SEQ. ID. No: 1 <400> 3 Met Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu 1 5 10 15 Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala 20 25 30 Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu 35 40 45 Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr 50 55 60 Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg 65 70 75 80 Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr 85 90 95 Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val 100 105 110 Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr 115 120 125 Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln 130 135 140 Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu 145 150 155 160 His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu 165 170 175 Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe 180 185 190 Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala 195 200 205 Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp 210 215 220 Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr 225 230 235 240 Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala 245 250 255 Lys Lys Ala Gln Ile Cys Gln Leu Gln Val Lys Tyr Leu Gly Tyr Leu 260 265 270 Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val 275 280 285 Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu 290 295 300 Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met 305 310 315 320 Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp 325 330 335 Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu 340 345 350 Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu 355 360 365 Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys 370 375 380 Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp 385 390 395 400 Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile 405 410 415 Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu 420 425 430 Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro 435 440 445 Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu 450 455 460 Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro 465 470 475 480 Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu 485 490 495 Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln 500 505 510 Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asp Gly Ser Ser Leu 515 520 525 Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr 530 535 540 Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg 545 550 555 560 Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys 565 570 575 Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His 580 585 590 Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly 595 600 605 Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu 610 615 620 Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys 625 630 635 640 Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala 645 650 655 Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ile 660 665 670 <210> 4 <211> 672 <212> PRT <213> Moloney Murine Leukemia Virus <220> <221> MUTAGEN <222> (295) <223> mutant reverse transcriptase having glutamine residue instead of lysine residue at 295 position of SEQ. ID. No: 1 <400> 4 Met Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu 1 5 10 15 Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala 20 25 30 Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu 35 40 45 Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr 50 55 60 Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg 65 70 75 80 Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr 85 90 95 Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val 100 105 110 Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr 115 120 125 Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln 130 135 140 Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu 145 150 155 160 His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu 165 170 175 Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe 180 185 190 Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala 195 200 205 Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp 210 215 220 Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr 225 230 235 240 Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala 245 250 255 Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu 260 265 270 Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val 275 280 285 Met Gly Gln Pro Thr Pro Gln Thr Pro Arg Gln Leu Arg Glu Phe Leu 290 295 300 Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met 305 310 315 320 Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp 325 330 335 Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu 340 345 350 Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu 355 360 365 Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys 370 375 380 Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp 385 390 395 400 Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile 405 410 415 Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu 420 425 430 Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro 435 440 445 Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu 450 455 460 Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro 465 470 475 480 Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu 485 490 495 Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln 500 505 510 Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asp Gly Ser Ser Leu 515 520 525 Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr 530 535 540 Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg 545 550 555 560 Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys 565 570 575 Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His 580 585 590 Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly 595 600 605 Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu 610 615 620 Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys 625 630 635 640 Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala 645 650 655 Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ile 660 665 670 <210> 5 <211> 672 <212> PRT <213> Moloney Murine Leukemia Virus <220> <221> MUTAGEN <222> (306) <223> mutant reverse transcriptase having leucine residue instead of threonine residue at 306 position of SEQ. ID. No: 1 <400> 5 Met Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu 1 5 10 15 Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala 20 25 30 Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu 35 40 45 Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr 50 55 60 Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg 65 70 75 80 Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr 85 90 95 Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val 100 105 110 Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr 115 120 125 Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln 130 135 140 Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu 145 150 155 160 His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu 165 170 175 Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe 180 185 190 Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala 195 200 205 Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp 210 215 220 Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr 225 230 235 240 Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala 245 250 255 Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu 260 265 270 Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val 275 280 285 Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu 290 295 300 Gly Leu Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met 305 310 315 320 Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp 325 330 335 Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu 340 345 350 Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu 355 360 365 Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys 370 375 380 Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp 385 390 395 400 Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile 405 410 415 Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu 420 425 430 Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro 435 440 445 Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu 450 455 460 Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro 465 470 475 480 Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu 485 490 495 Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln 500 505 510 Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asp Gly Ser Ser Leu 515 520 525 Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr 530 535 540 Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg 545 550 555 560 Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys 565 570 575 Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His 580 585 590 Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly 595 600 605 Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu 610 615 620 Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys 625 630 635 640 Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala 645 650 655 Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ile 660 665 670 <210> 6 <211> 672 <212> PRT <213> Moloney Murine Leukemia Virus <220> <221> MUTAGEN <222> (346) <223> mutant reverse transcriptase having methionine residue instead of glutamic acid residue at 346 position of SEQ. ID. No: 1 <400> 6 Met Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu 1 5 10 15 Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala 20 25 30 Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu 35 40 45 Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr 50 55 60 Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg 65 70 75 80 Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr 85 90 95 Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val 100 105 110 Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr 115 120 125 Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln 130 135 140 Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu 145 150 155 160 His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu 165 170 175 Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe 180 185 190 Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala 195 200 205 Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp 210 215 220 Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr 225 230 235 240 Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala 245 250 255 Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu 260 265 270 Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val 275 280 285 Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu 290 295 300 Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met 305 310 315 320 Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp 325 330 335 Gly Pro Asp Gln Gln Lys Ala Tyr Gln Met Ile Lys Gln Ala Leu Leu 340 345 350 Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu 355 360 365 Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys 370 375 380 Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp 385 390 395 400 Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile 405 410 415 Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu 420 425 430 Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro 435 440 445 Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu 450 455 460 Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro 465 470 475 480 Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu 485 490 495 Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln 500 505 510 Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asp Gly Ser Ser Leu 515 520 525 Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr 530 535 540 Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg 545 550 555 560 Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys 565 570 575 Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His 580 585 590 Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly 595 600 605 Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu 610 615 620 Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys 625 630 635 640 Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala 645 650 655 Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ile 660 665 670 <210> 7 <211> 672 <212> PRT <213> Moloney Murine Leukemia Virus <220> <221> MUTAGEN <222> (408) <223> mutant reverse transcriptase having glutamic acid residue instead of proline residue at 408 position of SEQ. ID. No: 1 <400> 7 Met Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu 1 5 10 15 Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala 20 25 30 Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu 35 40 45 Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr 50 55 60 Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg 65 70 75 80 Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr 85 90 95 Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val 100 105 110 Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr 115 120 125 Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln 130 135 140 Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu 145 150 155 160 His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu 165 170 175 Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe 180 185 190 Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala 195 200 205 Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp 210 215 220 Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr 225 230 235 240 Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala 245 250 255 Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu 260 265 270 Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val 275 280 285 Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu 290 295 300 Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met 305 310 315 320 Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp 325 330 335 Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu 340 345 350 Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu 355 360 365 Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys 370 375 380 Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp 385 390 395 400 Pro Val Ala Ala Gly Trp Pro Glu Cys Leu Arg Met Val Ala Ala Ile 405 410 415 Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu 420 425 430 Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro 435 440 445 Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu 450 455 460 Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro 465 470 475 480 Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu 485 490 495 Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln 500 505 510 Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asp Gly Ser Ser Leu 515 520 525 Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr 530 535 540 Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg 545 550 555 560 Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys 565 570 575 Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His 580 585 590 Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly 595 600 605 Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu 610 615 620 Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys 625 630 635 640 Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala 645 650 655 Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ile 660 665 670 <210> 8 <211> 672 <212> PRT <213> Moloney Murine Leukemia Virus <220> <221> MUTAGEN <222> (438) <223> mutant reverse transcriptase having tyrosine residue instead of histidine residue at 438 position of SEQ. ID. No: 1 <400> 8 Met Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu 1 5 10 15 Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala 20 25 30 Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu 35 40 45 Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr 50 55 60 Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg 65 70 75 80 Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr 85 90 95 Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val 100 105 110 Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr 115 120 125 Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln 130 135 140 Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu 145 150 155 160 His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu 165 170 175 Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe 180 185 190 Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala 195 200 205 Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp 210 215 220 Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr 225 230 235 240 Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala 245 250 255 Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu 260 265 270 Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val 275 280 285 Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu 290 295 300 Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met 305 310 315 320 Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp 325 330 335 Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu 340 345 350 Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu 355 360 365 Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys 370 375 380 Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp 385 390 395 400 Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile 405 410 415 Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu 420 425 430 Val Ile Leu Ala Pro Tyr Ala Val Glu Ala Leu Val Lys Gln Pro Pro 435 440 445 Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu 450 455 460 Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro 465 470 475 480 Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu 485 490 495 Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln 500 505 510 Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asp Gly Ser Ser Leu 515 520 525 Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr 530 535 540 Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg 545 550 555 560 Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys 565 570 575 Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His 580 585 590 Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly 595 600 605 Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu 610 615 620 Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys 625 630 635 640 Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala 645 650 655 Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ile 660 665 670 <210> 9 <211> 672 <212> PRT <213> Moloney Murine Leukemia Virus <220> <221> MUTAGEN <222> (454) <223> mutant reverse transcriptase having phenylalanine residue instead of asparagine residue at 454 position of SEQ. ID. No: 1 <400> 9 Met Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu 1 5 10 15 Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala 20 25 30 Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu 35 40 45 Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr 50 55 60 Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg 65 70 75 80 Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr 85 90 95 Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val 100 105 110 Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr 115 120 125 Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln 130 135 140 Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu 145 150 155 160 His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu 165 170 175 Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe 180 185 190 Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala 195 200 205 Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp 210 215 220 Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr 225 230 235 240 Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala 245 250 255 Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu 260 265 270 Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val 275 280 285 Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu 290 295 300 Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met 305 310 315 320 Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp 325 330 335 Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu 340 345 350 Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu 355 360 365 Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys 370 375 380 Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp 385 390 395 400 Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile 405 410 415 Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu 420 425 430 Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro 435 440 445 Asp Arg Trp Leu Ser Phe Ala Arg Met Thr His Tyr Gln Ala Leu Leu 450 455 460 Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro 465 470 475 480 Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu 485 490 495 Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln 500 505 510 Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asp Gly Ser Ser Leu 515 520 525 Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr 530 535 540 Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg 545 550 555 560 Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys 565 570 575 Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His 580 585 590 Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly 595 600 605 Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu 610 615 620 Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys 625 630 635 640 Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala 645 650 655 Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ile 660 665 670 <210> 10 <211> 2016 <212> DNA <213> Moloney Murine Leukemia Virus <220> <221> mat_peptide <222> (1)..(2016) <223> nucleotide sequence coding for the amino acid sequence of SEQ. ID. No: 1 <400> 10 atgctaaata tagaagatga gcatcggcta catgagacct caaaagagcc agatgtttct 60 ctagggtcca catggctgtc tgattttcct caggcctggg cggaaaccgg gggcatggga 120 ctggcagttc gccaagctcc tctgatcata cctctgaaag caacctctac ccccgtgtcc 180 ataaaacaat accccatgtc acaagaagcc agactgggga tcaagcccca catacagaga 240 ctgttggacc agggaatact ggtaccctgc cagtccccct ggaacacgcc cctgctaccc 300 gttaagaaac cagggactaa tgattatagg cctgtccagg atctgagaga agtcaacaag 360 cgggtggaag acatccaccc caccgtgccc aacccttaca acctcttgag cgggctccca 420 ccgtcccacc agtggtacac tgtgcttgat ttaaaggatg cctttttctg cctgagactc 480 caccccacca gtcagcctct cttcgccttt gagtggagag atccagagat gggaatctca 540 ggacaattga cctggaccag actcccacag ggtttcaaaa acagtcccac cctgtttgat 600 gaggcactgc acagagacct agcagacttc cggatccagc acccagactt gatcctgcta 660 cagtacgtgg atgacttact gctggccgcc acttctgagc tagactgcca acaaggtact 720 cgggccctgt tacaaaccct agggaacctc gggtatcggg cctcggccaa gaaagcccaa 780 atttgccaga aacaggtcaa gtatctgggg tatcttctaa aagagggtca gagatggctg 840 actgaggcca gaaaagagac tgtgatgggg cagcctactc cgaagacccc tcgacaacta 900 agggagttcc tagggacggc aggcttctgt cgcctctgga tccctgggtt tgcagaaatg 960 gcagccccct tgtaccctct caccaaaacg gggactctgt ttaattgggg cccagaccaa 1020 caaaaggcct atcaagaaat caagcaagct cttctaactg ccccagccct ggggttgcca 1080 gatttgacta agccctttga actctttgtc gacgagaagc agggctacgc caaaggtgtc 1140 ctaacgcaaa aactgggacc ttggcgtcgg ccggtggcct acctgtccaa aaagctagac 1200 ccagtagcag ctgggtggcc cccttgccta cggatggtag cagccattgc cgtactgaca 1260 aaggatgcag gcaagctaac catgggacag ccactagtca ttctggcccc ccatgcagta 1320 gaggcactag tcaaacaacc ccccgaccgc tggctttcca acgcccggat gactcactat 1380 caggccttgc ttttggacac ggaccgggtc cagttcggac cggtggtagc cctgaacccg 1440 gctacgctgc tcccactgcc tgaggaaggg ctgcaacaca actgccttga tatcctggcc 1500 gaagcccacg gaacccgacc cgacctaacg gaccagccgc tcccagacgc cgaccacacc 1560 tggtacacgg atggaagcag tctcttacaa gagggacagc gtaaggcggg agctgcggtg 1620 accaccgaga ccgaggtaat ctgggctaaa gccctgccag ccgggacatc cgctcagcgg 1680 gctgaactga tagcactcac ccaggcccta aagatggcag aaggtaagaa gctaaatgtt 1740 tatactgata gccgttatgc ttttgctact gcccatatcc atggagaaat atacagaagg 1800 cgtgggttgc tcacatcaga aggcaaagag atcaaaaata aagacgagat cttggcccta 1860 ctaaaagccc tctttctgcc caaaagactt agcataatcc attgtccagg acatcaaaag 1920 ggacacagcg ccgaggctag aggcaaccgg atggctgacc aagcggcccg aaaggcagcc 1980 atcacagaga ctccagacac ctctaccctc ctcata 2016 <210> 11 <211> 2016 <212> DNA <213> Moloney Murine Leukemia Virus <220> <221> mat_peptide <222> (1)..(2016) <223> nucleotide sequence coding for the amino acid sequence of SEQ. ID. No: 2 <220> <221> mutation <222> (187)..(189) <223> substitution of ctt instead of caa at 187-189 positions of SEQ. ID. No: 10 <400> 11 atgctaaata tagaagatga gcatcggcta catgagacct caaaagagcc agatgtttct 60 ctagggtcca catggctgtc tgattttcct caggcctggg cggaaaccgg gggcatggga 120 ctggcagttc gccaagctcc tctgatcata cctctgaaag caacctctac ccccgtgtcc 180 ataaaacttt accccatgtc acaagaagcc agactgggga tcaagcccca catacagaga 240 ctgttggacc agggaatact ggtaccctgc cagtccccct ggaacacgcc cctgctaccc 300 gttaagaaac cagggactaa tgattatagg cctgtccagg atctgagaga agtcaacaag 360 cgggtggaag acatccaccc caccgtgccc aacccttaca acctcttgag cgggctccca 420 ccgtcccacc agtggtacac tgtgcttgat ttaaaggatg cctttttctg cctgagactc 480 caccccacca gtcagcctct cttcgccttt gagtggagag atccagagat gggaatctca 540 ggacaattga cctggaccag actcccacag ggtttcaaaa acagtcccac cctgtttgat 600 gaggcactgc acagagacct agcagacttc cggatccagc acccagactt gatcctgcta 660 cagtacgtgg atgacttact gctggccgcc acttctgagc tagactgcca acaaggtact 720 cgggccctgt tacaaaccct agggaacctc gggtatcggg cctcggccaa gaaagcccaa 780 atttgccaga aacaggtcaa gtatctgggg tatcttctaa aagagggtca gagatggctg 840 actgaggcca gaaaagagac tgtgatgggg cagcctactc cgaagacccc tcgacaacta 900 agggagttcc tagggacggc aggcttctgt cgcctctgga tccctgggtt tgcagaaatg 960 gcagccccct tgtaccctct caccaaaacg gggactctgt ttaattgggg cccagaccaa 1020 caaaaggcct atcaagaaat caagcaagct cttctaactg ccccagccct ggggttgcca 1080 gatttgacta agccctttga actctttgtc gacgagaagc agggctacgc caaaggtgtc 1140 ctaacgcaaa aactgggacc ttggcgtcgg ccggtggcct acctgtccaa aaagctagac 1200 ccagtagcag ctgggtggcc cccttgccta cggatggtag cagccattgc cgtactgaca 1260 aaggatgcag gcaagctaac catgggacag ccactagtca ttctggcccc ccatgcagta 1320 gaggcactag tcaaacaacc ccccgaccgc tggctttcca acgcccggat gactcactat 1380 caggccttgc ttttggacac ggaccgggtc cagttcggac cggtggtagc cctgaacccg 1440 gctacgctgc tcccactgcc tgaggaaggg ctgcaacaca actgccttga tatcctggcc 1500 gaagcccacg gaacccgacc cgacctaacg gaccagccgc tcccagacgc cgaccacacc 1560 tggtacacgg atggaagcag tctcttacaa gagggacagc gtaaggcggg agctgcggtg 1620 accaccgaga ccgaggtaat ctgggctaaa gccctgccag ccgggacatc cgctcagcgg 1680 gctgaactga tagcactcac ccaggcccta aagatggcag aaggtaagaa gctaaatgtt 1740 tatactgata gccgttatgc ttttgctact gcccatatcc atggagaaat atacagaagg 1800 cgtgggttgc tcacatcaga aggcaaagag atcaaaaata aagacgagat cttggcccta 1860 ctaaaagccc tctttctgcc caaaagactt agcataatcc attgtccagg acatcaaaag 1920 ggacacagcg ccgaggctag aggcaaccgg atggctgacc aagcggcccg aaaggcagcc 1980 atcacagaga ctccagacac ctctaccctc ctcata 2016 <210> 12 <211> 2016 <212> DNA <213> Moloney Murine Leukemia Virus <220> <221> mat_peptide <222> (1)..(2016) <223> nucleotide sequence coding for the amino acid sequence of SEQ. ID. No: 3 <220> <221> mutation <222> (790)..(792) <223> substitution of ctt instead of aaa at 790-792 positions of SEQ. ID. No: 10 <400> 12 atgctaaata tagaagatga gcatcggcta catgagacct caaaagagcc agatgtttct 60 ctagggtcca catggctgtc tgattttcct caggcctggg cggaaaccgg gggcatggga 120 ctggcagttc gccaagctcc tctgatcata cctctgaaag caacctctac ccccgtgtcc 180 ataaaacaat accccatgtc acaagaagcc agactgggga tcaagcccca catacagaga 240 ctgttggacc agggaatact ggtaccctgc cagtccccct ggaacacgcc cctgctaccc 300 gttaagaaac cagggactaa tgattatagg cctgtccagg atctgagaga agtcaacaag 360 cgggtggaag acatccaccc caccgtgccc aacccttaca acctcttgag cgggctccca 420 ccgtcccacc agtggtacac tgtgcttgat ttaaaggatg cctttttctg cctgagactc 480 caccccacca gtcagcctct cttcgccttt gagtggagag atccagagat gggaatctca 540 ggacaattga cctggaccag actcccacag ggtttcaaaa acagtcccac cctgtttgat 600 gaggcactgc acagagacct agcagacttc cggatccagc acccagactt gatcctgcta 660 cagtacgtgg atgacttact gctggccgcc acttctgagc tagactgcca acaaggtact 720 cgggccctgt tacaaaccct agggaacctc gggtatcggg cctcggccaa gaaagcccaa 780 atttgccagc ttcaggtcaa gtatctgggg tatcttctaa aagagggtca gagatggctg 840 actgaggcca gaaaagagac tgtgatgggg cagcctactc cgaagacccc tcgacaacta 900 agggagttcc tagggacggc aggcttctgt cgcctctgga tccctgggtt tgcagaaatg 960 gcagccccct tgtaccctct caccaaaacg gggactctgt ttaattgggg cccagaccaa 1020 caaaaggcct atcaagaaat caagcaagct cttctaactg ccccagccct ggggttgcca 1080 gatttgacta agccctttga actctttgtc gacgagaagc agggctacgc caaaggtgtc 1140 ctaacgcaaa aactgggacc ttggcgtcgg ccggtggcct acctgtccaa aaagctagac 1200 ccagtagcag ctgggtggcc cccttgccta cggatggtag cagccattgc cgtactgaca 1260 aaggatgcag gcaagctaac catgggacag ccactagtca ttctggcccc ccatgcagta 1320 gaggcactag tcaaacaacc ccccgaccgc tggctttcca acgcccggat gactcactat 1380 caggccttgc ttttggacac ggaccgggtc cagttcggac cggtggtagc cctgaacccg 1440 gctacgctgc tcccactgcc tgaggaaggg ctgcaacaca actgccttga tatcctggcc 1500 gaagcccacg gaacccgacc cgacctaacg gaccagccgc tcccagacgc cgaccacacc 1560 tggtacacgg atggaagcag tctcttacaa gagggacagc gtaaggcggg agctgcggtg 1620 accaccgaga ccgaggtaat ctgggctaaa gccctgccag ccgggacatc cgctcagcgg 1680 gctgaactga tagcactcac ccaggcccta aagatggcag aaggtaagaa gctaaatgtt 1740 tatactgata gccgttatgc ttttgctact gcccatatcc atggagaaat atacagaagg 1800 cgtgggttgc tcacatcaga aggcaaagag atcaaaaata aagacgagat cttggcccta 1860 ctaaaagccc tctttctgcc caaaagactt agcataatcc attgtccagg acatcaaaag 1920 ggacacagcg ccgaggctag aggcaaccgg atggctgacc aagcggcccg aaaggcagcc 1980 atcacagaga ctccagacac ctctaccctc ctcata 2016 <210> 13 <211> 2016 <212> DNA <213> Moloney Murine Leukemia Virus <220> <221> mat_peptide <222> (1)..(2016) <223> nucleotide sequence coding for the amino acid sequence of SEQ. ID. No: 4 <220> <221> mutation <222> (883)..(885) <223> substitution of caa instead of aag at 883-885 positions of SEQ. ID. No: 10 <400> 13 atgctaaata tagaagatga gcatcggcta catgagacct caaaagagcc agatgtttct 60 ctagggtcca catggctgtc tgattttcct caggcctggg cggaaaccgg gggcatggga 120 ctggcagttc gccaagctcc tctgatcata cctctgaaag caacctctac ccccgtgtcc 180 ataaaacaat accccatgtc acaagaagcc agactgggga tcaagcccca catacagaga 240 ctgttggacc agggaatact ggtaccctgc cagtccccct ggaacacgcc cctgctaccc 300 gttaagaaac cagggactaa tgattatagg cctgtccagg atctgagaga agtcaacaag 360 cgggtggaag acatccaccc caccgtgccc aacccttaca acctcttgag cgggctccca 420 ccgtcccacc agtggtacac tgtgcttgat ttaaaggatg cctttttctg cctgagactc 480 caccccacca gtcagcctct cttcgccttt gagtggagag atccagagat gggaatctca 540 ggacaattga cctggaccag actcccacag ggtttcaaaa acagtcccac cctgtttgat 600 gaggcactgc acagagacct agcagacttc cggatccagc acccagactt gatcctgcta 660 cagtacgtgg atgacttact gctggccgcc acttctgagc tagactgcca acaaggtact 720 cgggccctgt tacaaaccct agggaacctc gggtatcggg cctcggccaa gaaagcccaa 780 atttgccaga aacaggtcaa gtatctgggg tatcttctaa aagagggtca gagatggctg 840 actgaggcca gaaaagagac tgtgatgggg cagcctactc cgcaaacccc tcgacaacta 900 agggagttcc tagggacggc aggcttctgt cgcctctgga tccctgggtt tgcagaaatg 960 gcagccccct tgtaccctct caccaaaacg gggactctgt ttaattgggg cccagaccaa 1020 caaaaggcct atcaagaaat caagcaagct cttctaactg ccccagccct ggggttgcca 1080 gatttgacta agccctttga actctttgtc gacgagaagc agggctacgc caaaggtgtc 1140 ctaacgcaaa aactgggacc ttggcgtcgg ccggtggcct acctgtccaa aaagctagac 1200 ccagtagcag ctgggtggcc cccttgccta cggatggtag cagccattgc cgtactgaca 1260 aaggatgcag gcaagctaac catgggacag ccactagtca ttctggcccc ccatgcagta 1320 gaggcactag tcaaacaacc ccccgaccgc tggctttcca acgcccggat gactcactat 1380 caggccttgc ttttggacac ggaccgggtc cagttcggac cggtggtagc cctgaacccg 1440 gctacgctgc tcccactgcc tgaggaaggg ctgcaacaca actgccttga tatcctggcc 1500 gaagcccacg gaacccgacc cgacctaacg gaccagccgc tcccagacgc cgaccacacc 1560 tggtacacgg atggaagcag tctcttacaa gagggacagc gtaaggcggg agctgcggtg 1620 accaccgaga ccgaggtaat ctgggctaaa gccctgccag ccgggacatc cgctcagcgg 1680 gctgaactga tagcactcac ccaggcccta aagatggcag aaggtaagaa gctaaatgtt 1740 tatactgata gccgttatgc ttttgctact gcccatatcc atggagaaat atacagaagg 1800 cgtgggttgc tcacatcaga aggcaaagag atcaaaaata aagacgagat cttggcccta 1860 ctaaaagccc tctttctgcc caaaagactt agcataatcc attgtccagg acatcaaaag 1920 ggacacagcg ccgaggctag aggcaaccgg atggctgacc aagcggcccg aaaggcagcc 1980 atcacagaga ctccagacac ctctaccctc ctcata 2016 <210> 14 <211> 2016 <212> DNA <213> Moloney Murine Leukemia Virus <220> <221> mat_peptide <222> (1)..(2016) <223> nucleotide sequence coding for the amino acid sequence of SEQ. ID. No: 5 <220> <221> mutation <222> (916)..(918) <223> substitution of ctt instead of acg at 916-918 positions of SEQ. ID. No: 10 <400> 14 atgctaaata tagaagatga gcatcggcta catgagacct caaaagagcc agatgtttct 60 ctagggtcca catggctgtc tgattttcct caggcctggg cggaaaccgg gggcatggga 120 ctggcagttc gccaagctcc tctgatcata cctctgaaag caacctctac ccccgtgtcc 180 ataaaacaat accccatgtc acaagaagcc agactgggga tcaagcccca catacagaga 240 ctgttggacc agggaatact ggtaccctgc cagtccccct ggaacacgcc cctgctaccc 300 gttaagaaac cagggactaa tgattatagg cctgtccagg atctgagaga agtcaacaag 360 cgggtggaag acatccaccc caccgtgccc aacccttaca acctcttgag cgggctccca 420 ccgtcccacc agtggtacac tgtgcttgat ttaaaggatg cctttttctg cctgagactc 480 caccccacca gtcagcctct cttcgccttt gagtggagag atccagagat gggaatctca 540 ggacaattga cctggaccag actcccacag ggtttcaaaa acagtcccac cctgtttgat 600 gaggcactgc acagagacct agcagacttc cggatccagc acccagactt gatcctgcta 660 cagtacgtgg atgacttact gctggccgcc acttctgagc tagactgcca acaaggtact 720 cgggccctgt tacaaaccct agggaacctc gggtatcggg cctcggccaa gaaagcccaa 780 atttgccaga aacaggtcaa gtatctgggg tatcttctaa aagagggtca gagatggctg 840 actgaggcca gaaaagagac tgtgatgggg cagcctactc cgaagacccc tcgacaacta 900 agggagttcc tagggcttgc aggcttctgt cgcctctgga tccctgggtt tgcagaaatg 960 gcagccccct tgtaccctct caccaaaacg gggactctgt ttaattgggg cccagaccaa 1020 caaaaggcct atcaagaaat caagcaagct cttctaactg ccccagccct ggggttgcca 1080 gatttgacta agccctttga actctttgtc gacgagaagc agggctacgc caaaggtgtc 1140 ctaacgcaaa aactgggacc ttggcgtcgg ccggtggcct acctgtccaa aaagctagac 1200 ccagtagcag ctgggtggcc cccttgccta cggatggtag cagccattgc cgtactgaca 1260 aaggatgcag gcaagctaac catgggacag ccactagtca ttctggcccc ccatgcagta 1320 gaggcactag tcaaacaacc ccccgaccgc tggctttcca acgcccggat gactcactat 1380 caggccttgc ttttggacac ggaccgggtc cagttcggac cggtggtagc cctgaacccg 1440 gctacgctgc tcccactgcc tgaggaaggg ctgcaacaca actgccttga tatcctggcc 1500 gaagcccacg gaacccgacc cgacctaacg gaccagccgc tcccagacgc cgaccacacc 1560 tggtacacgg atggaagcag tctcttacaa gagggacagc gtaaggcggg agctgcggtg 1620 accaccgaga ccgaggtaat ctgggctaaa gccctgccag ccgggacatc cgctcagcgg 1680 gctgaactga tagcactcac ccaggcccta aagatggcag aaggtaagaa gctaaatgtt 1740 tatactgata gccgttatgc ttttgctact gcccatatcc atggagaaat atacagaagg 1800 cgtgggttgc tcacatcaga aggcaaagag atcaaaaata aagacgagat cttggcccta 1860 ctaaaagccc tctttctgcc caaaagactt agcataatcc attgtccagg acatcaaaag 1920 ggacacagcg ccgaggctag aggcaaccgg atggctgacc aagcggcccg aaaggcagcc 1980 atcacagaga ctccagacac ctctaccctc ctcata 2016 <210> 15 <211> 2016 <212> DNA <213> Moloney Murine Leukemia Virus <220> <221> mat_peptide <222> (1)..(2016) <223> nucleotide sequence coding for the amino acid sequence of SEQ. ID. No: 6 <220> <221> mutation <222> (1036)..(1038) <223> substitution of atg instead of gaa at 1036-1038 positions of SEQ. ID. No: 10 <400> 15 atgctaaata tagaagatga gcatcggcta catgagacct caaaagagcc agatgtttct 60 ctagggtcca catggctgtc tgattttcct caggcctggg cggaaaccgg gggcatggga 120 ctggcagttc gccaagctcc tctgatcata cctctgaaag caacctctac ccccgtgtcc 180 ataaaacaat accccatgtc acaagaagcc agactgggga tcaagcccca catacagaga 240 ctgttggacc agggaatact ggtaccctgc cagtccccct ggaacacgcc cctgctaccc 300 gttaagaaac cagggactaa tgattatagg cctgtccagg atctgagaga agtcaacaag 360 cgggtggaag acatccaccc caccgtgccc aacccttaca acctcttgag cgggctccca 420 ccgtcccacc agtggtacac tgtgcttgat ttaaaggatg cctttttctg cctgagactc 480 caccccacca gtcagcctct cttcgccttt gagtggagag atccagagat gggaatctca 540 ggacaattga cctggaccag actcccacag ggtttcaaaa acagtcccac cctgtttgat 600 gaggcactgc acagagacct agcagacttc cggatccagc acccagactt gatcctgcta 660 cagtacgtgg atgacttact gctggccgcc acttctgagc tagactgcca acaaggtact 720 cgggccctgt tacaaaccct agggaacctc gggtatcggg cctcggccaa gaaagcccaa 780 atttgccaga aacaggtcaa gtatctgggg tatcttctaa aagagggtca gagatggctg 840 actgaggcca gaaaagagac tgtgatgggg cagcctactc cgaagacccc tcgacaacta 900 agggagttcc tagggacggc aggcttctgt cgcctctgga tccctgggtt tgcagaaatg 960 gcagccccct tgtaccctct caccaaaacg gggactctgt ttaattgggg cccagaccaa 1020 caaaaggcct atcaaatgat caagcaagct cttctaactg ccccagccct ggggttgcca 1080 gatttgacta agccctttga actctttgtc gacgagaagc agggctacgc caaaggtgtc 1140 ctaacgcaaa aactgggacc ttggcgtcgg ccggtggcct acctgtccaa aaagctagac 1200 ccagtagcag ctgggtggcc cccttgccta cggatggtag cagccattgc cgtactgaca 1260 aaggatgcag gcaagctaac catgggacag ccactagtca ttctggcccc ccatgcagta 1320 gaggcactag tcaaacaacc ccccgaccgc tggctttcca acgcccggat gactcactat 1380 caggccttgc ttttggacac ggaccgggtc cagttcggac cggtggtagc cctgaacccg 1440 gctacgctgc tcccactgcc tgaggaaggg ctgcaacaca actgccttga tatcctggcc 1500 gaagcccacg gaacccgacc cgacctaacg gaccagccgc tcccagacgc cgaccacacc 1560 tggtacacgg atggaagcag tctcttacaa gagggacagc gtaaggcggg agctgcggtg 1620 accaccgaga ccgaggtaat ctgggctaaa gccctgccag ccgggacatc cgctcagcgg 1680 gctgaactga tagcactcac ccaggcccta aagatggcag aaggtaagaa gctaaatgtt 1740 tatactgata gccgttatgc ttttgctact gcccatatcc atggagaaat atacagaagg 1800 cgtgggttgc tcacatcaga aggcaaagag atcaaaaata aagacgagat cttggcccta 1860 ctaaaagccc tctttctgcc caaaagactt agcataatcc attgtccagg acatcaaaag 1920 ggacacagcg ccgaggctag aggcaaccgg atggctgacc aagcggcccg aaaggcagcc 1980 atcacagaga ctccagacac ctctaccctc ctcata 2016 <210> 16 <211> 2016 <212> DNA <213> Moloney Murine Leukemia Virus <220> <221> mat_peptide <222> (1)..(2016) <223> nucleotide sequence coding for the amino acid sequence of SEQ. ID. No: 7 <220> <221> mutation <222> (1222)..(1224) <223> substitution of gaa instead of cct at 1222-1224 positions of SEQ. ID. No: 10 <400> 16 atgctaaata tagaagatga gcatcggcta catgagacct caaaagagcc agatgtttct 60 ctagggtcca catggctgtc tgattttcct caggcctggg cggaaaccgg gggcatggga 120 ctggcagttc gccaagctcc tctgatcata cctctgaaag caacctctac ccccgtgtcc 180 ataaaacaat accccatgtc acaagaagcc agactgggga tcaagcccca catacagaga 240 ctgttggacc agggaatact ggtaccctgc cagtccccct ggaacacgcc cctgctaccc 300 gttaagaaac cagggactaa tgattatagg cctgtccagg atctgagaga agtcaacaag 360 cgggtggaag acatccaccc caccgtgccc aacccttaca acctcttgag cgggctccca 420 ccgtcccacc agtggtacac tgtgcttgat ttaaaggatg cctttttctg cctgagactc 480 caccccacca gtcagcctct cttcgccttt gagtggagag atccagagat gggaatctca 540 ggacaattga cctggaccag actcccacag ggtttcaaaa acagtcccac cctgtttgat 600 gaggcactgc acagagacct agcagacttc cggatccagc acccagactt gatcctgcta 660 cagtacgtgg atgacttact gctggccgcc acttctgagc tagactgcca acaaggtact 720 cgggccctgt tacaaaccct agggaacctc gggtatcggg cctcggccaa gaaagcccaa 780 atttgccaga aacaggtcaa gtatctgggg tatcttctaa aagagggtca gagatggctg 840 actgaggcca gaaaagagac tgtgatgggg cagcctactc cgaagacccc tcgacaacta 900 agggagttcc tagggacggc aggcttctgt cgcctctgga tccctgggtt tgcagaaatg 960 gcagccccct tgtaccctct caccaaaacg gggactctgt ttaattgggg cccagaccaa 1020 caaaaggcct atcaagaaat caagcaagct cttctaactg ccccagccct ggggttgcca 1080 gatttgacta agccctttga actctttgtc gacgagaagc agggctacgc caaaggtgtc 1140 ctaacgcaaa aactgggacc ttggcgtcgg ccggtggcct acctgtccaa aaagctagac 1200 ccagtagcag ctgggtggcc cgaatgccta cggatggtag cagccattgc cgtactgaca 1260 aaggatgcag gcaagctaac catgggacag ccactagtca ttctggcccc ccatgcagta 1320 gaggcactag tcaaacaacc ccccgaccgc tggctttcca acgcccggat gactcactat 1380 caggccttgc ttttggacac ggaccgggtc cagttcggac cggtggtagc cctgaacccg 1440 gctacgctgc tcccactgcc tgaggaaggg ctgcaacaca actgccttga tatcctggcc 1500 gaagcccacg gaacccgacc cgacctaacg gaccagccgc tcccagacgc cgaccacacc 1560 tggtacacgg atggaagcag tctcttacaa gagggacagc gtaaggcggg agctgcggtg 1620 accaccgaga ccgaggtaat ctgggctaaa gccctgccag ccgggacatc cgctcagcgg 1680 gctgaactga tagcactcac ccaggcccta aagatggcag aaggtaagaa gctaaatgtt 1740 tatactgata gccgttatgc ttttgctact gcccatatcc atggagaaat atacagaagg 1800 cgtgggttgc tcacatcaga aggcaaagag atcaaaaata aagacgagat cttggcccta 1860 ctaaaagccc tctttctgcc caaaagactt agcataatcc attgtccagg acatcaaaag 1920 ggacacagcg ccgaggctag aggcaaccgg atggctgacc aagcggcccg aaaggcagcc 1980 atcacagaga ctccagacac ctctaccctc ctcata 2016 <210> 17 <211> 2016 <212> DNA <213> Moloney Murine Leukemia Virus <220> <221> mat_peptide <222> (1)..(2016) <223> nucleotide sequence coding for the amino acid sequence of SEQ. ID. No: 8 <220> <221> mutation <222> (1312)..(1314) <223> substitution of tac instead of cat at 1312-1314 positions of SEQ. ID. No: 10 <400> 17 atgctaaata tagaagatga gcatcggcta catgagacct caaaagagcc agatgtttct 60 ctagggtcca catggctgtc tgattttcct caggcctggg cggaaaccgg gggcatggga 120 ctggcagttc gccaagctcc tctgatcata cctctgaaag caacctctac ccccgtgtcc 180 ataaaacaat accccatgtc acaagaagcc agactgggga tcaagcccca catacagaga 240 ctgttggacc agggaatact ggtaccctgc cagtccccct ggaacacgcc cctgctaccc 300 gttaagaaac cagggactaa tgattatagg cctgtccagg atctgagaga agtcaacaag 360 cgggtggaag acatccaccc caccgtgccc aacccttaca acctcttgag cgggctccca 420 ccgtcccacc agtggtacac tgtgcttgat ttaaaggatg cctttttctg cctgagactc 480 caccccacca gtcagcctct cttcgccttt gagtggagag atccagagat gggaatctca 540 ggacaattga cctggaccag actcccacag ggtttcaaaa acagtcccac cctgtttgat 600 gaggcactgc acagagacct agcagacttc cggatccagc acccagactt gatcctgcta 660 cagtacgtgg atgacttact gctggccgcc acttctgagc tagactgcca acaaggtact 720 cgggccctgt tacaaaccct agggaacctc gggtatcggg cctcggccaa gaaagcccaa 780 atttgccaga aacaggtcaa gtatctgggg tatcttctaa aagagggtca gagatggctg 840 actgaggcca gaaaagagac tgtgatgggg cagcctactc cgaagacccc tcgacaacta 900 agggagttcc tagggacggc aggcttctgt cgcctctgga tccctgggtt tgcagaaatg 960 gcagccccct tgtaccctct caccaaaacg gggactctgt ttaattgggg cccagaccaa 1020 caaaaggcct atcaagaaat caagcaagct cttctaactg ccccagccct ggggttgcca 1080 gatttgacta agccctttga actctttgtc gacgagaagc agggctacgc caaaggtgtc 1140 ctaacgcaaa aactgggacc ttggcgtcgg ccggtggcct acctgtccaa aaagctagac 1200 ccagtagcag ctgggtggcc cccttgccta cggatggtag cagccattgc cgtactgaca 1260 aaggatgcag gcaagctaac catgggacag ccactagtca ttctggcccc ctacgcagta 1320 gaggcactag tcaaacaacc ccccgaccgc tggctttcca acgcccggat gactcactat 1380 caggccttgc ttttggacac ggaccgggtc cagttcggac cggtggtagc cctgaacccg 1440 gctacgctgc tcccactgcc tgaggaaggg ctgcaacaca actgccttga tatcctggcc 1500 gaagcccacg gaacccgacc cgacctaacg gaccagccgc tcccagacgc cgaccacacc 1560 tggtacacgg atggaagcag tctcttacaa gagggacagc gtaaggcggg agctgcggtg 1620 accaccgaga ccgaggtaat ctgggctaaa gccctgccag ccgggacatc cgctcagcgg 1680 gctgaactga tagcactcac ccaggcccta aagatggcag aaggtaagaa gctaaatgtt 1740 tatactgata gccgttatgc ttttgctact gcccatatcc atggagaaat atacagaagg 1800 cgtgggttgc tcacatcaga aggcaaagag atcaaaaata aagacgagat cttggcccta 1860 ctaaaagccc tctttctgcc caaaagactt agcataatcc attgtccagg acatcaaaag 1920 ggacacagcg ccgaggctag aggcaaccgg atggctgacc aagcggcccg aaaggcagcc 1980 atcacagaga ctccagacac ctctaccctc ctcata 2016 <210> 18 <211> 2016 <212> DNA <213> Moloney Murine Leukemia Virus <220> <221> mat_peptide <222> (1)..(2016) <223> nucleotide sequence coding for the amino acid sequence of SEQ. ID. No: 9 <220> <221> mutation <222> (1360)..(1362) <223> substitution of ttc instead of aac at 1360-1362 positions of SEQ. ID. No: 10 <400> 18 atgctaaata tagaagatga gcatcggcta catgagacct caaaagagcc agatgtttct 60 ctagggtcca catggctgtc tgattttcct caggcctggg cggaaaccgg gggcatggga 120 ctggcagttc gccaagctcc tctgatcata cctctgaaag caacctctac ccccgtgtcc 180 ataaaacaat accccatgtc acaagaagcc agactgggga tcaagcccca catacagaga 240 ctgttggacc agggaatact ggtaccctgc cagtccccct ggaacacgcc cctgctaccc 300 gttaagaaac cagggactaa tgattatagg cctgtccagg atctgagaga agtcaacaag 360 cgggtggaag acatccaccc caccgtgccc aacccttaca acctcttgag cgggctccca 420 ccgtcccacc agtggtacac tgtgcttgat ttaaaggatg cctttttctg cctgagactc 480 caccccacca gtcagcctct cttcgccttt gagtggagag atccagagat gggaatctca 540 ggacaattga cctggaccag actcccacag ggtttcaaaa acagtcccac cctgtttgat 600 gaggcactgc acagagacct agcagacttc cggatccagc acccagactt gatcctgcta 660 cagtacgtgg atgacttact gctggccgcc acttctgagc tagactgcca acaaggtact 720 cgggccctgt tacaaaccct agggaacctc gggtatcggg cctcggccaa gaaagcccaa 780 atttgccaga aacaggtcaa gtatctgggg tatcttctaa aagagggtca gagatggctg 840 actgaggcca gaaaagagac tgtgatgggg cagcctactc cgaagacccc tcgacaacta 900 agggagttcc tagggacggc aggcttctgt cgcctctgga tccctgggtt tgcagaaatg 960 gcagccccct tgtaccctct caccaaaacg gggactctgt ttaattgggg cccagaccaa 1020 caaaaggcct atcaagaaat caagcaagct cttctaactg ccccagccct ggggttgcca 1080 gatttgacta agccctttga actctttgtc gacgagaagc agggctacgc caaaggtgtc 1140 ctaacgcaaa aactgggacc ttggcgtcgg ccggtggcct acctgtccaa aaagctagac 1200 ccagtagcag ctgggtggcc cccttgccta cggatggtag cagccattgc cgtactgaca 1260 aaggatgcag gcaagctaac catgggacag ccactagtca ttctggcccc ccatgcagta 1320 gaggcactag tcaaacaacc ccccgaccgc tggctttcct tcgcccggat gactcactat 1380 caggccttgc ttttggacac ggaccgggtc cagttcggac cggtggtagc cctgaacccg 1440 gctacgctgc tcccactgcc tgaggaaggg ctgcaacaca actgccttga tatcctggcc 1500 gaagcccacg gaacccgacc cgacctaacg gaccagccgc tcccagacgc cgaccacacc 1560 tggtacacgg atggaagcag tctcttacaa gagggacagc gtaaggcggg agctgcggtg 1620 accaccgaga ccgaggtaat ctgggctaaa gccctgccag ccgggacatc cgctcagcgg 1680 gctgaactga tagcactcac ccaggcccta aagatggcag aaggtaagaa gctaaatgtt 1740 tatactgata gccgttatgc ttttgctact gcccatatcc atggagaaat atacagaagg 1800 cgtgggttgc tcacatcaga aggcaaagag atcaaaaata aagacgagat cttggcccta 1860 ctaaaagccc tctttctgcc caaaagactt agcataatcc attgtccagg acatcaaaag 1920 ggacacagcg ccgaggctag aggcaaccgg atggctgacc aagcggcccg aaaggcagcc 1980 atcacagaga ctccagacac ctctaccctc ctcata 2016 <210> 19 <211> 2019 <212> DNA <213> Artificial Sequence <220> <223> Optimized Moloney Murine Leukemia Virus reverse transcriptase in accordance with codon usage <220> <221> mat_peptide <222> (1)..(2016) <223> nucleotide sequence coding for the amino acid sequence of SEQ. ID. No: 1 <400> 19 atgctgaaca tcgaagacga acaccgtctg cacgaaacct ctaaagaacc ggacgtttct 60 ctgggttcta cctggctgtc tgacttcccg caggcttggg ctgaaaccgg tggtatgggt 120 ctggctgttc gtcaggctcc gctgatcatc ccgctgaaag ctacctctac cccggtttct 180 atcaaacagt acccgatgtc tcaggaagct cgtctgggta tcaaaccgca catccagcgt 240 ctgctggacc agggtatcct ggttccgtgc cagtctccgt ggaacacccc gctgctgccg 300 gttaaaaaac cgggtaccaa cgactaccgt ccggttcagg acctgcgtga agttaacaaa 360 cgtgttgaag acatccaccc gaccgttccg aacccgtaca acctgctgtc tggtctgccg 420 ccgtctcacc agtggtacac cgttctggac ctgaaagacg ctttcttctg cctgcgtctg 480 cacccgacct ctcagccgct gttcgctttc gaatggcgtg acccggaaat gggtatctct 540 ggtcagctga cctggacccg tctgccgcag ggtttcaaaa actctccgac cctgttcgac 600 gaagctctgc accgtgacct ggctgacttc cgtatccagc acccggacct gatcctgctg 660 cagtacgttg acgacctgct gctggctgct acctctgaac tggactgcca gcagggtacc 720 cgtgctctgc tgcagaccct gggtaacctg ggttaccgtg cttctgctaa aaaagctcag 780 atctgccaga aacaggttaa atacctgggt tacctgctga aagaaggtca gcgttggctg 840 accgaagctc gtaaagaaac cgttatgggt cagccgaccc cgaaaacccc gcgtcagctg 900 cgtgaattcc tgggtaccgc tggtttctgc cgtctgtgga taccgggttt cgctgaaatg 960 gctgctccgc tgtacccgct gaccaaaacc ggtaccctgt tcaactgggg tccggaccag 1020 cagaaagcgt accaggaaat caaacaggct ctgctgaccg ctccggctct gggtctgccg 1080 gacctgacca aaccgttcga actgttcgtt gacgaaaaac agggttacgc taaaggtgtt 1140 ctgacccaga aactgggtcc gtggcgtcgt ccggttgctt acctgtctaa aaaactggac 1200 ccggttgctg ctggttggcc gccgtgcctg cgtatggttg ctgctatcgc tgttctgacc 1260 aaagacgctg gtaaactgac catgggtcag ccgctggtta tcctggctcc gcacgctgtt 1320 gaagctctgg ttaaacagcc gccggaccgt tggctgtcta acgctcgtat gacccactac 1380 caggctctgc tgctggacac cgaccgtgtt cagttcggtc cggttgttgc tctgaacccg 1440 gctaccctgc tgccgctgcc ggaagaaggt ctgcagcaca actgcctgga catcctggct 1500 gaagctcacg gtacccgtcc ggacctgacc gaccagccgc tgccggacgc tgaccacacc 1560 tggtacaccg acggttcttc tctgctgcag gaaggtcagc gtaaagctgg tgctgctgtt 1620 accaccgaaa ccgaagttat ctgggctaaa gctctgccgg ctggtacctc tgctcagcgt 1680 gctgaactga tcgctctgac ccaggctctg aaaatggctg aaggtaaaaa actgaacgtt 1740 tacaccgact ctcgttacgc tttcgctacc gctcacatcc acggtgaaat ctaccgtcgt 1800 cgtggtctgc tgacctctga aggtaaagaa atcaaaaaca aagacgaaat cctggctctg 1860 ctgaaagctc tgttcctgcc gaaacgtctg tctatcatcc actgcccggg tcaccagaaa 1920 ggtcactctg ctgaagctcg tggtaaccgt atggctgacc aggctgctcg taaagctgct 1980 atcaccgaaa ccccggacac ctctaccctg ctgatctaa 2019 <210> 20 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> R0 oligonucleotide <400> 20 ttcagggata gagggagta 19 <210> 21 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> F0 oligonucleotide <400> 21 tactccctct atccctgaac atcgaagacg aacacc 36 <210> 22 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> R19 oligonucleotide <400> 22 gaggtttcgt gcagacggtg ttcgtcttcg atg 33 <210> 23 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> F36 oligonucleotide <400> 23 gtctgcacga aacctctaaa gaaccggacg tttc 34 <210> 24 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> R52 oligonucleotide <400> 24 ccaggtagaa cccagagaaa cgtccggttc ttta 34 <210> 25 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> F70 oligonucleotide <400> 25 tctgggttct acctggctgt ctgacttccc gc 32 <210> 26 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R86 oligonucleotide <400> 26 ttcagcccaa gcctgcggga agtcagacag 30 <210> 27 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> F102 oligonucleotide <400> 27 aggcttgggc tgaaaccggt ggtatgggt 29 <210> 28 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R116 oligonucleotide <400> 28 ctgacgaaca gccagaccca taccaccggt 30 <210> 29 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> F131 oligonucleotide <400> 29 ctggctgttc gtcaggctcc gctgatcatc 30 <210> 30 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R146 oligonucleotide <400> 30 ggtagctttc agcgggatga tcagcggagc 30 <210> 31 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> F161 oligonucleotide <400> 31 ccgctgaaag ctacctctac cccggtttct atc 33 <210> 32 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> R176 oligonucleotide <400> 32 gacatcgggt actgtttgat agaaaccggg gtaga 35 <210> 33 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> F194 oligonucleotide <400> 33 aaacagtacc cgatgtctca ggaagctcgt ctg 33 <210> 34 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> R211 oligonucleotide <400> 34 tgtgcggttt gatacccaga cgagcttcct ga 32 <210> 35 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> F227 oligonucleotide <400> 35 ggtatcaaac cgcacatcca gcgtctgctg 30 <210> 36 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R243 oligonucleotide <400> 36 ccaggatacc ctggtccagc agacgctgga 30 <210> 37 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> F257 oligonucleotide <400> 37 gaccagggta tcctggttcc gtgccagtct c 31 <210> 38 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> R273 oligonucleotide <400> 38 cggggtgttc cacggagact ggcacggaa 29 <210> 39 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> F288 oligonucleotide <400> 39 cgtggaacac cccgctgctg ccggttaaa 29 <210> 40 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> R302 oligonucleotide <400> 40 cgttggtacc cggtttttta accggcagca g 31 <210> 41 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> F317 oligonucleotide <400> 41 aaaccgggta ccaacgacta ccgtccggtt c 31 <210> 42 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R333 oligonucleotide <400> 42 cttcacgcag gtcctgaacc ggacggtagt 30 <210> 43 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> F348 oligonucleotide <400> 43 aggacctgcg tgaagttaac aaacgtgttg aagac 35 <210> 44 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> R363 oligonucleotide <400> 44 acggtcgggt ggatgtcttc aacacgtttg ttaa 34 <210> 45 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> F383 oligonucleotide <400> 45 atccacccga ccgttccgaa cccgtacaa 29 <210> 46 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R397 oligonucleotide <400> 46 agaccagaca gcaggttgta cgggttcgga 30 <210> 47 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> F412 oligonucleotide <400> 47 cctgctgtct ggtctgccgc cgtctca 27 <210> 48 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> R427 oligonucleotide <400> 48 acggtgtacc actggtgaga cggcggc 27 <210> 49 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> F439 oligonucleotide <400> 49 ccagtggtac accgttctgg acctgaaaga cg 32 <210> 50 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> R454 oligonucleotide <400> 50 cgcaggcaga agaaagcgtc tttcaggtcc aga 33 <210> 51 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> F471 oligonucleotide <400> 51 ctttcttctg cctgcgtctg cacccgacct 30 <210> 52 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> R487 oligonucleotide <400> 52 cgaacagcgg ctgagaggtc gggtgcaga 29 <210> 53 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> F501 oligonucleotide <400> 53 ctcagccgct gttcgctttc gaatggcgtg a 31 <210> 54 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> R516 oligonucleotide <400> 54 agatacccat ttccgggtca cgccattcga aag 33 <210> 55 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> F532 oligonucleotide <400> 55 cccggaaatg ggtatctctg gtcagctgac ct 32 <210> 56 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> R549 oligonucleotide <400> 56 ggcagacggg tccaggtcag ctgaccag 28 <210> 57 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> F564 oligonucleotide <400> 57 ggacccgtct gccgcagggt ttcaaaaact c 31 <210> 58 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> R577 oligonucleotide <400> 58 cgaacagggt cggagagttt ttgaaaccct gc 32 <210> 59 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> F595 oligonucleotide <400> 59 tccgaccctg ttcgacgaag ctctgcacc 29 <210> 60 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R609 oligonucleotide <400> 60 agtcagccag gtcacggtgc agagcttcgt 30 <210> 61 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> F624 oligonucleotide <400> 61 gtgacctggc tgacttccgt atccagcacc 30 <210> 62 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R639 oligonucleotide <400> 62 gcaggatcag gtccgggtgc tggatacgga 30 <210> 63 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> F654 oligonucleotide <400> 63 cggacctgat cctgctgcag tacgttgacg a 31 <210> 64 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> R669 oligonucleotide <400> 64 agccagcagc aggtcgtcaa cgtactgca 29 <210> 65 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> F685 oligonucleotide <400> 65 cctgctgctg gctgctacct ctgaactgga 30 <210> 66 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R698 oligonucleotide <400> 66 accctgctgg cagtccagtt cagaggtagc 30 <210> 67 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> F715 oligonucleotide <400> 67 ctgccagcag ggtacccgtg ctctgc 26 <210> 68 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> R728 oligonucleotide <400> 68 tacccagggt ctgcagcaga gcacgggt 28 <210> 69 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> F741 oligonucleotide <400> 69 tgcagaccct gggtaacctg ggttaccgtg 30 <210> 70 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> R756 oligonucleotide <400> 70 ctgagctttt ttagcagaag cacggtaacc caggt 35 <210> 71 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> F771 oligonucleotide <400> 71 cttctgctaa aaaagctcag atctgccaga aacaggt 37 <210> 72 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> R791 oligonucleotide <400> 72 gcaggtaacc caggtattta acctgtttct ggcagat 37 <210> 73 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> F808 oligonucleotide <400> 73 taaatacctg ggttacctgc tgaaagaagg tcagcgt 37 <210> 74 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> R828 oligonucleotide <400> 74 gcttcggtca gccaacgctg accttctttc a 31 <210> 75 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> F845 oligonucleotide <400> 75 tggctgaccg aagctcgtaa agaaaccgtt atgg 34 <210> 76 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> R859 oligonucleotide <400> 76 ggggtcggct gacccataac ggtttcttta cga 33 <210> 77 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> F879 oligonucleotide <400> 77 gtcagccgac cccgaaaacc ccgcgtc 27 <210> 78 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> R892 oligonucleotide <400> 78 ggaattcacg cagctgacgc ggggttttc 29 <210> 79 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> F906 oligonucleotide <400> 79 agctgcgtga attcctgggt accgctggt 29 <210> 80 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> R921 oligonucleotide <400> 80 ccacagacgg cagaaaccag cggtaccca 29 <210> 81 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> F935 oligonucleotide <400> 81 ttctgccgtc tgtggatacc gggtttcgct 30 <210> 82 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R950 oligonucleotide <400> 82 cggagcagcc atttcagcga aacccggtat 30 <210> 83 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> F965 oligonucleotide <400> 83 gaaatggctg ctccgctgta cccgctgacc 30 <210> 84 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> R980 oligonucleotide <400> 84 acagggtacc ggttttggtc agcgggtaca g 31 <210> 85 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> F995 oligonucleotide <400> 85 aaaaccggta ccctgttcaa ctggggtccg 30 <210> 86 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> R1011 oligonucleotide <400> 86 cgctttctgc tggtccggac cccagttga 29 <210> 87 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> F1025 oligonucleotide <400> 87 gaccagcaga aagcgtacca ggaaatcaaa cagg 34 <210> 88 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> R1040 oligonucleotide <400> 88 agcggtcagc agagcctgtt tgatttcctg gta 33 <210> 89 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> F1059 oligonucleotide <400> 89 ctctgctgac cgctccggct ctgggtc 27 <210> 90 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> R1073 oligonucleotide <400> 90 ggtcaggtcc ggcagaccca gagccgg 27 <210> 91 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> F1086 oligonucleotide <400> 91 tgccggacct gaccaaaccg ttcgaactgt t 31 <210> 92 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> R1100 oligonucleotide <400> 92 cctgtttttc gtcaacgaac agttcgaacg gttt 34 <210> 93 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> F1117 oligonucleotide <400> 93 cgttgacgaa aaacagggtt acgctaaagg tgttct 36 <210> 94 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> R1134 oligonucleotide <400> 94 acccagtttc tgggtcagaa cacctttagc gtaac 35 <210> 95 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> F1153 oligonucleotide <400> 95 gacccagaaa ctgggtccgt ggcgtcgt 28 <210> 96 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> R1169 oligonucleotide <400> 96 caggtaagca accggacgac gccacgg 27 <210> 97 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> F1181 oligonucleotide <400> 97 ccggttgctt acctgtctaa aaaactggac ccg 33 <210> 98 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> R1196 oligonucleotide <400> 98 ccaaccagca gcaaccgggt ccagtttttt aga 33 <210> 99 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> F1214 oligonucleotide <400> 99 gttgctgctg gttggccgcc gtgcctg 27 <210> 100 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> R1229 oligonucleotide <400> 100 atagcagcaa ccatacgcag gcacggcgg 29 <210> 101 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> F1241 oligonucleotide <400> 101 cgtatggttg ctgctatcgc tgttctgacc aaa 33 <210> 102 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> R1258 oligonucleotide <400> 102 gtcagtttac cagcgtcttt ggtcagaaca gcg 33 <210> 103 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> F1274 oligonucleotide <400> 103 gacgctggta aactgaccat gggtcagccg c 31 <210> 104 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R1291 oligonucleotide <400> 104 ggagccagga taaccagcgg ctgacccatg 30 <210> 105 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> F1305 oligonucleotide <400> 105 tggttatcct ggctccgcac gctgttgaag c 31 <210> 106 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> R1321 oligonucleotide <400> 106 gcggctgttt aaccagagct tcaacagcgt gc 32 <210> 107 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> F1336 oligonucleotide <400> 107 tctggttaaa cagccgccgg accgttggct 30 <210> 108 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> R1353 oligonucleotide <400> 108 gtcatacgag cgttagacag ccaacggtcc g 31 <210> 109 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> F1366 oligonucleotide <400> 109 gtctaacgct cgtatgaccc actaccaggc tctg 34 <210> 110 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R1384 oligonucleotide <400> 110 tcggtgtcca gcagcagagc ctggtagtgg 30 <210> 111 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> F1400 oligonucleotide <400> 111 ctgctggaca ccgaccgtgt tcagttcgg 29 <210> 112 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R1414 oligonucleotide <400> 112 agagcaacaa ccggaccgaa ctgaacacgg 30 <210> 113 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> F1429 oligonucleotide <400> 113 tccggttgtt gctctgaacc cggctaccc 29 <210> 114 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> R1444 oligonucleotide <400> 114 gcagcggcag cagggtagcc gggttc 26 <210> 115 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> F1458 oligonucleotide <400> 115 tgctgccgct gccggaagaa ggtctgca 28 <210> 116 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R1470 oligonucleotide <400> 116 ccaggcagtt gtgctgcaga ccttcttccg 30 <210> 117 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> F1486 oligonucleotide <400> 117 gcacaactgc ctggacatcc tggctgaagc 30 <210> 118 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R1500 oligonucleotide <400> 118 gacgggtacc gtgagcttca gccaggatgt 30 <210> 119 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> F1516 oligonucleotide <400> 119 tcacggtacc cgtccggacc tgaccgac 28 <210> 120 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> R1530 oligonucleotide <400> 120 cggcagcggc tggtcggtca ggtccg 26 <210> 121 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> F1544 oligonucleotide <400> 121 cagccgctgc cggacgctga ccacacc 27 <210> 122 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> R1556 oligonucleotide <400> 122 ccgtcggtgt accaggtgtg gtcagcgtc 29 <210> 123 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> F1571 oligonucleotide <400> 123 tggtacaccg acggttcttc tctgctgcag g 31 <210> 124 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> R1585 oligonucleotide <400> 124 gctttacgct gaccttcctg cagcagagaa gaa 33 <210> 125 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> F1602 oligonucleotide <400> 125 aaggtcagcg taaagctggt gctgctgtta cca 33 <210> 126 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> R1618 oligonucleotide <400> 126 cagataactt cggtttcggt ggtaacagca gcacca 36 <210> 127 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> F1635 oligonucleotide <400> 127 ccgaaaccga agttatctgg gctaaagctc tgccg 35 <210> 128 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> R1654 oligonucleotide <400> 128 gagcagaggt accagccggc agagctttag cc 32 <210> 129 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> F1670 oligonucleotide <400> 129 gctggtacct ctgctcagcg tgctgaactg at 32 <210> 130 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> R1686 oligonucleotide <400> 130 gcctgggtca gagcgatcag ttcagcacgc t 31 <210> 131 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> F1702 oligonucleotide <400> 131 cgctctgacc caggctctga aaatggctga aggta 35 <210> 132 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> R1717 oligonucleotide <400> 132 ggtgtaaacg ttcagttttt taccttcagc cattttcaga 40 <210> 133 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> F1737 oligonucleotide <400> 133 aaaaactgaa cgtttacacc gactctcgtt acgctttc 38 <210> 134 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> R1757 oligonucleotide <400> 134 ggatgtgagc ggtagcgaaa gcgtaacgag agtc 34 <210> 135 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> F1775 oligonucleotide <400> 135 gctaccgctc acatccacgg tgaaatctac cgt 33 <210> 136 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> R1791 oligonucleotide <400> 136 cagcagacca cgacgacggt agatttcacc gt 32 <210> 137 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> F1808 oligonucleotide <400> 137 cgtcgtggtc tgctgacctc tgaaggtaaa gaaat 35 <210> 138 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> R1823 oligonucleotide <400> 138 ggatttcgtc tttgtttttg atttctttac cttcagaggt 40 <210> 139 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> F1843 oligonucleotide <400> 139 caaaaacaaa gacgaaatcc tggctctgct gaaagct 37 <210> 140 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> R1863 oligonucleotide <400> 140 gtttcggcag gaacagagct ttcagcagag cca 33 <210> 141 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> F1880 oligonucleotide <400> 141 ctgttcctgc cgaaacgtct gtctatcatc cactgc 36 <210> 142 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> R1896 oligonucleotide <400> 142 ttctggtgac ccgggcagtg gatgatagac agac 34 <210> 143 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> F1916 oligonucleotide <400> 143 ccgggtcacc agaaaggtca ctctgctgaa g 31 <210> 144 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> R1930 oligonucleotide <400> 144 catacggtta ccacgagctt cagcagagtg acct 34 <210> 145 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> F1947 oligonucleotide <400> 145 ctcgtggtaa ccgtatggct gaccaggctg c 31 <210> 146 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> R1964 oligonucleotide <400> 146 ggtgatagca gctttacgag cagcctggtc agc 33 <210> 147 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> F1978 oligonucleotide <400> 147 tcgtaaagct gctatcaccg aaaccccgga cacc 34 <210> 148 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> R1997 oligonucleotide <400> 148 ttagatcagc agggtagagg tgtccggggt ttc 33 <210> 149 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> F2012 oligonucleotide <400> 149 tctaccctgc tgatctaa 18 <210> 150 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> forward primer for the synthesis of K295Q mutant <400> 150 gtcagccgac cccgcaaacc ccgcgtc 27 <210> 151 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for the synthesis of K295Q mutant <400> 151 ggaattcacg cagctgacgc ggggtttgc 29 <210> 152 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer for the amplification of synthesized gene <400> 152 ctgaacatcg aagacgaa 18 <210> 153 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for the amplification of synthesized gene <400> 153 ttagatcagc agggtaga 18 <210> 154 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> forward primer for site-directed mutagenesis of Q63L <400> 154 caaactttac ccgatgtctc aggaagctcg 30 <210> 155 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for site-directed mutagenesis of Q63L <400> 155 atagaaaccg gggtagaggt agctttcagc 30 <210> 156 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> forward primer for site-directed mutagenesis of K264L <400> 156 ccagcttcag gttaaatacc tgggttacct g 31 <210> 157 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for site-directed mutagenesis of K264L <400> 157 cagatctgag cttttttagc agaagcacgg 30 <210> 158 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> forward primer for site-directed mutagenesis of T306L <400> 158 ggtcttgctg gtttctgccg tctgtg 26 <210> 159 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for site-directed mutagenesis of T306L <400> 159 caggaattca cgcagctgac gcgg 24 <210> 160 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> forward primer for site-directed mutagenesis of E346M <400> 160 cagatgatca aacaggctct gctgaccg 28 <210> 161 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for site-directed mutagenesis of E346M <400> 161 gtacgctttc tgctggtccg gacc 24 <210> 162 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> forward primer for site-directed mutagenesis of P408E <400> 162 ccggaatgcc tgcgtatggt tgctg 25 <210> 163 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for site-directed mutagenesis of P408E <400> 163 ccaaccagca gcaaccgggt cc 22 <210> 164 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> forward primer for site-directed mutagenesis of H438Y <400> 164 cgtacgctgt tgaagctctg gttaaacagc 30 <210> 165 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for site-directed mutagenesis of H438Y <400> 165 gagccaggat aaccagcggc tgacc 25 <210> 166 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> forward primer for site-directed mutagenesis of N454F <400> 166 ctgtctttcg ctcgtatgac ccactaccag 30 <210> 167 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for site-directed mutagenesis of N454F <400> 167 ccaacggtcc ggcggctgtt taac 24 <210> 168 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> primer for sense strand of Q63L, K264L, T306L and E346M mutant genes <400> 168 gcgcgccata tgctgaacat cgaagacgaa caccgtctgc acgaaac 47 <210> 169 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> primer for antisense strand of Q63L, K264L, T306L and E346M mutant genes <400> 169 gcgcgcgcgg ccgcttagat cagcagggta gaggtgtccg gggtttc 47 <210> 170 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> primer for sense strand of K295Q, P408E, H438Y and N454F mutant genes <400> 170 gcgcgccata tgctgaacat cgaagacgaa caccgtctgc acgaaac 47 <210> 171 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> primer for antisense strand of K295Q, P408E, H438Y and N454F mutant genes <400> 171 gcgcgcgcgg ccgcgatcag cagggtagag gtgtccgggg tttc 44 <210> 172 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for sense strand of T7 promoter <400> 172 taatacgact cactataggg 20 <210> 173 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer for antisense strand of T7 promoter <400> 173 gctagttatt gctcagcgg 19 <210> 174 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer for sense strand of GAPDH gene <400> 174 gaaggtgaag gtcggagtca acg 23 <210> 175 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer for antisense strand of GAPDH gene <400> 175 agtccttcca cgataccaaa gttg 24 <110> BIONEER CORPORATION <120> Reverse Transcriptase Having Improved Thermostability <130> 2013-dpa-0708 <160> 175 <170> KopatentIn 2.0 <210> 1 <211> 672 <212> PRT <213> Moloney Murine Leukemia Virus <400> 1 Met Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu 1 5 10 15 Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala 20 25 30 Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu 35 40 45 Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr 50 55 60 Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg 65 70 75 80 Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr 85 90 95 Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val 100 105 110 Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr 115 120 125 Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln 130 135 140 Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu 145 150 155 160 His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu 165 170 175 Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe 180 185 190 Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala 195 200 205 Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp 210 215 220 Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr 225 230 235 240 Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala 245 250 255 Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu 260 265 270 Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val 275 280 285 Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu 290 295 300 Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met 305 310 315 320 Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp 325 330 335 Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu 340 345 350 Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu 355 360 365 Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys 370 375 380 Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp 385 390 395 400 Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile 405 410 415 Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu 420 425 430 Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro 435 440 445 Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu 450 455 460 Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro 465 470 475 480 Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu 485 490 495 Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln 500 505 510 Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asp Gly Ser Ser Leu 515 520 525 Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr 530 535 540 Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg 545 550 555 560 Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys 565 570 575 Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His 580 585 590 Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly 595 600 605 Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu 610 615 620 Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys 625 630 635 640 Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala 645 650 655 Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ile 660 665 670 <210> 2 <211> 672 <212> PRT <213> Moloney Murine Leukemia Virus <220> <221> MUTAGEN <222> (63) <223> mutant reverse transcriptase having leucine residue instead of glutamine residue at 63 position of SEQ. ID. No: 1 <400> 2 Met Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu 1 5 10 15 Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala 20 25 30 Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu 35 40 45 Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Leu Tyr 50 55 60 Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg 65 70 75 80 Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr 85 90 95 Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val 100 105 110 Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr 115 120 125 Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln 130 135 140 Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu 145 150 155 160 His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu 165 170 175 Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe 180 185 190 Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala 195 200 205 Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp 210 215 220 Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr 225 230 235 240 Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala 245 250 255 Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu 260 265 270 Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val 275 280 285 Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu 290 295 300 Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met 305 310 315 320 Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp 325 330 335 Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu 340 345 350 Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu 355 360 365 Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys 370 375 380 Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp 385 390 395 400 Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile 405 410 415 Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu 420 425 430 Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro 435 440 445 Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu 450 455 460 Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro 465 470 475 480 Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu 485 490 495 Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln 500 505 510 Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asp Gly Ser Ser Leu 515 520 525 Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr 530 535 540 Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg 545 550 555 560 Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys 565 570 575 Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His 580 585 590 Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly 595 600 605 Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu 610 615 620 Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys 625 630 635 640 Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala 645 650 655 Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ile 660 665 670 <210> 3 <211> 672 <212> PRT <213> Moloney Murine Leukemia Virus <220> <221> MUTAGEN <222> (264) <223> mutant reverse transcriptase having leucine residue instead of lysine residue at 264 position of SEQ. ID. No: 1 <400> 3 Met Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu 1 5 10 15 Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala 20 25 30 Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu 35 40 45 Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr 50 55 60 Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg 65 70 75 80 Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr 85 90 95 Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val 100 105 110 Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr 115 120 125 Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln 130 135 140 Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu 145 150 155 160 His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu 165 170 175 Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe 180 185 190 Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala 195 200 205 Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp 210 215 220 Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr 225 230 235 240 Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala 245 250 255 Lys Lys Ala Gln Ile Cys Gln Leu Gln Val Lys Tyr Leu Gly Tyr Leu 260 265 270 Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val 275 280 285 Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu 290 295 300 Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met 305 310 315 320 Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp 325 330 335 Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu 340 345 350 Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu 355 360 365 Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys 370 375 380 Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp 385 390 395 400 Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile 405 410 415 Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu 420 425 430 Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro 435 440 445 Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu 450 455 460 Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro 465 470 475 480 Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu 485 490 495 Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln 500 505 510 Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asp Gly Ser Ser Leu 515 520 525 Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr 530 535 540 Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg 545 550 555 560 Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys 565 570 575 Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His 580 585 590 Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly 595 600 605 Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu 610 615 620 Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys 625 630 635 640 Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala 645 650 655 Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ile 660 665 670 <210> 4 <211> 672 <212> PRT <213> Moloney Murine Leukemia Virus <220> <221> MUTAGEN <222> (295) <223> mutant reverse transcriptase having glutamine residue instead of lysine residue at 295 position of SEQ. ID. No: 1 <400> 4 Met Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu 1 5 10 15 Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala 20 25 30 Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu 35 40 45 Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr 50 55 60 Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg 65 70 75 80 Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr 85 90 95 Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val 100 105 110 Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr 115 120 125 Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln 130 135 140 Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu 145 150 155 160 His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu 165 170 175 Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe 180 185 190 Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala 195 200 205 Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp 210 215 220 Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr 225 230 235 240 Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala 245 250 255 Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu 260 265 270 Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val 275 280 285 Met Gly Gln Pro Thr Pro Gln Thr Pro Arg Gln Leu Arg Glu Phe Leu 290 295 300 Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met 305 310 315 320 Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp 325 330 335 Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu 340 345 350 Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu 355 360 365 Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys 370 375 380 Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp 385 390 395 400 Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile 405 410 415 Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu 420 425 430 Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro 435 440 445 Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu 450 455 460 Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro 465 470 475 480 Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu 485 490 495 Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln 500 505 510 Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asp Gly Ser Ser Leu 515 520 525 Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr 530 535 540 Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg 545 550 555 560 Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys 565 570 575 Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His 580 585 590 Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly 595 600 605 Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu 610 615 620 Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys 625 630 635 640 Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala 645 650 655 Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ile 660 665 670 <210> 5 <211> 672 <212> PRT <213> Moloney Murine Leukemia Virus <220> <221> MUTAGEN <222> (306) <223> mutant reverse transcriptase having leucine residue instead of threonine residue at 306 position of SEQ. ID. No: 1 <400> 5 Met Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu 1 5 10 15 Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala 20 25 30 Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu 35 40 45 Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr 50 55 60 Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg 65 70 75 80 Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr 85 90 95 Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val 100 105 110 Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr 115 120 125 Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln 130 135 140 Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu 145 150 155 160 His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu 165 170 175 Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe 180 185 190 Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala 195 200 205 Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp 210 215 220 Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr 225 230 235 240 Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala 245 250 255 Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu 260 265 270 Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val 275 280 285 Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu 290 295 300 Gly Leu Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met 305 310 315 320 Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp 325 330 335 Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu 340 345 350 Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu 355 360 365 Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys 370 375 380 Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp 385 390 395 400 Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile 405 410 415 Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu 420 425 430 Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro 435 440 445 Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu 450 455 460 Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro 465 470 475 480 Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu 485 490 495 Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln 500 505 510 Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asp Gly Ser Ser Leu 515 520 525 Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr 530 535 540 Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg 545 550 555 560 Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys 565 570 575 Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His 580 585 590 Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly 595 600 605 Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu 610 615 620 Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys 625 630 635 640 Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala 645 650 655 Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ile 660 665 670 <210> 6 <211> 672 <212> PRT <213> Moloney Murine Leukemia Virus <220> <221> MUTAGEN <222> (346) <223> mutant reverse transcriptase having methionine residue instead of glutamic acid residue at 346 position of SEQ. ID. No: 1 <400> 6 Met Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu 1 5 10 15 Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala 20 25 30 Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu 35 40 45 Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr 50 55 60 Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg 65 70 75 80 Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr 85 90 95 Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val 100 105 110 Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr 115 120 125 Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln 130 135 140 Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu 145 150 155 160 His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu 165 170 175 Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe 180 185 190 Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala 195 200 205 Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp 210 215 220 Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr 225 230 235 240 Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala 245 250 255 Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu 260 265 270 Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val 275 280 285 Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu 290 295 300 Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met 305 310 315 320 Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp 325 330 335 Gly Pro Asp Gln Gln Lys Ala Tyr Gln Met Ile Lys Gln Ala Leu Leu 340 345 350 Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu 355 360 365 Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys 370 375 380 Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp 385 390 395 400 Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile 405 410 415 Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu 420 425 430 Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro 435 440 445 Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu 450 455 460 Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro 465 470 475 480 Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu 485 490 495 Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln 500 505 510 Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asp Gly Ser Ser Leu 515 520 525 Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr 530 535 540 Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg 545 550 555 560 Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys 565 570 575 Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His 580 585 590 Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly 595 600 605 Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu 610 615 620 Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys 625 630 635 640 Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala 645 650 655 Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ile 660 665 670 <210> 7 <211> 672 <212> PRT <213> Moloney Murine Leukemia Virus <220> <221> MUTAGEN <222> (408) <223> mutant reverse transcriptase having glutamic acid residue instead of proline residue at 408 position of SEQ. ID. No: 1 <400> 7 Met Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu 1 5 10 15 Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala 20 25 30 Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu 35 40 45 Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr 50 55 60 Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg 65 70 75 80 Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr 85 90 95 Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val 100 105 110 Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr 115 120 125 Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln 130 135 140 Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu 145 150 155 160 His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu 165 170 175 Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe 180 185 190 Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala 195 200 205 Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp 210 215 220 Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr 225 230 235 240 Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala 245 250 255 Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu 260 265 270 Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val 275 280 285 Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu 290 295 300 Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met 305 310 315 320 Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp 325 330 335 Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu 340 345 350 Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu 355 360 365 Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys 370 375 380 Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp 385 390 395 400 Pro Val Ala Ala Gly Trp Pro Glu Cys Leu Arg Met Val Ala Ala Ile 405 410 415 Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu 420 425 430 Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro 435 440 445 Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu 450 455 460 Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro 465 470 475 480 Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu 485 490 495 Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln 500 505 510 Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asp Gly Ser Ser Leu 515 520 525 Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr 530 535 540 Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg 545 550 555 560 Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys 565 570 575 Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His 580 585 590 Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly 595 600 605 Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu 610 615 620 Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys 625 630 635 640 Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala 645 650 655 Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ile 660 665 670 <210> 8 <211> 672 <212> PRT <213> Moloney Murine Leukemia Virus <220> <221> MUTAGEN <222> (438) <223> mutant reverse transcriptase having tyrosine residue instead of histidine residue at 438 position of SEQ. ID. No: 1 <400> 8 Met Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu 1 5 10 15 Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala 20 25 30 Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu 35 40 45 Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr 50 55 60 Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg 65 70 75 80 Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr 85 90 95 Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val 100 105 110 Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr 115 120 125 Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln 130 135 140 Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu 145 150 155 160 His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu 165 170 175 Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe 180 185 190 Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala 195 200 205 Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp 210 215 220 Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr 225 230 235 240 Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala 245 250 255 Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu 260 265 270 Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val 275 280 285 Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu 290 295 300 Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met 305 310 315 320 Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp 325 330 335 Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu 340 345 350 Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu 355 360 365 Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys 370 375 380 Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp 385 390 395 400 Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile 405 410 415 Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu 420 425 430 Val Ile Leu Ala Pro Tyr Ala Val Glu Ala Leu Val Lys Gln Pro Pro 435 440 445 Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu 450 455 460 Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro 465 470 475 480 Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu 485 490 495 Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln 500 505 510 Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asp Gly Ser Ser Leu 515 520 525 Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr 530 535 540 Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg 545 550 555 560 Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys 565 570 575 Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His 580 585 590 Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly 595 600 605 Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu 610 615 620 Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys 625 630 635 640 Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala 645 650 655 Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ile 660 665 670 <210> 9 <211> 672 <212> PRT <213> Moloney Murine Leukemia Virus <220> <221> MUTAGEN <222> (454) <223> mutant reverse transcriptase having phenylalanine residue instead of asparagine residue at 454 position of SEQ. ID. No: 1 <400> 9 Met Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu 1 5 10 15 Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala 20 25 30 Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu 35 40 45 Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr 50 55 60 Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg 65 70 75 80 Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr 85 90 95 Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val 100 105 110 Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr 115 120 125 Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln 130 135 140 Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu 145 150 155 160 His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu 165 170 175 Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe 180 185 190 Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala 195 200 205 Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp 210 215 220 Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr 225 230 235 240 Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala 245 250 255 Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu 260 265 270 Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val 275 280 285 Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu 290 295 300 Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met 305 310 315 320 Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp 325 330 335 Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu 340 345 350 Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu 355 360 365 Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys 370 375 380 Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp 385 390 395 400 Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile 405 410 415 Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu 420 425 430 Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro 435 440 445 Asp Arg Trp Leu Ser Phe Ala Arg Met Thr His Tyr Gln Ala Leu Leu 450 455 460 Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro 465 470 475 480 Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu 485 490 495 Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln 500 505 510 Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asp Gly Ser Ser Leu 515 520 525 Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr 530 535 540 Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg 545 550 555 560 Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys 565 570 575 Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr Ala His 580 585 590 Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly 595 600 605 Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu 610 615 620 Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys 625 630 635 640 Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala 645 650 655 Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ile 660 665 670 <210> 10 <211> 2016 <212> DNA <213> Moloney Murine Leukemia Virus <220> <221> mat_peptide <222> (1)..(2016) <223> nucleotide sequence coding for the amino acid sequence of SEQ. ID. No: 1 <400> 10 atgctaaata tagaagatga gcatcggcta catgagacct caaaagagcc agatgtttct 60 ctagggtcca catggctgtc tgattttcct caggcctggg cggaaaccgg gggcatggga 120 ctggcagttc gccaagctcc tctgatcata cctctgaaag caacctctac ccccgtgtcc 180 ataaaacaat accccatgtc acaagaagcc agactgggga tcaagcccca catacagaga 240 ctgttggacc agggaatact ggtaccctgc cagtccccct ggaacacgcc cctgctaccc 300 gttaagaaac cagggactaa tgattatagg cctgtccagg atctgagaga agtcaacaag 360 cgggtggaag acatccaccc caccgtgccc aacccttaca acctcttgag cgggctccca 420 ccgtcccacc agtggtacac tgtgcttgat ttaaaggatg cctttttctg cctgagactc 480 caccccacca gtcagcctct cttcgccttt gagtggagag atccagagat gggaatctca 540 ggacaattga cctggaccag actcccacag ggtttcaaaa acagtcccac cctgtttgat 600 gaggcactgc acagagacct agcagacttc cggatccagc acccagactt gatcctgcta 660 cagtacgtgg atgacttact gctggccgcc acttctgagc tagactgcca acaaggtact 720 cgggccctgt tacaaaccct agggaacctc gggtatcggg cctcggccaa gaaagcccaa 780 atttgccaga aacaggtcaa gtatctgggg tatcttctaa aagagggtca gagatggctg 840 actgaggcca gaaaagagac tgtgatgggg cagcctactc cgaagacccc tcgacaacta 900 agggagttcc tagggacggc aggcttctgt cgcctctgga tccctgggtt tgcagaaatg 960 gcagccccct tgtaccctct caccaaaacg gggactctgt ttaattgggg cccagaccaa 1020 caaaaggcct atcaagaaat caagcaagct cttctaactg ccccagccct ggggttgcca 1080 gatttgacta agccctttga actctttgtc gacgagaagc agggctacgc caaaggtgtc 1140 ctaacgcaaa aactgggacc ttggcgtcgg ccggtggcct acctgtccaa aaagctagac 1200 ccagtagcag ctgggtggcc cccttgccta cggatggtag cagccattgc cgtactgaca 1260 aaggatgcag gcaagctaac catgggacag ccactagtca ttctggcccc ccatgcagta 1320 gaggcactag tcaaacaacc ccccgaccgc tggctttcca acgcccggat gactcactat 1380 caggccttgc ttttggacac ggaccgggtc cagttcggac cggtggtagc cctgaacccg 1440 gctacgctgc tcccactgcc tgaggaaggg ctgcaacaca actgccttga tatcctggcc 1500 gaagcccacg gaacccgacc cgacctaacg gaccagccgc tcccagacgc cgaccacacc 1560 tggtacacgg atggaagcag tctcttacaa gagggacagc gtaaggcggg agctgcggtg 1620 accaccgaga ccgaggtaat ctgggctaaa gccctgccag ccgggacatc cgctcagcgg 1680 gctgaactga tagcactcac ccaggcccta aagatggcag aaggtaagaa gctaaatgtt 1740 tatactgata gccgttatgc ttttgctact gcccatatcc atggagaaat atacagaagg 1800 cgtgggttgc tcacatcaga aggcaaagag atcaaaaata aagacgagat cttggcccta 1860 ctaaaagccc tctttctgcc caaaagactt agcataatcc attgtccagg acatcaaaag 1920 ggacacagcg ccgaggctag aggcaaccgg atggctgacc aagcggcccg aaaggcagcc 1980 atcacagaga ctccagacac ctctaccctc ctcata 2016 <210> 11 <211> 2016 <212> DNA <213> Moloney Murine Leukemia Virus <220> <221> mat_peptide <222> (1)..(2016) <223> nucleotide sequence coding for the amino acid sequence of SEQ. ID. No: 2 <220> <221> mutation <222> (187)..(189) <223> substitution of ctt instead of caa at 187-189 positions of SEQ. ID. No: 10 <400> 11 atgctaaata tagaagatga gcatcggcta catgagacct caaaagagcc agatgtttct 60 ctagggtcca catggctgtc tgattttcct caggcctggg cggaaaccgg gggcatggga 120 ctggcagttc gccaagctcc tctgatcata cctctgaaag caacctctac ccccgtgtcc 180 ataaaacttt accccatgtc acaagaagcc agactgggga tcaagcccca catacagaga 240 ctgttggacc agggaatact ggtaccctgc cagtccccct ggaacacgcc cctgctaccc 300 gttaagaaac cagggactaa tgattatagg cctgtccagg atctgagaga agtcaacaag 360 cgggtggaag acatccaccc caccgtgccc aacccttaca acctcttgag cgggctccca 420 ccgtcccacc agtggtacac tgtgcttgat ttaaaggatg cctttttctg cctgagactc 480 caccccacca gtcagcctct cttcgccttt gagtggagag atccagagat gggaatctca 540 ggacaattga cctggaccag actcccacag ggtttcaaaa acagtcccac cctgtttgat 600 gaggcactgc acagagacct agcagacttc cggatccagc acccagactt gatcctgcta 660 cagtacgtgg atgacttact gctggccgcc acttctgagc tagactgcca acaaggtact 720 cgggccctgt tacaaaccct agggaacctc gggtatcggg cctcggccaa gaaagcccaa 780 atttgccaga aacaggtcaa gtatctgggg tatcttctaa aagagggtca gagatggctg 840 actgaggcca gaaaagagac tgtgatgggg cagcctactc cgaagacccc tcgacaacta 900 agggagttcc tagggacggc aggcttctgt cgcctctgga tccctgggtt tgcagaaatg 960 gcagccccct tgtaccctct caccaaaacg gggactctgt ttaattgggg cccagaccaa 1020 caaaaggcct atcaagaaat caagcaagct cttctaactg ccccagccct ggggttgcca 1080 gatttgacta agccctttga actctttgtc gacgagaagc agggctacgc caaaggtgtc 1140 ctaacgcaaa aactgggacc ttggcgtcgg ccggtggcct acctgtccaa aaagctagac 1200 ccagtagcag ctgggtggcc cccttgccta cggatggtag cagccattgc cgtactgaca 1260 aaggatgcag gcaagctaac catgggacag ccactagtca ttctggcccc ccatgcagta 1320 gaggcactag tcaaacaacc ccccgaccgc tggctttcca acgcccggat gactcactat 1380 caggccttgc ttttggacac ggaccgggtc cagttcggac cggtggtagc cctgaacccg 1440 gctacgctgc tcccactgcc tgaggaaggg ctgcaacaca actgccttga tatcctggcc 1500 gaagcccacg gaacccgacc cgacctaacg gaccagccgc tcccagacgc cgaccacacc 1560 tggtacacgg atggaagcag tctcttacaa gagggacagc gtaaggcggg agctgcggtg 1620 accaccgaga ccgaggtaat ctgggctaaa gccctgccag ccgggacatc cgctcagcgg 1680 gctgaactga tagcactcac ccaggcccta aagatggcag aaggtaagaa gctaaatgtt 1740 tatactgata gccgttatgc ttttgctact gcccatatcc atggagaaat atacagaagg 1800 cgtgggttgc tcacatcaga aggcaaagag atcaaaaata aagacgagat cttggcccta 1860 ctaaaagccc tctttctgcc caaaagactt agcataatcc attgtccagg acatcaaaag 1920 ggacacagcg ccgaggctag aggcaaccgg atggctgacc aagcggcccg aaaggcagcc 1980 atcacagaga ctccagacac ctctaccctc ctcata 2016 <210> 12 <211> 2016 <212> DNA <213> Moloney Murine Leukemia Virus <220> <221> mat_peptide <222> (1)..(2016) <223> nucleotide sequence coding for the amino acid sequence of SEQ. ID. No: 3 <220> <221> mutation <222> (790)..(792) <223> substitution of ctt instead of aaa at 790-792 positions of SEQ. ID. No: 10 <400> 12 atgctaaata tagaagatga gcatcggcta catgagacct caaaagagcc agatgtttct 60 ctagggtcca catggctgtc tgattttcct caggcctggg cggaaaccgg gggcatggga 120 ctggcagttc gccaagctcc tctgatcata cctctgaaag caacctctac ccccgtgtcc 180 ataaaacaat accccatgtc acaagaagcc agactgggga tcaagcccca catacagaga 240 ctgttggacc agggaatact ggtaccctgc cagtccccct ggaacacgcc cctgctaccc 300 gttaagaaac cagggactaa tgattatagg cctgtccagg atctgagaga agtcaacaag 360 cgggtggaag acatccaccc caccgtgccc aacccttaca acctcttgag cgggctccca 420 ccgtcccacc agtggtacac tgtgcttgat ttaaaggatg cctttttctg cctgagactc 480 caccccacca gtcagcctct cttcgccttt gagtggagag atccagagat gggaatctca 540 ggacaattga cctggaccag actcccacag ggtttcaaaa acagtcccac cctgtttgat 600 gaggcactgc acagagacct agcagacttc cggatccagc acccagactt gatcctgcta 660 cagtacgtgg atgacttact gctggccgcc acttctgagc tagactgcca acaaggtact 720 cgggccctgt tacaaaccct agggaacctc gggtatcggg cctcggccaa gaaagcccaa 780 atttgccagc ttcaggtcaa gtatctgggg tatcttctaa aagagggtca gagatggctg 840 actgaggcca gaaaagagac tgtgatgggg cagcctactc cgaagacccc tcgacaacta 900 agggagttcc tagggacggc aggcttctgt cgcctctgga tccctgggtt tgcagaaatg 960 gcagccccct tgtaccctct caccaaaacg gggactctgt ttaattgggg cccagaccaa 1020 caaaaggcct atcaagaaat caagcaagct cttctaactg ccccagccct ggggttgcca 1080 gatttgacta agccctttga actctttgtc gacgagaagc agggctacgc caaaggtgtc 1140 ctaacgcaaa aactgggacc ttggcgtcgg ccggtggcct acctgtccaa aaagctagac 1200 ccagtagcag ctgggtggcc cccttgccta cggatggtag cagccattgc cgtactgaca 1260 aaggatgcag gcaagctaac catgggacag ccactagtca ttctggcccc ccatgcagta 1320 gaggcactag tcaaacaacc ccccgaccgc tggctttcca acgcccggat gactcactat 1380 caggccttgc ttttggacac ggaccgggtc cagttcggac cggtggtagc cctgaacccg 1440 gctacgctgc tcccactgcc tgaggaaggg ctgcaacaca actgccttga tatcctggcc 1500 gaagcccacg gaacccgacc cgacctaacg gaccagccgc tcccagacgc cgaccacacc 1560 tggtacacgg atggaagcag tctcttacaa gagggacagc gtaaggcggg agctgcggtg 1620 accaccgaga ccgaggtaat ctgggctaaa gccctgccag ccgggacatc cgctcagcgg 1680 gctgaactga tagcactcac ccaggcccta aagatggcag aaggtaagaa gctaaatgtt 1740 tatactgata gccgttatgc ttttgctact gcccatatcc atggagaaat atacagaagg 1800 cgtgggttgc tcacatcaga aggcaaagag atcaaaaata aagacgagat cttggcccta 1860 ctaaaagccc tctttctgcc caaaagactt agcataatcc attgtccagg acatcaaaag 1920 ggacacagcg ccgaggctag aggcaaccgg atggctgacc aagcggcccg aaaggcagcc 1980 atcacagaga ctccagacac ctctaccctc ctcata 2016 <210> 13 <211> 2016 <212> DNA <213> Moloney Murine Leukemia Virus <220> <221> mat_peptide <222> (1)..(2016) <223> nucleotide sequence coding for the amino acid sequence of SEQ. ID. No: 4 <220> <221> mutation <222> (883)..(885) <223> substitution of caa instead of aag at 883-885 positions of SEQ. ID. No: 10 <400> 13 atgctaaata tagaagatga gcatcggcta catgagacct caaaagagcc agatgtttct 60 ctagggtcca catggctgtc tgattttcct caggcctggg cggaaaccgg gggcatggga 120 ctggcagttc gccaagctcc tctgatcata cctctgaaag caacctctac ccccgtgtcc 180 ataaaacaat accccatgtc acaagaagcc agactgggga tcaagcccca catacagaga 240 ctgttggacc agggaatact ggtaccctgc cagtccccct ggaacacgcc cctgctaccc 300 gttaagaaac cagggactaa tgattatagg cctgtccagg atctgagaga agtcaacaag 360 cgggtggaag acatccaccc caccgtgccc aacccttaca acctcttgag cgggctccca 420 ccgtcccacc agtggtacac tgtgcttgat ttaaaggatg cctttttctg cctgagactc 480 caccccacca gtcagcctct cttcgccttt gagtggagag atccagagat gggaatctca 540 ggacaattga cctggaccag actcccacag ggtttcaaaa acagtcccac cctgtttgat 600 gaggcactgc acagagacct agcagacttc cggatccagc acccagactt gatcctgcta 660 cagtacgtgg atgacttact gctggccgcc acttctgagc tagactgcca acaaggtact 720 cgggccctgt tacaaaccct agggaacctc gggtatcggg cctcggccaa gaaagcccaa 780 atttgccaga aacaggtcaa gtatctgggg tatcttctaa aagagggtca gagatggctg 840 actgaggcca gaaaagagac tgtgatgggg cagcctactc cgcaaacccc tcgacaacta 900 agggagttcc tagggacggc aggcttctgt cgcctctgga tccctgggtt tgcagaaatg 960 gcagccccct tgtaccctct caccaaaacg gggactctgt ttaattgggg cccagaccaa 1020 caaaaggcct atcaagaaat caagcaagct cttctaactg ccccagccct ggggttgcca 1080 gatttgacta agccctttga actctttgtc gacgagaagc agggctacgc caaaggtgtc 1140 ctaacgcaaa aactgggacc ttggcgtcgg ccggtggcct acctgtccaa aaagctagac 1200 ccagtagcag ctgggtggcc cccttgccta cggatggtag cagccattgc cgtactgaca 1260 aaggatgcag gcaagctaac catgggacag ccactagtca ttctggcccc ccatgcagta 1320 gaggcactag tcaaacaacc ccccgaccgc tggctttcca acgcccggat gactcactat 1380 caggccttgc ttttggacac ggaccgggtc cagttcggac cggtggtagc cctgaacccg 1440 gctacgctgc tcccactgcc tgaggaaggg ctgcaacaca actgccttga tatcctggcc 1500 gaagcccacg gaacccgacc cgacctaacg gaccagccgc tcccagacgc cgaccacacc 1560 tggtacacgg atggaagcag tctcttacaa gagggacagc gtaaggcggg agctgcggtg 1620 accaccgaga ccgaggtaat ctgggctaaa gccctgccag ccgggacatc cgctcagcgg 1680 gctgaactga tagcactcac ccaggcccta aagatggcag aaggtaagaa gctaaatgtt 1740 tatactgata gccgttatgc ttttgctact gcccatatcc atggagaaat atacagaagg 1800 cgtgggttgc tcacatcaga aggcaaagag atcaaaaata aagacgagat cttggcccta 1860 ctaaaagccc tctttctgcc caaaagactt agcataatcc attgtccagg acatcaaaag 1920 ggacacagcg ccgaggctag aggcaaccgg atggctgacc aagcggcccg aaaggcagcc 1980 atcacagaga ctccagacac ctctaccctc ctcata 2016 <210> 14 <211> 2016 <212> DNA <213> Moloney Murine Leukemia Virus <220> <221> mat_peptide <222> (1)..(2016) <223> nucleotide sequence coding for the amino acid sequence of SEQ. ID. No: 5 <220> <221> mutation <222> (916)..(918) <223> substitution of ctt instead of acg at 916-918 positions of SEQ. ID. No: 10 <400> 14 atgctaaata tagaagatga gcatcggcta catgagacct caaaagagcc agatgtttct 60 ctagggtcca catggctgtc tgattttcct caggcctggg cggaaaccgg gggcatggga 120 ctggcagttc gccaagctcc tctgatcata cctctgaaag caacctctac ccccgtgtcc 180 ataaaacaat accccatgtc acaagaagcc agactgggga tcaagcccca catacagaga 240 ctgttggacc agggaatact ggtaccctgc cagtccccct ggaacacgcc cctgctaccc 300 gttaagaaac cagggactaa tgattatagg cctgtccagg atctgagaga agtcaacaag 360 cgggtggaag acatccaccc caccgtgccc aacccttaca acctcttgag cgggctccca 420 ccgtcccacc agtggtacac tgtgcttgat ttaaaggatg cctttttctg cctgagactc 480 caccccacca gtcagcctct cttcgccttt gagtggagag atccagagat gggaatctca 540 ggacaattga cctggaccag actcccacag ggtttcaaaa acagtcccac cctgtttgat 600 gaggcactgc acagagacct agcagacttc cggatccagc acccagactt gatcctgcta 660 cagtacgtgg atgacttact gctggccgcc acttctgagc tagactgcca acaaggtact 720 cgggccctgt tacaaaccct agggaacctc gggtatcggg cctcggccaa gaaagcccaa 780 atttgccaga aacaggtcaa gtatctgggg tatcttctaa aagagggtca gagatggctg 840 actgaggcca gaaaagagac tgtgatgggg cagcctactc cgaagacccc tcgacaacta 900 agggagttcc tagggcttgc aggcttctgt cgcctctgga tccctgggtt tgcagaaatg 960 gcagccccct tgtaccctct caccaaaacg gggactctgt ttaattgggg cccagaccaa 1020 caaaaggcct atcaagaaat caagcaagct cttctaactg ccccagccct ggggttgcca 1080 gatttgacta agccctttga actctttgtc gacgagaagc agggctacgc caaaggtgtc 1140 ctaacgcaaa aactgggacc ttggcgtcgg ccggtggcct acctgtccaa aaagctagac 1200 ccagtagcag ctgggtggcc cccttgccta cggatggtag cagccattgc cgtactgaca 1260 aaggatgcag gcaagctaac catgggacag ccactagtca ttctggcccc ccatgcagta 1320 gaggcactag tcaaacaacc ccccgaccgc tggctttcca acgcccggat gactcactat 1380 caggccttgc ttttggacac ggaccgggtc cagttcggac cggtggtagc cctgaacccg 1440 gctacgctgc tcccactgcc tgaggaaggg ctgcaacaca actgccttga tatcctggcc 1500 gaagcccacg gaacccgacc cgacctaacg gaccagccgc tcccagacgc cgaccacacc 1560 tggtacacgg atggaagcag tctcttacaa gagggacagc gtaaggcggg agctgcggtg 1620 accaccgaga ccgaggtaat ctgggctaaa gccctgccag ccgggacatc cgctcagcgg 1680 gctgaactga tagcactcac ccaggcccta aagatggcag aaggtaagaa gctaaatgtt 1740 tatactgata gccgttatgc ttttgctact gcccatatcc atggagaaat atacagaagg 1800 cgtgggttgc tcacatcaga aggcaaagag atcaaaaata aagacgagat cttggcccta 1860 ctaaaagccc tctttctgcc caaaagactt agcataatcc attgtccagg acatcaaaag 1920 ggacacagcg ccgaggctag aggcaaccgg atggctgacc aagcggcccg aaaggcagcc 1980 atcacagaga ctccagacac ctctaccctc ctcata 2016 <210> 15 <211> 2016 <212> DNA <213> Moloney Murine Leukemia Virus <220> <221> mat_peptide <222> (1)..(2016) <223> nucleotide sequence coding for the amino acid sequence of SEQ. ID. No: 6 <220> <221> mutation <222> (1036)..(1038) <223> substitution of atg instead of gaa at 1036-1038 positions of SEQ. ID. No: 10 <400> 15 atgctaaata tagaagatga gcatcggcta catgagacct caaaagagcc agatgtttct 60 ctagggtcca catggctgtc tgattttcct caggcctggg cggaaaccgg gggcatggga 120 ctggcagttc gccaagctcc tctgatcata cctctgaaag caacctctac ccccgtgtcc 180 ataaaacaat accccatgtc acaagaagcc agactgggga tcaagcccca catacagaga 240 ctgttggacc agggaatact ggtaccctgc cagtccccct ggaacacgcc cctgctaccc 300 gttaagaaac cagggactaa tgattatagg cctgtccagg atctgagaga agtcaacaag 360 cgggtggaag acatccaccc caccgtgccc aacccttaca acctcttgag cgggctccca 420 ccgtcccacc agtggtacac tgtgcttgat ttaaaggatg cctttttctg cctgagactc 480 caccccacca gtcagcctct cttcgccttt gagtggagag atccagagat gggaatctca 540 ggacaattga cctggaccag actcccacag ggtttcaaaa acagtcccac cctgtttgat 600 gaggcactgc acagagacct agcagacttc cggatccagc acccagactt gatcctgcta 660 cagtacgtgg atgacttact gctggccgcc acttctgagc tagactgcca acaaggtact 720 cgggccctgt tacaaaccct agggaacctc gggtatcggg cctcggccaa gaaagcccaa 780 atttgccaga aacaggtcaa gtatctgggg tatcttctaa aagagggtca gagatggctg 840 actgaggcca gaaaagagac tgtgatgggg cagcctactc cgaagacccc tcgacaacta 900 agggagttcc tagggacggc aggcttctgt cgcctctgga tccctgggtt tgcagaaatg 960 gcagccccct tgtaccctct caccaaaacg gggactctgt ttaattgggg cccagaccaa 1020 caaaaggcct atcaaatgat caagcaagct cttctaactg ccccagccct ggggttgcca 1080 gatttgacta agccctttga actctttgtc gacgagaagc agggctacgc caaaggtgtc 1140 ctaacgcaaa aactgggacc ttggcgtcgg ccggtggcct acctgtccaa aaagctagac 1200 ccagtagcag ctgggtggcc cccttgccta cggatggtag cagccattgc cgtactgaca 1260 aaggatgcag gcaagctaac catgggacag ccactagtca ttctggcccc ccatgcagta 1320 gaggcactag tcaaacaacc ccccgaccgc tggctttcca acgcccggat gactcactat 1380 caggccttgc ttttggacac ggaccgggtc cagttcggac cggtggtagc cctgaacccg 1440 gctacgctgc tcccactgcc tgaggaaggg ctgcaacaca actgccttga tatcctggcc 1500 gaagcccacg gaacccgacc cgacctaacg gaccagccgc tcccagacgc cgaccacacc 1560 tggtacacgg atggaagcag tctcttacaa gagggacagc gtaaggcggg agctgcggtg 1620 accaccgaga ccgaggtaat ctgggctaaa gccctgccag ccgggacatc cgctcagcgg 1680 gctgaactga tagcactcac ccaggcccta aagatggcag aaggtaagaa gctaaatgtt 1740 tatactgata gccgttatgc ttttgctact gcccatatcc atggagaaat atacagaagg 1800 cgtgggttgc tcacatcaga aggcaaagag atcaaaaata aagacgagat cttggcccta 1860 ctaaaagccc tctttctgcc caaaagactt agcataatcc attgtccagg acatcaaaag 1920 ggacacagcg ccgaggctag aggcaaccgg atggctgacc aagcggcccg aaaggcagcc 1980 atcacagaga ctccagacac ctctaccctc ctcata 2016 <210> 16 <211> 2016 <212> DNA <213> Moloney Murine Leukemia Virus <220> <221> mat_peptide <222> (1)..(2016) <223> nucleotide sequence coding for the amino acid sequence of SEQ. ID. No: 7 <220> <221> mutation <222> (1222)..(1224) <223> substitution of gaa instead of cct at 1222-1224 positions of SEQ. ID. No: 10 <400> 16 atgctaaata tagaagatga gcatcggcta catgagacct caaaagagcc agatgtttct 60 ctagggtcca catggctgtc tgattttcct caggcctggg cggaaaccgg gggcatggga 120 ctggcagttc gccaagctcc tctgatcata cctctgaaag caacctctac ccccgtgtcc 180 ataaaacaat accccatgtc acaagaagcc agactgggga tcaagcccca catacagaga 240 ctgttggacc agggaatact ggtaccctgc cagtccccct ggaacacgcc cctgctaccc 300 gttaagaaac cagggactaa tgattatagg cctgtccagg atctgagaga agtcaacaag 360 cgggtggaag acatccaccc caccgtgccc aacccttaca acctcttgag cgggctccca 420 ccgtcccacc agtggtacac tgtgcttgat ttaaaggatg cctttttctg cctgagactc 480 caccccacca gtcagcctct cttcgccttt gagtggagag atccagagat gggaatctca 540 ggacaattga cctggaccag actcccacag ggtttcaaaa acagtcccac cctgtttgat 600 gaggcactgc acagagacct agcagacttc cggatccagc acccagactt gatcctgcta 660 cagtacgtgg atgacttact gctggccgcc acttctgagc tagactgcca acaaggtact 720 cgggccctgt tacaaaccct agggaacctc gggtatcggg cctcggccaa gaaagcccaa 780 atttgccaga aacaggtcaa gtatctgggg tatcttctaa aagagggtca gagatggctg 840 actgaggcca gaaaagagac tgtgatgggg cagcctactc cgaagacccc tcgacaacta 900 agggagttcc tagggacggc aggcttctgt cgcctctgga tccctgggtt tgcagaaatg 960 gcagccccct tgtaccctct caccaaaacg gggactctgt ttaattgggg cccagaccaa 1020 caaaaggcct atcaagaaat caagcaagct cttctaactg ccccagccct ggggttgcca 1080 gatttgacta agccctttga actctttgtc gacgagaagc agggctacgc caaaggtgtc 1140 ctaacgcaaa aactgggacc ttggcgtcgg ccggtggcct acctgtccaa aaagctagac 1200 ccagtagcag ctgggtggcc cgaatgccta cggatggtag cagccattgc cgtactgaca 1260 aaggatgcag gcaagctaac catgggacag ccactagtca ttctggcccc ccatgcagta 1320 gaggcactag tcaaacaacc ccccgaccgc tggctttcca acgcccggat gactcactat 1380 caggccttgc ttttggacac ggaccgggtc cagttcggac cggtggtagc cctgaacccg 1440 gctacgctgc tcccactgcc tgaggaaggg ctgcaacaca actgccttga tatcctggcc 1500 gaagcccacg gaacccgacc cgacctaacg gaccagccgc tcccagacgc cgaccacacc 1560 tggtacacgg atggaagcag tctcttacaa gagggacagc gtaaggcggg agctgcggtg 1620 accaccgaga ccgaggtaat ctgggctaaa gccctgccag ccgggacatc cgctcagcgg 1680 gctgaactga tagcactcac ccaggcccta aagatggcag aaggtaagaa gctaaatgtt 1740 tatactgata gccgttatgc ttttgctact gcccatatcc atggagaaat atacagaagg 1800 cgtgggttgc tcacatcaga aggcaaagag atcaaaaata aagacgagat cttggcccta 1860 ctaaaagccc tctttctgcc caaaagactt agcataatcc attgtccagg acatcaaaag 1920 ggacacagcg ccgaggctag aggcaaccgg atggctgacc aagcggcccg aaaggcagcc 1980 atcacagaga ctccagacac ctctaccctc ctcata 2016 <210> 17 <211> 2016 <212> DNA <213> Moloney Murine Leukemia Virus <220> <221> mat_peptide <222> (1)..(2016) <223> nucleotide sequence coding for the amino acid sequence of SEQ. ID. No: 8 <220> <221> mutation <222> (1312)..(1314) <223> substitution of tac instead of cat at 1312-1314 positions of SEQ. ID. No: 10 <400> 17 atgctaaata tagaagatga gcatcggcta catgagacct caaaagagcc agatgtttct 60 ctagggtcca catggctgtc tgattttcct caggcctggg cggaaaccgg gggcatggga 120 ctggcagttc gccaagctcc tctgatcata cctctgaaag caacctctac ccccgtgtcc 180 ataaaacaat accccatgtc acaagaagcc agactgggga tcaagcccca catacagaga 240 ctgttggacc agggaatact ggtaccctgc cagtccccct ggaacacgcc cctgctaccc 300 gttaagaaac cagggactaa tgattatagg cctgtccagg atctgagaga agtcaacaag 360 cgggtggaag acatccaccc caccgtgccc aacccttaca acctcttgag cgggctccca 420 ccgtcccacc agtggtacac tgtgcttgat ttaaaggatg cctttttctg cctgagactc 480 caccccacca gtcagcctct cttcgccttt gagtggagag atccagagat gggaatctca 540 ggacaattga cctggaccag actcccacag ggtttcaaaa acagtcccac cctgtttgat 600 gaggcactgc acagagacct agcagacttc cggatccagc acccagactt gatcctgcta 660 cagtacgtgg atgacttact gctggccgcc acttctgagc tagactgcca acaaggtact 720 cgggccctgt tacaaaccct agggaacctc gggtatcggg cctcggccaa gaaagcccaa 780 atttgccaga aacaggtcaa gtatctgggg tatcttctaa aagagggtca gagatggctg 840 actgaggcca gaaaagagac tgtgatgggg cagcctactc cgaagacccc tcgacaacta 900 agggagttcc tagggacggc aggcttctgt cgcctctgga tccctgggtt tgcagaaatg 960 gcagccccct tgtaccctct caccaaaacg gggactctgt ttaattgggg cccagaccaa 1020 caaaaggcct atcaagaaat caagcaagct cttctaactg ccccagccct ggggttgcca 1080 gatttgacta agccctttga actctttgtc gacgagaagc agggctacgc caaaggtgtc 1140 ctaacgcaaa aactgggacc ttggcgtcgg ccggtggcct acctgtccaa aaagctagac 1200 ccagtagcag ctgggtggcc cccttgccta cggatggtag cagccattgc cgtactgaca 1260 aaggatgcag gcaagctaac catgggacag ccactagtca ttctggcccc ctacgcagta 1320 gaggcactag tcaaacaacc ccccgaccgc tggctttcca acgcccggat gactcactat 1380 caggccttgc ttttggacac ggaccgggtc cagttcggac cggtggtagc cctgaacccg 1440 gctacgctgc tcccactgcc tgaggaaggg ctgcaacaca actgccttga tatcctggcc 1500 gaagcccacg gaacccgacc cgacctaacg gaccagccgc tcccagacgc cgaccacacc 1560 tggtacacgg atggaagcag tctcttacaa gagggacagc gtaaggcggg agctgcggtg 1620 accaccgaga ccgaggtaat ctgggctaaa gccctgccag ccgggacatc cgctcagcgg 1680 gctgaactga tagcactcac ccaggcccta aagatggcag aaggtaagaa gctaaatgtt 1740 tatactgata gccgttatgc ttttgctact gcccatatcc atggagaaat atacagaagg 1800 cgtgggttgc tcacatcaga aggcaaagag atcaaaaata aagacgagat cttggcccta 1860 ctaaaagccc tctttctgcc caaaagactt agcataatcc attgtccagg acatcaaaag 1920 ggacacagcg ccgaggctag aggcaaccgg atggctgacc aagcggcccg aaaggcagcc 1980 atcacagaga ctccagacac ctctaccctc ctcata 2016 <210> 18 <211> 2016 <212> DNA <213> Moloney Murine Leukemia Virus <220> <221> mat_peptide <222> (1)..(2016) <223> nucleotide sequence coding for the amino acid sequence of SEQ. ID. No: 9 <220> <221> mutation <222> (1360)..(1362) <223> substitution of ttc instead of aac at 1360-1362 positions of SEQ. ID. No: 10 <400> 18 atgctaaata tagaagatga gcatcggcta catgagacct caaaagagcc agatgtttct 60 ctagggtcca catggctgtc tgattttcct caggcctggg cggaaaccgg gggcatggga 120 ctggcagttc gccaagctcc tctgatcata cctctgaaag caacctctac ccccgtgtcc 180 ataaaacaat accccatgtc acaagaagcc agactgggga tcaagcccca catacagaga 240 ctgttggacc agggaatact ggtaccctgc cagtccccct ggaacacgcc cctgctaccc 300 gttaagaaac cagggactaa tgattatagg cctgtccagg atctgagaga agtcaacaag 360 cgggtggaag acatccaccc caccgtgccc aacccttaca acctcttgag cgggctccca 420 ccgtcccacc agtggtacac tgtgcttgat ttaaaggatg cctttttctg cctgagactc 480 caccccacca gtcagcctct cttcgccttt gagtggagag atccagagat gggaatctca 540 ggacaattga cctggaccag actcccacag ggtttcaaaa acagtcccac cctgtttgat 600 gaggcactgc acagagacct agcagacttc cggatccagc acccagactt gatcctgcta 660 cagtacgtgg atgacttact gctggccgcc acttctgagc tagactgcca acaaggtact 720 cgggccctgt tacaaaccct agggaacctc gggtatcggg cctcggccaa gaaagcccaa 780 atttgccaga aacaggtcaa gtatctgggg tatcttctaa aagagggtca gagatggctg 840 actgaggcca gaaaagagac tgtgatgggg cagcctactc cgaagacccc tcgacaacta 900 agggagttcc tagggacggc aggcttctgt cgcctctgga tccctgggtt tgcagaaatg 960 gcagccccct tgtaccctct caccaaaacg gggactctgt ttaattgggg cccagaccaa 1020 caaaaggcct atcaagaaat caagcaagct cttctaactg ccccagccct ggggttgcca 1080 gatttgacta agccctttga actctttgtc gacgagaagc agggctacgc caaaggtgtc 1140 ctaacgcaaa aactgggacc ttggcgtcgg ccggtggcct acctgtccaa aaagctagac 1200 ccagtagcag ctgggtggcc cccttgccta cggatggtag cagccattgc cgtactgaca 1260 aaggatgcag gcaagctaac catgggacag ccactagtca ttctggcccc ccatgcagta 1320 gaggcactag tcaaacaacc ccccgaccgc tggctttcct tcgcccggat gactcactat 1380 caggccttgc ttttggacac ggaccgggtc cagttcggac cggtggtagc cctgaacccg 1440 gctacgctgc tcccactgcc tgaggaaggg ctgcaacaca actgccttga tatcctggcc 1500 gaagcccacg gaacccgacc cgacctaacg gaccagccgc tcccagacgc cgaccacacc 1560 tggtacacgg atggaagcag tctcttacaa gagggacagc gtaaggcggg agctgcggtg 1620 accaccgaga ccgaggtaat ctgggctaaa gccctgccag ccgggacatc cgctcagcgg 1680 gctgaactga tagcactcac ccaggcccta aagatggcag aaggtaagaa gctaaatgtt 1740 tatactgata gccgttatgc ttttgctact gcccatatcc atggagaaat atacagaagg 1800 cgtgggttgc tcacatcaga aggcaaagag atcaaaaata aagacgagat cttggcccta 1860 ctaaaagccc tctttctgcc caaaagactt agcataatcc attgtccagg acatcaaaag 1920 ggacacagcg ccgaggctag aggcaaccgg atggctgacc aagcggcccg aaaggcagcc 1980 atcacagaga ctccagacac ctctaccctc ctcata 2016 <210> 19 <211> 2019 <212> DNA <213> Artificial Sequence <220> <223> Optimized Moloney Murine Leukemia Virus reverse transcriptase in accordance with codon usage <220> <221> mat_peptide <222> (1)..(2016) <223> nucleotide sequence coding for the amino acid sequence of SEQ. ID. No: 1 <400> 19 atgctgaaca tcgaagacga acaccgtctg cacgaaacct ctaaagaacc ggacgtttct 60 ctgggttcta cctggctgtc tgacttcccg caggcttggg ctgaaaccgg tggtatgggt 120 ctggctgttc gtcaggctcc gctgatcatc ccgctgaaag ctacctctac cccggtttct 180 atcaaacagt acccgatgtc tcaggaagct cgtctgggta tcaaaccgca catccagcgt 240 ctgctggacc agggtatcct ggttccgtgc cagtctccgt ggaacacccc gctgctgccg 300 gttaaaaaac cgggtaccaa cgactaccgt ccggttcagg acctgcgtga agttaacaaa 360 cgtgttgaag acatccaccc gaccgttccg aacccgtaca acctgctgtc tggtctgccg 420 ccgtctcacc agtggtacac cgttctggac ctgaaagacg ctttcttctg cctgcgtctg 480 cacccgacct ctcagccgct gttcgctttc gaatggcgtg acccggaaat gggtatctct 540 ggtcagctga cctggacccg tctgccgcag ggtttcaaaa actctccgac cctgttcgac 600 gaagctctgc accgtgacct ggctgacttc cgtatccagc acccggacct gatcctgctg 660 cagtacgttg acgacctgct gctggctgct acctctgaac tggactgcca gcagggtacc 720 cgtgctctgc tgcagaccct gggtaacctg ggttaccgtg cttctgctaa aaaagctcag 780 atctgccaga aacaggttaa atacctgggt tacctgctga aagaaggtca gcgttggctg 840 accgaagctc gtaaagaaac cgttatgggt cagccgaccc cgaaaacccc gcgtcagctg 900 cgtgaattcc tgggtaccgc tggtttctgc cgtctgtgga taccgggttt cgctgaaatg 960 gctgctccgc tgtacccgct gaccaaaacc ggtaccctgt tcaactgggg tccggaccag 1020 cagaaagcgt accaggaaat caaacaggct ctgctgaccg ctccggctct gggtctgccg 1080 gacctgacca aaccgttcga actgttcgtt gacgaaaaac agggttacgc taaaggtgtt 1140 ctgacccaga aactgggtcc gtggcgtcgt ccggttgctt acctgtctaa aaaactggac 1200 ccggttgctg ctggttggcc gccgtgcctg cgtatggttg ctgctatcgc tgttctgacc 1260 aaagacgctg gtaaactgac catgggtcag ccgctggtta tcctggctcc gcacgctgtt 1320 gaagctctgg ttaaacagcc gccggaccgt tggctgtcta acgctcgtat gacccactac 1380 caggctctgc tgctggacac cgaccgtgtt cagttcggtc cggttgttgc tctgaacccg 1440 gctaccctgc tgccgctgcc ggaagaaggt ctgcagcaca actgcctgga catcctggct 1500 gaagctcacg gtacccgtcc ggacctgacc gaccagccgc tgccggacgc tgaccacacc 1560 tggtacaccg acggttcttc tctgctgcag gaaggtcagc gtaaagctgg tgctgctgtt 1620 accaccgaaa ccgaagttat ctgggctaaa gctctgccgg ctggtacctc tgctcagcgt 1680 gctgaactga tcgctctgac ccaggctctg aaaatggctg aaggtaaaaa actgaacgtt 1740 tacaccgact ctcgttacgc tttcgctacc gctcacatcc acggtgaaat ctaccgtcgt 1800 cgtggtctgc tgacctctga aggtaaagaa atcaaaaaca aagacgaaat cctggctctg 1860 ctgaaagctc tgttcctgcc gaaacgtctg tctatcatcc actgcccggg tcaccagaaa 1920 ggtcactctg ctgaagctcg tggtaaccgt atggctgacc aggctgctcg taaagctgct 1980 atcaccgaaa ccccggacac ctctaccctg ctgatctaa 2019 <210> 20 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> R0 oligonucleotide <400> 20 ttcagggata gagggagta 19 <210> 21 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> F0 oligonucleotide <400> 21 tactccctct atccctgaac atcgaagacg aacacc 36 <210> 22 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> R19 oligonucleotide <400> 22 gaggtttcgt gcagacggtg ttcgtcttcg atg 33 <210> 23 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> F36 oligonucleotide <400> 23 gtctgcacga aacctctaaa gaaccggacg tttc 34 <210> 24 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> R52 oligonucleotide <400> 24 ccaggtagaa cccagagaaa cgtccggttc ttta 34 <210> 25 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> F70 oligonucleotide <400> 25 tctgggttct acctggctgt ctgacttccc gc 32 <210> 26 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R86 oligonucleotide <400> 26 ttcagcccaa gcctgcggga agtcagacag 30 <210> 27 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> F102 oligonucleotide <400> 27 aggcttgggc tgaaaccggt ggtatgggt 29 <210> 28 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R116 oligonucleotide <400> 28 ctgacgaaca gccagaccca taccaccggt 30 <210> 29 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> F131 oligonucleotide <400> 29 ctggctgttc gtcaggctcc gctgatcatc 30 <210> 30 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R146 oligonucleotide <400> 30 ggtagctttc agcgggatga tcagcggagc 30 <210> 31 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> F161 oligonucleotide <400> 31 ccgctgaaag ctacctctac cccggtttct atc 33 <210> 32 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> R176 oligonucleotide <400> 32 gacatcgggt actgtttgat agaaaccggg gtaga 35 <210> 33 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> F194 oligonucleotide <400> 33 aaacagtacc cgatgtctca ggaagctcgt ctg 33 <210> 34 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> R211 oligonucleotide <400> 34 tgtgcggttt gatacccaga cgagcttcct ga 32 <210> 35 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> F227 oligonucleotide <400> 35 ggtatcaaac cgcacatcca gcgtctgctg 30 <210> 36 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R243 oligonucleotide <400> 36 ccaggatacc ctggtccagc agacgctgga 30 <210> 37 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> F257 oligonucleotide <400> 37 gaccagggta tcctggttcc gtgccagtct c 31 <210> 38 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> R273 oligonucleotide <400> 38 cggggtgttc cacggagact ggcacggaa 29 <210> 39 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> F288 oligonucleotide <400> 39 cgtggaacac cccgctgctg ccggttaaa 29 <210> 40 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> R302 oligonucleotide <400> 40 cgttggtacc cggtttttta accggcagca g 31 <210> 41 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> F317 oligonucleotide <400> 41 aaaccgggta ccaacgacta ccgtccggtt c 31 <210> 42 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R333 oligonucleotide <400> 42 cttcacgcag gtcctgaacc ggacggtagt 30 <210> 43 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> F348 oligonucleotide <400> 43 aggacctgcg tgaagttaac aaacgtgttg aagac 35 <210> 44 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> R363 oligonucleotide <400> 44 acggtcgggt ggatgtcttc aacacgtttg ttaa 34 <210> 45 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> F383 oligonucleotide <400> 45 atccacccga ccgttccgaa cccgtacaa 29 <210> 46 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R397 oligonucleotide <400> 46 agaccagaca gcaggttgta cgggttcgga 30 <210> 47 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> F412 oligonucleotide <400> 47 cctgctgtct ggtctgccgc cgtctca 27 <210> 48 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> R427 oligonucleotide <400> 48 acggtgtacc actggtgaga cggcggc 27 <210> 49 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> F439 oligonucleotide <400> 49 ccagtggtac accgttctgg acctgaaaga cg 32 <210> 50 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> R454 oligonucleotide <400> 50 cgcaggcaga agaaagcgtc tttcaggtcc aga 33 <210> 51 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> F471 oligonucleotide <400> 51 ctttcttctg cctgcgtctg cacccgacct 30 <210> 52 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> R487 oligonucleotide <400> 52 cgaacagcgg ctgagaggtc gggtgcaga 29 <210> 53 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> F501 oligonucleotide <400> 53 ctcagccgct gttcgctttc gaatggcgtg a 31 <210> 54 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> R516 oligonucleotide <400> 54 agatacccat ttccgggtca cgccattcga aag 33 <210> 55 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> F532 oligonucleotide <400> 55 cccggaaatg ggtatctctg gtcagctgac ct 32 <210> 56 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> R549 oligonucleotide <400> 56 ggcagacggg tccaggtcag ctgaccag 28 <210> 57 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> F564 oligonucleotide <400> 57 ggacccgtct gccgcagggt ttcaaaaact c 31 <210> 58 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> R577 oligonucleotide <400> 58 cgaacagggt cggagagttt ttgaaaccct gc 32 <210> 59 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> F595 oligonucleotide <400> 59 tccgaccctg ttcgacgaag ctctgcacc 29 <210> 60 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R609 oligonucleotide <400> 60 agtcagccag gtcacggtgc agagcttcgt 30 <210> 61 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> F624 oligonucleotide <400> 61 gtgacctggc tgacttccgt atccagcacc 30 <210> 62 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R639 oligonucleotide <400> 62 gcaggatcag gtccgggtgc tggatacgga 30 <210> 63 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> F654 oligonucleotide <400> 63 cggacctgat cctgctgcag tacgttgacg a 31 <210> 64 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> R669 oligonucleotide <400> 64 agccagcagc aggtcgtcaa cgtactgca 29 <210> 65 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> F685 oligonucleotide <400> 65 cctgctgctg gctgctacct ctgaactgga 30 <210> 66 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R698 oligonucleotide <400> 66 accctgctgg cagtccagtt cagaggtagc 30 <210> 67 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> F715 oligonucleotide <400> 67 ctgccagcag ggtacccgtg ctctgc 26 <210> 68 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> R728 oligonucleotide <400> 68 tacccagggt ctgcagcaga gcacgggt 28 <210> 69 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> F741 oligonucleotide <400> 69 tgcagaccct gggtaacctg ggttaccgtg 30 <210> 70 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> R756 oligonucleotide <400> 70 ctgagctttt ttagcagaag cacggtaacc caggt 35 <210> 71 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> F771 oligonucleotide <400> 71 cttctgctaa aaaagctcag atctgccaga aacaggt 37 <210> 72 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> R791 oligonucleotide <400> 72 gcaggtaacc caggtattta acctgtttct ggcagat 37 <210> 73 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> F808 oligonucleotide <400> 73 taaatacctg ggttacctgc tgaaagaagg tcagcgt 37 <210> 74 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> R828 oligonucleotide <400> 74 gcttcggtca gccaacgctg accttctttc a 31 <210> 75 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> F845 oligonucleotide <400> 75 tggctgaccg aagctcgtaa agaaaccgtt atgg 34 <210> 76 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> R859 oligonucleotide <400> 76 ggggtcggct gacccataac ggtttcttta cga 33 <210> 77 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> F879 oligonucleotide <400> 77 gtcagccgac cccgaaaacc ccgcgtc 27 <210> 78 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> R892 oligonucleotide <400> 78 ggaattcacg cagctgacgc ggggttttc 29 <210> 79 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> F906 oligonucleotide <400> 79 agctgcgtga attcctgggt accgctggt 29 <210> 80 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> R921 oligonucleotide <400> 80 ccacagacgg cagaaaccag cggtaccca 29 <210> 81 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> F935 oligonucleotide <400> 81 ttctgccgtc tgtggatacc gggtttcgct 30 <210> 82 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R950 oligonucleotide <400> 82 cggagcagcc atttcagcga aacccggtat 30 <210> 83 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> F965 oligonucleotide <400> 83 gaaatggctg ctccgctgta cccgctgacc 30 <210> 84 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> R980 oligonucleotide <400> 84 acagggtacc ggttttggtc agcgggtaca g 31 <210> 85 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> F995 oligonucleotide <400> 85 aaaaccggta ccctgttcaa ctggggtccg 30 <210> 86 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> R1011 oligonucleotide <400> 86 cgctttctgc tggtccggac cccagttga 29 <210> 87 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> F1025 oligonucleotide <400> 87 gaccagcaga aagcgtacca ggaaatcaaa cagg 34 <210> 88 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> R1040 oligonucleotide <400> 88 agcggtcagc agagcctgtt tgatttcctg gta 33 <210> 89 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> F1059 oligonucleotide <400> 89 ctctgctgac cgctccggct ctgggtc 27 <210> 90 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> R1073 oligonucleotide <400> 90 ggtcaggtcc ggcagaccca gagccgg 27 <210> 91 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> F1086 oligonucleotide <400> 91 tgccggacct gaccaaaccg ttcgaactgt t 31 <210> 92 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> R1100 oligonucleotide <400> 92 cctgtttttc gtcaacgaac agttcgaacg gttt 34 <210> 93 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> F1117 oligonucleotide <400> 93 cgttgacgaa aaacagggtt acgctaaagg tgttct 36 <210> 94 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> R1134 oligonucleotide <400> 94 acccagtttc tgggtcagaa cacctttagc gtaac 35 <210> 95 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> F1153 oligonucleotide <400> 95 gacccagaaa ctgggtccgt ggcgtcgt 28 <210> 96 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> R1169 oligonucleotide <400> 96 caggtaagca accggacgac gccacgg 27 <210> 97 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> F1181 oligonucleotide <400> 97 ccggttgctt acctgtctaa aaaactggac ccg 33 <210> 98 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> R1196 oligonucleotide <400> 98 ccaaccagca gcaaccgggt ccagtttttt aga 33 <210> 99 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> F1214 oligonucleotide <400> 99 gttgctgctg gttggccgcc gtgcctg 27 <210> 100 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> R1229 oligonucleotide <400> 100 atagcagcaa ccatacgcag gcacggcgg 29 <210> 101 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> F1241 oligonucleotide <400> 101 cgtatggttg ctgctatcgc tgttctgacc aaa 33 <210> 102 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> R1258 oligonucleotide <400> 102 gtcagtttac cagcgtcttt ggtcagaaca gcg 33 <210> 103 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> F1274 oligonucleotide <400> 103 gacgctggta aactgaccat gggtcagccg c 31 <210> 104 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R1291 oligonucleotide <400> 104 ggagccagga taaccagcgg ctgacccatg 30 <210> 105 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> F1305 oligonucleotide <400> 105 tggttatcct ggctccgcac gctgttgaag c 31 <210> 106 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> R1321 oligonucleotide <400> 106 gcggctgttt aaccagagct tcaacagcgt gc 32 <210> 107 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> F1336 oligonucleotide <400> 107 tctggttaaa cagccgccgg accgttggct 30 <210> 108 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> R1353 oligonucleotide <400> 108 gtcatacgag cgttagacag ccaacggtcc g 31 <210> 109 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> F1366 oligonucleotide <400> 109 gtctaacgct cgtatgaccc actaccaggc tctg 34 <210> 110 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R1384 oligonucleotide <400> 110 tcggtgtcca gcagcagagc ctggtagtgg 30 <210> 111 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> F1400 oligonucleotide <400> 111 ctgctggaca ccgaccgtgt tcagttcgg 29 <210> 112 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R1414 oligonucleotide <400> 112 agagcaacaa ccggaccgaa ctgaacacgg 30 <210> 113 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> F1429 oligonucleotide <400> 113 tccggttgtt gctctgaacc cggctaccc 29 <210> 114 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> R1444 oligonucleotide <400> 114 gcagcggcag cagggtagcc gggttc 26 <210> 115 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> F1458 oligonucleotide <400> 115 tgctgccgct gccggaagaa ggtctgca 28 <210> 116 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R1470 oligonucleotide <400> 116 ccaggcagtt gtgctgcaga ccttcttccg 30 <210> 117 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> F1486 oligonucleotide <400> 117 gcacaactgc ctggacatcc tggctgaagc 30 <210> 118 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R1500 oligonucleotide <400> 118 gacgggtacc gtgagcttca gccaggatgt 30 <210> 119 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> F1516 oligonucleotide <400> 119 tcacggtacc cgtccggacc tgaccgac 28 <210> 120 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> R1530 oligonucleotide <400> 120 cggcagcggc tggtcggtca ggtccg 26 <210> 121 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> F1544 oligonucleotide <400> 121 cagccgctgc cggacgctga ccacacc 27 <210> 122 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> R1556 oligonucleotide <400> 122 ccgtcggtgt accaggtgtg gtcagcgtc 29 <210> 123 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> F1571 oligonucleotide <400> 123 tggtacaccg acggttcttc tctgctgcag g 31 <210> 124 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> R1585 oligonucleotide <400> 124 gctttacgct gaccttcctg cagcagagaa gaa 33 <210> 125 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> F1602 oligonucleotide <400> 125 aaggtcagcg taaagctggt gctgctgtta cca 33 <210> 126 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> R1618 oligonucleotide <400> 126 cagataactt cggtttcggt ggtaacagca gcacca 36 <210> 127 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> F1635 oligonucleotide <400> 127 ccgaaaccga agttatctgg gctaaagctc tgccg 35 <210> 128 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> R1654 oligonucleotide <400> 128 gagcagaggt accagccggc agagctttag cc 32 <210> 129 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> F1670 oligonucleotide <400> 129 gctggtacct ctgctcagcg tgctgaactg at 32 <210> 130 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> R1686 oligonucleotide <400> 130 gcctgggtca gagcgatcag ttcagcacgc t 31 <210> 131 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> F1702 oligonucleotide <400> 131 cgctctgacc caggctctga aaatggctga aggta 35 <210> 132 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> R1717 oligonucleotide <400> 132 ggtgtaaacg ttcagttttt taccttcagc cattttcaga 40 <210> 133 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> F1737 oligonucleotide <400> 133 aaaaactgaa cgtttacacc gactctcgtt acgctttc 38 <210> 134 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> R1757 oligonucleotide <400> 134 ggatgtgagc ggtagcgaaa gcgtaacgag agtc 34 <210> 135 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> F1775 oligonucleotide <400> 135 gctaccgctc acatccacgg tgaaatctac cgt 33 <210> 136 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> R1791 oligonucleotide <400> 136 cagcagacca cgacgacggt agatttcacc gt 32 <210> 137 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> F1808 oligonucleotide <400> 137 cgtcgtggtc tgctgacctc tgaaggtaaa gaaat 35 <210> 138 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> R1823 oligonucleotide <400> 138 ggatttcgtc tttgtttttg atttctttac cttcagaggt 40 <210> 139 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> F1843 oligonucleotide <400> 139 caaaaacaaa gacgaaatcc tggctctgct gaaagct 37 <210> 140 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> R1863 oligonucleotide <400> 140 gtttcggcag gaacagagct ttcagcagag cca 33 <210> 141 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> F1880 oligonucleotide <400> 141 ctgttcctgc cgaaacgtct gtctatcatc cactgc 36 <210> 142 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> R1896 oligonucleotide <400> 142 ttctggtgac ccgggcagtg gatgatagac agac 34 <210> 143 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> F1916 oligonucleotide <400> 143 ccgggtcacc agaaaggtca ctctgctgaa g 31 <210> 144 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> R1930 oligonucleotide <400> 144 catacggtta ccacgagctt cagcagagtg acct 34 <210> 145 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> F1947 oligonucleotide <400> 145 ctcgtggtaa ccgtatggct gaccaggctg c 31 <210> 146 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> R1964 oligonucleotide <400> 146 ggtgatagca gctttacgag cagcctggtc agc 33 <210> 147 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> F1978 oligonucleotide <400> 147 tcgtaaagct gctatcaccg aaaccccgga cacc 34 <210> 148 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> R1997 oligonucleotide <400> 148 ttagatcagc agggtagagg tgtccggggt ttc 33 <210> 149 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> F2012 oligonucleotide <400> 149 tctaccctgc tgatctaa 18 <210> 150 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> forward primer for the synthesis of K295Q mutant <400> 150 gtcagccgac cccgcaaacc ccgcgtc 27 <210> 151 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for the synthesis of K295Q mutant <400> 151 ggaattcacg cagctgacgc ggggtttgc 29 <210> 152 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer for the amplification of synthesized gene <400> 152 ctgaacatcg aagacgaa 18 <210> 153 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for the amplification of synthesized gene <400> 153 ttagatcagc agggtaga 18 <210> 154 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> forward primer for site-directed mutagenesis of Q63L <400> 154 caaactttac ccgatgtctc aggaagctcg 30 <210> 155 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for site-directed mutagenesis of Q63L <400> 155 atagaaaccg gggtagaggt agctttcagc 30 <210> 156 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> forward primer for site-directed mutagenesis of K264L <400> 156 ccagcttcag gttaaatacc tgggttacct g 31 <210> 157 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for site-directed mutagenesis of K264L <400> 157 cagatctgag cttttttagc agaagcacgg 30 <210> 158 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> forward primer for site-directed mutagenesis of T306L <400> 158 ggtcttgctg gtttctgccg tctgtg 26 <210> 159 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for site-directed mutagenesis of T306L <400> 159 caggaattca cgcagctgac gcgg 24 <210> 160 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> forward primer for site-directed mutagenesis of E346M <400> 160 cagatgatca aacaggctct gctgaccg 28 <210> 161 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for site-directed mutagenesis of E346M <400> 161 gtacgctttc tgctggtccg gacc 24 <210> 162 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> forward primer for site-directed mutagenesis of P408E <400> 162 ccggaatgcc tgcgtatggt tgctg 25 <210> 163 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for site-directed mutagenesis of P408E <400> 163 ccaaccagca gcaaccgggt cc 22 <210> 164 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> forward primer for site-directed mutagenesis of H438Y <400> 164 cgtacgctgt tgaagctctg gttaaacagc 30 <210> 165 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for site-directed mutagenesis of H438Y <400> 165 gagccaggat aaccagcggc tgacc 25 <210> 166 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> forward primer for site-directed mutagenesis of N454F <400> 166 ctgtctttcg ctcgtatgac ccactaccag 30 <210> 167 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for site-directed mutagenesis of N454F <400> 167 ccaacggtcc ggcggctgtt taac 24 <210> 168 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> primer for sense strand of Q63L, K264L, T306L and E346M mutant genes <400> 168 gcgcgccata tgctgaacat cgaagacgaa caccgtctgc acgaaac 47 <210> 169 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> primer for antisense strand of Q63L, K264L, T306L and E346M mutant genes <400> 169 gcgcgcgcgg ccgcttagat cagcagggta gaggtgtccg gggtttc 47 <210> 170 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> primer for sense strand of K295Q, P408E, H438Y and N454F mutant genes <400> 170 gcgcgccata tgctgaacat cgaagacgaa caccgtctgc acgaaac 47 <210> 171 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> primer for antisense strand of K295Q, P408E, H438Y and N454F mutant genes <400> 171 gcgcgcgcgg ccgcgatcag cagggtagag gtgtccgggg tttc 44 <210> 172 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer for sense strand of T7 promoter <400> 172 taatacgact cactataggg 20 <210> 173 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer for antisense strand of T7 promoter <400> 173 gctagttatt gctcagcgg 19 <210> 174 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer for sense strand of GAPDH gene <400> 174 gaaggtgaag gtcggagtca acg 23 <210> 175 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer for antisense strand of GAPDH gene <400> 175 agtccttcca cgataccaaa gttg 24

Claims (10)

서열번호 1로 기재되는 M-MLV(Moloney-Murine Leukemia Virus, M-MLV) 유래 역전사효소의 아미노산 서열의 408번째 프롤린이 글루탐산으로 치환(P408E)되어 열안정성이 증가된 역전사효소.A reverse transcriptase in which the 408th proline of the amino acid sequence of Moloney-Murine Leukemia Virus (M-MLV) -mediated reverse transcriptase described in SEQ ID NO: 1 is replaced with glutamic acid (P408E) to increase thermal stability. 청구항 1에 있어서,
서열번호 1로 기재되는 M-MLV 유래 역전사효소의 아미노산 서열의 63번째 글루타민에서 루이신으로의 치환(Q63L), 264번째 리신에서 루이신으로의 치환(K264L), 346번째 글루탐산에서 메티오닌으로의 치환(E346M), 438번째 히스티딘에서 티로신으로의 치환(H438Y) 및 454번째 아스파라긴에서 페닐알라닌으로의 치환(N454F)으로 이루어진 군으로부터 선택되는 하나 이상의 아미노산 치환을 추가로 포함하는 역전사효소.The method according to claim 1,
(Q63L) from the 63rd glutamine to the leucine in the amino acid sequence of the M-MLV-derived reverse transcriptase represented by SEQ ID NO: 1, the substitution from the 264th lysine to the leucine (K264L), the substitution with methionine from the 346th glutamic acid (E346M), substitution of tyrosine at 438th histidine (H438Y), and substitution of phenylalanine at 454th asparagine (N454F). 청구항 1에 있어서,
서열번호 7로 기재되는 아미노산 서열로 이루어진 역전사효소.The method according to claim 1,
A reverse transcriptase comprising the amino acid sequence set forth in SEQ ID NO: 7. 청구항 1 내지 청구항 3 중 어느 한 항에 있어서,
60℃ 내지 65℃의 온도에서 서열번호 1로 기재되는 아미노산 서열로 이루어진 야생형 M-MLV 역전사효소와 비교하여 뛰어난 역전사 활성을 나타내는 역전사효소.The method according to any one of claims 1 to 3,
A reverse transcriptase exhibiting excellent reverse transcription activity as compared to the wild-type M-MLV reverse transcriptase comprising the amino acid sequence set forth in SEQ ID NO: 1 at a temperature of 60 ° C to 65 ° C. 청구항 1의 열안정성이 증가된 역전사효소를 코딩하는 유전자.A gene encoding a reverse transcriptase with increased thermostability of claim 1. 청구항 5에 있어서,
서열번호 1로 기재되는 야생형 역전사효소의 63번째 글루타민에서 루이신으로의 치환(Q63L), 264번째 리신에서 루이신으로의 치환(K264L), 346번째 글루탐산에서 메티오닌으로의 치환(E346M), 438번째 히스티딘에서 티로신으로의 치환(H438Y) 및 454번째 아스파라긴에서 페닐알라닌으로의 치환(N454F)으로 이루어진 군으로부터 선택되는 하나 이상의 아미노산 치환을 추가로 포함하는 돌연변이 역전사효소를 코딩하는 유전자.The method of claim 5,
(Q63L) from the 63rd glutamine to the lysine of the wild-type reverse transcriptase shown in SEQ ID NO: 1, substitution with lysine (K264L) from the 264th lysine, substitution with methionine from the 346th glutamic acid (E346M) A gene coding for a mutant reverse transcriptase further comprising at least one amino acid substitution selected from the group consisting of substitution of histidine to tyrosine (H438Y) and substitution of phenylalanine to phenylalanine (N454F) at 454th asparagine. 청구항 5에 있어서,
서열번호 16으로 기재되는 염기서열로 이루어진 유전자.The method of claim 5,
A gene consisting of the nucleotide sequence shown in SEQ ID NO: 16. 청구항 5의 유전자를 포함하는 발현 벡터.An expression vector comprising the gene of claim 5. 청구항 8의 발현 벡터로 형질전환된 형질전환체.A transformant transformed with the expression vector of claim 8. 청구항 1의 열안정성이 증가된 역전사효소를 포함하는 역전사 반응용 키트.A kit for a reverse reaction comprising the thermostable reverse transcriptase of claim 1.
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