用基于SNP分型的实时PCR定量检测造血干细胞移植后的嵌合体
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摘要
【背景】
造血干细胞移植是目前临床上治疗白血病、骨髓瘤、淋巴瘤、重型再生障碍性贫血和地中海贫血等血液系统疾病的最有效手段之一。根据造血干细胞的来源可以分为自体、异体同基因(如双胞胎)、以及异基因(如父母、同胞或无血缘关系的供者)移植。随着移植理论和技术的进步，以及骨髓库的完善，HLA相合或半相合的异基因造血干细胞移植在临床上的应用越来越广泛。异基因干细胞移植的目的是建立供者型的正常造血与免疫功能，以取代受者原有的异常的造血及免疫系统。为判断移植是否成功并及时地实施免疫抑制治疗、预后等，人们需要对移植后受体体内形成的供、受者外周血细胞嵌合体进行检测并观察其变化趋势。此外，嵌合体分析还可为移植相关的其他免疫学研究提供良好的实验手段。
从遗传学的角度来说，异基因的供受者外周血细胞嵌合体本质上是两个基因型不同的个体之细胞的混合物。因此，利用人体染色体上的一些遗传标记可以区分供受者细胞并对供受者细胞的比例进行定量分析。传统的嵌合体分析手段包括基于第一代和第二代遗传标记检测的RFLP-PCR和VNTR／STR-PCR技术，以及基于染色体片段分析的FISH技术，这些方法存在着一些局限性，如灵敏度低(如STR-PCR仅能达到5％-1％)，应用面窄(如FISH仅能用于性别不合的供受者)等缺陷。近年来，随着嵌合体分析在白血病复发及白血病微残余灶监测上的广泛应用，临床实践对嵌合体分析技术的检测灵敏度和定量的精确性也提出了更高的要求。
SNP做为第三代遗传标记较RFLP、VNTR／STR有不可取代的优势：①SNP
[Background]
Now, hematopoietic stem cell transplantation is one of the most effective methods for curing many diseases of blood system, such as leukemia, myeloma, lymphoma, acute aplastic anemia and thalassemia, etc. Based on the sources of the hematopoietic stem cell, transplantation can be classified into three categories, i.e. autologous transplantation, isogeneic transplantation and heterogous transplantation. Because of the progress in the theory and technology of transplantation and the expansion of Marrow Bank, the allogeneic transplantation with HLA matched or HLA semi-matched was used extensively in clinical practice. The objective of allogeneic transplantation is to establish a normal hemopoiesis and immune function of donor type to substitute the intrinsic abnormal ones of recipient. Following transplantation, doctors must estimate the success or failure of transplantation, carry out immunosuppressive therapy timely and infer prognosis. The best method is to quantitatively detect the proportion of donor cells existed in chimera fathermore dynamicly monitor its change trend. In addition, the chimera study provided an excellent tool for developing the immunology and transplantation theory.
From the point of view of genetics, the chimera was a mixture containing two types of cell with different genotype. Therefore, some genetic markers could be used to discriminate donor cells or recipient cells and to quantitate their proportion. Some technology and methods, such as FISH and RFLP-PCR, VNTR/STR-PCR, etc.which are based on the first and second generation of genetic markers, were used previously. The disadvantages of these methods were lower sensitivity (5%-1% of STR-PCR method) and narrow detection limit (FISH can only differentiate sex). Recently, the sensitivity for chimera analysis was needed to reach more height point when people used it in monitoring prognosis and minimal residual disease of leukemia.
造血干细胞移植是目前临床上治疗白血病、骨髓瘤、淋巴瘤、重型再生障碍性贫血和地中海贫血等血液系统疾病的最有效手段之一。根据造血干细胞的来源可以分为自体、异体同基因(如双胞胎)、以及异基因(如父母、同胞或无血缘关系的供者)移植。随着移植理论和技术的进步，以及骨髓库的完善，HLA相合或半相合的异基因造血干细胞移植在临床上的应用越来越广泛。异基因干细胞移植的目的是建立供者型的正常造血与免疫功能，以取代受者原有的异常的造血及免疫系统。为判断移植是否成功并及时地实施免疫抑制治疗、预后等，人们需要对移植后受体体内形成的供、受者外周血细胞嵌合体进行检测并观察其变化趋势。此外，嵌合体分析还可为移植相关的其他免疫学研究提供良好的实验手段。
从遗传学的角度来说，异基因的供受者外周血细胞嵌合体本质上是两个基因型不同的个体之细胞的混合物。因此，利用人体染色体上的一些遗传标记可以区分供受者细胞并对供受者细胞的比例进行定量分析。传统的嵌合体分析手段包括基于第一代和第二代遗传标记检测的RFLP-PCR和VNTR／STR-PCR技术，以及基于染色体片段分析的FISH技术，这些方法存在着一些局限性，如灵敏度低(如STR-PCR仅能达到5％-1％)，应用面窄(如FISH仅能用于性别不合的供受者)等缺陷。近年来，随着嵌合体分析在白血病复发及白血病微残余灶监测上的广泛应用，临床实践对嵌合体分析技术的检测灵敏度和定量的精确性也提出了更高的要求。
SNP做为第三代遗传标记较RFLP、VNTR／STR有不可取代的优势：①SNP
[Background]
Now, hematopoietic stem cell transplantation is one of the most effective methods for curing many diseases of blood system, such as leukemia, myeloma, lymphoma, acute aplastic anemia and thalassemia, etc. Based on the sources of the hematopoietic stem cell, transplantation can be classified into three categories, i.e. autologous transplantation, isogeneic transplantation and heterogous transplantation. Because of the progress in the theory and technology of transplantation and the expansion of Marrow Bank, the allogeneic transplantation with HLA matched or HLA semi-matched was used extensively in clinical practice. The objective of allogeneic transplantation is to establish a normal hemopoiesis and immune function of donor type to substitute the intrinsic abnormal ones of recipient. Following transplantation, doctors must estimate the success or failure of transplantation, carry out immunosuppressive therapy timely and infer prognosis. The best method is to quantitatively detect the proportion of donor cells existed in chimera fathermore dynamicly monitor its change trend. In addition, the chimera study provided an excellent tool for developing the immunology and transplantation theory.
From the point of view of genetics, the chimera was a mixture containing two types of cell with different genotype. Therefore, some genetic markers could be used to discriminate donor cells or recipient cells and to quantitate their proportion. Some technology and methods, such as FISH and RFLP-PCR, VNTR/STR-PCR, etc.which are based on the first and second generation of genetic markers, were used previously. The disadvantages of these methods were lower sensitivity (5%-1% of STR-PCR method) and narrow detection limit (FISH can only differentiate sex). Recently, the sensitivity for chimera analysis was needed to reach more height point when people used it in monitoring prognosis and minimal residual disease of leukemia.
引文
[1]．高纯，谢佩蓉．嵌合体的检测及临床应用．国外医学临床生物化学与检验学分册．2003，24(1)：38-40
[2]. Starzl TE, Demetris AJ, Murase N, et al. Lancet 1992;339:1579-82
[3]．李素霞，达万明．异基因造血干细胞移植后嵌合体的检测及临床意义．中华器官移植杂志．2004，25(4)：253-254．
[4]. Thiede C, Bomhauser M,Ehninger G. Strategies and clinical implica-tions of chimerism diagnostics after allogeneic hematopoietic stem cell transplantation. Acta Haematol. 2004; 112(1-2):16-23.
[5]. Palka G, Stuppia L et al. FISH detection of mixed chimerism in 33 patients submitted to bone marrow transplantation. Bone Marrow Transplant 1996,17 (2):231-6.
[6]. Blazar BR, Orr HT, Arthur DC, Kersey JH, Filipovich AH Restriction fragment length polymorphisms as markers of engraftment in allogeneic marrow transplanttation. Blood 1985, 66:1436-1444
[7]. Scharf SJ, Smith AG, Hansen JA, McFadand C, Erlich HA Quantitative determination of bone marrow transplant engraftment using fluorescent polymerase chain reaction primers for human identity markers. Blood 1995, 85:1954-1963
[8]. Frankel W, Chan A, Corringham RE, Shepherd S, Rearden A, Wang-Rodrignez J: Detection of chimerism and early engraftment after allogeneic peripheral blood stem cell or bone marrow transplantation by short tandem repeats. Am J Hematol 1996, 52:281-287
[9]. Ephraim P. Hochberg, David B. Miklos, et al. A novel rapid single nucleotide polymorphism (SNP)-based method for assessment of hematopoietic chimerism after allogeneic stem cell transplantation. Blood.2003, 101:363-369.
[10]. M Fredriksson, G Barbany, et al. Assessing hematopoietic chimerism after allogeneic stem cell transplantation by multiplexed SNP genotyping using microarrays and quantitative analysis of SNP alleles Leukemia 2004,18:255-266
[11] Stock W, Yu D, Karrison T, Sher D, Stone RM, Larson RA, Bloomfield CD Quantitative real-time RT-PCR monitoring of BCR-ABL in chronic myelogenous leukemia shows lack of agreement in blood and bone marrow samples. Int J oncol 2006, 28(5):1099-103.
[12] van Tol MJ, Langlois van den Bergh R, Mesker W, Ouwerkerk-van Velzen MC, Vossen JM, Tanke HJ: Simultaneous detection of X and Y chromosomes by two- colour fluorescence in situ hybridization in combination with immunopheno- typing of single cells to document chimaerism after sex-mismatched bone marrow transplantation. Bone Marrow Transplant 1998,21:497-503.
[13] Thiede C. Diagnostic chimerism analysis after allogeneic stem celltransplantation: new methods and markers. Am J Pharmacogenomics. 2004,4(3):177-87.
[14] Tania N. Masmas, Hans O. Madsen, et al. Evaluation and Automation of Hemato- poietic Chimerism Analysis Based on Real-Time Quantitative Polymerase Chain Reaction Biology of Blood and Marrow Transplantation 2005,11:558-566
[15]. F Maasl, N Schaap2, S Kolen et al. Quantification of donor and recipient hemopoietic cells by real-time PCR of singlenucleotide polymorphisms. Leukemia 2003,17:621-629
[16]. Mehdi Alizadeh, Marc Bernard, et al. Quantitative assessment of hematopoietic chimerism after bone marrow transplantation by real-time quantitative polymerase chain reaction. Blood 2002,15:4618-25
[17] Dwight H. Oliver, Richard E. Thompson, Constance A. Griffin, James R.、 Eshleman Use of Single Nucleotide Polymorphisms (SNP) and Real-Time、 Polymerase Chain Reaction for BoneMarrow Engraftment Analysis. Journal of Molecular Diagnostics 2000,2 (4):202-208
[18] Venkatesan R, Sarkar R, Old JM beta-Thalassaemia mutations and their linkage to beta-haplotypes in Tamil Nadu in southern India. Clin Genet 1992 Nov;42(5):251-6.
[19] S0ren Germer, Michael J. Holland, and Russell Higuchi High-Throughput SNP、 Allele-FrequencyDetermination in Pooled DNA Samplesby Kinetic PCR. Genome Research 2000,10:258-266
[20] C.R.Newton, A.Graham. PCR, Second Edition. BIOS Scientific Publishers Limited 1997:135-137
[21] C.W.迪芬巴赫, G.S.德维克斯勒. PCR技术实验指南。科学出版社。96-97
[22] Stein CA, Benimetskaya L, Mani S. Antisense strategies for oncogene inactivation. Semin Oncol 2005 Dec;32(6): 563-72.
[23] Lu Y. Recent advances in the stereocontrolled synthesis of antisense phosphorothioates. Mini Rev Med Chem 2006 Mar;6(3):319-30.
[24] Gebski BL, Errington SJ et al. Terminal antisense oligonucleotide modifications can enhance induced exon skipping. Neuromuscul Disord. 2005 Oct; 15(9-10): 622-9.
[25] Gale JM. Tafoya GB. Evaluation of 15 polymerases and phosphorothioate primer modify- cation for detection of UV-induced C:G to T:A mutations by allele-specific PCR. Photochem Photobiol. 2004 May;79(5):461-9.
[26] Sandeep Verma and Fritz Eckstein. MODIFIED OLIGONUCLEOTIDES: Synthesis and Strategy for Users. Annu. Rev. Biochem. 1998. 67:99-134
[27]. Burgers, P. M. J.; Eckstein, F.; Hunneman, D. H. Stereochemistry of Hydrolysis by Snake-Venom Phosphodiesterase. Journal of Biological Chemistry 1979, 254, 7476-7478.
[28]. Scott D. Putney, Stephen J. Benkovic, Paul. R. Schimmel. A DNA fragment with an a-phosphorothioate nucleotide at one endis asymmetrically blocked from digestion by exonuclease III and can be replicated in vivo. Proc. Nati Acadi Sci. 1981,78(12):7350-54.
[29]. Burgers, P. M. J.; Eckstein, F. Study of the Mechanism of DNA-Polymerase-I fromEscherichia-Coli with Diastereomeric Phosphorothioate Analogs of DeoxyadenosineTriphosphate. Journal of Biological Chemistry 1979, 254, 6889-6893.
[30]. Brautigam, C. A.; Steitz, T. A. Structural principles for the inhibition of the 3'-5' exonuclease activity of Escherichia coli DNA polymerase I by phosphorothioates. Journal of Molecular Biology 1998, 277, 363-311.
[31] Gupta, A., DeBrosses, C, and Benkovic, S. J. Template-Primer-dependent Turnover of (S,)-dATPaS by T4 DNAPolymerase. J. Biol. Chem. 1982, 257: 7689-92
[32]. Harry Thorogood, Jane A. Grasby, Bernard A. Connolly. Influence of the Phosphate Backbone on the Recognition and Hydrolysis of DNA by the EcoRV Restriction Endonuclease.The journal of biological chemistry.l996,271(15)8855- 62
[33]. J R Sayers, D B Olsen, and F Eckstein. Inhibition of restriction endonuclease hydrolysis by phosphorothioate-containing DNA. Nucleic Acids Res. 1989 November 25; 17(22): 9495
[34].A Skerra.Phosphorothioate primers improve the amplification of DNA sequences by DNA polymerases with proofreading activity. Nucleic Acids Research. 1992,20(14):3551-3554,
[35]. Burgers, P. M. J.; Eckstein, F. Diastereomers of 5'-O-Adenosyl 3'-O-Uridyl Phosphorothioate - Chemical Synthesis and Enzymatic Properties. Biochemistry 1979, 18, 592-596.
[36]. Potter, B. V. L.; Connolly, B. A.; Eckstein, F. Synthesis and Configurational Analysis of a Dinucleoside Phosphate Isotopically Chiral at Phosphorus - Stereochemical Course ofPenicillium-Citrum Nuclease P1 Reaction. Biochemistry 1983, 22,1369-1377.
[37].Burgers PM, Sathyanarayana BK, Saenger W, Eckstein F. Crystal and molecular structure of adenosine 5'-O-phosphorothioate O-p-nitrophenyl ester (Sp diastereomer). Substrate stereospecificity of snake venom phosphodiesterase. Eur. J. Biochem.1979 Oct 15;100(2):585-91.
[38]. Chad A. Brautigam Thomas A. Steitz. Structural Principles for the Inhibition of the 3'-5'Exonuclease Activity of Escherichia coli DNAPolymerase I by Phosphorothioates J. Mol. Biol. 1998, 277:363-377
[39]. Daniel Di Giusto and Garry C. King. Single base extension (SBE) with proofreading polymerases and phosphorothioate primers:improved fidelity in single-substrate assaysNucleic Acids Research, 2003, Vol. 31, No. 3 e7
[40]. Jia Zhang, Kai Li, Duanfang Liao, Jose R. Pardinas, Linling Chen, and Xu\ Zhang. Different Applications of Polymerases With andWithout Proofreading Activity in Single-Nucleotide Polymorphism Analysis. Laboratory Investigation. 2003, 83 (8):1147-54
[41]. Hui-Ling Yang , Hu-Jun Jiang, Wei-Yi Fang, Yang-Yan Xua, Kai Lia, Jia Zhang,Duan-Fang Liao, Fu-Chu He. High fidelity PCR with an off/on switch mediated by proofreadingpolymerases combining with phosphorothioate- modified primer. Biochemical and Biophysical Research Communications 2005,328:265-272
[42]. Takashi Uemori, Yoshizumi Ishino, Hiroyuki Toh, Kiyozo Asada, Ikunoshin Kato. Organization and nucleotide sequence of the DNA polymerase gene from the archaeon Pyrococcus furiosus. Nucleic Acid Research.1993,21(2):259-265
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[47]. Michael Liew, Robert Pryor, Robert Palais, Cindy Meadows, Maria Erali, Elaine Lyon, Carl Wittwer. Genotyping of Single-Nucleotide Polymorphisms by High- Resolution Melting of Small Amplicons.Clinical Chemistry 2004, 50:71156-1164
[48]．卢圣栋．现代分子生物学实验技术．第二版．北京：中国协和医科大学出版社，1999．
[49]. Harry Thorogood, Jane A. Grasby, Bernard A. Connolly. Influence of the Phosphate Backbone on the Recognition and Hydrolysis of DNA by the EcoRV Restriction Endonuclease. The Journal of Biological Chemistry. 1996,271 (15) :8855-62
[50]. Stefan Loverix, Anna Winqvist, Roger Stromberg and Jan Steyaert. Mechanism of RNase T1: concerted triester-like phosphoryl transfer via a catalytic three-centered hydrogen bond Chemistry & Biology 2000, 7:651-658
[2]. Starzl TE, Demetris AJ, Murase N, et al. Lancet 1992;339:1579-82
[3]．李素霞，达万明．异基因造血干细胞移植后嵌合体的检测及临床意义．中华器官移植杂志．2004，25(4)：253-254．
[4]. Thiede C, Bomhauser M,Ehninger G. Strategies and clinical implica-tions of chimerism diagnostics after allogeneic hematopoietic stem cell transplantation. Acta Haematol. 2004; 112(1-2):16-23.
[5]. Palka G, Stuppia L et al. FISH detection of mixed chimerism in 33 patients submitted to bone marrow transplantation. Bone Marrow Transplant 1996,17 (2):231-6.
[6]. Blazar BR, Orr HT, Arthur DC, Kersey JH, Filipovich AH Restriction fragment length polymorphisms as markers of engraftment in allogeneic marrow transplanttation. Blood 1985, 66:1436-1444
[7]. Scharf SJ, Smith AG, Hansen JA, McFadand C, Erlich HA Quantitative determination of bone marrow transplant engraftment using fluorescent polymerase chain reaction primers for human identity markers. Blood 1995, 85:1954-1963
[8]. Frankel W, Chan A, Corringham RE, Shepherd S, Rearden A, Wang-Rodrignez J: Detection of chimerism and early engraftment after allogeneic peripheral blood stem cell or bone marrow transplantation by short tandem repeats. Am J Hematol 1996, 52:281-287
[9]. Ephraim P. Hochberg, David B. Miklos, et al. A novel rapid single nucleotide polymorphism (SNP)-based method for assessment of hematopoietic chimerism after allogeneic stem cell transplantation. Blood.2003, 101:363-369.
[10]. M Fredriksson, G Barbany, et al. Assessing hematopoietic chimerism after allogeneic stem cell transplantation by multiplexed SNP genotyping using microarrays and quantitative analysis of SNP alleles Leukemia 2004,18:255-266
[11] Stock W, Yu D, Karrison T, Sher D, Stone RM, Larson RA, Bloomfield CD Quantitative real-time RT-PCR monitoring of BCR-ABL in chronic myelogenous leukemia shows lack of agreement in blood and bone marrow samples. Int J oncol 2006, 28(5):1099-103.
[12] van Tol MJ, Langlois van den Bergh R, Mesker W, Ouwerkerk-van Velzen MC, Vossen JM, Tanke HJ: Simultaneous detection of X and Y chromosomes by two- colour fluorescence in situ hybridization in combination with immunopheno- typing of single cells to document chimaerism after sex-mismatched bone marrow transplantation. Bone Marrow Transplant 1998,21:497-503.
[13] Thiede C. Diagnostic chimerism analysis after allogeneic stem celltransplantation: new methods and markers. Am J Pharmacogenomics. 2004,4(3):177-87.
[14] Tania N. Masmas, Hans O. Madsen, et al. Evaluation and Automation of Hemato- poietic Chimerism Analysis Based on Real-Time Quantitative Polymerase Chain Reaction Biology of Blood and Marrow Transplantation 2005,11:558-566
[15]. F Maasl, N Schaap2, S Kolen et al. Quantification of donor and recipient hemopoietic cells by real-time PCR of singlenucleotide polymorphisms. Leukemia 2003,17:621-629
[16]. Mehdi Alizadeh, Marc Bernard, et al. Quantitative assessment of hematopoietic chimerism after bone marrow transplantation by real-time quantitative polymerase chain reaction. Blood 2002,15:4618-25
[17] Dwight H. Oliver, Richard E. Thompson, Constance A. Griffin, James R.、 Eshleman Use of Single Nucleotide Polymorphisms (SNP) and Real-Time、 Polymerase Chain Reaction for BoneMarrow Engraftment Analysis. Journal of Molecular Diagnostics 2000,2 (4):202-208
[18] Venkatesan R, Sarkar R, Old JM beta-Thalassaemia mutations and their linkage to beta-haplotypes in Tamil Nadu in southern India. Clin Genet 1992 Nov;42(5):251-6.
[19] S0ren Germer, Michael J. Holland, and Russell Higuchi High-Throughput SNP、 Allele-FrequencyDetermination in Pooled DNA Samplesby Kinetic PCR. Genome Research 2000,10:258-266
[20] C.R.Newton, A.Graham. PCR, Second Edition. BIOS Scientific Publishers Limited 1997:135-137
[21] C.W.迪芬巴赫, G.S.德维克斯勒. PCR技术实验指南。科学出版社。96-97
[22] Stein CA, Benimetskaya L, Mani S. Antisense strategies for oncogene inactivation. Semin Oncol 2005 Dec;32(6): 563-72.
[23] Lu Y. Recent advances in the stereocontrolled synthesis of antisense phosphorothioates. Mini Rev Med Chem 2006 Mar;6(3):319-30.
[24] Gebski BL, Errington SJ et al. Terminal antisense oligonucleotide modifications can enhance induced exon skipping. Neuromuscul Disord. 2005 Oct; 15(9-10): 622-9.
[25] Gale JM. Tafoya GB. Evaluation of 15 polymerases and phosphorothioate primer modify- cation for detection of UV-induced C:G to T:A mutations by allele-specific PCR. Photochem Photobiol. 2004 May;79(5):461-9.
[26] Sandeep Verma and Fritz Eckstein. MODIFIED OLIGONUCLEOTIDES: Synthesis and Strategy for Users. Annu. Rev. Biochem. 1998. 67:99-134
[27]. Burgers, P. M. J.; Eckstein, F.; Hunneman, D. H. Stereochemistry of Hydrolysis by Snake-Venom Phosphodiesterase. Journal of Biological Chemistry 1979, 254, 7476-7478.
[28]. Scott D. Putney, Stephen J. Benkovic, Paul. R. Schimmel. A DNA fragment with an a-phosphorothioate nucleotide at one endis asymmetrically blocked from digestion by exonuclease III and can be replicated in vivo. Proc. Nati Acadi Sci. 1981,78(12):7350-54.
[29]. Burgers, P. M. J.; Eckstein, F. Study of the Mechanism of DNA-Polymerase-I fromEscherichia-Coli with Diastereomeric Phosphorothioate Analogs of DeoxyadenosineTriphosphate. Journal of Biological Chemistry 1979, 254, 6889-6893.
[30]. Brautigam, C. A.; Steitz, T. A. Structural principles for the inhibition of the 3'-5' exonuclease activity of Escherichia coli DNA polymerase I by phosphorothioates. Journal of Molecular Biology 1998, 277, 363-311.
[31] Gupta, A., DeBrosses, C, and Benkovic, S. J. Template-Primer-dependent Turnover of (S,)-dATPaS by T4 DNAPolymerase. J. Biol. Chem. 1982, 257: 7689-92
[32]. Harry Thorogood, Jane A. Grasby, Bernard A. Connolly. Influence of the Phosphate Backbone on the Recognition and Hydrolysis of DNA by the EcoRV Restriction Endonuclease.The journal of biological chemistry.l996,271(15)8855- 62
[33]. J R Sayers, D B Olsen, and F Eckstein. Inhibition of restriction endonuclease hydrolysis by phosphorothioate-containing DNA. Nucleic Acids Res. 1989 November 25; 17(22): 9495
[34].A Skerra.Phosphorothioate primers improve the amplification of DNA sequences by DNA polymerases with proofreading activity. Nucleic Acids Research. 1992,20(14):3551-3554,
[35]. Burgers, P. M. J.; Eckstein, F. Diastereomers of 5'-O-Adenosyl 3'-O-Uridyl Phosphorothioate - Chemical Synthesis and Enzymatic Properties. Biochemistry 1979, 18, 592-596.
[36]. Potter, B. V. L.; Connolly, B. A.; Eckstein, F. Synthesis and Configurational Analysis of a Dinucleoside Phosphate Isotopically Chiral at Phosphorus - Stereochemical Course ofPenicillium-Citrum Nuclease P1 Reaction. Biochemistry 1983, 22,1369-1377.
[37].Burgers PM, Sathyanarayana BK, Saenger W, Eckstein F. Crystal and molecular structure of adenosine 5'-O-phosphorothioate O-p-nitrophenyl ester (Sp diastereomer). Substrate stereospecificity of snake venom phosphodiesterase. Eur. J. Biochem.1979 Oct 15;100(2):585-91.
[38]. Chad A. Brautigam Thomas A. Steitz. Structural Principles for the Inhibition of the 3'-5'Exonuclease Activity of Escherichia coli DNAPolymerase I by Phosphorothioates J. Mol. Biol. 1998, 277:363-377
[39]. Daniel Di Giusto and Garry C. King. Single base extension (SBE) with proofreading polymerases and phosphorothioate primers:improved fidelity in single-substrate assaysNucleic Acids Research, 2003, Vol. 31, No. 3 e7
[40]. Jia Zhang, Kai Li, Duanfang Liao, Jose R. Pardinas, Linling Chen, and Xu\ Zhang. Different Applications of Polymerases With andWithout Proofreading Activity in Single-Nucleotide Polymorphism Analysis. Laboratory Investigation. 2003, 83 (8):1147-54
[41]. Hui-Ling Yang , Hu-Jun Jiang, Wei-Yi Fang, Yang-Yan Xua, Kai Lia, Jia Zhang,Duan-Fang Liao, Fu-Chu He. High fidelity PCR with an off/on switch mediated by proofreadingpolymerases combining with phosphorothioate- modified primer. Biochemical and Biophysical Research Communications 2005,328:265-272
[42]. Takashi Uemori, Yoshizumi Ishino, Hiroyuki Toh, Kiyozo Asada, Ikunoshin Kato. Organization and nucleotide sequence of the DNA polymerase gene from the archaeon Pyrococcus furiosus. Nucleic Acid Research.1993,21(2):259-265
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