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    Recent Progress of Life Science Technology in Japan
    Recent Progress of Life Science Technology in Japan
    Recent Progress of Life Science Technology in Japan
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    Recent Progress of Life Science Technology in Japan

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    Recent Progress of Life Science Technology in Japan discusses developments in cancer research technologies in Japan. In June 1983 an intra-cabinet panel of the Japanese Government drafted a 10-year strategy for cancer control, recognizing the importance of this field of research. A scientific research group was organized to comprise two sections—the first concerning the development and evaluation of DNA technologies, and the second on protein-related technologies. In the promotion of fundamental cancer research, the development and refinement of basic technologies for each component of the ""triangle of bio-sciences""—DNA, protein, and antibody—are essential, particularly in the elucidation of tumor-inducing and tumor-suppressing genes, tumor-specific antigens, and so forth. Part I of the book details the achievements of the first group in developing automated instrumentations for DNA sequencing. The second scientific research group worked on three major subareas: (1) gene transfer and expression technologies; (2) technologies for extraction, purification, and structural analysis of cancer-related proteins; and (3) technologies for analysis and synthesis of saccharide chains. Reports from these areas are respectively grouped in Part II, Part III, and Part IV of this monograph.
    LanguageEnglish
    PublisherAcademic Press
    Release dateJun 28, 2014
    ISBN9781483273259
    Recent Progress of Life Science Technology in Japan

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      Recent Progress of Life Science Technology in Japan - Yoji Ikawa

      Wada

      PART I

      DNA SEQUENCING TECHNOLOGY

      Outline

      Chapter 1: TEMPERATURE–GRADIENT DNA–PROBE COLUMN CHROMATOGRAPHY: A NEW METHOD FOR DETECTION AND PURIFICATION OF PARTICULAR DNAS OR RNAS

      Chapter 2: Development of Automatic Technics of Purification Process of M13 Single Strand DNA and Sequence Reaction Based on Sanger Method

      Chapter 3: Development of Electrophoresis Precast Gel for DNA Sequencing

      Chapter 4: REAL-TIME DNA DETECTION SYSTEMS

      Chapter 5: TWO DIMENSIONAL IMAGE PROCESSING OF ELECTRON MICROGRAPHS OF tRNA THIN CRYSTALS

      TEMPERATURE–GRADIENT DNA–PROBE COLUMN CHROMATOGRAPHY: A NEW METHOD FOR DETECTION AND PURIFICATION OF PARTICULAR DNAS OR RNAS

      Akira Suyama¹ and Hiromichi Tsurui

      Mitsuru Yoneyama and Akiyoshi Wada²,³,     Department of Physics, Faculty of Science, The University of Tokyo, Tokyo, Japan

      Nobuyuki Baba²,     Scientific Instrument Division, Toyo Soda Manufacturing Co., Ltd., Ayase, Japan

      Publisher Summary

      This chapter describes the physical basis of the equilibrium thermal stability of hybrid duplexes formed between DNA probes and sample DNAs or RNAs. Tetraalkylammonium salt is useful for detecting the base sequence difference between DNA probes and sample DNAs on the basis of the thermal stability difference of the hybrid duplex, because this salt eliminates the stability difference between two complementary (A-T and G-C) base pairs. As a result, the thermal stability of the hybrid duplex depends only on base mismatches created by the base sequence difference, provided that DNA probes of the same length are used. This is required to establish the quantitative and high sequence-resolution DNA probing method. The chapter also describes the principle, instrumentation, performance, and application of the new DNA probe method.

      I INTRODUCTION

      DNA base sequences are somewhat like fingerprints and unique to individual genes or genomes. As a consequce, the sequences can be used to distinguish particular genes or genomes from others. At the present time a fairly large number of base sequences have been determined for various genes and genomes which cause diseases, and the number is increasing. Therefore, a method which enables one to detect particular base sequences would provide a good tool for (i) the diagnosis of various diseases, including genetic disease, cancer, and infectious disease, (ii) the detection of bacteria in foods, and (iii) the screening or the purification of particular DNAs and RNAs.

      DNA-probes have been devised for the purpose of discriminating particular base sequences from others. Various methods using DNA probes have been developed and have been applied for the diagnosis of diseases, human chromosome mapping, and screening of particular DNAs and RNAs(1–3). Some of them are very powerful, and capable of detecting single-base changes. Their detection procedures rely on changes in restriction enzyme cleavage sites (4–7), on S1 nuclease (8, 9) or RNase A cleavage (10) of base mismatches caused by single-base changes, on chemical modification of base mis-matches (11), or on reduction in DNA duplex stability caused by base mismatches (12–22). However, the procedure always seems to be fairly complicated for non-experts, and at least one day is required to finish the protocol. In addition, none of them could perform simultaneous discrimination and purification, though this would supply very powerful basic methods to molecular biology and genetic engineering. For these reasons, we have developed a new method, temperature-gradient DNA-probe column chromatography, which provides accurate and reproducible DNA probing. The method is easy and rapid (within two hours) to perform, and simultaneous purification of samples is available. Its sequence resolution is extremely high so that single-base mismatch can be detected. The present method is a kind of affinity chromatography using immobilized DNA probes so that simultaneous discrimination and purification can be accomplished. However, no former affinity chromatography using immobilized nucleic acids (23–27) have succeeded in single-base mismatch detection and rapid manipulation.

      In the present report, we first describe the physical basis of the equilibrium thermal stability of hybrid duplexes formed between DNA probes and sample DNAs or RNAs. This is required to establish the quantitative and high sequence-resolution DNA probing method. Then we describe the principle, the instrumentation, the performance and the application of our new DNA probe method.

      II THERMAL STABILITY OF PROBE-SAMPLE HYBRID DUPLEX

      A Hybrid Stability as the Basis of DNA Probing

      DNA probes and sample DNAs or RNAs are capable of hybridizing each other through the complementary base-pairing and result in double-stranded hybrid formation. The strength of DNA probe hybridization to sample molecules is determined by the stability of the hybrid duplex formed. The other parts of each sample molecule, which is usually longer than DNA probe, remains on either side of the hybrid duplex and does not affect the hybridization strength since they have no interactions with the probe.

      The formed hybrid duplexes may be melted into single-stranded random coils by elevating temperature. Their thermal stability under a given solvent condition is determined by three factors: (i) the G+C content and base sequence of DNA probes, (ii) the length of hybrid duplexes, and (iii) base mismatches in hybrid duplexes. The last factor allows the detection of base sequence difference between DNA probes and sample DNAs or RNAs using the thermal stability of the hybrid duplex, and thus provides the basis of DNA probing.

      B Elimination of Base-Pair Stability Difference

      Tetraalkylammonium salt is useful for detecting the base sequence difference between DNA probes and sample DNAs on the basis of the thermal stability difference of the hybrid duplex, since this salt eliminates the stability difference between two complementary (A-T and G-C) base pairs (28,29). As a consequence, the thermal stability of the hybrid duplex depends only on base mismatches created by the base sequence difference, provided that DNA probes of the same length are used.

      In the case of DNA hybrid duplexes, the salt concentration required for elimination of the stability difference is 2.4 M for tetraetylammonium chloride (TEACl) and 3.0 M for tetramethylammonium chloride (TMACl) (28). FIGURE 1 clearly shows the elimination effect on the melting of long natural DNA. The melting transition of ColE1 DNA occurs over a wide range of 15°C in the absence of TEACl due to the stability difference. In the presence of 2.4 M TEACl, on the other hand, the melting is remarkably sharpened and occurs within 1°C at a temperature which is independent of the G+C content and determined by the duplex length because of the elimination of the stability difference. Removal of the fine structures in broad melting profiles caused by the stability difference facilitates the detection of base sequence difference on the basis of the hybrid melting (30).

      FIGURE 1 Melting profiles of ColE1 DNA linearized with EcoR1 in 19.5 mM sodium-acetate buffer, pH 6.0, in the presence (TEACl+) or in the absence (TEACl–) of 2.4 M tetramethylammonium chloride (TEACl).

      C Thermal Stability of Hybrid with Base Mismatches

      When sample DNAs or RNAs lack the completely complementary base sequence to the DNA probe, base mismatches are created in the hybrid duplex formed. The base mismatches are lacking in the duplex-stabilizing interactions of the complementary base-pairs and as a consequence they destabilize the hybrid duplex (31–33). In order to use this stability reduction to detect the base sequence difference between DNA probes and sample DNAs or RNAs, it is necessary to clarify the quantitative effect of base mismatches on the thermal stability of the hybrid duplex.

      Base mismatches generally result in formation of internal or bulge loops or single-stranded tails in the hybrid duplex, as shown in Fig. 2. When the base sequence difference of sample DNAs or RNAs from the completely complementary base sequence of the DNA probe is caused by base insertion or deletion, bulge loops or single-stranded tails are formed in the hybrid duplex. When samples have different base sequences caused by base replacement, internal loops or single-stranded tails are formed. Although all types of base mismatches have the effect of reducing the hybrid duplex stability regardless of the presence of the tetraalkylammonium salt, it is not easy to derive quantitative relationship between base mismatches and the stability reduction which is sufficiently general to apply to all types of base

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