Chemical apparatus and process disinfecting – deodorizing – preser – Analyzer – structured indicator – or manipulative laboratory... – Means for analyzing liquid or solid sample
Patent
1984-10-29
1992-04-21
Elmore, Carolyn S.
Chemical apparatus and process disinfecting, deodorizing, preser
Analyzer, structured indicator, or manipulative laboratory...
Means for analyzing liquid or solid sample
422 70, 422116, 422131, 422187, G01N 3002, G05B 1700
Patent
active
051065851
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
This invention relates to a procedure for chemically determining the base sequence of deoxyribonucleic acid and, in particular, its step of modifying radioactive phosphorus-labeled deoxyribonucleic acid base-selectively with chemical reagents and then cleaving the deoxyribonucleic acid strand at the position of the modified base. More particularly, it relates to a method for cleaving deoxyribonucleic acid by subjecting a reaction mixture containing deoxyribonucleic acid to a chemical modification reaction, separating the chemically modified deoxyribonucleic acid from the reaction mixture and then cleaving it at the position of the modified base and to apparatus for carrying out this method.
BACKGROUND ART
Recently, a number of procedures for determining the base sequence of deoxyribonucleic acid (hereinafter referred to as DNA) directly have been developed. Among others, the chemical sequence determination procedure developed by Maxam and Gilbert is being widely used because it has the advantages of involving relatively simple experimental operations and comparing favorably in rapidity and accuracy with other determination procedures. Details of this procedure are described in Proc. Natl. Acad. Sci., Vol. 74, p. 560, 1977, Tanpakushitsu-Kakusan-Koso [Protein,, Nucleic Acid and Enzyme (Japan)], Vol. 23, p. 182, 1978 and the like. Briefly stated, a DNA sample having one end labeled with radioactive phosphorus is prepared and the four bases constituting the DNA (i.e., guanine, adenine, thymine and cytosine which will hereinafter be referred to as G, A, T and C, respectively) are modified selectively with different chemical reagents. Then, with respect to each base, the DNA strand is cleaved at the position of the modified base and the resulting DNA fragments are subjected to gel electrophoresis in which the fragments are developed and separated in order of molecular chain length. By autoradiography, the developed and separated bands are recorded on an X-ray film and the bases corresponding to the bands on the X-ray film are identified to determine the base sequence of the original DNA sample.
Broadly divided, the above-described procedure can be said to comprise the following four steps: phosphorus; manner and cleaving the DNA strand at the position of the modified base; electrophoresis and recording the bands on an X-ray film; and sequence.
This Maxam-Gilbert procedure is being widely used because of its above-described advantages. However, since it has been conventional practice to carry out all of the foregoing steps by hand, determination of the base sequences of many DNA samples requires very troublesome operations and consumes much time. Therefore, it would be desirable to enhance the operating efficiency by mechanization of the steps.
Among the above-described four steps, the present invention is directed to the second step, i.e., the step of chemically modifying the labeled DNA in a base-selective manner and cleaving the DNA strand at the position of the modified base, and is concerned with a method and apparatus for mechanizing this step to enhance the operating efficiency. Conventionally, the second step has been carried out by injecting a sample solution containing the labeled DNA into four tubes made of resin, subjecting them separately to chemical modification reactions specific for four bases (i.e., G, A, T and C) using their respective reagents and reaction conditions, adding ethanol to each tube, cooling the mixture to a temperature of -40.degree. to -70.degree. C., centrifuging it and sucking out the supernatant, washing the precipitate repeatedly (by adding ethanol thereto, centrifuging the mixture and sucking out the supernatant) to remove therefrom any excess reagents used for the chemical modification, adding an aqueous solution of a cleavage reagent (sodium hydroxide or piperidine) to the precipitate and then heating the resulting mixture to cleave the DNA strand at the position of the modified base. The present inventors have concluded that, among the above-described
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Chemical Engineers' Handbook, Fifth Edition, (1973), pp. 21-60-21-62.
Proc. Natl. Acad. Sci. U.S.A., vol. 74, No. 2, pp. 560-564.
Kusuhara Nobumi
Minami Tamotsu
Onofusa Mitsuio
Sasaki Toshihiro
Elmore Carolyn S.
Mitsui Toatsu Chemicals Inc.
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