Method and apparatus for cleaving deoxyribonucleic acid

• US 5,106,585 A
• Filed: 10/29/1984
• Issued: 04/21/1992
• Est. Priority Date: 03/02/1983
• Status: Expired due to Fees

• Current Assignee: Mitsui Chemicals Incorporated (Mitsui Chemicals)
• Original Assignee: Mitsui Chemicals Incorporated (Mitsui Chemicals)
• Inventors: Sasaki, Toshihiro, Kusuhara, Nobumi, Onofusa, Mitsuio, Minami, Tamotsu
• Primary Examiner(s): Elmore, Carolyn S.
• Application Number:
  US06/672,253
• Time in Patent Office: 2,731 Days
• Field of Search: 435/287, 435/91, 422/131, 422/134, 422/116, 422/68.1, 422/70, 422/187, 935/88
• US Class Current: 422/68.1
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/6869 Methods for sequencing

Abstract

The invention relates to a procedure for chemically determining the base sequence of DNA and, in particular, its step of cleaving the labeled DNA having undergone a base-selective chemical modification at the position of the modified base. More particularly, it relates to a method for cleaving DNA which comprises modifying DNA base-selectively with chemical reagents, bringing the resulting reaction mixture into contact with an adsorbent to adsorb the chemically modified DNA on the adsorbent, washing the adsorbent with a washing fluid to remove any chemical modification reagents from the adsorbent, bringing the adsorbent into contact with an eluent to elute the chemically modified DNA adsorbed thereon, concentrating the resulting eluate and then heating it together with a cleavage reagent.

This invention also relates to apparatus for carrying out the above-described method which comprises a reactor 1 for receiving the reaction mixture having undergone a chemical modification reaction of DNA, a transfer pump 4, an adsorber 2 containing an adsorbent, a three-way valve 5 and a concentrator 3, these units being interconnected by conduits in the order mentioned.

Claims

1. Apparatus for cleaving deoxyribonucleic acid, which comprises:

(a) a reactor containing a reaction mixture comprising deoxyribonucleic acid which is to be subjected to a chemical modification reaction, whereby at least one base of the deoxyribonucleic acid is chemically modified;

(b) a transfer pump connected by conduit to said reactor for bringing said reaction mixture having undergone a chemical modification reaction into contact with an adsorber;

(c) an adsorber connected by conduit to said transfer pump, said adsorber comprising an adsorbent for adsorbing and retaining the chemically modified deoxyribonucleic acid, said adsorber further comprising washing means for washing the adsorbent with a washing fluid to remove any chemical modification reagents from said adsorbent, said adsorber further comprising eluting means for eluting chemically modified deoxyribonucleic acid from the adsorbent; and

(d) a concentrator connected by conduit to said adsorber for concentrating DNA eluates received from the adsorber, said concentrator further comprising cleavage reagent supplying means for adding cleavage reagent to the concentrator, said concentrator further comprising heating means for heating the cleavage reagent and DNA eluates, whereby the deoxyribonucleic acid is cleaved at the position of the modified base.

2. Apparatus as claimed in claim 1 wherein said adsorber is packed with an adsorbent selected from the group consisting of ion exchanges, nonionic porous resins and fine-granular materials adapted for reversed phase partition.

3. Apparatus as claimed in claim 1 wherein said concentrator is adapted to be capable of controlling the concentration vessel at an arbitrarily selected temperature and subatmospheric pressure and permit rotation, revolution, precession and vibration of said vessel.

4. Apparatus as claimed in claim 1 wherein the operation of the apparatus is automated by means of a sequencer for elements (a) to (d) of the apparatus.

5. Apparatus for the selective cleavage of DNA for four bases simultaneously, comprising four reaction vessels in concentrator (d) of claim 1 and three sets of elements (a) to (c) of claim 1 arranged in parallel.

6. An apparatus for determining the base sequence of deoxyribonucleic acid comprising:

(a) a reaction vessel containing a reaction mixture comprising radioactive phosphorous-labeled DNA;

(b) a DNA adsorber attached to said vessel, said DNA adsorber further comprising an adsorbent for adsorbing DNA;

(c) means for washing adsorbed DNA to remove any reagents used for chemical modification;

(d) means for eluting the adsorbed DNA from the adsorbent, whereby an eluate is obtained;

(e) a concentrator attached to said adsorber for collecting and concentrating the eluate;

(f) means for introducing a cleavage reagent attached to said concentrator whereby modified DNA strands located in said concentrator can be cleaved where modified bases are positioned to provide DNA fragments.

7. The apparatus of claim 6 further comprising a conduit between said vessel and said adsorber and a transfer pump located on said conduit for transferring fluids to said adsorber.

8. The apparatus of claim 7 further comprising a waste liquid tank connected by conduit to said adsorber for collecting effluent from said adsorber.

9. The apparatus of claim 8 wherein said adsorber has inlet and outlet means, and wherein said conduit attached to said waste liquid tank is connected to said outlet means.

10. The apparatus of claim 9 wherein said conduit attached to said waste liquid tank is provided with a three-way valve.

11. The apparatus of claim 10 wherein said concentrator is connected to said adsorber by a conduit connected to said three-way valve.

12. The apparatus of claim 11 where the concentrator further comprises a vessel for collecting an eluate from said adsorber, a vacuum pump, a vibrator, heating means and a trap for collecting evaporated liquid.

13. The apparatus of claim 12 wherein said means for washing is a washing fluid tank containing washing fluid connected by conduit to said reaction vessel, said means for washing further comprising a two-way valve located on the conduit connecting said washing fluid tank to said reaction vessel, whereby flow of the washing fluid is controlled.

14. The apparatus of claim 13 wherein said means for eluting is an eluent tank containing eluent connected by conduit to said reaction vessel, said eluent tank further comprising a two-way valve located on the conduit connecting said eluent tank to the reaction vessel, whereby flow of the eluent is controlled.

15. The apparatus of claim 14 wherein said means for introducing a cleavage reagent is a cleavage reagent tank containing a cleavage reagent connected by conduit to said concentrator, said cleavage reagent tank further comprising a two-way valve located on said conduit connecting said cleavage reagent tank to said concentrator, whereby flow of the cleavage reagent is controlled.

16. The apparatus of claim 6 wherein the adsorbent comprises cation, anion or nonionic porous resins.

17. The apparatus of claim 6 wherein the adsorbent comprises polystyrene as base material, cellulose as base material, AMBERLITE XAD-2 or LEVACIT OC1031.

18. Apparatus as claimed in claim 6 wherein the operation of the apparatus is automated by means of a sequencer for elements (a) to (f) of the apparatus.

19. Apparatus for the selective cleavage of DNA for four bases simultaneously, comprising four reaction vessels in concentrator (e) of claim 6, means (f) of claim 6 can separately deliver to each of the four reaction vessels in concentrator (e), and three sets of elements (a) to (d) of claim 6 arranged in parallel.