DNA sequencing apparatus

• US 5,221,518 A
• Filed: 08/13/1991
• Issued: 06/22/1993
• Est. Priority Date: 12/14/1984
• Status: Expired due to Fees

• Current Assignee: Randell L. Mills
• Original Assignee: Randell L. Mills
• Inventors: Mills, Randell L.
• Primary Examiner(s): Housel, James C.
• Assistant Examiner(s): REDDING, DAVID A
• Application Number:
  US07/744,697
• Time in Patent Office: 679 Days
• Field of Search: 422/62, 422/67, 422/82.05, 436/89, 436/94, 436/161, 436/173, 435/6, 435/291
• US Class Current: 422/62
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: G01N 35/00 Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
• CPC Class Codes: G01R 33/465 applied to biological material, e.g. in vitro testing

Abstract

The DNA sequencing apparatus contains the following components. A first reaction vessel contains a reaction chamber into and out of which can be transferred reagents, reactants and reaction products. A device for separating individual oligonucleotides and polynucleotides on the basis of length, such as an electrophoretic unit, receives reaction products from the first reaction vessel. A second reaction vessel is designed for oxidizing pentose sugars. It comprises a reaction chamber having a device for transferring reactants and reagents into the reaction chamber and gaseous by-products of a reaction out of the reaction chamber. Separated oligonucleotides and polynucleotides can be selectably transferred from the separating device alternatively into the first or the second reaction vessel. The device also includes a second transfer device for transferring the gaseous by-products out of the second reaction vessel and a collection chamber for collecting the gaseous by-products. The collection chamber is in communication with the second transfer device. Finally, the apparatus contains an analyzer for analyzing the relative abundance of the components of the gaseous by-product by mass such as a mass spectrometer, the analyzer being in communication with a transfer device for transferring gaseous by-products from the collection chamber to the analyzer.

Claims

1. A DNA sequencing apparatus, comprising:

a) a first reaction vessel comprising a reaction chamber having a first means for transfer of reagents, reactants and reaction products into and out of the chamber;

b) means for separating individual oligonucelotides and polynucleotides on the basis of length, the separating means having means for receiving reaction products from the first reaction vessel;

c) a second reaction vessel for oxidizing pentose sugars comprising a second reaction chamber having a second means for transferring reactants and reagents into the reaction chamber and gaseous by-products of a reaction out of the reaction chamber;

d) means for selectably transferring separated oligonucelotides and polynucleotides from the separating means alternatively into the first or the second reaction vessel;

e) a third transfer means for transferring the gaseous by-products out of the second reaction vessel;

f) a collection chamber for collecting the gaseous by-products, the collection chamber being in communication with the third transfer means; and

g) means for analyzing the relative abundance of the components of the gaseous by-product by mass, the analyzing means being in communication with a fourth transfer means for transferring gaseous by-products from the collection chamber to the analyzing means.

2. A DNA sequencing apparatus of claim 1, further comprising a programmable control means for controlling;

a) the transfer of reagents into and out of the first and second reaction vessels;

b) the delivery of reaction products from the first reaction vessel to the separating means;

c) the separation of oligonucleotides and polynucleotides by the separating means;

d) the collection of separated oligonucleotides and polynucleotides from the separating means;

e) the delivery of collected, separated oligonucleotides and polynucleotides to the first and second reaction vessels;

f) the collection of gaseous by-product by the collection chamber;

g) the transfer of gaseous by-products to the analyzing means; and

h) the storing and processing the data generated by the analyzing means.

3. A DNA sequencing apparatus of claim 1, wherein said first means for transferring reagents and reactants into the first reaction vessel and transferring reaction products out of the first reaction vessel is a robotic dispenser.

4. A DNA sequencing apparatus of claim 1, wherein the separating means is an agarose or polyacrylamide gel electrophoresis unit.

5. A DNA sequencing apparatus of claim 1, wherein the means for separating includes means for monitoring the separated oligo and polynucleotides.

6. A DNA sequencing apparatus of claim 5, wherein the monitoring means comprises a uv absorption detector, a fluorescence detector or a scintillation detector.

7. A DNA sequencing apparatus of claim 1, wherein the means for selectably transferring comprises a selective two way valve communicating with a fluid pump.

8. A DNA sequencing apparatus of claim 1, wherein the collection chamber for collecting gaseous by-products is a gas cylinder having a piston injector.

9. A DNA sequencing apparatus of claim 1, wherein the analyzing means is a mass spectrometer.

10. A DNA sequencing apparatus of claim 9, wherein the mass spectrometer has means for analyzing the entire mass range of the gaseous by-products simultaneously.

11. A DNA sequencing apparatus of claim 1, wherein the analyzing means is an electro-optical ion detector array mass spectrometer.

12. A DNA sequencing apparatus of claim 2, wherein the programmable control means is a computer.

13. A DNA sequencing apparatus;

comprising;

a) a reaction vessel comprising a reaction chamber and a programmable robotic dispensing means for delivering reagents and reactants into the chamber and for transferring reaction products out of the chamber;

b) an electrophoretic unit having means for accepting reaction products from the reaction vessel;

c) a periodic acid oxidation cell having means for transferring reactants and reagents into the cell and gaseous by-products out of the cell;

d) a fluid pump in communication with a selectable two way valve, the electrophoretic unit, the reaction vessel, and the periodic acid oxidation cell;

e) a formaldehyde gas collection cell and piston injector in communication with the periodic acid oxidation cell;

f) a one way valve in communication with the formaldehyde gas collection cell;

g) a mass spectrometer in communication with the one way valve; and

a

h) programmable computer for controlling;

i) the reaction vessel;

ii) the electrophoretic unit;

iii) the pump and selectable two way valve;

iv) the formaldehyde gas collection cell and piston injector;

v) the one way valve; and

vi) the mass spectrometer.