Apparatus for robotically performing sanger dideoxynucleotide DNA sequencing reactions using controlled pipet

• US 5,455,008 A
• Filed: 08/08/1994
• Issued: 10/03/1995
• Est. Priority Date: 10/16/1992
• Status: Expired due to Fees

• Current Assignee: Thomas Jefferson University (Main Line Health System)
• Original Assignee: Thomas Jefferson University (Main Line Health System)
• Inventors: Earley, James J., Kuivaniemi, Sisko H., Tromp, Gerardus C., Prockop, Darwin J.
• Primary Examiner(s): Housel, James C.
• Assistant Examiner(s): Ludlow, Jan M.
• Application Number:
  US08/287,105
• Time in Patent Office: 421 Days
• Field of Search: 422/63, 422/67, 422/100, 935/87, 73/863.01, 73/864.24, 73/864.25, 395/93, 901/10, 901/46, 318/568.16
• US Class Current: 422/511
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00315 Microtiter plates
• CPC Class Codes: B01J 2219/00355 peristaltic
• CPC Class Codes: B01J 2219/00364 Pipettes
• CPC Class Codes: B01J 2219/00495 Means for heating or cooling the reaction vessels
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00689 using computers
• CPC Class Codes: B01J 2219/00691 using robots
• CPC Class Codes: B01J 2219/00695 Synthesis control routines, e.g. using computer programs
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 60/14 for creating libraries
• CPC Class Codes: G01N 2035/00445 Other cooling arrangements
• CPC Class Codes: G01N 2035/00455 Controlling humidity in analyser
• CPC Class Codes: G01N 2035/042 moved independently, e.g. by fork manipulator
• CPC Class Codes: G01N 2035/0425 Stacks, magazines or elevators for plates
• CPC Class Codes: G01N 35/0099 comprising robots or similar manipulators robots per se B25J
• CPC Class Codes: G01N 35/028 having reaction cells in the form of microtitration plates
• CPC Class Codes: G01N 35/1002 Reagent dispensers
• CPC Class Codes: G01N 35/1011 : Control of the position or alignment of the transfer device
• CPC Class Codes: G01N 35/1065 : Multiple transfer devices

Abstract

A robotic system for performing nucleic acid sequencing reactions in microtiter plate format. The robot has a robot arm and its controller, robot hands with which it can manipulate microtiter plates with lids and microtiter plates without lids and with which it can perform pipetting in the 5 to 200 microliter range, cooled storage chambers with electronically controllable doors for storage of microtiter plates, cooled storage with an electronically controllable cover for storage of nucleic acid sequencing reagents, heating blocks to incubate the reactions at appropriate temperatures above ambient temperature, storage for boxes of micropipet tips, storage for microtiter plates in which sequence reactions will be performed and software for controlling the operation of the robot during pipetting.

Claims

1. A robotic system for automatically performing DNA sequencing, comprising:

a robot having an arm mounted for motion in vertical and horizontal planes;

a hand for said robot arm having means for pipetting a fluid from a microtiter plate well;

a microtiter plate having a well formed therein, said well having a bottom, a reagent solution contained in said well;

a plurality of work surfaces, each having means for supporting a microtiter plate;

a plurality of reservoirs, each having means for holding a reagent;

first storage means for storing microtiter plates;

sensing means for generating a signal in response to said pipetting means making contact with said microtiter plate well bottom; and

a programmable controller having (i) first logic means for instructing said robot to move said pipetting means toward said microtiter plate well bottom by a series of discrete incremental movements, each of said discrete incremental movements in said series being separated by a finite period of time, said series of incremental movements including a set of incremental movements in which each discrete incremental movement is successively smaller than each previous discrete incremental movement in said set, and (ii) second logic means for determining whether said pipetting means has made contact with said microtiter plate well bottom based on said signal from said sensing means during said finite period of time between each of said discrete incremental movements in said set of movements.

2. The robotic system according to claim 1, wherein said first logic means further comprises means for instructing said robot to move said pipetting means toward said microtiter plate well bottom by at least one additional set of incremental movements, each incremental movement in any such additional sets being successively smaller than each previous incremental movement in its respective set, additional sets of incremental movements being successively performed until said second logic means has determined that said pipetting means has contacted said microtiter plate well bottom.

3. The robotic system according to claim 1, wherein said set of incremental movements comprises at least three incremental movements.

4. The robotic system according to claim 1, wherein said sensing means comprises means for sensing a force acting on said hand, and wherein said second logic means comprises:

means for instructing said robot to sense an initial force acting on said hand prior to said set of incremental movements;

means for determining if said force sensed differs from said initial force by more than a first predetermined value after each of said incremental movements in said set of movements.

5. The robotic system according to claim 4, wherein said first predetermined value is approximately equal to the difference in said forced sensed when said hand is hanging freely from said robot arm immediately after said hand has been moved in a downward direction and said force sensed when said hand is hanging freely from said robot arm immediately after said hand has been moved in an upward direction.

6. The robotic system according to claim 1, wherein said reagent solution contained in said well includes a non-ionic surfactant.

7. The robotic system according to claim 6, wherein said non-ionic surfactant in said reagent solution has a concentration of at least 0.02%.

8. The robotic system according to claim 6, wherein said non-ionic surfactant in said reagent solution has a concentration of about 0.02%.

9. The robotic system according to claim 1, further comprising means for holding and heating said microtiter plate.

10. The robotic system according to claim 9, wherein said pipetting means comprises a plurality of pipet tips capable of being coupled and uncoupled from said hand.

11. The robotic system according to claim 10, further comprising:

means for storing said pipet tips;

means for storing a plurality of empty microtiter plates; and

means for heating said microtiter plate.

12. The robotic system according to claim 11, wherein said means for holding and heating said microtiter plate, said work surfaces, said reservoirs, and each of said storage means are arranged in a approximately circular array, and wherein said robot is centrally disposed within said array.

13. The robotic system according to claim 9, wherein said microtiter plate well defines a surface through which heat can flow into said well, and wherein said means for holding and heating said microtiter plate has a heat transfer surface formed therein that conforms to said microtiter plate well surface.

14. The robotic system according to claim 10, further comprising means for washing said pipet tips.

15. The robotic system according to claim 1, wherein said programmable controller has third logic means for instructing said robot to move said pipetting means away from said microtiter plate well bottom to allow aspiration of substantially all of said reagent solution from said microtiter plate well after said second logic means has determined that said pipetting means has made contact with said microtiter plate well bottom.

16. The robotic system according to claim 15, wherein said third logic means comprises means for instructing said robot to move said pipetting means in a direction away from said microtiter plate well bottom by a succession of incremental movements until said means for determining whether said pipetting means is still in contact with said microtiter plate well bottom has determined that said pipetting means is not still in contact with said microtiter plate well bottom.

17. The robotic system according to claim 15, wherein said third logic means comprises:

means for instructing said robot to move said pipetting means in a direction away from said microtiter plate well bottom by a first predetermined increment; and

means for determining whether said sensed has changed by more than a second predetermined pipetting means is still in contact with said microtiter plate well bottom after said pipetting means has been moved away from said plate well bottom by said first predetermined increment.

18. The robotic system according to claim 17, wherein:

said first logic means further comprises means for instructing said robot to move said pipetting means toward said microtiter plate well bottom by at least one additional set of incremental movements, each incremental movement in any such additional sets being successively smaller than each previous incremental movement in its respective set, additional sets of incremental movements being performed in succession until said second logic means has determined that said pipetting means has contacted said microtiter plate well bottom; and

the value of said first predetermined increment is variable, said third logic means having means for varying the magnitude of said first predetermined increment as a function of the number of said sets of incremental movements performed.

19. The robotic system according to claim 18, wherein said third logic means has means for varying the magnitude of said first predetermined increment so that said first predetermined increment increases as the number of said sets of incremental movements performed increases.

20. The robotic system according to claim 17, wherein said sensing means comprises means for sensing a force acting on said hand, and wherein said means for determining whether said pipetting means is still in contact with said microtiter plate well bottom further comprises:

means for instructing said robot to sense an initial force acting on said hand prior to said movement by said first predetermined increment; and

means for determining if said force sensed differs from said initial force by more than a first predetermined value after said pipetting means has been moved by said first predetermined increment.

21. The robotic system according to claim 20, wherein said first predetermined value is approximately equal to the difference in said forced sensed when said hand is hanging freely from said robot arm immediately after said hand has been moved in a downward direction and said force sensed when said hand is hanging freely from said robot arm immediately after said hand has been moved in an upward direction.

22. A robotic system for automatically performing reactions in sequence, comprising:

means for containing a fluid, said fluid containing means having a bottom;

a robot having means for drawing a fluid from said fluid containing means, said fluid drawing means normally spaced from said fluid containing means bottom by a distance;

means for causing relative motion between said fluid containing means and said fluid drawing means;

sensing means for generating a signal in response to said fluid drawing means making contact with said bottom of said fluid containing means; and

a programmable controller having (i) first logic means for instructing said relative motion causing means to reduce said distance between said bottom of said fluid containing means and said fluid drawing means by a series of discrete incremental movements, each of said discrete incremental movements in said series being separated by a finite period of time, said series of discrete incremental movements including a set of discrete incremental movements in which each discrete incremental movement is successively smaller than each previous discrete incremental movement in said set, and (ii) second logic means for determining whether said fluid drawing means has made contact with said bottom of said fluid containing means based on said signal from said sensing means during said finite period of time between each of said discrete incremental movements in said set of movements.

23. The robotic system according to claim 22, wherein said sensing means comprises means for sensing a force acting on said fluid drawing means, and wherein said second logic means comprises:

means for instructing said robot to sense an initial force acting on said fluid drawing means prior to said set of incremental movements;

means for determining if said force sensed differs from said initial force by more than a first predetermined value after each of said incremental movements in said set of movements.

24. The robotic system according to claim 22, wherein said programmable controller has third logic means for instructing said relative motion causing means to move said fluid drawing means in a direction away from said bottom of said fluid containing means by a first predetermined increment after said second logic means has determined that said fluid drawing means has made contact with said bottom of said fluid containing means.

25. The robotic system according to claim 24, wherein said third logic means comprises means for determining whether said fluid drawing means is still in contact with said bottom of said fluid containing means after said fluid drawing means has been moved in said direction away from said bottom of said fluid containing means by said first predetermined increment.

26. The robotic system according to claim 25, wherein said sensing means comprises means for sensing a force acting on said fluid drawing means, and wherein said means for determining whether said fluid drawing means is still in contact with said bottom of said fluid containing means further comprises:

means for instructing said robot to sense an initial force acting on said fluid drawing means prior to said movement by said first predetermined increment; and

means for determining if said force sensed differs from said initial force by more than a first predetermined value after said fluid drawing means has been moved by said first predetermined increment.