Method for automated flourescent tagged sample detecting

• US 6,210,973 B1
• Filed: 02/05/1999
• Issued: 04/03/2001
• Est. Priority Date: 12/05/1994
• Status: Expired due to Term

• Current Assignee: Microlab Diagnostics Limited
• Original Assignee: John W. Pettit
• Inventors: Pettit, John W.
• Primary Examiner(s): ALEXANDER, LYLE
• Application Number:
  US09/245,220
• Time in Patent Office: 788 Days
• Field of Search: 436/172, 436/91, 436/94, 436/56
• US Class Current: 436/172
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: G01N 27/44721 by optical means
• CPC Class Codes: Y10T 436/14 Heterocyclic carbon compound [i.e. , O, S, N, Se, Te, as only ring hetero atom]
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

An automated DNA sequencing apparatus and method is disclosed in which a laser beam having a predetermined wavelength is sequentially focused on a plurality of lanes of DNA fragments migrating in a polyacrylamide gel and in which the DNA fragments have been tagged with fluorescent compounds. The resulting fluorescent light given off by each of the tagged DNA fragments is collected and diffracted by means of an acousto-optic tunable filter and the level of the diffracted light is then detected and analyzed in order to determine the DNA sequences of the analyzed DNA fragments. The automatic DNA sequencing system of the present invention operates under computer control to repeat a series of measurements of the fluorescent data at a multiple number of wavelengths such that the entire spectrum of fluorescence emissions from all four fluorescent tagged DNA bases can be measured at any number of wavelengths.

Claims

1. A method for automating the detecting of flourescent tagged samples, comprising the steps of:

tagging each of a predetermined plurality of samples with a fluorescent tag;

stimulating the emission of light at its characteristic wavelength from said fluorescent tagged plurality of samples;

focusing the emission of fluorescent light from said fluorescent tagged plurality of samples as substantially parallel rays of light onto an acousto-optic tunable filter;

controlling the operation of said acousto-optic tunable filter under control of a digital data processor such that only light at a predetermined wavelength is diffracted and exits from said acoustic-optic tunable filter at at least one angle to a main beam of light exiting from said acousto-optic tunable filter; and

detecting at least one diffracted light beam exiting said acoustic-optic tunable filter to produce an electrical representation of said fluorescent light emissions of said plurality of samples.

2. The method of claim 1, further including the step of storing said electrical representations of said fluorescent light emissions of said plurality of samples.

3. The method of claim 1, wherein said step of controlling the operation of said acousto-optic tunable filter includes sequentially stepping said acousto-optic tunable filter through a plurality of wavelengths, wherein each of said plurality of wavelengths corresponds to a different one of said fluorescent tags.

4. A method for automating the detecting of flourescent tagged samples, comprising the steps of:

tagging a predetermined plurality of samples to be detected with a fluorescent tag;

generating a beam of light for stimulating the emission of fluorescent light from said fluorescent tagged plurality of samples;

optimizing the wavelength of the stimulating beam of light prior to its impingement on said fluorescent tagged plurality of samples;

focussing the emission of fluorescent light from said fluorescent tagged plurality of samples as substantially parallel rays of light onto a first acousto-optic tunable filter;

controlling the operation of said acousto-optic tunable filter using a digital data processor such that only light at a predetermined wavelength is diffracted and exits from said acoustic-optic tunable filter at at least one angle to a main beam of light exiting from said acousto-optic tunable filter; and

detecting at least one diffracted light beam exiting said acoustic-optic tunable filter to produce an electrical representation of said fluorescent light emissions of said plurality of samples.

5. The method of claim 4, further including the step of storing said electrical representations of said fluorescent light emissions of said plurality of samples.

6. The method of claim 4, wherein said step of controlling the operation of said acousto-optic tunable filter includes sequentially stepping said acousto-optic tunable filter through a plurality of wavelengths, wherein each of said plurality of wavelengths corresponds to a different one of said fluorescent tags.

7. The method of claim 4, further including the steps of:

modulating said diffracted light which exits said first acousto-optic tunable filter; and

synchronously detecting said modulated diffracted light in said detecting step.

8. The method of claim 4, wherein said step of optimizing comprises the step of filtering said beam of light through a second acousto-optic tunable filter which is commanded to a predetermined setting by said digital data processor dependent upon the expected wavelength of the emission of fluorescent light by each of said fluorescent tagged plurality of samples.

9. The method of claim 8, further including the step of modulating said beam of light passing through said second acousto-optic tunable filter.

10. The method of claim 8, further including the steps of:

modulating said beam of light passing through said second acousto-optic tunable filter;

modulating said diffracted light which exits said first acousto-optic tunable filter; and

synchronously detecting said modulated diffracted light in said detecting step.

11. The method of claim 10, wherein said synchronously detecting step includes the steps of:

generating first and second modulating signals suitable for modulating said diffracted light exiting from said first acousto-optic tunable filter and said beam of light passing through said second acousto-optic tunable filter;

combining said first and second modulating signals to form a combined modulating signal; and

using said combined modulating signal to synchronously detect said modulated diffracted light in said detecting step.

12. A method for automating the detecting of flourescent tagged samples, comprising the steps of:

tagging each of a predetermined plurality of samples to be used to identify a different predetermined characteristic with a different fluorescent tag;

stimulating the emission of light at its characteristic wavelength from said fluorescent tagged plurality of samples;

focusing the emission of fluorescent light from said fluorescent tagged plurality of samples as substantially parallel rays of light onto an acousto-optic tunable filter;

controlling the operation of said acousto-optic tunable filter using a digital data processor such that only light at a predetermined wavelength is diffracted and exits from said acoustic-optic tunable filter at at least one angle to a main beam of light exiting from said acousto-optic tunable filter; and

detecting at least one diffracted light beam exiting said acoustic-optic tunable filter to produce an electrical representation of said fluorescent light emissions of said plurality of samples.

13. The method of claim 12, further including the step of storing said electrical representations of said fluorescent light emissions of said plurality of samples.

14. The method of claim 12, wherein said step of controlling the operation of said acousto-optic tunable filter includes sequentially stepping said acousto-optic tunable filter through a plurality of wavelengths, wherein each of said plurality of wavelengths corresponds to a different one of said fluorescent tags.

15. A method for automating the detecting of flourescent tagged samples, comprising the steps of:

tagging each of a predetermined plurality of samples to be used to identify a different predetermined characteristic with a different fluorescent tag;

generating a beam of light for stimulating the emission of fluorescent light from said fluorescent tagged plurality of samples;

optimizing the wavelength of the stimulating beam of light prior to its impingement on said fluorescent tagged plurality of samples;

focussing the emission of fluorescent light from said fluorescent tagged plurality of samples as substantially parallel rays of light onto a first acousto-optic tunable filter;

controlling the operation of said acousto-optic tunable filter using a digital data processor such that only light at a predetermined wavelength is diffracted and exits from said acoustic-optic tunable filter at at least one angle to a main beam of light exiting from said acousto-optic tunable filter; and

detecting at least one diffracted light beam exiting said acoustic-optic tunable filter to produce an electrical representation of said fluorescent light emissions of said plurality of samples.

16. The method of claim 15, further including the step of storing said electrical representations of said fluorescent light emissions of said plurality of samples.

17. The method of claim 15, wherein said step of controlling the operation of said acousto-optic tunable filter includes sequentially stepping said acousto-optic tunable filter through a plurality of wavelengths, wherein each of said plurality of wavelengths corresponds to a different one of said fluorescent tags.

18. The method of claim 15, further including the steps of:

modulating said diffracted light which exits said first acousto-optic tunable filter; and

synchronously detecting said modulated diffracted light in said detecting step.

19. The method of claim 15, wherein said step of optimizing comprises the step of filtering said beam of light through a second acousto-optic tunable filter which is commanded to a predetermined setting by said digital data processor dependent upon the expected wavelength of the emission of fluorescent light by each of said fluorescent tagged plurality of samples.

20. The method of claim 19, further including the step of modulating said beam of light passing through said second acousto-optic tunable filter.

21. The method of claim 19, further including the steps of:

modulating said beam of light passing through said second acousto-optic tunable filter;

modulating said diffracted light which exits said first acousto-optic tunable filter; and

synchronously detecting said modulated diffracted light in said detecting step.

22. The method of claim 21, wherein said synchronously detecting step includes the steps of:

generating first and second modulating signals suitable for modulating said diffracted light exiting from said first acousto-optic tunable filter and said beam of light passing through said second acousto-optic tunable filter;

combining said first and second modulating signals to form a combined modulating signal; and

using said combined modulating signal to synchronously detect said modulated diffracted light in said detecting step.