High dynamic range apparatus for separation and detection of polynucleotide fragments

• US 6,014,213 A
• Filed: 03/18/1997
• Issued: 01/11/2000
• Est. Priority Date: 12/12/1994
• Status: Expired due to Fees

• Current Assignee: Bayer Healthcare LLC (Bayer AG)
• Original Assignee: Visible Genetics, Inc. (Bayer AG)
• Inventors: Zaleski, Henryk, Renfrew, John A., Waterhouse, Paul, Izmailov, Alexandre M., Cassidy, James W.
• Primary Examiner(s): Font, Frank G.
• Assistant Examiner(s): Punnoose, Roy M.
• Application Number:
  US08/819,910
• Time in Patent Office: 1,029 Days
• Field of Search: 356/344, 356/244, 204/229 R, 204/461, 204/466, 204/612, 436/172
• US Class Current: 356/344
• CPC Class Codes: G01N 27/44721 by optical means
• CPC Class Codes: G01N 30/8603 with integration or differentiation

Abstract

A high dynamic range apparatus for separation and detection of polynucleotide fragments has a housing adapted to receive an electrophoresis gel holder containing an electrophoresis gel loaded with fluorophore-labeled samples; one or more laser diodes for providing radiation of a frequency suitable for excitation of the fluorophore which irradiates a an array of excitation/detection sites on the electrophoresis gel; an array of detectors aligned with the excitation/detection sites for collecting fluorescent emissions; and one or more components for increasing the dynamic range of the instrument by at least an order of magnitude. These components, which can be used individually or in combination include detectors that are connected to a signal processing system that modulates the period of signal integration employed so that large signals are totaled at short time intervals and smaller signals are totaled at longer time intervals; the use of a beam splitter to produces a high intensity beam of emitted light and a low intensity beam of emitted light from each excitation/detection site; and a device for modulating the intensity of the excitation beam can be used to improve the dynamic range of the instrument.

Claims

1. An electrophoresis separation and real-time detection apparatus comprising:

(a) a housing adapted to receive an electrophoresis gel holder containing an electrophoresis gel loaded with fluorophore-labeled samples;

(b) one or more laser diodes for providing radiation of a frequency suitable for excitation of the fluorophore;

(c) means for directing the radiation to a plurality of excitation/detection sites on the electrophoresis gel;

(d) an array of detectors aligned with the excitation/detection sites for collecting fluorescent emissions and producing an electrical output signal indicative of the intensity of the fluorescent emissions; and

(e) means for increasing the dynamic range of the apparatus by at least one order of magnitude.

2. The apparatus according to claim 1, wherein the means for increasing the dynamic range comprises an integrator for producing an integrated output signal from the detector, said integrator integrating over alternating long intervals and short intervals, the long intervals being at least ten times longer than the short intervals.

3. The apparatus according to claim 2, wherein the long intervals are at least 100 times longer than the short intervals.

4. The apparatus according to claim 1, wherein the means for increasing the dynamic range comprises a beam splitter which produces a high intensity beam of emitted light and a low intensity beam of emitted from each excitation/detection site.

5. The apparatus according to claim 4, wherein the high intensity beam is at least ten times as intense as low intensity beam.

6. The apparatus according to claim 4, wherein two detectors in the array of detectors are aligned with each excitation/detection site, a first of said detectors being aligned to detect the high intensity beam and a second of said detectors being aligned to detect the low intensity beam.

7. The apparatus according to claim 4, wherein the means for increasing the dynamic range further comprises an integrator responsive to the output signal and producing an integrated output signal therefrom, said integrator integrating over alternating long intervals and short intervals, the long intervals being at least ten times longer than the short intervals.

8. The apparatus according to claim 7, wherein the long intervals are at least 100 times longer than the short intervals.

9. The apparatus according to claim 1, wherein the means for increasing the dynamic range comprises means for modulating the intensity of the excitation source between a high intensity level and a low intensity level.

10. The apparatus according to claim 9, wherein the intensity of the excitation source is modulated in a square wave function.

11. The apparatus according to claim 9, wherein the means for increasing the dynamic range further comprises an integrator responsive to the output signal and producing an integrated output signal therefrom, said integrator integrating over alternating long intervals and short intervals, the long intervals being at least ten times longer than the short intervals.

12. The apparatus according to claim 11, wherein the long intervals are at least 100 times longer than the short intervals.

13. The apparatus according to claim 11, wherein the means for increasing the dynamic range further comprises a beam splitter which produces a high intensity beam of emitted light and a low intensity beam of emitted from each excitation/detection site.

14. The apparatus according to claim 13, wherein the high intensity beam is at least ten times as intense as low intensity beam.

15. The apparatus according to claim 13, wherein two detectors in the array of detectors are aligned with each excitation/detection site, a first of said detectors being aligned to detect the high intensity beam and a second of said detectors being aligned to detect the low intensity beam.

16. The apparatus according to claim 13, wherein the means for increasing the dynamic range further comprises an integrator responsive to the output signal and producing an integrated output signal therefrom, said integrator integrating over alternating long intervals and short intervals, the long intervals being at least ten times longer than the short intervals.

17. The apparatus according to claim 16, wherein the long intervals are at least 100 times longer than the short intervals.

18. The apparatus according to claim 9, wherein the means for increasing the dynamic range comprises a beam splitter which produces a high intensity beam of emitted light and a low intensity beam of emitted from each excitation/detection site.

19. The apparatus according to claim 18, wherein the high intensity beam is at least ten times as intense as low intensity beam.

20. The apparatus according to claim 18, wherein two detectors in the array of detectors are aligned with each excitation/detection site, a first of said detectors being aligned to detect the high intensity beam and a second of said detectors being aligned to detect the low intensity beam.

21. The apparatus according to claim 2, wherein the means for increasing the dynamic range comprises a beam splitter which produces a high intensity beam of emitted light and a low intensity beam of emitted from each excitation/detection site.

22. The apparatus according to claim 21, wherein the high intensity beam is at least ten times as intense as low intensity beam.

23. The apparatus according to claim 21, wherein two detectors in the array of detectors are aligned with each excitation/detection site, a first of said detectors being aligned to detect the high intensity beam and a second of said detectors being aligned to detect the low intensity beam.