Method for analyzing an organic sample

• US 5,210,412 A
• Filed: 01/31/1991
• Issued: 05/11/1993
• Est. Priority Date: 01/31/1991
• Status: Expired due to Fees

• Current Assignee: Wayne State University
• Original Assignee: Wayne State University
• Inventors: Levis, Robert J., Romano, Louis J.
• Primary Examiner(s): Anderson, Bruce C.
• Application Number:
  US07/648,282
• Time in Patent Office: 831 Days
• Field of Search: 250/288, 250/282, 250/423 P, 250/287
• US Class Current: 250/288
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: H01J 49/0463 Desorption by laser or particle beam, followed by ionisation as a separate step sample holder per se H01J49/0418
• CPC Class Codes: H01J 49/162 Direct photo-ionisation, e.g. single photon or multi-photon ionisation

Abstract

Described is a method and apparatus for analyzing an organic sample. In the preferred embodiment, this method and apparatus allows the determination of the base sequence of a nucleic acid by determining the molecular weights of the components of a biological sample. The method uses either a pre-existing chromophore or the covalent attachment of an ionizable chromophore to a biological sample followed by the vaporization of these molecules by exposure to an intense pulse of electromagnetic radiation in the presence of a matrix which strongly absorbs the radiation. The gaseous molecules are subsequently extracted into an evacuated ionization chamber and then exposed to electromagnetic radiation at a wavelength which specifically excites the chromophore covalently attached to the biological sample. The molecular weights of these ionized species are then determined by mass spectroscopic analysis. This method of molecular weight determination allows for a DNA sequencing method. Four samples of DNA molecules are prepared such that each is covalently linked to an ionizable chromophore and each is fragmented by a means which results in fragments within each of the four samples that terminate at a different one of the nucleotides A, C, G or T. Each of these four fragmented samples is subsequently introduced into the said apparatus in order to determine their molecular weights hence providing the strand length of the fragments. The data generated may be analyzed by high-speed computer, the four data sets correlated, and the sequence deduced.

Claims

1. A method analyzing an organic sample, comprising the steps of:

a) providing an organic sample in a medium which absorbs lights wherein the sample is a nucleic acid that has been fragmented such that each fragment terminates with one of the bases adenosine (A), cytosine (C), quanosine (G), or thymidine (T);

b) vaporizing the sample and the medium into the gaseous state;

c) inoizing the vaporized sample; and

d) detecting the contents of the vaporized, ionized sample.

2. The method of claim 1 wherein the organic sample to be analyzed has an ionizable chromophore covalently bonded to the sample.

3. The method of claim 2 wherein the chromophore to be bonded to the sample is a dye.

4. The method of claim 2 wherein the chromophore to be bonded to the sample is a fluorescent dye.

5. The method of claim 2 wherein the chromophore to be bonded to the sample is selected from the group consisting of fluorescein, rhodamine, tetramethyl rhodamine, sulforhodamine, nitrobenzo-2-oxa-1-diazole and derivatives thereof which can covalently react with a biomolecule sample.

6. The method of claim 5 wherein the derivatives are selected from the group consisting of iodoacetamide, maleimide, isothiocyanate and succinimidyl carboxylate.

7. The method of claim 1 wherein the light absorbing medium is an organic dye.

8. The method of claim 1 wherein the light absorbing medium is in the liquid state.

9. The method of claim 1 wherein the light absorbing medium is in the solid state.

10. The method of claim 1 wherein the contents of the vaporized, ionized sample is determined by utilizing a mass spectrometer of the time-of-flight type.

11. The method of claim 1 wherein the sample is a protein.

12. The method of claim 1 wherein the vaporizing step is performed by a laser.

13. The method of claim 1 wherein the ionizing step is performed by a laser.

14. The method of claim 1 wherein the light absorbing medium has the ability to absorb light at a wavelength greater than 500 nanometers.

15. The method of claim 1 wherein the light absorbing medium is comprised of a dye.

16. The method of claim 1 wherein the amount of light absorbing medium used ranges from about 100 to about 100,000 parts by weight for each part of the sample in the light absorbing medium.

17. A method for determining the base sequence of a nucleic acid strand wherein four separate nucleic acid samples derived from the strand are fragmented such that each fragment in a particular sample terminates at one of the bases adenosine (A), cytosine (C), guanosine (G), or thymidine (T), comprising the steps of:

(a) providing an organic sample comprised of the four samples of the nucleic acid in a light absorbing medium which absorbs light;

(b) vaporizing the sample and the light absorbing medium into the gaseous state;

(c) ionizing the vaporized sample; and

(d) detecting the contents of the vaporized, ionized sample.

18. The method of claim 17 wherein the nucleic acid sample to be analyzed has an ionizable chromophore covalently bonded to the sample.

19. The method of claim 17 wherein the light absorbing medium is an organic dye.

20. The method of claim 17 wherein the light absorbing medium is in the liquid state.

21. The method of claim 17 wherein the light absorbing medium is in the solid state.

22. The method of claim 17 wherein the contents of the vaporized, ionized sample is determined by utilizing a mass spectrometer of the time-of-flight type.

23. The method of claim 17 wherein the vaporizing step is performed by a laser.

24. The method of claim 17 wherein the ionizing step is performed by a laser.

25. The method of claim 17 wherein the light absorbing medium has the ability to absorb light at a wave length less than 500 nanometers.

26. The method of claim 17 wherein the light absorbing medium is comprised of a dye.

27. The method of claim 17 wherein the chromophore to be bonded to the sample is a dye.

28. The method of claim 17 wherein the chromophore to be bonded to the sample is a fluorescent dye.

29. The method of claim 17 wherein the chromophore to be bonded to the sample is selected from the group consisting of fluorescein, rhodamine, tetramethyl rhodamine, sulforhodamine, nitrobenzo-2-oxa-1-diazole and derivatives thereof which can covalently react with a biomolecule sample.

30. The method of claim 17 wherein the derivatives are selected from the group consisting of iodoacetamide, maleimide, isothiocyanate and succinimidyl carboxylate.

31. The method of claim 17 wherein the amount of light absorbing medium used ranges from about 100 to about 100,000 parts by weight for each part of the sample in the light absorbing medium.

32. An apparatus that does analyze nucleic acid material present in a visible light absorbing medium comprising:

an optical source for generating electromagnetic radiation in the visible light region capable of vaporizing the nucleic acid sample material in the visible light absorbing medium which absorbs visible light;

a source of ionizing radiation capable of ionizing the vaporized sample; and

an apparatus, juxtaposed to the vaporized, ionized sample, capable of determining the contents of the vaporized, ionized sample.

33. The apparatus of claim 32 further comprising a pulsed nozzle positioned between the sample and the output of the source of vaporizing energy which is timed to open in concert with the exposure of the sample to the electromagnetic radiation and capable of remaining open for a sufficient of period of time necessary to allow the vaporized sample to enter the ionizing chamber.

34. The apparatus of claim 32 wherein the apparatus that is capable of determining the contents of the vaporized, ionized sample is a Channeltron.

35. The apparatus of claim 34 wherein the apparatus capable of determining the contents of the vaporized ionized sample is a mass spectrometer.

36. The apparatus of claim 35 wherein the mass spectrometer is a time-of-flight type mass spectrometer.

37. The apparatus of claim 32 wherein the source of vaporizing radiation is a laser.

38. The apparatus of claim 32 wherein the source of ionizing radiation is a laser.