IR-MALDI mass spectrometry of nucleic acids using liquid matrices

• US 6,706,530 B2
• Filed: 05/10/1999
• Issued: 03/16/2004
• Est. Priority Date: 05/07/1998
• Status: Expired due to Fees

• Current Assignee: Agena Bioscience Inc. (Mesa Laboratories Inc.)
• Original Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Hillenkamp, Franz
• Primary Examiner(s): Soderquist, Arlen
• Application Number:
  US09/309,125
• Publication Number:
  US 20030148528A1
• Time in Patent Office: 1,772 Days
• Field of Search: 436/43, 436/91, 436/86, 436/93-94, 436/173-174, 436/181
• US Class Current: 436/94
• CPC Class Codes: B01J 2219/00702 Processes involving means for analysing and characterising the products
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: H01J 49/164 Laser desorption/ionisation, e.g. matrix-assisted laser desorption/ionisation [MALDI] sample holders H01J49/0418
• CPC Class Codes: Y10T 436/11 Automated chemical analysis
• 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 436/142222 Hetero-O [e.g., ascorbic acid, etc.]
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]
• CPC Class Codes: Y10T 436/24 Nuclear magnetic resonance, electron spin resonance or other spin effects or mass spectrometry
• CPC Class Codes: Y10T 436/25 including sample preparation
• CPC Class Codes: Y10T 436/25875 Gaseous sample or with change of physical state

Abstract

Mass spectrometry of large nucleic acids by infrared Matrix-Assisted Laser Desorption/Ionization (MALDI) using a liquid matrix is reported.

Claims

1. A method for detecting the presence or absence of a nucleic acid in a sample, comprising:

(a) mixing the nucleic acid with a liquid matrix;

(b) analyzing the mixture using infrared matrix-assisted laser desorption/ionization mass spectrometry;

(c) detecting the nucleic acid by determining the mass of the nucleic acid by a mass spectrometric format for separation and detection of desorbed and ionized molecules,

wherein;

the nucleic acid is detected if present in the sample;

the nucleic acid comprises at least 280 nucleotides; and

the accuracy for determining the mass of the nucleic acid is within about 1%.

2. The method of claim 1, wherein the sample is a biological sample.

3. The method of claim 1, wherein the nucleic acid comprises at least 2000 nucleotides.

4. The method of claim 1, wherein the nucleic acid is DNA.

5. The method of claim 1, wherein the nucleic acid comprises at least 1200 nucleotides.

6. The method of claim 1, wherein the nucleic acid is RNA.

7. The method of claim 1, wherein the presence or absence of the nucleic acid is indicative of the presence or absence of a genetic disease.

8. The method of claim 1, wherein the presence or absence of the nucleic acid is indicative of the presence or absence of a birth defect.

9. The method of claim 1, wherein the presence or absence of the nucleic acid is indicative of the presence or absence of an infectious organism.

10. The method of claim 1, wherein the presence or absence of the nucleic acid is indicative of the identity of a subject.

11. The method of claim 1, wherein delayed ion extraction is used in the matrix-assisted laser desorption/ionization mass spectrometry.

12. The method of claim 1, wherein the matrix has at least one of the following properties:

(1) is miscible with a nucleic acid compatible solvent (ii) is vacuum stable, and (iii) is of an appropriate viscosity to facilitate dispensing of micro- to nenoliter volumes of matrix alone or mixed with a nucleic acid compatible solvent.

13. The method of claim 1, wherein the matrix is selected from the group consisting of:

an alcohol, a carboxylic acid, a primary or secondary amine, a primary or secondary amide, a nitrile, a hydrazine and a hydrazide.

14. The method of claim 13, wherein the matrix is a substituted or unsubstituted alcohol.

15. The method of claim 14, wherein the alcohol is selected from the group consisting of glycerol, 1,2- or 1,3-propane diol, 1,2-, 1,3- or 1,4-butane diol and triethanolamine.

16. The method of claim 14, wherein the matrix is glycerol.

17. The method of claim 1, wherein the nucleic acid/matrix mixture is deposited onto a substrate selected from the group consisting of beads, capillaries, flat supports, pins and wafers.

18. The method of claim 1, wherein the nucleic acid/matrix mixture is deposited onto a substrate comprising a material selected from the group consisting of steel, gold, silver, aluminum, copper or silicon.

19. The method of claim 1, wherein the nucleic acid/matrix mixture is deposited onto a substrate comprising a material selected from the group consisting of polyethylene, polyprdpylene, polyamide and polyvinylidenefluoride.

20. The method of claim 1, wherein the nucleic acid/matrix mixture is deposited onto fields of a chip array, arrays of pins or beads in pits of flat surfaces.

21. The method of claim 1, wherein the sample is cooled to a temperature below about 20°

C.

22. A method for detecting the presence or absence of a nucleic acid in a sample, comprising:

(a) mixing the nucleic acid with a matrix which forms a homogeneous solution with the nucleic acid;

(b) analyzing the mixture using infrared matrix-assisted laser desorption/ionization mass spectrometry; and

(c) detecting the nucleic acid by determining the mass of the nucleic acid by a mass spectrometric format for separation and detection of desorbed and ionized molecules,

wherein;

the nucleic acid is detected if present in the sample;

the nucleic acid comprises at least 280 nucleotides; and

the accuracy for determining the mass of the nucleic acid is within about 1%.