Method and apparatus for mass spectrometric genotyping

• US 20020146735A1
• Filed: 03/15/2002
• Published: 10/10/2002
• Est. Priority Date: 03/15/2001
• Status: Active Grant

• Current Assignee: Bruker Daltonik GmbH (Bruker Corporation)
• Original Assignee: Bruker Daltonik GmbH (Bruker Corporation)
• Inventors: Franzen, Jochen
• Granted Patent: US 6,770,443 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2523/319 Photocleavage, photolysis, photoactivation
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer
• CPC Class Codes: H01J 49/04 Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components
• CPC Class Codes: Y10T 436/24 Nuclear magnetic resonance, electron spin resonance or other spin effects or mass spectrometry

Abstract

Methods and equipment are provided for the mass spectrometric measurement of a large number of genotyping profiles, each formed by several tens to several hundreds of SNPs (single nucleotide polymorphisms). A multitude of chips each carrying an array of surface-bound oligonucleotide probes for mutations are synchronously processed. The chips are attached to plates such that they can be immersed in a multitude of wells with DNA samples requiring analysis while also serving directly as sample carriers in mass spectrometers. The multitude of wells can, for instance, take the form of microtitre plates. Primers may be used which possess a photolytically or chemically cleavable linker that bridges one base pair and does not hinder either the possibility of hybridization or enzymatic extension. Light or chemicals can then be used to cleave short chains particularly suitable for ionization by matrix assisted laser desorption and mass spectrometric analysis using time-of-flight mass spectrometers.

Claims

1. Method for mass spectrometric analysis of large numbers of genotyping profiles through the measurement of several mutations on one chip, wherein a multitude of chips is arranged together on a chip carrier in such a way that the chips may be processed individually in a matching multitude of different wells, and can be analyzed jointly on the chip carrier by mass spectrometry.

2. Method as in claim 1 wherein the chips are located on stems on a base plate of the chip carrier, and are arranged in an array in such a way that they may be immersed for processing in a matching array of wells.

3. Method as in claim 2 wherein the array of wells is a microtitre plate.

4. Method as in claim 2 wherein for the mass spectrometric measurement, the chip carrier is covered by a metallic frame that fills the gaps between the chips and contacts them electrically.

5. Method as in claim 4 wherein the chips are located on elastic stems on the chip carrier and have an appropriate shape so that their height and separation are adjusted by the complementary form in the frame to prepare them for mass spectrometric analysis.

6. Method as in claim 1 wherein various primers are bonded in various compartments on each chip, and wherein the primers are processed together with the amplification products of a single DNA sample and supply a genotyping profile for this DNA sample by mass spectrometric analysis.

7. Method as in claim 6 wherein the processing consists in a mutation-dependent primer extension of the various types of bonded primers that function as mutation probes.

8. Method as in claim 7 wherein the primers have cleavable linkers, that these linkers are cleaved prior to the mass spectrometric analysis, and the cleavage fragments are subjected to mass spectrometric analysis.

9. Method as in claim 8 wherein the cleavable linkers are located three, four or five nucleotides from the 3′

end.

10. Method as in claim 8 wherein the linkers are photo-cleavable.

11. Method as in claim 1 wherein the samples are ionized for mass spectrometric analysis by laser desorption.

12. Method as in claim 11 wherein the ionization is performed by matrix assisted laser desorption (MALDI).

14. A chip carrier as in claim 13 wherein the stems are elastic and the chips each have an appropriate shape that fits into the corresponding opening in a cover frame.

15. A chip carrier as in claim 13 wherein the chip array corresponds to the array of wells in a microtitre plate.

16. A chip carrier as in claim 13 wherein each chip contains arrays with primers bonded to the surface as mutation-specific probes for mass spectrometric analysis of a genotyping profile.

17. A chip carrier as in claim 16 wherein the primers contain cleavable linkers.

18. A chip carrier as in claim 16 wherein all the chips of the chip carrier are prepared for the same genotyping profile.

19. Application kit, consisting of several chip carriers as in claim 16 and the associated mixtures of primer pairs for PCR amplification of the DNA samples in preparation for further processing of the chips for analysis of the genotyping profile.

20. Application kit as in claim 19 wherein the package also contains the data for the mass spectrometric measurements of the end products of the primers in the compartments and their association with the alleles of the mutations in computer-readable form.

21. Application kit as in claim 19 wherein the package contains an executable computer program which processes the measured data and presents the genotyping profile.

13. Chip carrier for the mass spectrometric analysis of genotyping profiles on individual chips wherein the chips are located on stems on a base plate of the chip carrier and are arranged in an array in such a way that they may be immersed for processing in a matching array of wells.