Method and apparatus for mass spectrometric genotyping

• US 6,770,443 B2
• Filed: 03/15/2002
• Issued: 08/03/2004
• 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
• Primary Examiner(s): Fredman, Jeffrey
• Assistant Examiner(s): Chakrabarti, Arun
• Application Number:
  US10/099,157
• Publication Number:
  US 20020146735A1
• Time in Patent Office: 872 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/24.3, 935/6, 436/518
• US Class Current: 435/6.12
• 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, the method comprising the steps of:

(a) providing a sample carrier plate configured to hold a plurality of sample chips mounted in an array on stems or pillars, with each chip carrying DNA probes bonded to the chip surface;

(b) providing a well plate with a plurality of wells arranged in an array that matches the chip array;

(c) locating processing liquid in each of the wells;

(d) contacting the processing liquid of each of the wells with a different one of the chips;

processing the DNA robes in the processing liquid by chemical or enzymatic reactions;

(f) locating the sample carrier plate in a mass spectrometer; and

(g) analyzing products of the reactions by mass spectrometry.

2. Method as in claim 1 wherein the array of wells is part of a microtitre plate.

3. Method as in claim 1 wherein for the mass spectrometric measurement, the chip carrier is embedded in a metallic frame that fills the gaps between the chips, contacts them electrically and forms an even surface with the surfaces of the chips.

4. Method as in claim 3, wherein the chips are fixed on elastic stems on the chip carrier and have an appropriate shape so that their height and separation may be adjusted by a complementary form in the embedding frame.

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

6. Method as in claim 5 wherein the processing consists in a mutation-dependent primer extension of the various types of bonded primers that function as mutation probes.

7. Method as in claim 5 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.

8. Method as in claim 7 wherein the cleavable linkers are located three, four or five nucleotides from the 3′

end.

9. Method as in claim 7 wherein the linkers are photo-cleavable.

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

11. Method as in claim 10 wherein the ionization is performed by matrix assisted laser desorption (MALDI).

12. Sample carrier plate for the mass spectrometric analysis of genotyping profiles on individual chips, the sample carrier plate comprising:

a base plate;

stems or pillars fixed to a base plate in an array;

chips fixed to the tops of the stems or pillars; and

DNA probes bonded to the surfaces of the chips in defined locations.

13. A sample carrier plate as in claim 12 wherein the stems or pillars of the chips are elastic, and wherein the chips each have an appropriate shape that fits into a corresponding opening in an embedding frame.

14. A sample carrier plate as in claim 12 wherein an arrangement of the chip array corresponds to an arrangement of the array of wells in a microtitre plate.

15. A sample carrier plate as in claim 12 wherein each chip contains arrays with primers bonded to the surface as mutation-specific probes for mass spectrometric analysis of a genotyping profile.

16. A sample carrier plate as in claim 15 wherein the primers contain cleavable linkers.

17. A sample carrier plate as in claim 15 wherein all the chips of the chip carrier are prepared for the same genotyping profile.

18. An application kit comprising:

sample carrier plates according to claim 15; and

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.

19. An application kit as in claim 18 wherein the kit 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.

20. An application kit as in claim 18 wherein the kit contains an executable computer program which processes the measured data and presents the genotyping profile.