Methods for generating databases and databases for identifying polymorphic genetic markers

• US 20030180749A1
• Filed: 10/15/2002
• Published: 09/25/2003
• Est. Priority Date: 10/13/1999
• Status: Active Grant

• Current Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Original Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Boom, Dirk Van Den, Yip, Ping, Braun, Andreas, Koster, Hubert, He, Liyan, Chiu, Norman, Rodi, Charlie, Jurinke, Christian
• Granted Patent: US 7,668,658 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 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2561/125 Ligase Detection Reaction [LDR]
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: Y10T 436/24 Nuclear magnetic resonance, electron spin resonance or other spin effects or mass spectrometry

Abstract

Processes and methods for creating a database of genomic samples from healthy human donors, methods that use the database to identify and correlate polymorphic genetic markers and other markers with diseases and conditions are provided.

Claims

1. A method for determining whether a polymorphism correlates with suceptibility to morbidity, early mortality, or morbidity and early mortality, comprising;

identifying a polymorphism; and

determining the frequency of the polymorphism with increasing age in a healthy population.

2. The method of claim 1, wherein the polymorphism is identified by detecting the presence of target nucleic acids in a sample by a method, comprising the steps of:

a) hybridizing a first oligonucleotide to the target nucleic acid;

b) hybridizing a second oligonucleotide to an adjacent region of the target nucleic acid;

c) ligating the hybridized oligonucleotides; and

d) detecting hybridized first oligonucleotide by mass spectrometry as an indication of the presence of the target nucleic acid.

3. The method of claim 1, wherein the polymorphism is identified by detecting target nucleic acids in a sample by a method, comprising the steps of:

a) hybridizing a first oligonucleotide to the target nucleic acid and hybridizing a second oligonucleotide to an adjacent region of the target nucleic acid;

b) contacting the hybridized first and second oligonucleotides with a cleavage enzyme to form a cleavage product; and

c) detecting the cleavage product by mass spectrometry as an indication of the presence of the target nucleic acid.

4. The method of claim 3 wherein the samples are from subjects in a healthy database.

5. The method of claim 1, wherein the polymorphism is identified by identifying target nucleic acids in a sample by primer oligo base extension (probe).

10. The method of claim 1, wherein the polymorphism is identified by a method, comprising:

sorting the database according to a selected parameter to identify samples that match the selected parameter, wherein the database comprises;

datapoints representative of a plurality of healthy organisms from whom biological samples are obtained, wherein each

datapoint is associated with data representative of the organism type and other identifying information.;

isolating a biopolymer from each identified sample;

optionally pooling each isolated biopolymer;

optionally amplifying the amount of biopolymer;

cleaving the pooled biopolymers to produce fragments thereof;

obtaining a mass spectrum of the resulting fragments and comparing the mass spectrum with a control mass spectrum to identify differences between the spectra and thereby identifing any polymorphisms;

wherein;

the control mass spectrum is obtained from unsorted samples in the collection or samples sorted according to a different parameter.

11. The method of claim 10, wherein cleaving is effected by contacting the biopolymer with an enzyme.

12. The method of claim 11, wherein the enzyme is selected from the group consisting of nucleotide glycosylase, a nickase and a type IIS restriction enzyme.

13. The method of claim 10, wherein the biopolymer is a nucleic acid or a protein.

14. The method of claim 10, wherein the the mass spectrometric format is selected from among Matrix-Assisted Laser Desorption/Ionization, Time-of-Flight (MALDI-TOF), Electrospray (ES), IR-MALDI, Ion Cyclotron Resonance (ICR), Fourier Transform and combinations thereof.

15. The method of claim 1, wherein the polymorphism is identified by a method, comprising:

obtaining samples of body tissue or fluid from a plurality of organisms;

isolating a biopolymer from each sample;

pooling each isolated biopolymer;

optionally amplifying the amount of biopolymer;

cleaving the pooled biopolymers to produce fragments thereof;

obtaining a mass spectrum of the resulting fragments;

comparing the frequency of each fragment to identify fragments present in amounts lower than the average frequency, thereby identifying any polymorphisms.

16. The method of claim 15, wherein cleaving is effected by contacting the biopolymer with an enzyme.

17. The method of claim 16, wherein the enzyme is selected from the group consisting of nucleotide glycosylase, a nickase and a type IIS restriction enzyme.

18. The method of claim 15, wherein the biopolymer is a nucleic acid or a protein.

19. The method of claim 15, wherein the the mass spectrometric format is selected from among Matrix-Assisted Laser Desorption/Ionization, Time-of-Flight (MALDI-TOF), Electrospray (ES), IR-MALDI, Ion Cyclotron Resonance (ICR), Fourier Transform and combinations thereof.

20. The method of claim 15, wherein the samples are obtained from healthy subjects.

6. The method of 5, wherein primer oligo base extension, comprises:

a) obtaining a nucleic acid molecule that contains a target nucleotide;

b) optionally immobilizing the nucleic acid molecule onto a solid support, to produce an immobilized nucleic acid molecule;

c) hybridizing the nucleic acid molecule with a primer oligonucleotide that is complementary to the nucleic acid molecule at a site adjacent to the target nucleotide;

d) contacting the product of step c) with a composition comprising a dideoxynucleoside triphosphate or a 3′

-deoxynucleoside triphosphate and a polymerase, so that only a dideoxynucleoside or 3′

-deoxynucleoside triphosphate that is complementary to the target nucleotide is extended onto the primer; and

e) detecting the extended primer, thereby identifying the target nucleotide.

7. The method of claim 6, wherein detection of the extended primer is effected by mass spectrometry, comprising:

ionizing and volatizing the product of step d) ; and

detecting the extended primer by mass spectrometry, thereby identifying the target nucleotide.

8. The method of claim 7, wherein;

samples are presented to the mass spectrometer as arrays on chips; and

each sample occupies a volume that is about the size of the laser spot projected by the laser in a mass spectrometer used in matrix-assisted laser desorption/ionization (MALDI) spectrometry.

9. A method for determining a polymorphism that correlates with age, ethnicity or gender, comprising:

identifying a polymorphism; and

determining the frequency of the polymorphism with increasing age, with ethnicity or with gender in a healthy population.