Methods for generating databases and databases for identifying polymorphic genetic markers

• US 7,668,658 B2
• Filed: 10/15/2002
• Issued: 02/23/2010
• Est. Priority Date: 10/13/1999
• Status: Expired due to Fees

• Current Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Original Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Braun, Andreas, Koster, Hubert, He, Liyan, Van Den Boom, Dirk, Chiu, Norman, Ping, Yip, Rodi, Charlie, Jurinke, Christian
• Primary Examiner(s): Clow; Lori A
• Application Number:
  US10/273,321
• Publication Number:
  US 20030180749A1
• Time in Patent Office: 2,688 Days
• Field of Search: 702/19, 702/32, 435/6, 435/183, 435/174, 530/300, 530/350, 536/24.3
• US Class Current: 702/19
• 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 correlating a change in the frequency of a polymorphism in a gene or a non-coding portion of a genome with increasing age, comprising;

obtaining samples from healthy individuals within an age range;

pooling the samples from healthy individuals within the age range;

determining the frequency of a polymorphism in said samples, wherein said polymorphism or said gene have not been correlated with susceptibility to morbidity, early mortality, or morbidity and early mortality; and

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

2. The method of claim 1, wherein the frequency of said polymorphism in said samples is in a database, and said database is sorted to identify correlations between the frequency of said allele and age.

3. The method of claim 1, wherein said polymorphism comprises a SNP.

4. The method of claim 1, further comprising correlating said polymorphism with a gene or pathway in which said polymorphism is involved.

5. The method of claim 4, comprising using a database to correlate said polymorphism with said gene or pathway.

6. The method of claim 1, further comprising identifying the locus of said polymorphism and assessing or deducing the function of a gene at said locus.

7. The method of claim 6, comprising using a database to assess or deduce the function of said gene.

8. The method of claim 1, wherein the frequency of said polymorphism decreases with increasing age.

9. The method of claim 1, wherein the frequency of said polymorphism increases with increasing age.

10. The method of claim 1, wherein said sample comprises body tissue or fluid from said organism.

11. The method of claim 1, wherein the frequency of the polymorphism is determined using mass spectrometry.

12. The method of claim 11, wherein 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.

13. The method of claim 11, wherein samples are presented to the mass spectrometer as arrays on chips.

14. The method of claim 1, wherein said method comprises obtaining genomic nucleic acid from a sample from a healthy organism.

15. The method of claim 14, further comprising amplifying a portion of the genomic nucleic acid to produce amplified fragments thereof.

16. The method of claim 1, wherein the presence of the polymorphism is identified by a method comprising primer oligo base extension.

17. The method of claim 16, wherein primer oligo base extension comprises hybridizing a nucleic acid molecule from a sample from a healthy organism with a primer oligonucleotide that is complementary to the nucleic acid molecule at a site adjacent to the polymorphic marker.

18. The method of claim 17, further comprising:

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

b) contacting the optionally-immobilized nucleic acid molecule 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 polymorphic marker is extended onto the primer; and

c) detecting the extended primer, thereby identifying the presence of the polymorphism.

19. The method of claim 18, wherein detection of the extended primer is effected by mass spectrometry, comprising:

ionizing and volatizing the product of step c); and

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

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

identifying samples by sorting a database comprising 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, wherein said database is sorted according to a selected parameter to identify samples that match the selected parameter;

isolating a nucleic acid molecule from each identified sample;

pooling each isolated nucleic acid molecule; and

identifying the presence of a polymorphism in the nucleic acid molecule by a method comprising primer oligo base extension.

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

identifying samples by sorting a database comprising 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, wherein said database is sorted according to a selected parameter to identify samples that match the selected parameter;

isolating a biopolymer from each identified sample;

pooling each isolated 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 identifying any polymorphisms;

wherein;

the control mass spectrum is obtained from either

a) samples represented by datapoints in said database that were not selected by sorting said database;

or

b) samples identified by sorting said database according to a different selected parameter.

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

isolating a biopolymer from samples of body tissue or fluid from a plurality of organisms;

pooling each isolated biopolymer;

cleaving the pooled biopolymers to produce fragments thereof;

obtaining a mass spectrum of the resulting fragments;

determining a frequency of each fragment, whereby an average frequency is calculated; and

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

23. The method of claim 1, further comprising using genetic mapping to identify a gene or pathway that correlates with said polymorphism.

24. The method of claim 1, further comprising identifying the locus of the polymorphism and assessing or deducing its function.

25. A method for identifying a gene or gene product comprising a polymorphism whose frequency decreases with increasing age, comprising:

determining the frequency of a polymorphism in pooled samples from healthy individuals within an age range, wherein said polymorphism has not been correlated with susceptibility to morbidity, early mortality, or morbidity and early mortality;

correlating a change in frequency of the polymorphism with increasing age in a healthy population; and

identifying the gene that comprises said polymorphism.

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

identifying the presence of a polymorphism, wherein said polymorphism has not been correlated with susceptibility to morbidity, early mortality, or morbidity and early mortality;

obtaining samples from individuals of a given age range, ethnicity, or gender;

pooling samples from individuals of a given age range, ethnicity, or gender;

determining the frequency of the polymorphism in said samples; and

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

27. The method of claim 26, wherein said biopolymers comprise genomic nucleic acid molecules.

28. The method of claim 26, wherein the frequency of the polymorphism is determined using mass spectrometry.

29. The method of claim 26, wherein the presence of the polymorphism is identified by a method comprising primer oligo base extension.

30. A method for identifying a genetic marker in a gene or a non-coding portion of a genome that correlates with age, comprising

a. identifying a previously unknown genetic marker using mapping, sequencing, or amplification, of pooled samples, wherein the gene has not been correlated with susceptibility to morbidity, early mortality, or morbidity and early mortality;

b. analyzing the presence of the genetic marker among a population;

c. entering the analysis of the presence of the genetic marker into a database;

d. determining whether the frequency of the presence of the genetic marker correlates with age; and

e. determining the locus of the genetic marker.

31. The method of claim 30, wherein the database is sorted by age.

32. The method of claim 30, further comprising assessing the function or the effect of the genetic marker.

33. The method of claim 30, wherein the frequency of the genetic marker decreases with age.