Analysis of genetic polymorphisms and gene copy number

• US 6,468,744 B1
• Filed: 11/17/1999
• Issued: 10/22/2002
• Est. Priority Date: 01/03/1997
• Status: Expired due to Term

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Hubbell, Earl A., Lipshutz, Robert J., Miyada, Charles G., Cronin, Maureen T., Chee, Mark, Fodor, Stephen P. A., Huang, Xiaohua C., Lobban, Peter E., Sheldon, Edward L., Morris, MacDonald S.
• Primary Examiner(s): Brusca, John S.
• Application Number:
  US09/341,399
• Time in Patent Office: 1,070 Days
• Field of Search: 435/6
• US Class Current: 435/6.11
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00432 Photolithographic masks
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00617 by chemical means
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00644 the porous medium being present in discrete locations, e.g. gel pads
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00711 Light-directed synthesis
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2565/513 : characterised by the pattern of the arrayed oligonucleotides
• CPC Class Codes: C12Q 2600/106 : Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
• CPC Class Codes: C12Q 2600/156 : Polymorphic or mutational markers
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 60/14 : for creating libraries

Abstract

The invention provides methods for detecting variations in polymorphic sites and/or variations in gene copy number. The methods are particularly useful for analysis of biotransformation genes, such as cytochromes P450.

Claims

1. A method of determining copy number of a gene present in an individual, comprising:

analyzing a plurality of polymorphic sites in a chromosome containing a gene from an individual to determine the number of different polymorphic forms present at each site; and

assigning the copy number of the gene as the highest number of polymorphic forms present at a single site.

2. The method of claim 1, wherein the plurality of polymorphic sites are in a noncoding segment of the gene.

3. The method of claim 1, wherein the plurality of polymorphic sites are silent polymorphisms.

4. The method of claim 3, wherein the at least one polymorphic site is present in an intronic segment of the gene.

5. The method of claim 1, wherein the pluralility of polymorphic sites comprises at least 10 sites.

6. The method of claim 1, wherein the plurality of polymprhic sites comprises at least 50 sites.

7. The method of claim 1, further comprising:

obtaining a tissue sample from the individual containing the gene and amplifying the gene or a fragment thereof.

8. The method of claim 1, wherein the analyzing comprises:

contacting a nucleic acid comprising the gene or a fragment thereof with an array of oligonucleotides, the array comprising a plurality of subarrays, each subarray spanning a polymorphic site and complementarity to at least one polymorphic form of the gene at the site;

detecting hybridization intensities of the nucleic acid to the oligonucleotides in the array, whereby the pattern of hybridization indicates the number of polymorphic forms present at each polymorphic site.

9. The method of claim 8, wherein the subarrays each comprise a plurality of probe groups, each probe group complementarity to a different polymorphic form at the site.

10. The method of claim 9, wherein a probe group comprises

(a) a first probe set comprising a plurality of probes spanning a polymorphic site of the gene, each probe comprising a segment of at least six nucleotides exactly complementary to a polymorphic form of the gene at the site, the segment including at least one interrogation position complementary to a corresponding nucleotide in the polymorphic form,

(b) a second probe set comprising a corresponding probe for each probe in the first probe set, the corresponding probe in the second probe set being identical to a sequence comprising the corresponding probe from the first probe set or a subsequence of at least six nucleotides thereof that includes the at least one interrogation position, except that the at least one interrogation position is occupied by a different nucleotide in each of the two corresponding probes from the first and second probe sets.

11. The method of claim 9, wherein a probe group comprises

(a) a first probe set comprising a plurality of probes spanning a polymorphic site, each probe comprising a segment of at least six nucleotides exactly complementary to a subsequence of a polymorphic form at the site, the segment including at least one interrogation position complementary to a corresponding nucleotide in the polymorphic form,

(b) second, third and fourth probe sets, each comprising a corresponding probe for each probe in the first probe set, the probes in the second, third and fourth probe sets being identical to a sequence comprising the corresponding probe from the first probe set or a subsequence of at least six nucleotides thereof that includes the at least one interrogation position, except that the at least one interrogation position is occupied by a different nucleotide in each of the four corresponding probes from the four probe sets.

12. The method of claim 1, wherein a single polymorphic form is present at each of the plurality of sites and the copy number of the gene is assigned as 1.

13. The method of claim 1, wherein two polymorphic forms are present at one site and a single polymorphic form is present at each other of the plurality of sites, and the copy number of the gene is assigned as 2.

14. The method of claim 1, wherein three polymorphic forms are present at a first polymorphic site, a single polymorphic form is present at a second polymorphic site and two polymorphic forms are present at a third polymorphic site and the copy number of the gene is assigned as 3.

15. The method of claim 1, further comprising analyzing a phenotype-determining polymorphic site in the gene to determine which polymorphic form(s) are present at the site.

16. The method of claim 15, further comprising diagnosing a phenotype of the patient based on the polymorphic form(s) present at the phenotype-determining polymorphic site.