Polymorphism detection

• US 6,300,063 B1
• Filed: 05/08/1997
• Issued: 10/09/2001
• Est. Priority Date: 11/29/1995
• Status: Expired due to Term

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Sapolsky, Ronald, Lipshutz, Robert J., Ghandour, Ghassan
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US08/853,370
• Time in Patent Office: 1,615 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/22.1, 536/24.2, 536/24.3, 536/24.321
• 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 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00695 Synthesis control routines, e.g. using computer programs
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• 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: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof

Abstract

The present invention generally provides a rapid efficient method for analyzing polymorphic or biallelic markers, and arrays for carrying out these analyses. In general, the methods of the present invention employ arrays of oligonucleotide probes that are complementary to target nucleic acids which correspond to the marker sequences of an individual. The probes are typically arranged in detection blocks, each block being capable of discriminating the three genotypes for a given marker, e.g., the heterozygote or either of the two homozygotes. The method allows for rapid, automatable analysis of genetic linkage to even complex polygenic traits.

Claims

1. A method of identifying whether a target nucleic acid sequence includes a polymorphism, comprising:

hybridizing said target nucleic acid sequence to an array comprising at least one detection block of probes, said detection block including a first group of probes that are complementary to said target nucleic acid sequence except that the first group of probes includes a plurality of monosubstitutions of positions in said sequence that are within n bases of a base in said sequence that is complementary to said polymorphism, wherein n is from 0 to 5, and a second and third group of probes complementary to marker-specific regions upstream and downstream of the target nucleic acid sequence, wherein the third group of probes differs from the second set of probes at single bases corresponding to known mismatch positions; and

determining hybridization intensities of the target nucleic acid and the marker-specific regions to identify said polymorphism.

2. The method of claim 1, wherein said target nucleic acid comprises a detectable label.

3. The method of claim 2, wherein said detectable label is a fluorescent group.

6. The method of claim 1, wherein the polymorphism is identified as a result of Principal Component Analysis of hybridization intensities of the array of probes.

7. The method of claim 1, wherein at least two alleles of the polymorphism are known.

8. The method of claim 1, wherein said step of determining comprises:

a) calculating the control difference between the average of the hybridization intensities of the second group of probes, the hybridization intensities comprising control perfect matches (PM), minus the average of the hybridization intensities, the hybridization intensities comprising control single-base mismatches (MN);

b) calculating the possible perfect match intensity and a heteromismatch intensity from the hybridization intensities for each position of monosubstitutions of the first group of probes;

c) calculating the difference between the possible perfect match intensity and the heteromismatch intensity for each position of monosubstitutions of the first group of probes;

d) calculating a normalized difference (ND) by dividing the difference of step c) by control difference; and

e) using principal component analysis, identifying a polymorphism by comparing normalized differences between individuals in a population.

9. The method of claim 8, wherein an ND is calculated for at least two alleles of a polymorphism.

10. The method of claim 9, wherein homozygotes and heterozygotes are detected by combining principal component analysis for the at least two alleles.

4. A method of identifying whether a target nucleic acid sequence includes a polymorphism, comprising:

hybridizing said target nucleic acid sequence to an array comprising at least one detection block of probes, said detection block including a first group of probes that are complementary to said target nucleic acid sequence except that the first group of probes includes a plurality of monosubstitutions of positions in said sequence that are within n bases of a base in said sequence that is complementary to said polymorphism, wherein n is from 0 to 5, and a second and third group of probes complementary to marker-specific regions upstream and downstream of the target nucleic acid sequence, wherein the third group of probes differs from the second set of probes at single bases corresponding to known mismatch positions, wherein said target nucleic acid comprises a detectable label that is a binding group; and

determining hybridization intensities of the target nucleic acid and the marker-specific regions to identify said polymorphism.

5. The method of claim 4, wherein said binding group is selected from biotin, avidin and streptavidin.

11. A method of determining a genotype of a sample of nucleic acid sequences, comprising:

receiving a first hybridization intensity indicating hybridization affinity between a first set of probes and a first allele, the first set of probes being perfectly complementary to a portion of the first allele;

receiving a second hybridization intensity indicating hybridization affinity between a second set of probes and the first allele, the second set of probes having a single base mismatch with a portion of the first allele;

calculating a first difference between the first and second hybridization intensities;

receiving a third hybridization intensity indicating hybridization affinity between a third set of probes and a second allele, the third set of probes being perfectly complementary to a portion of the second allele;

receiving a fourth hybridization intensity indicating hybridization affinity between a fourth set of probes and the second allele, the fourth set of probes having a single base mismatch with a portion of the second allele;

calculating a second difference between the third and fourth hybridization intensities; and

comparing the first and second differences in order to determine the genotype of the sample of nucleic acid sequences.

12. The method of claim 11, wherein the first hybridization intensity is an average of hybridization intensities for perfect match probes for the first allele.

13. The method of claim 11, wherein the third hybridization intensity is an average of hybridization intensities for perfect match probes for the second allele.

14. The method of claim 11, wherein the comparing the first and second differences includes calculating a ratio of the first difference to the second difference.

15. The method of claim 14, wherein the comparing the first and second differences includes calculating a logarithm of the ratio of the first difference to the second difference.

16. The method of claim 15, further comprising determining the sample of nucleic acids is homozygous for the first allele if the logarithm is a positive number above a threshold.

17. The method of claim 15, further comprising determining the sample of nucleic acids is homozygous for the second allele if the logarithm is a negative number below a threshold.

18. The method of claim 15, further comprising determining the sample of nucleic acids is heterozygous for the first and second alleles if the logarithm is approximately zero.

19. The method of claim 11, further comprising determining the genotype of the sample of nucleic acid sequences by combining principal component analysis (PCA) for the first and second alleles.

20. The method of claim 11, further comprising performing a statistical check on the hybridization intensities to determine if the hybridization intensities will likely produce good results.