Identifying a base in a nucleic acid

• US 6,852,488 B2
• Filed: 02/06/2001
• Issued: 02/08/2005
• Est. Priority Date: 06/25/1993
• Status: Expired due to Fees

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Huang, Xiaohua, Lipshutz, Robert J., Fodor, Stephen P. A.
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): CALAMITA, HEATHER
• Application Number:
  US09/776,768
• Publication Number:
  US 20020177131A1
• Time in Patent Office: 1,463 Days
• Field of Search: 435/6, 435/7.1, 435/91.1, 435/91.2, 435/285.2, 435/287.2, 500/22.1, 500/23.1, 500/24.3, 500/33
• US Class Current: 435/6.14
• 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 2219/00497 Features relating to the solid phase supports
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/00576 fluorophore
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• 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 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00675 In-situ synthesis on the substrate
• CPC Class Codes: B01J 2219/00689 using computers
• 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 1/6869 : Methods for sequencing
• CPC Class Codes: C12Q 1/6874 : involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/161 : incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/186 : incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2525/204 : specific length of the oligonucleotides
• CPC Class Codes: C12Q 2565/501 : being an array of oligonucleotides
• CPC Class Codes: C12Q 2565/513 : characterised by the pattern of the arrayed oligonucleotides
• CPC Class Codes: C12Q 2565/518 : characterised by the immobilisation of the nucleic acid sample or target
• 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
• CPC Class Codes: G16B 25/00 : ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression

Abstract

Devices and techniques for hybridization of nucleic acids and for determining the sequence of nucleic acids. Arrays of nucleic acids are formed by techniques, preferably high resolution, light-directed techniques. Positions of hybridization of a target nucleic acid are determined by, e.g., epifluorescence microscopy. Devices and techniques are proposed to determine the sequence of a target nucleic acid more efficiently and more quickly through such synthesis and detection techniques.

Claims

1. A method of detecting a mutation in a target nucleic acid sequence versus a known sequence comprising:

a) exposing the target sequence to at least one known core sequence probe;

b) determining the binding affinity of the target sequence to the at least one known core sequence probe;

c) determining the binding affinity of the target sequence to at least one probe that has a single nucleotide variation as compared to the at least one known core sequence probe; and

d) comparing the binding affinity determined in step b) to the binding affinity determined in step c);

thereby detecting a mutation in a target nucleic acid sequence versus a known sequence.

2. A method of claim 1, wherein the at least one known core sequence probe is an array of known core sequence probes.

3. A method of claim 1, further comprising before step a), identifying one or more core sequences which are present within the known sequence and would be expected to have high affinity to the target sequence if the target sequence does not contain a mutation.

4. A method of claim 1, wherein the binding affinity to the known core sequence probe is plotted as affinity versus mismatch position, and normalized to the affinity of a perfect complement of the known core sequence probe.

5. A method of claim 4, further comprising determining that the target sequence comprises a mutation versus the known sequence if the pattern formed by the normalized affinity plot of the target sequence does not match the pattern formed by an affinity plot of the perfect complement.

6. A method of claim 1, further comprising determining if the binding affinity of the target sequence to the at least one known core sequence probe is weaker than the binding affinity of the known sequence to the at least one known core sequence probe.

7. A method of claim 1, wherein the at least one probe is between about 5 and 100 bases in length.

8. A method of claim 7, wherein the at least one probe is between about 5 and 50 bases in length.

9. A method of claim 8, wherein the at least one probe is between about 8 and 30 bases in length.

10. A method of claim 9, wherein the at least one probe is between about 8 and 15 bases in length.

11. A method of claim 1, wherein the mutation in the target sequence is indicative of a genetic disease.

12. A method of claim 11, wherein the genetic disease is sickle cell anemia.

13. A method of claim 11, wherein the genetic disease is cystic fibrosis.

14. A method of claim 11, wherein the genetic disease is associated with a P-53 mutation.

15. A method of claim 1, wherein the mutation in the target sequence is indicative of a genetic predisposition.

16. A method of claim 15, wherein the genetic predisposition is associated with a particular HLA Class I or HLA Class II gene.

17. A method of claim 15, wherein the ELA Class II gene is selected from the group consisting of DP, DQ and DR beta.

18. A method of claim 1, wherein detecting a mutation in the target sequence is comprised in a genetic evaluation.

19. A method of claim 18, wherein the genetic evaluation is a forensics evaluation.

20. The method of claim 1, wherein the binding affinity in step b) and c) is absolute binding affinity.

22. A method of claim 1, wherein the at least one core sequence probe or the at least one probe is attached to a bead.

21. A method of claim 25, wherein said at least one probe having a single nucleotide variation as compared to the at least one known core sequence probe is made by substituting A, C, T, U or G at each position of the at least one known core sequence probe.