Computer-aided analysis system for sequencing by hybridization

• US 6,316,191 B1
• Filed: 12/04/1998
• Issued: 11/13/2001
• Est. Priority Date: 04/01/1987
• Status: Expired due to Fees

• Current Assignee: Callida Genomics, Inc. (SBH Genomics, Inc.)
• Original Assignee: Hyseq, Inc. (Arca Biopharma, Inc.)
• Inventors: Crkvenjakov, Radomir B., Drmanac, Radoje T.
• Primary Examiner(s): KETTER, JAMES S
• Application Number:
  US09/205,865
• Time in Patent Office: 1,075 Days
• Field of Search: 435/6
• US Class Current: 435/6.18
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2537/165 Mathematical modelling, e.g. logarithm, ratio
• CPC Class Codes: C12Q 2545/114 involving a quantitation step
• CPC Class Codes: C12Q 2565/50 Detection characterised by immobilisation to a surface
• 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: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/10 Sequence alignment; Homology search
• CPC Class Codes: G16B 30/20 Sequence assembly

Abstract

The conditions under which oligonucleotide probes hybridize preferentially with entirely complementary and homologous nucleic acid targets are described. Using these hybridization conditions, overlapping oligonucleotide probes associate with a target nucleic acid. Following washes, positive hybridization signals are used to assemble the sequence of a given nucleic acid fragment. Representative target nucleic acids are applied as dots. Up to 100,000 probes of the type (A,T,C,G)(A,T,C,G)N8(A,T,C,G) are used to determine sequence information by simultaneous hybridization with nucleic acid molecules bound to a filter. Additional hybridization conditions are provided that allow stringent hybridization of 6-10 nucleotide long oligomers which extends the utility of the invention. A computer process determines the information sequence of the target nucleic acid which can include targets with the complexity of mammalian genomes. Sequence generation can be obtained for a large complex mammalian genome in a single process.

Claims

1. A method of analyzing a target nucleic acid sequence, comprising:

obtaining a plurality of H values, each H value corresponding to an extent of hybridization of an oligonucleotide probe with at least one nucleic acid sequence including said target sequence and each oligonucleotide probe differing from each other by at least a single base; and

comparing said H values to each other, thereby analyzing the target nucleic sequence.

5. The method of any one of claims 1, 2, 3, or 4, wherein said method is performed on a computer.

6. The method according to claim 5, wherein the oligonucleotide probes are in an array.

2. A method of analyzing a target nucleic acid sequence, comprising:

obtaining a plurality of H values, each H value corresponding to an extent of hybridization of an oligonucleotide probe with said target nucleic acid sequence and each oligonucleotide probe differing from each other by at least a single base; and

calculating a D value from the ratio of a higher H value to a lower H value, thereby analyzing the target nucleic sequence.

3. A method of analyzing a target nucleic acid sequence, comprising:

obtaining a first set of H values, each H value in said first set corresponding to an extent of hybridization of an oligonucleotide probe with a control nucleic acid sequence and each probe differing from each other by at least a single base;

obtaining a second set of H values, each H value in said second set corresponding to an extent of hybridization of an oligonucleotide probe with said target nucleic acid sequence and each probe differing from each other by at least a single base; and

comparing at least one of said H values in said first set with at least one of said H values in said second set,

thereby analyzing the target nucleic sequence.

4. A method of analyzing a target nucleic acid sequence, comprising:

obtaining a statistical data set corresponding to statistics about a plurality of experiments producing H values, each H value in said statistical data set corresponding to an extent of hybridization of an oligonucleotide probe with a control nucleic acid sequence and each oligonucleotide probe differing from each other by at least a single base;

obtaining a plurality of H values, each H value in said plurality corresponding to an extent of hybridization of an oligonucleotide probe with said target sequence and each probe differing from each other by at least a single base; and

comparing at least one of said H values in said plurality with said statistical data set, thereby analyzing the target nucleic sequence.