Computer-aided analysis system for sequencing by hybridization

• US 5,972,619 A
• Filed: 10/22/1998
• Issued: 10/26/1999
• Est. Priority Date: 04/01/1987
• Status: Expired due to Fees

• Current Assignee: Hyseq, Inc. (Arca Biopharma, Inc.)
• Original Assignee: Hyseq, Inc. (Arca Biopharma, Inc.)
• Inventors: Drmanac, Radoje T., Crkvenjakov, Radomir B.
• Primary Examiner(s): KETTER, JAMES S
• Application Number:
  US09/176,819
• Time in Patent Office: 369 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/24.33
• 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 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 identifying a base in 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;

comparing said H values to each other; and

identifying said base according to results of said comparisons.

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

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

2. A method of identifying an unknown base in 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;

comparing said H values to each other; and

identifying said unknown base according to results of said comparisons.

3. A method of identifying an unknown base in 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;

calculating a D value from the ratio of a higher H value to a lower H value; and

identifying said unknown base according to results of said calculations if said D value is greater than a predetermined ratio value.

4. A method of identifying an unknown base in a target nucleic acid sequence, comprising:

obtaining a first set of H values, each H value 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 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;

comparing at least one of said H values in said first set and at least one of said H values in said second set; and

identifying said unknown base according to results of said comparisons.

5. A method of identifying an unknown base in 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 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 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;

comparing at least one of said plurality of H values with said statistical data set; and

identifying said unknown base of said target according to results of said comparisons.