Method of determining an ordered sequence of subfragments of a nucleic acid fragment by hybridization of oligonucleotide probes

• US 5,492,806 A
• Filed: 04/12/1993
• Issued: 02/20/1996
• 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): Zitomer, Stephanie W.
• Application Number:
  US08/045,912
• Time in Patent Office: 1,044 Days
• Field of Search: 435/5, 435/6
• US Class Current: 435/5
• 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 2525/204 specific length of the oligonucleotides
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The sequence of a given nucleic acid fragment is read by the hybridization and assembly of positively hybridizing exactly complementary oligonucleotide probes through overlapping subfragments. By simultaneous hybridization of nucleic acid subfragments bound onto a filter, representing single-stranded phage vector with a cloned insert, with about 50,000 to 100,000 groups of probes, the main type of which is (A,T,C,G)(A,T,C,G)N8(A,T,C,G), information for computer determination of a sequence of DNA having the complexity of a mammalian genome are obtained in one step. To obtain a maximally completed sequence, three libraries cloned into the phage vector, M13, are used. The process can be easily and entirely robotized for factory reading of complex genomic fragments or DNA molecules.

Claims

1. A method for determining an ordered sequence of one or more subfragments of a nucleic acid fragment by ordering oligonucleotide probe sequences which are complementary to subfragments of said nucleic acid fragment, comprising the steps of:

(a) contacting said nucleic acid fragment with a set of oligonucleotide probes, each having a unique internal portion selected from the group consisting of 5-mers, 6-mers, 7-mers, and 8-mers, which may be in tandem, under conditions which distinguish a subset of oligonucleotide probes which are exactly complementary to one or more subfragments of said nucleic acid fragment from oligonucleotide probes which are not exactly complementary to one or more subfragments of said nucleic acid fragment;

(b) detecting the subset of oligonucleotide probes which are exactly complementary to one or more subfragments of said nucleic acid fragment; and

(c) ordering said subset of oligonucleotide probes by compiling overlapping sequences of said subset of oligonucleotide probes which are exactly complementary to one or more subfragments of said nucleic acid fragment, thereby determining the ordered sequence of one or more subfragments of a nucleic acid fragment.