Method of sequencing DNA

• US 5,405,746 A
• Filed: 09/22/1993
• Issued: 04/11/1995
• Est. Priority Date: 03/23/1988
• Status: Expired due to Term

• Current Assignee: Qiagen GmbH (Qiagen NV)
• Original Assignee: Cemu Bioteknik AB
• Inventors: Uhlen, Mathias
• Primary Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US08/124,628
• Time in Patent Office: 566 Days
• Field of Search: 435/6, 435/91.1, 435/91.2
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 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 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

The invention relates to a method of sequencing target DNA in which said DNA is provided in double stranded form immobilized on a solid support via one terminus of one of the two strands thereof and is then subjected to strand separation whereby the unattached strand is removed prior to sequencing the immobilized strand. The target DNA, e.g genomic DNA, may initially be amplified by the PCR method using at least one oligonucleotide primer which is either attached to a solid support or carries means for attachment to a solid support.

Claims

1. A method for solid-phase sequencing of one strand of double-stranded DNA comprising the steps of:

(A) providing said double-stranded DNA;

(B) immobilizing on a solid support one terminus of one of the two strands of said double-stranded DNA;

(C) subjecting said immobilized double-stranded DNA to strand separation, thereby providing an immobilized strand and an unattached strand;

(D) removing said unattached strand, thereby providing an immobilized single-stranded DNA;

(E) sequencing said immobilized single-stranded DNA.

2. The method of claim 1 wherein strand separation is effected by melting.

3. The method of claim 1 wherein prior to step (B), a functional group which binds to the solid support is introduced at only one terminus of one of the strands of the double stranded DNA.

4. The method of claim 1 wherein the double stranded DNA is a plasmid containing a target sequence;

the plasmid is cleaved at a restriction site outside the target sequence;

a functional group is introduced at at least one terminus of one strand only of said DNA;

the functionality DNA is cleaved at a further restriction site outside the target sequence; and

the functionalized target sequence is contacted with a solid support carrying a substance with affinity for said functional group.

5. The method of claim 1 wherein the double stranded DNA is a plasmid containing a target sequence;

the plasmid DNA is cleaved at a restriction site outside the target sequence and at one or more restriction sites within the target sequence; and

oligonucleotides are ligated at both termini of the plasmid DNA generated by said cleavage, wherein one of said oligonucleotides carries a functional group which binds to a solid support and the other oligonucleotide is capable of hybridizing to a general sequencing primer,

6. The method of claim 1 wherein the immobilized double stranded DNA is produced by the polymerase chain reaction method using at least one oligonucleotide primer carrying means for attachment to a solid support.

7. The method of claim 6 wherein the immobilized double stranded DNA is produced by amplification of genomic DNA by the polymerase chain reaction method.

8. The method of claim 1 wherein said immobilized single stranded DNA is sequenced by the primer extension method,

9. The method of claim 3 wherein said functional group is a biotin, avidin, streptavidin or thiol group and said solid support carries groups binding thereto.

10. The method of claim 1 wherein the solid support is in particulate form or is a microtitre well.

11. The method of claim 1 wherein stand separation is effectuated by treatment with aqueous alkali.

12. The method of claim 9, wherein said functional group is biotin.

13. The method of claim 11 wherein said strand separation is effectuated by treatment with 0.15M NaOH.