Labeling by simultaneous ligation and restriction

• US 5,093,245 A
• Filed: 01/26/1988
• Issued: 03/03/1992
• Est. Priority Date: 01/26/1988
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Kronick, Mel N., McBride, Lincoln J., Whiteley, Norman M., Keith, Douglas H.
• Primary Examiner(s): NOT, DEFINED
• Application Number:
  US07/148,757
• Time in Patent Office: 1,498 Days
• Field of Search: 435/6, 435/91, 435/35, 435/810, 536/27
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

Termini of restricted double-stranded DNA fragments are modified by ligating the fragments with terminal phosphate-free double-stranded oligonucleotides having a complementary terminus in the presence of a restriction enzyme and a ligase, where joining of the complementary ends results in loss of the restriction enzyme recognition sequence.

Claims

1. A method for extending DNA fragments with a ligand comprising:

combining in a reaction mixture said fragments in the simultaneous presence of ligase and a restriction enzyme which cuts in or near the recognition site, with dsDNA comprising said ligand and having a terminus complementary to a terminus of said fragments, wherein said fragments have predetermined termini as a result of digestions with said restriction enzyme, said fragments have terminal phosphates and said complementary termini of said dsDNA lack a terminal phosphate and ligation of said dsDNA results in sequence different from the sequence recognized and cleaved by said restriction enzyme,

whereby said fragments and dsDNA are covalently joined.

2. A method according to claim 1, wherein said ligand is a label capable of providing a detectable signal.

3. A method according to claim 1, wherein said ligand is a DNA sequence of interest.

4. A method according to claim 3, including the additional steps of:

selecting a fraction of said covalently joined fragments and dsDNA;

adding to said selected fraction a DNA polymerase, nucleotide triphosphates and sequences complementary to the strands of said dsDNA; and

carrying out a polymerase chain reaction to amplify said selected fraction.

5. A method according to claim 4, wherein said selecting is size selection by electrophoresis.

6. A method according to claim 1, wherein said DNA fragments are obtained by adding precursor DNA to be fragmented to said reaction mixture, whereby said precursor DNA is cleaved by said restriction enzyme.

7. A method for labeling DNA fragments with a ligand or radionuclide comprising:

combining said fragments in the simultaneous presence of ligase and a restriction enzyme which cuts in or near the recognition site, with ligand or radionuclide labeled dsDNA of fewer than about 200 bp and having a terminus complementary to a terminus of said fragments, wherein said fragments have predetermined termini as a result of digestion with said restriction enzyme, said fragments have terminal phosphates and said complementary termini of said dsDNA lack a terminal phosphate and ligation of said dsDNA results in a sequence different from the sequence recognized and cleaved by said restriction enzyme,

whereby said fragments and dsDNA are covalently joined to label said fragments.

8. A method according to claim 7, wherein said ligand is biotin.

9. A method according to claim 7, wherein said ligand is a radionuclide, fluorescer or an enzyme.

10. A method according to claim 7, wherein said termini have cohesive ends.

11. A method according to claim 7, wherein said fragments are obtained by the step of:

digesting genomic DNA with at least one restriction enzyme to provide fragments of less than about 200 kbp.

12. A labeling composition for binding to a restricted dsDNA fragment resulting in loss of the restriction recognition site by simultaneous restriction and ligation, said composition comprising:

a bridging strand and first and second strands hybridized to said bridging strand, wherein said strands hybridize contiguously to said bridging strand;

said first strand is a labeling strand providing for a double-stranded DNA sequence of interest and a fluorescent label;

said second strand is an adapter strand hybridizing to said bridging strand to provide a cohesive end or blunt end with said bridging strand, and wherein said adapter strand contains a particular sequence necessary to destroy the recognition site of a restriction enzyme of interest.