Method of detecting mutations in DNA and RNA

• US 4,656,127 A
• Filed: 04/23/1984
• Issued: 04/07/1987
• Est. Priority Date: 04/22/1983
• Status: Expired due to Fees

• Current Assignee: Amersham International PLC White Lion Road Amersham Buckinghamshire HP7 9LL England
• Original Assignee: Amersham International Plc (General Electric Company)
• Inventors: Mundy, Christopher R.
• Primary Examiner(s): NOT, DEFINED
• Assistant Examiner(s): Jay, Jeremy
• Application Number:
  US06/602,720
• Time in Patent Office: 1,079 Days
• Field of Search: 435/6, 435/29, 435/34, 435/91, 435/172.1, 435/172.3, 435/193, 435/199, 436/63, 436/94, 436/501, 536/27, 935/9, 935/10, 935/76, 935/77, 935/78
• US Class Current: 435/6.12
• CPC Class Codes: C12N 15/00 Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2521/539 Deaminase
• CPC Class Codes: C12Q 2525/125 incorporating agents resulting in resistance to degradation
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

A method of detecting a mutation of a specific nucleotide base in a target nucleic acid chain comprises: (a) hybridizing a labelled probe to the target to form a hybrid in which one end of the probe is positioned adjacent the specific base; (b) adding a nucleotide derivative, e.g. a thionucleotide, under conditions to cause it to join to the end of the probe if it is complementary to the specific base; (c) digesting the hybrid using an exonuclease enzyme under conditions such that the nucleotide derivative protects the probe from digestion; and observing the presence or absence of label attached to the target. The method can be used to detect mutations even when these are not present at restriction enzyme cleavage sites, and does not require the preliminary steps of restriction digestion, gel electrophoresis and DNA (or RNA) blotting.

Claims

1. A method of detecting a mutation of a specific nucleotide base in a target nucleic acid chain by providing a linear probe complementary to a part of the nucleic acid chain extending in one direction from the specific base,

(a) hybridizing the probe to the target to form a nucleic acid hybrid, whereby one end of the probe becomes hybridized to the nucleic acid chain substantially adjacent the specific base,

(b) admixing with the hybrid a nucleotide derivative, under conditions appropriate for probe extension, so as to cause the nucleotide derivative to join on to the end of the probe only if the specific base in the target is, or is not, the mutation to be detected, a probe carrying said nucleotide derivative being resistant to digestion under particular conditions, wherein one of the probe and the nucleotide derivative is labelled,

(c) subjecting the hybrid to digestion by an exonuclease enzyme under the said conditions whereby the double-stranded portion thereof is progressively digested starting at the said end of the probe unless the end has had said nucleotide derivative joined to it,

(d) removing portions of the probe which are no longer hybridized to the nucleic acid chain,

(e) and using the presence or absence of the probe remaining after digestion to detect a mutation of the specific nucleotide base in the target.

2. The method as claimed in claim 1, wherein the probe is provided such that in step (a) one end becomes hybridized to the nucleic acid chain immediately adjacent the specific base.

3. The method as claimed in claim 2, wherein step (b) is:

(b) admixing with the hybrid a nucleotide derivative under conditions appropriate for probe extension so as to cause the nucleotide derivative to join on to the end of the probe if it is complementary to the specific base, a probe carrying said nucleotide derivative being resistant to digestion under particular conditions.

4. The method as claimed in claim 2, wherein step (b) is:

(b)(i) admixing with the hybrid a chain-terminating nucleotide compound under conditions appropriate for probe extension so as to cause it to join on to the end of the probe if it is complementary to the specific base,

(b)(ii) admixing with the resulting hybrid one or more nucleotide derivatives under conditions appropriate for probe extension so as to cause them to join on to the end of the probe if a chain-terminating nucleotide compound is not already present, a probe carrying said one or more nucleotide derivatives being resistant to digestion under particular conditions.

5. The method as claimed in claim 1, wherein the probe is provided such that in step (a) one end becomes hybridized to the nucleic acid chain a few bases away from the specific base.

6. The method as claimed in claim 5, wherein step (b) is:

(b) admixing the hybrid a nucleotide derivative, together with one or two other different nucleotides, under conditions appropriate for probe extension so as to cause them to join on to the end of the probe, including the nucleotide derivative if it is complementary to the specific base, a probe carrying said nucleotide derivative being resistant to digestion under particular conditions.

7. The method as claimed in claim 5, wherein step (b) is:

(b)(i) admixing with the hybrid a chain-terminating nucleotide compound, together with one or two other different nucleotides, under conditions appropriate for probe extension so as to cause them to join on to the end of the probe, including the chain-terminating nucleotide compound if it is complementary to the specific base,

(b)(ii) admixing with the resulting hybrid one or more nucleotide derivatives under conditions appropriate for probe extension so as to cause them to join on to the end of the probe if a chain-terminating nucleotide compound is not already present, a probe carrying said one or more nucleotide derivatives being resistant to digestion under particular conditions.

8. The method as claimed in claim 1, wherein the target is DNA or RNA.

9. The method as claimed in claim 1, wherein the probe is labelled.

10. The method as claimed in claim 1, wherein the probe is DNA or RNA.

11. The method as claimed in claim 1, wherein the nucleotide derivative used in step (b) is a thionucleotide.

12. A method as claimed in claim 1, wherein there is used in step (c) an exonuclease enzyme that digests double-stranded nucleic acid chains only from the 3'"'"' ends.

13. The method as claimed in claim 1, wherein the probe extension is achieved by the addition of calf thymus DNA polymerase under polymerisation conditions.

14. A method as claimed in claim 1, wherein the target is immobilised.