Detection of base pair mismatches using RNAase A

• US 4,946,773 A
• Filed: 12/23/1985
• Issued: 08/07/1990
• Est. Priority Date: 12/23/1985
• Status: Expired due to Term

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Myers, Richard M., Maniatis, Thomas P.
• Primary Examiner(s): Kepplinger, Esther M.
• Assistant Examiner(s): NOT, DEFINED
• Application Number:
  US06/812,261
• Time in Patent Office: 1,688 Days
• Field of Search: 435/6, 435/172.3, 435/803, 436/501, 436/504, 436/63, 436/813, 935/2, 935/4, 935/21, 935/23, 935/78
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: Y10S 435/803 Physical recovery methods, e.g. chromatography, grinding
• CPC Class Codes: Y10S 436/813 Cancer

Abstract

A method for detecting and localizing single base substitutions in RNA or DNA that involves RNAase A cleavage of single base mismatches in RNA:RNA or RNA:DNA heteroduplexes. A RNA probe complementary to wild type DNA is annealed to the test DNA containing a single base substitution. Many of the possible single base mismatches can be cleaved by RNAase A. The location of the single base substitution can be determined by analyzing the sizes of the RNA cleavage products.

Claims

1. A method to detect at least one single base pair mismatch between a single stranded DNA or RNA sample and a single stranded RNA probe wherein said method comprises:

annealing together said single stranded DNA or RNA sample and said single stranded RNA probe to form a sample duplex,

providing a control duplex comprising said single stranded RNA probe and a polynucleotide strand, wherein said duplex is free from mismatches,

mixing said sample duplex and said control duplex with RNAase A,

separating by size the products of said RNAase A treatment,

and comparing the number and sizes of the products from said sample duplex with the products from identically treated said control duplex, as an indication of the presence or absence of a mismatch in said sample duplex.

2. The method of claim 1 wherein said separation is by gel electrophoresis.

3. The method of claim 2 wherein said electrophoresis is through agarose or polyacrylamide.

4. The method of claim 1 wherein said single stranded RNA probe is labelled.

5. The method of claim 4 wherein said label is radioactive or nonradioactive.

6. The method of claim 5 wherein said non-isotopic label is biotin.

7. The method of claim 1 wherein said method detects a single base deletion in said sample.

8. The method of claim 1 wherein said single stranded RNA probe is present at 10 fold or greater excess in comparison to said single stranded DNA or RNA sample.

9. The method of claim 8 wherein said excess is between 10 fold to 1,000 fold.

10. The method of claim 1 wherein said single stranded RNA probe is synthesized from an SP6 or T7 promoter.

11. The method of claim 1 wherein said single stranded RNA probe is less than 20 kb in size.

12. The method of claim 1 wherein said single stranded sample is less than 20 kb in size.

13. The method of claim 1 wherein said single stranded sample is derived from genomic DNA.

14. The method of claim 1 wherein said RNA sample is derived from somatic cells.